Leeuwen, J.P.T.M. van

Bone fragility and decline in stem cells in prematurely aging DNA repair deficient trichothiodystrophy mice (Article)

2012-08-01T00:00:00Z

Trichothiodystrophy (TTD) is a rare, autosomal recessive nucleotide excision repair (NER) disorder caused by mutations in components of the dual functional NER/basal transcription factor TFIIH. TTD mice, carrying a patient-based point mutation in the Xpd gene, strikingly resemble many features of the human syndrome and exhibit signs of premature aging. To examine to which extent TTD mice resemble the normal process of aging, we thoroughly investigated the bone phenotype. Here, we show that female TTD mice exhibit accelerated bone aging from 39 weeks onwards as well as lack of periosteal apposition leading to reduced bone strength. Before 39 weeks have passed, bones of wild-type and TTD mice are identical excluding a developmental defect. Albeit that bone formation is decreased, osteoblasts in TTD mice retain bone-forming capacity as in vivo PTH treatment leads to increased cortical thickness. In vitro bone marrow cell cultures showed that TTD osteoprogenitors retain the capacity to differentiate into osteoblasts. However, after 13 weeks of age TTD females show decreased bone nodule formation. No increase in bone resorption or the number of osteoclasts was detected. In conclusion, TTD mice show premature bone aging, which is preceded by a decrease in mesenchymal stem cells/osteoprogenitors and a change in systemic factors, identifying DNA damage and repair as key determinants for bone fragility by influencing osteogenesis and bone metabolism.

Age-related skeletal dynamics and decrease in bone strength in DNA repair deficient male trichothiodystrophy mice (Article)

2012-04-10T00:00:00Z

Accumulation of DNA damage caused by oxidative stress is thought to be one of the main contributors of human tissue aging. Trichothiodystrophy (TTD) mice have a mutation in the Ercc2 DNA repair gene, resulting in accumulation of DNA damage and several features of segmental accelerated aging. We used male TTD mice to study the impact of DNA repair on bone metabolism with age. Analysis of bone parameters, measured by micro-computed tomography, displayed an earlier decrease in trabecular and cortical bone as well as a loss of periosteal apposition and a reduction in bone strength in TTD mice with age compared to wild type mice. Ex vivo analysis of bone marrow differentiation potential showed an accelerated reduction in the number of osteogenic and osteoprogenitor cells with unaltered differentiation capacity. Adipocyte differentiation was normal. Early in life, osteoclast number tended to be increased while at 78 weeks it was significantly lower in TTD mice. Our findings reveal the importance of genome stability and proper DNA repair for skeletal homeostasis with age and support the idea that accumulation of damage interferes with normal skeletal maintenance, causing reduction in the number of osteoblast precursors that are required for normal bone remodeling leading to a loss of bone structure and strength.

Opposing actions of rosiglitazone and resveratrol on mineralization in human vascular smooth muscle cells (Article)

2011-11-01T00:00:00Z

Arteriosclerotic vascular disease is a major cardiac health problem in westernized countries and the primary cause of mortality in diabetic patients. Recent data have raised serious safety concerns with the antidiabetic rosiglitazone, a thiazolidinedione with peroxisome proliferator-activated receptor γ (PPAR-γ) agonistic activity, in regard to cardiovascular risks. A common feature of atherosclerosis is vascular mineralization. The latter is formed by vascular smooth muscle cells (VSMC) through complex processes that are similar to mineralization in bone. The aim of the current study was to investigate the effect of rosiglitazone on mineralization in cultured human VSMCs. We found that rosiglitazone stimulated mineralization by, at least in part, induction of caspase-dependent apoptosis. Furthermore, rosiglitazone-induced oxidative stress was correlated with stimulated osteoblast-like differentiation of VSMCs. Treatment of rosiglitazone-supplemented VSMC cultures with the caloric restriction mimetic and antioxidant resveratrol diminished rosiglitazone-induced oxidative stress, osteoblast-like differentiation and mineralization. In conclusion, this study reveals novel insights into the relationship of rosiglitazone and cardiovascular events by providing a model that links rosiglitazone-induced osteoblast-like differentiation, oxidative stress and apoptosis with mineralization in VSMCs. In addition, we position resveratrol in this model acting to reduce rosiglitazone-induced oxidative stress, osteoblast-like VSMC differentiation and mineralization.

Unraveling the human bone microenvironment beyond the classical extracellular matrix proteins: A human bone protein library (Article)

2011-10-07T00:00:00Z

A characteristic feature of bone, differentiating it from other connective tissues, is the mineralized extracellular matrix (ECM). Mineral accounts for the majority of the bone tissue volume, being the remainder organic material mostly derived from collagen. This, and the fact that only a limited number of noncollagenous ECM proteins are described, provides a limited view of the bone tissue composition and bone metabolism, the more so considering the increasing understanding of ECM significance for cellular form and function. For this reason, we set out to analyze and extensively characterize the human bone proteome using large-scale mass spectrometry-based methods. Bone samples of four individuals were analyzed identifying 3038 unique proteins. A total of 1213 of these were present in at least 3 out of 4 bone samples. For quantification purposes, we were limited to noncollagenous proteins (NCPs) and we could quantify 1051 NCPs. Most classical bone matrix proteins mentioned in literature were detected but were not among the highly abundant ones. Gene ontology analyses identified high-abundance groups of proteins with a functional link to mineralization and mineral metabolism such as transporters, pyrophosphatase activity, and Ca2+-dependent phospholipid binding proteins. ECM proteins were as well overrepresented together with nucleosome and antioxidant activity proteins, which have not been extensively characterized as being important for bone. In conclusion, our data clearly demonstrates that human bone tissue is a reservoir of a wide variety of proteins. In addition to the classical osteoblast-derived ECM, we have identified many proteins from different sources and of unknown function in bone. Thus, this study represents an informative library of bone proteins forming a source for novel bone formation modulators as well as biomarkers for bone diseases such as osteoporosis.

Pro-osteogenic trophic effects by PKA activation in human mesenchymal stromal cells (Article)

2011-09-01T00:00:00Z

Human mesenchymal stromal cells (hMSCs) are able to differentiate into a wide variety of cell types, which makes them an interesting source for tissue engineering applications. On the other hand, these cells also secrete a broad panel of growth factors and cytokines that can exert trophic effects on surrounding tissues. In bone tissue engineering applications, the general assumption is that direct differentiation of hMSCs into osteoblasts accounts for newly observed bone formation in vivo. However, the secretion of bone-specific growth factors, but also pro-angiogenic factors, could also contribute to this process. We recently demonstrated that secretion of bone specific growth factors can be enhanced by treatment of hMSCs with the small molecule db-cAMP (cAMP) and here we investigate the biological activity of these secreted factors. We demonstrate that conditioned medium contains a variety of secreted growth factors, with differences between medium from basic-treated and cAMP-treated hMSCs. We show that conditioned medium from cAMP-treated hMSCs increases proliferation of various cell types and also induces osteogenic differentiation, whereas it has differential effects on migration. Microarray analysis on hMSCs exposed to conditioned medium confirmed upregulation of pathways involved in proliferation as well as osteogenic differentiation. Our data suggests that trophic factors secreted by hMSCs can be tuned for specific applications and that a good balance between differentiation on the one hand and secretion of bone trophic factors on the other, could potentially enhance bone formation for bone tissue engineering applications.

GPM6B regulates osteoblast function and induction of mineralization by controlling cytoskeleton and matrix vesicle release (Article)

2011-09-01T00:00:00Z

Neuronal membrane glycoprotein gene (GPM6B) encodes a membrane glycoprotein that belongs to the proteolipid protein family. We identified GPM6B as a gene that is strongly upregulated during osteoblast differentiation. To investigate the role of GPM6B in the process of bone formation, we silenced GPM6B expression during osteogenic differentiation of human mesenchymal stem cells (hMSCs). GPM6B silencing in hMSCs resulted in reduced alkaline phosphate (ALP) activity along with reduced mineralization of extracellular matrix (ECM). Microarray expression analysis of GPM6B-depleted osteogenic hMSCs revealed significant changes in genes involved in cytoskeleton organization and biogenesis. Immunocytochemistry results confirm changes in the distribution of actin filaments, as well as the shape and size of focal adhesions on GPM6B silencing. Moreover, we demonstrated that production and release of ALP-positive matrix vesicles (MVs) were reduced. In conclusion, we identified GPM6B as a novel regulator of osteoblast function and bone formation. This finding demonstrates the significance of cytoskeleton organization for MV production and subsequent mineralization.

Better knowledge on vitamin D and calcium in older people is associated with a higher serum vitamin D level and a higher daily dietary calcium intake (Article)

2011-06-01T00:00:00Z

Abstract: Objective: The objective of the present study was to examine knowledge on vitamin D and calcium in a cohort of older adults and to test the association between health knowledge, vitamin D status and dietary calcium intake. Methods: The participants of this cross-sectional survey consisted of 426 individuals (≥65 years), living in residential homes. Participants were tested for their knowledge on vitamin D and calcium using a standardized questionnaire. Serum 25-hydroxyvitamin D3 (25(OH)D3) levels and dietary calcium intake were measured. Results: The mean serum 25(OH)D3 level was 39.1 (±21.4) nmol/l and the mean daily dietary calcium intake was 826 (±242) mg/day. Of the participants, only 38 per cent indicated that they knew or had heard of vitamin D. Participants overestimated their daily calcium intake. Better knowledge on vitamin D and calcium was associated with both higher vitamin D levels (P < 0.0001) and a higher daily dietary calcium intake (P < 0.0001). Conclusion: Given the poor knowledge on vitamin D and calcium and the observed associations, improving health knowledge could be a possible intervention to improve vitamin D status and calcium intake in older people. Further studies are needed to assess whether education will indeed lead to improvement of vitamin D levels and calcium intake in this age group.

Preliminary validation of assays to measure parameters of calcium metabolism in captive asian and african elephants in Western Europe (Article)

2011-05-01T00:00:00Z

Hypocalcemia is a well-known cause of dystocia in animals, including elephants in captivity. In order to study calcium metabolism in elephants, it is of utmost importance to use properly validated assays, as these might be prone to specific matrix effects in elephant blood. The aim of the current study was to conduct preliminary work for validation of various parameters involved in calcium metabolism in both blood and urine of captive elephants. Basal values of these parameters were compared between Asian elephants (Elephas maximus) and African elephants (Loxodonta africana). Preliminary testing of total calcium, inorganic phosphorus, and creatinine appeared valid for use in plasma and creatinine in urine in both species. Furthermore, measurements of bone alkaline phosphatase and N-terminal telopeptide of type I collagen appeared valid for use in Asian elephants. Mean heparinized plasma ionized calcium concentration and pH were not significantly affected by 3 cycles of freezing and thawing. Storage at 4°C, room temperature, and 37°C for 6, 12, and 24 hr did not alter the heparinized plasma ionized calcium concentration in Asian elephants. The following linear regression equation using pH (range: 6.858- 7.887) and ionized calcium concentration in heparinized plasma was utilized: iCa7.4(mmol/l) = -2.1075 + 0.3130·pHactual+ 0.8296·iCaactual(mmol/l). Mean basal values for pH and plasma in Asian elephant whole blood were 7.40 ± 0.048 and 7.49 ± 0.077, respectively. The urinary specific gravity and creatinine concentrations in both Asian and African elephants were significantly correlated and both were significantly lower in Asian elephants.

Osteoarthritis induction leads to early and temporal subchondral plate porosity in the tibial plateau of mice: an in vivo micro CT study. (Article)

2011-02-01T00:00:00Z

Abstract: In osteoarthritis (OA) changes occur both in cartilage and subchondral bone. The subchondral bone plate facilitates normal crosstalk between articular cartilage and trabecular subchondral bone, and adaptive changes in the plate due to OA may therefore disturb crosstalk homeostasis. To investigate these changes over time we examined the cartilage-subchondral bone interface using a combined approach of histology and in vivo micro CT. Male C57Bl/6 mice (n=8), aged 16 weeks, received intra-articular injections with collagenase in one joint to induce instability-related OA and saline into the contralateral knee joint (controls). At 2, 4, 6, 10 and 14 weeks post-injection, changes in the tibial subchondral bone plate and subchondral trabeculae were analyzed. At two weeks post-injection, collagenase injected joints had significantly more cartilage damage and osteophytosis than control joints. Osteoclast activity directly underneath the subchondral bone plate was significantly elevated (Oc.S./BS controls: 7.60± 0.81%; OA: 11.07± 0.79%), causing the plate to become thinner and creating a large increase in subchondral bone plate porosity (cumulative porosity volume controls: 0.05e-3 ± 0.04e-3 mm(3) ; OA: 2.52e-3 ± 0.69e-3 mm(3) ). At four weeks post-injection, the previously formed perforations disappeared, coinciding with a significant rise in osteoblast activity in the subchondral trabecular bone (bone formation rate controls: 0.30± 0.03, OA: 0.62± 0.13 μm(2) /μm(3) *day). The current study provides for the first time quantitative longitudinal data on the dynamic changes in the subchondral bone plate after OA induction. The development of plate perforations may enhance mutual interaction between subchondral trabeculae, bone marrow cells and the articular cartilage in OA.

Design principles of nuclear receptor signaling: How complex networking improves signal transduction (Article)

2010-12-01T00:00:00Z

The topology of nuclear receptor (NR) signaling is captured in a systems biological graphical notation. This enables us to identify a number of design aspects of the topology of these networks that might appear unnecessarily complex or even functionally paradoxical. In realistic kinetic models of increasing complexity, calculations show how these features correspond to potentially important design principles, e.g.: (i) cytosolic nuclear receptor may shuttle signal molecules to the nucleus, (ii) the active export of NRs may ensure that there is sufficient receptor protein to capture ligand at the cytoplasmic membrane, (iii) a three conveyor belts design dissipating GTP-free energy, greatly aids response, (iv) the active export of importins may prevent sequestration of NRs by importins in the nucleus and (v) the unspecific nature of the nuclear pore may ensure signal-flux robustness. In addition, the models developed are suitable for implementation in specific cases of NR-mediated signaling, to predict individual receptor functions and differential sensitivity toward physiological and pharmacological ligands.

Oestrogen is important for maintenance of cartilage and subchondral bone in a murine model of knee osteoarthritis (Article)

2010-10-05T00:00:00Z

Introduction: Oestrogen depletion may influence onset and/or progression of osteoarthritis. We investigated in an ovariectomized mouse model the impact of oestrogen loss and oestrogen supplementation on articular cartilage and subchondral bone in tibia and patella, and assessed bone changes in osteoarthritis development.Methods: C3H/HeJ mice were divided into four groups: sham-operated, oestrogen depletion by ovariectomy (OVX), OVX with estradiol supplementation (OVX+E) and OVX with bisphosphonate (OVX+BP). Each mouse had one knee injected with low-dose iodoacetate (IA), and the contralateral knee was injected with saline. Cartilage was analysed histologically 12 weeks postsurgery; bone changes were monitored over time using in vivo micro-computed tomography.Results: In tibiae, OVX alone failed to induce cartilage damage, but OVX and IA combination significantly induced cartilage damage. In patellae, OVX alone induced significant cartilage damage, which was enhanced by IA. In both tibiae and patellae, OVX in combination with IA significantly decreased subchondral cortical thickness in an additive manner. OVX+E and OVX+BP inhibited tibial and patellar subchondral cortical thinning, inhibited patellar and tended to diminish tibial cartilage damage. In patellae, IA interacted with BP, leading to increased subchondral cortical and trabecular bone.Conclusions: This study demonstrates the significance of oestrogen for articular cartilage and subchondral bone and maintenance of healthy joints, supporting an etiological role for altered oestrogen signaling in osteoarthritis either by directly affecting cartilage or increasing susceptibility for an osteoarthritis trigger. The data strongly support the concept of involvement of subchondral bone plate in osteoarthritis.

Analysis of osteoarthritis in a mouse model of the progeroid human DNA repair syndrome trichothiodystrophy (Article)

2010-09-07T00:00:00Z

The increasing average age in developed societies is paralleled by an increase in the prevalence of many age-related diseases such as osteoarthritis (OA), which is characterized by deformation of the joint due to cartilage damage and increased turnover of subchondral bone. Consequently, deficiency in DNA repair, often associated with premature aging, may lead to increased pathology of these two tissues. To examine this possibility, we analyzed the bone and cartilage phenotype of male and female knee joints derived from 52- to 104-week-old WT C57Bl/6 and trichothiodystrophy (TTD) mice, who carry a defect in the nucleotide excision repair pathway and display many features of premature aging. Using micro-CT, we found bone loss in all groups of 104-week-old compared to 52-week-old mice. Cartilage damage was mild to moderate in all mice. Surprisingly, female TTD mice had less cartilage damage, proteoglycan depletion, and osteophytosis compared to WT controls. OA severity in males did not significantly differ between genotypes, although TTD males had less osteophytosis. These results indicate that in premature aging TTD mice age-related changes in cartilage were not more severe compared to WT mice, in striking contrast with bone and many other tissues. This segmental aging character may be explained by a difference in vasculature and thereby oxygen load in cartilage and bone. Alternatively, a difference in impact of an anti-aging response, previously found to be triggered by accumulation of DNA damage, might help explain why female mice were protected from cartilage damage. These findings underline the exceptional segmental nature of progeroid conditions and provide an explanation for pro- and anti-aging features occurring in the same individual.

Proteomic Analysis of Human Osteoblastic Cells: Relevant Proteins and Functional Categories for Differentiation (Article)

2010-09-01T00:00:00Z

Abstract Osteoblasts are the bone forming cells, capable of secreting an extracellular matrix with mineralization potential. The exact mechanism by which osteoblasts differentiate and form a mineralized extracellular matrix is presently not fully understood. To increase our knowledge about this process, we conducted proteomics analysis in human immortalized preosteoblasts (SV-HFO) able to differentiate and mineralize. We identified 381 proteins expressed during the time course of osteoblast differentiation. Gene ontology analysis revealed an overrepresentation of protein categories established as important players for osteoblast differentiation, bone formation, and mineralization such as pyrophosphatases. Proteins involved in antigen presentation, energy metabolism and cytoskeleton rearrangement constitute other overrepresented processes, whose function, albeit interesting, is not fully understood in the context of osteoblast differentiation and bone formation. Correlation analysis, based on quantitative data, revealed a biphasic osteoblast differentiation, encompassing a premineralization and a mineralization period. Identified differentially expressed proteins between mineralized and nonmineralized cells include cytoskeleton (e.g., CCT2, PLEC1, and FLNA) and extracellular matrix constituents (FN1, ANXA2, and LGALS1) among others. FT-ICR-MS data obtained for FN1, ANXA2, and LMNA shows a specific regulation of these proteins during the different phases of osteoblast differentiation. Taken together, this study increases our understanding of the proteomics changes that accompany osteoblast differentiation and may permit the discovery of novel modulators of bone formation.

The T-13910C polymorphism in the lactase phlorizin hydrolase gene is associated with differences in serum calcium levels and calcium intake (Article)

2010-09-01T00:00:00Z

The C-variant of a T-13910C polymorphism (rs4988235; NT-022135.15:g. 25316568G>A) upstream of the lactase phlorizin hydrolase (LPH) gene causes lactose intolerance. Association studies with differences in bone parameters and fracture risk have been inconclusive. The objective of this study was to examine the association of LPH rs4988235 with body height and bone parameters and calcium homeostasis in two elderly populations of Dutch Caucasians and assess interaction with vitamin D receptor (VDR) polymorphisms. Genotyping of LPH and VDR polymorphisms was performed in 6367 individuals from the Rotterdam Study and 844 from the Longitudinal Aging Study Amsterdam (LASA). Associations with age, height, weight, bone mineral density (BMD), skeletal morphometric parameters and serum vitamin D and calcium levels, and dietary calcium intake were assessed using ANOVA or analysis of covariance, and allele dose effect was assessed using linear regression analysis. Fracture risk was analyzed using Cox's proportional hazard regression analysis. Associations with body height (p=2.7 x 10-8) and vertebral area (p=.048) found in the Rotterdam Study were explained by population stratification, as assessed by principal-component analyses, and disappeared after additional adjustments. No associations with femoral neck or lumbar spine BMD or with fracture risk were detected. Calcium intake and serum ionized serum calcium were significantly lower in C-homozygotes (p=9.2 x 10-7, p=.02, respectively). For none of the parameters studied was interaction between the T-13910C polymorphism and VDR block 5 haplotype 1 observed. We show that the C allele of the T-13910C polymorphism causing lactose intolerance is associated with lower dietary calcium intake and serum calcium levels but not with BMD or fractures. The associations observed with height and vertebral area were the result of population stratification. This demonstrates the impact of population stratification and urges researchers to carefully take this into account in genetic associations, in particular, in dietary intake-related phenotypes, of which LPH and lactose intolerance are a strong example.

Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: An in vitro study (Article)

2010-08-26T00:00:00Z

Background: Although pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC) metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation. Methods: BMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition), expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK) signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p < 0.05. Results: Biochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control. Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and-3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-B ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation. Conclusions: PEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo. © 2010 Jansen et al; licensee BioMed Central Ltd.

Synergistic induction of local glucocorticoid generation by inflammatory cytokines and glucocorticoids: Implications for inflammation associated bone loss (Article)

2010-06-01T00:00:00Z

Objectives: Synovial fibroblasts and osteoblasts generate active glucocorticoids by means of the 11aβ-hydroxysteroid dehydrogenase type 1 (11aβ-HSD1) enzyme. This activity increases in response to proinflammatory cytokines or glucocorticoids. During inflammatory arthritis synovium and bone are exposed to both these factors. This study hypothesised that glucocorticoids magnify the effects of inflammatory cytokines on local glucocorticoid production in both synovium and bone. Methods: The effects of inflammatory cytokines (IL-1aβ/tumour necrosis factor alpha; TNFα) and glucocorticoids, alone or combined, were assessed on the expression and activity of 11β-HSD1 in primary synovial fibroblasts, primary human osteoblasts and MG-63 osteosarcoma cells. A range of other target genes and cell types were used to examine the specificity of effects. Functional consequences were assessed using IL-6 ELISA. Results: In synovial fibroblasts and osteoblasts, treatment with cytokines or glucocorticoids in isolation induced 11β-HSD1 expression and activity. However, in combination, 11β-HSD1 expression, activity and functional consequences were induced synergistically to a level not seen with isolated treatments. This effect was seen in normal skin fibroblasts but not foreskin fibroblasts or adipocytes and was only seen for the 11β-HSD1 gene. Synergistic induction had functional consequences on IL-6 production. Conclusions: Combined treatment with inflammatory cytokines and glucocorticoids synergistically induces 11aβ-HSD1 expression and activity in synovial fibroblasts and osteoblasts, providing a mechanism by which synovium and bone can interact to enhance anti-inflammatory responses by increasing localised glucocorticoid levels. However, the synergistic induction of 11β-HSD1 might also cause detrimental glucocorticoid accumulation in bone or surrounding tissues.

Effects of vitamin D3 supplementation and UVb exposure on the growth and plasma concentration of vitamin D3 metabolites in juvenile bearded dragons (Pogona vitticeps) (Article)

2010-06-01T00:00:00Z

The effectiveness of dietary vitamin D3and UVb exposure on plasma vitamin D metabolites in growing bearded dragons (Pogona vitticeps) was studied. A total of 84 (40 males and 44 females) newly hatched bearded dragons were allocated to six levels of oral vitamin D3supplementation (0 to 400%) or six UVb exposure times (2 to 12h). At 3 and 6months of age, blood samples were obtained from each animal and analysed for 25(OH)D3and 1,25(OH)2D3. At 3months of age, plasma concentrations of 25(OH)D3did not increase with increasing vitamin D3supplementation unlike the 1,25(OH)2D3. At 6months of age, plasma concentrations of both 25(OH)D3and 1,25(OH)2D3increased with increasing vitamin D3supplementation. Plasma concentrations in UVb-exposed animals were 18 times higher for 25(OH)D3(178.4±9.0 vs. 9.9±1.3nmol/L) and 5.3 times higher for 1,25(OH)2D3(1.205±0.100 vs. 0.229±0.025nmol/L) than in vitamin D3supplemented animals at 6months of age. This study shows that 2h of UVb exposure enables adequate physiological concentrations of plasma vitamin D metabolites to be maintained in growing bearded dragons. Oral supplementation of vitamin D3is ineffective in raising plasma concentrations of 25(OH)D3and 1,25(OH)2D3to concentrations observed in UVb-exposed animals.

1α,25-(OH)2D3 acts in the early phase of osteoblast differentiation to enhance mineralization via accelerated production of mature matrix vesicles (Article)

2010-05-19T00:00:00Z

1α,25-dihydroxyitamin D3 (1,25D3) deficiency leads to impaired bone mineralization. We used the human pre-osteoblastic cell line SV-HFO, which forms within 19 days of culture an extracellular matrix that starts to mineralize around day 12, to examine the mechanism by which 1,25D3 regulates osteoblasts and directly stimulates mineralization. Time phase studies showed that 1,25D3 treatment prior to the onset of mineralization, rather than during mineralization led to accelerated and enhanced mineralization. This is supported by the observation of unaltered stimulation by 1,25D3 even when osteoblasts were devitalized just prior to onset of mineralization and after 1,25D3 treatment. Gene Chip expression profiling identified the pre-mineralization and mineralization phase as two strongly distinctive transcriptional periods with only 0.6% overlap of genes regulated by 1,25D3. In neither phase 1,25D3 significantly altered expression of extracellular matrix genes. 1,25D3 significantly accelerated the production of mature matrix vesicles (MVs) in the pre-mineralization. Duration rather than timing determined the extent of the 1,25D3 effect. We propose the concept that besides indirect effects via intestinal calcium uptake 1,25D3 directly accelerates osteoblast-mediated mineralization via increased production of mature MVs in the period prior to mineralization. The accelerated deposition of mature MVs leads to an earlier onset and higher rate of mineralization. These effects are independent of changes in extracellular matrix protein composition. These data on 1,25D3, mineralization, and MV biology add new insights into the role of 1,25D3 in bone metabolism and emphasize the importance of MVs in bone and maintaining bone health and strength by optimal mineralization status.

Estrogen Modulates Iodoacetate-Induced Gene Expression in Bovine Cartilage Explants (Article)

2010-05-01T00:00:00Z

Abstract Estrogen loss may be involved in onset or progression of osteoarthritis. Estrogen receptors are present in chondrocytes, thus estrogen may exert effects directly on cartilage. However, studies on direct estrogen effects on cartilage are limited. We investigated, in an in vitro cartilage explant model, whether estrogen prevents damage or stimulates repair after damage induced by addition of iodoacetate (IA), as an experimental model for osteoarthritis. We used healthy bovine cartilage explants. Prevention experiment: Explants precultured with/without estradiol (E) for 3 days were cultured with IA for 4 h on day 0, and subsequently cultured as in preculture: with/without E. Explants were harvested at day 2 for gene expression analysis. Repair experiment: At day 0, explants were cultured with IA for 4 h on day 0, and subsequently cultured without E or with E. Explants were harvested at days 2, 10, and 14 for gene expression analysis. IA transiently downregulated most genes tested, whereas vascular endothelial growth factor (VEGF) was upregulated on day 2. On day 14, transforming growth factor beta (TGFB)1 and TGFB3 were upregulated, and matrix metalloproteinase (MMP)13 and VEGF downregulated. Estradiol affected gene expression of aggrecan (AGC)1, MMP2, MMP14, tissue inhibitor of metalloproteinase (TIMP)2, TGFB2, and TGFB3. Prevention experiment: Estradiol did not significantly affect IA-induced changes in gene expression (no significant interaction). Repair experiment: Estradiol affected IA-induced changes in expression of collagen (COL)2, MMP2, MMP3, MMP13, MMP14, TIMP2, TGFB2, TGFB3, and VEGF. Estradiol affects expression of anabolic and catabolic genes in bovine cartilage explants and modulates the effects of IA. These effects of estradiol may be beneficial for cartilage maintenance and repair.

1α,25-(OH)2D3 acts in the early phase of osteoblast differentiation to enhance mineralization via accelerated production of mature matrix vesicles (Article)

2010-05-01T00:00:00Z

A new concept underlying stem cell lineage skewing that explains the detrimental effects of thiazolidinediones on bone (Article)

2010-03-08T00:00:00Z

Bone-marrow adipogenesis is an aging-related phenomenon and correlated with osteoporosis. The latter is a prevalent bone disease in the elderly leading to increased fracture risk and mortality. It is widely hypothesized that the underlying molecular mechanism includes a shift in the commitment of mesenchymal stem cells (MSC) from the osteogenic lineage to the adipogenic lineage. Lineage skewing is at least partially a result of transcriptional changes. The nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPAR-gamma) has been proposed as a major decision factor in MSC lineage commitment promoting adipogenesis at the expense of osteogenesis. Here we found that PPAR-gamma acted unexpectedly to stimulate osteoblast differentiation from human bone marrow-derived MSCs. Both rosiglitazone-mediated activation and overexpression of PPAR-gamma caused acceleration of osteoblast differentiation. Conversely, shRNAi-mediated PPAR-gamma knockdown diminished osteoblast differentiation. MSCs that were treated with rosiglitazone did not preferentially differentiate into adipocytes. However, the rosiglitazone-mediated acceleration of osteoblast differentiation was followed by increased accumulation of reactive oxygen species and apoptosis. In contrast to the osteogenic lineage, cells of the adipogenic lineage were protected from this. Our data support a new concept on bone health that adds to the explanation of the clinically observed suppressive action of activated PPAR-gamma on bone and the associated phenomenon of bone marrow adipogenesis. This concept is based on a higher susceptibility of the osteogenic than the adipogenic lineage to oxidative stress and apoptosis that is preferentially triggered in the osteoblasts by activated PPAR-gamma.

Stretch-Induced Inhibition of Wnt/b-Catenin Signaling in Mineralizing Osteoblasts (Article)

2010-03-01T00:00:00Z

Wnt signaling is important for bone formation and osteoblastic differentiation. Recent findings indicate a stimulating role of Wnt signaling in bone mechanotransduction. However, negative effects of Wnt signaling on osteoblast differentiation and mineralization have been described as well. We conducted in vitro stretch experiments using human pre-osteoblasts to study short- and long-term effects of mechanical loading on Wnt/beta-catenin signaling. As the extracellular regulated kinase (ERK) pathway is known to be involved in mechanotransduction in osteoblasts, we also evaluated its role in Wnt/beta-catenin signaling. Stretch experiments up to 21 days (using stretch episodes of 15 min, alternated with 90 min rest) resulted in higher mineralization compared to static control cultures. We found that 15 min of stretch initially increased nuclear beta-catenin, but ultimately resulted in significant decrease at 12 and 40 h after stretch. Downregulation of Wnt-responsive element activity 16 h after stretch, using a luciferase construct, further supported these findings. The presence of the ERK inhibitor U0126 did not alter the stretch-induced decrease of beta-catenin levels. Our data indicate a biphasic effect of mechanical loading on beta-catenin in mineralizing human differentiating osteoblasts, which is independent of the ERK pathway. The osteogenic potential of our loading regime was confirmed by an increase in osteogenic differentiation markers such as alkaline phosphatase activity and calcium deposition after 3 weeks of culture. We conjecture that the biphasic aspect of Wnt/beta-catenin signaling with a strong decrease up to 40 h after the stretch induction, is important for the anabolic effects of mechanical stretch on bone.

The role of body mass index, insulin, and adiponectin in the relation between fat distribution and bone mineral density (Article)

2010-02-01T00:00:00Z

Despite the positive association between body mass index (BMI) and bone mineral density (BMD) and content (BMC), the role of fat distribution in BMD/BMC remains unclear. We examined relationships between BMD/BMC and various measurements of fat distribution and studied the role of BMI, insulin, and adiponectin in these relations. Using a cross-sectional investigation of 2631 participants from the Erasmus Rucphen Family study, we studied associations between BMD (using dual-energy X-ray absorptiometry (DXA]) at the hip, lumbar spine, total body (BMD and BMC), and fat distribution by the waist-to-hip ratio (WHR), waist-to-thigh ratio (WTR), and DXA-based trunk-to-leg fat ratio and android-to-gynoid fat ratio. Analyses were stratified by gender and median age (48.0 years in women and 49.2 years in men) and were performed with and without adjustment for BMI, fasting insulin, and adiponectin. Using linear regression (adjusting for age, height, smoking, and use of alcohol), most relationships between fat distribution and BMD and BMC were positive, except for WTR. After BMI adjustment, most correlations were negative except for trunk-to-leg fat ratio in both genders. No consistent influence of age or menopausal status was found. Insulin and adiponectin levels did not explain either positive or negative associations. In conclusion, positive associations between android fat distribution and BMD/BMC are explained by higher BMI but not by higher insulin and/or lower adiponectin levels. Inverse associations after adjustment for BMI suggest that android fat deposition as measured by the WHR, WTR, and DXA-based android-to-gynoid fat ratio is not beneficial and possibly even deleterious for bone.

1,25-Dihydroxyvitamin D3 modulates Th17 polarization and interleukin-22 expression by memory T cells from patients with early rheumatoid arthritis (Article)

2010-01-01T00:00:00Z

Objective. To examine the immunologic mechanism by which 1,25-dihydroxyvitamin D3(1,25[OH]2D3) may prevent corticosteroid-induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology. Methods. Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO- (non-memory) T cells separated by fluorescence-activated cell sorting (FACS) from treatment-naive patients with early RA were stimulated with anti-CD3/anti-CD28 in the absence or presence of various concentrations of 1,25(OH)2D3, dexamethasone (DEX), and 1,25(OH)2D3and DEX combined. Levels of T cell cytokines were determined by enzyme-linked immunosorbent assay and flow cytometry. Results. The presence of 1,25(OH)2D3reduced interleukin-17A (IL-17A) and interferon-γ levels and increased IL-4 levels in stimulated PBMCs from treatment-naive patients with early RA. In addition, 1,25(OH)2D3had favorable effects on tumor necrosis factor α (TNFα):IL-4 and IL-17A:IL-4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL-17A- and IL-22-expressing CD4+ T cells and IL-17A-expressing memory T cells were observed in PBMCs from treatment-naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO- cells between these 2 groups. Interestingly, 1,25(OH)2D3, in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL-17A, IL-17F, TNFα, and IL-22 production by memory T cells sorted by FACS from patients with early RA. Conclusion. These data indicate that 1,25(OH)2D3may contribute its bone-sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL-4.

Klotho prevents renal calcium loss (Article)

2009-11-01T00:00:00Z

Disturbed calcium (Ca2+) homeostasis, which is implicit to the aging phenotype of klotho-deficient mice, has been attributed to altered vitamin D metabolism, but alternative possibilities exist. We hypothesized that failed tubular Ca2+absorption is primary, which causes increased urinary Ca2+excretion, leading to elevated 1,25-dihydroxyvitamin D3[1,25(OH)2D3] and its sequelae. Here, we assessed intestinal Ca2+absorption, bone densitometry, renal Ca2+excretion, and renal morphology via energy-dispersive x-ray microanalysis in wild-type and klotho-/-mice. We observed elevated serum Ca2+and fractional excretion of Ca2+(FECa) in klotho-/-mice. Klotho-/-mice also showed intestinal Ca2+hyperabsorption, osteopenia, and renal precipitation of calcium-phosphate. Duodenal mRNA levels of transient receptor potential vanilloid 6 (TRPV6) and calbindin-D9Kincreased. In the kidney, klotho-/-mice exhibited increased expression of TRPV5 and decreased expression of the sodium/calcium exchanger (NCX1) and calbindin-D28K, implying a failure to absorb Ca2+through the distal convoluted tubule/connecting tubule (DCT/CNT) via TRPV5. Gene and protein expression of the vitamin D receptor (VDR), 25-hydroxyvitamin D-1-α-hydroxylase (1αOHase), and calbindin-D9Kexcluded renal vitamin D resistance. By modulating the diet, we showed that the renal Ca2+wasting was not secondary to hypercalcemia and/or hypervitaminosis D. In summary, these findings illustrate a primary defect in tubular Ca2+handling that contributes to the precipitation of calcium-phosphate in DCT/CNT. This highlights the importance of klotho to the prevention of renal Ca2+loss, secondary hypervitaminosis D, osteopenia, and nephrocalcinosis. Copyright

Development of osteoarthritic features in estrogen receptor knockout mice (Article)

2009-10-01T00:00:00Z

Objective: Estrogens are suggested to play a role in the development of osteoarthritis as indicated by the increased prevalence in women after menopause. We studied whether deletion of the estrogen receptor (ER) α, β, or both in female mice results in cartilage damage, osteophytosis, and changes in subchondral bone of skeletally mature animals. Methods: We studied knee joints of 6-month-old female ERα-/-, ERβ-/-, and (double) ERα-/-β-/- mice and their wild type (wt) littermates. The presence and size of osteophytes and osteoarthritic changes in cartilage were analyzed using histology. Changes in subchondral plate and trabecular bone were studied using micro-CT. Results: In ERα-/-β-/- mice, we observed an increase in number and/or size of osteophytes and thinning of the lateral subchondral plate. However, cartilage damage was not different from wt. In ERα-/- or ERβ-/- mice, no significant differences in cartilage damage score, osteophyte formation, or subchondral plate thickness were found. The bone volume fraction of the epiphyseal trabecular bone was unchanged in ERα-/- mice, increased in ERβ-/- mice, and decreased in ERα-/-β-/- mice. Conclusions: We conclude that deletion of both ERs leads to increased osteophytosis, but deletion of one or both ERs does not lead to overt cartilage damage in 6-month-old mice.

Vitamin D Status, Bone Mineral Density, and the Development of Radiographic Osteoarthritis of the Knee (Article)

2009-08-01T00:00:00Z

OBJECTIVE: To study the association between baseline vitamin D status, bone mineral density (BMD), and the development of radiographic osteoarthritis (ROA) of the knee in a large population-based cohort of men and women. METHODS: A sample of 1248 subjects (728 women and 520 men) was drawn from the Rotterdam Study, a prospective population-based cohort study of the elderly. At baseline, vitamin D dietary intake was determined, and BMD and 25-hydroxy vitamin D (25(OH)D) serum levels were measured. After a mean follow-up time of 6.5 years incidence and progression of knee ROA of was assessed. RESULTS: The mean vitamin D intake in our study population was 64 IU/d and the mean 25(OH)D level 66 nmol/L. Vitamin D levels were associated with baseline BMD, particularly in subjects with baseline knee ROA. Progressive ROA occurred in 5.1% of the participants in the highest tertile of vitamin D intake against 12.6% in the lowest tertile, resulting in an adjusted odds ratio of 7.7 (95% CI: 1.3-43.5). Both intake and levels of 25(OH)D were not significantly related to incident ROA. However, we found a significant interaction between vitamin D intake and BMD in the association with incident knee ROA (P = 0.03): in subjects with low lumbar spine BMD at baseline we observe an increasing incidence of knee ROA with decreasing vitamin D intake and serum levels. CONCLUSIONS: Low dietary vitamin D intake increases the risk of progression of knee ROA. Particularly in subjects with low baseline BMD, vitamin D status seems to influence the incidence and progression of knee ROA. Thus, improving the vitamin D status in the elderly could protect against the development and worsening of knee OA, especially in those with low BMD.

Vitamin D binding protein genotype and osteoporosis (Article)

2009-08-01T00:00:00Z

Osteoporosis is a bone disease leading to an increased fracture risk. It is considered a complex multifactorial genetic disorder with interaction of environmental and genetic factors. As a candidate gene for osteoporosis, we studied vitamin D binding protein (DBP, or group-specific component, Gc), which binds to and transports vitamin D to target tissues to maintain calcium homeostasis through the vitamin D endocrine system. DBP can also be converted to DBP-macrophage activating factor (DBP-MAF), which mediates bone resorption by directly activating osteoclasts. We summarized the genetic linkage structure of the DBP gene. We genotyped two single-nucleotide polymorphisms (SNPs, rs7041 = Glu416Asp and rs4588 = Thr420Lys) in 6,181 elderly Caucasians and investigated interactions of the DBP genotype with vitamin D receptor (VDR) genotype and dietary calcium intake in relation to fracture risk. Haplotypes of the DBP SNPs correspond to protein variations referred to as Gc1s (haplotype 1), Gc2 (haplotype 2), and Gc1f (haplotype3). In a subgroup of 1,312 subjects, DBP genotype was found to be associated with increased and decreased serum 25-(OH)D3for haplotype 1 (P = 3 × 10-4) and haplotype 2 (P = 3 × 10-6), respectively. Similar associations were observed for 1,25-(OH)2D3. The DBP genotype was not significantly associated with fracture risk in the entire study population. Yet, we observed interaction between DBP and VDR haplotypes in determining fracture risk. In the DBP haplotype 1-carrier group, subjects of homozygous VDR block 5-haplotype 1 had 33% increased fracture risk compared to noncarriers (P = 0.005). In a subgroup with dietary calcium intake <1.09 g/day, the hazard ratio (95% confidence interval) for fracture risk of DBP hap1-homozygote versus noncarrier was 1.47 (1.06-2.05). All associations were independent of age and gender. Our study demonstrated that the genetic effect of the DBP gene on fracture risk appears only in combination with other genetic and environmental risk factors for bone metabolism.

Evidence for direct effects of prolactin on human osteoblasts: Inhibition of cell growth and mineralization (Article)

2009-07-01T00:00:00Z

Hyperprolactinemia is one of the risk factor of decrease in bone mass which has been believed to be mediated by hypogonadism. However, the presence of prolactin receptor in human osteosarcoma cell line and primary bone cell culture from mouse calvariae supported the hypothesis of a direct prolactin (PRL) action on bone cells. Therefore, the aim of this study was to investigate the role of PRL and its signal transduction pathway in the regulation of bone metabolism via osteoblast differentiation. Human pre-osteoblasts (SV-HFO) that differentiate in a 3-week period from proliferating pre-osteoblasts (days 2-7) to extracellular matrix producing cells (days 7-14) which is eventually mineralized (days 14-21) were used. Concentration of PRL mimicked a lactating period (100 ng/ml) was used to incubate SV-HFO for 21 days in osteogenic medium. Human prolactin receptor mRNA and protein are expressed in SV-HFO. PRL significantly decreased osteoblast number (DNA content) which was due to a decrease in proliferation. PRL increased osteogenic markers, RUNX2 and ALP in early stage of osteoblast differentiation while decreasing it later suggesting a bi-directional effect. Calcium measurement and Alizarin red staining showed a reduction of mineralization by PRL while having neither an effect on osteoblast activity nor RANKL/OPG mRNA ratio. We also demonstrated that PRL action on mineralization was not via PI-3 kinase pathway. The present study provides evidence of a direct effect of prolactin on osteoblast differentiation and in vitro mineralization.

Ageing and vitamin D deficiency: Effects on calcium homeostasis and considerations for vitamin D supplementation (Article)

2009-04-28T00:00:00Z

Vitamin D is a fat-soluble, seco-steroid hormone. In man, the vitamin D receptor is expressed in almost all tissues, enabling effects in multiple systems of the human body. These effects can be endocrine, paracrine and autocrine. The present review summarises the effects of ageing on the vitamin D endocrine system and on Ca homeostasis. Furthermore, consequences for vitamin D supplementation are discussed.

SIRT1 genetic variation and mortality in type 2 diabetes: interaction with smoking and dietary niacin (Article)

2009-03-15T00:00:00Z

SIRT1 protects cells against oxidative stress and aging. Its activity may be modulated by dietary niacin (vitamin B3) intake. We studied the association of SIRT1 genetic variation with mortality in subjects with increased oxidative stress (type 2 diabetes and smokers) in relation to dietary niacin. In 4573 participants from the Rotterdam Study, including 413 subjects with prevalent and 378 with incident type 2 diabetes, three SIRT1 tagging SNPs were genotyped and all-cause mortality was studied (average follow-up12 years). We found no association between SIRT1 variation and mortality in the total population or in smokers. In subjects with prevalent type 2 diabetes, homozygous carriers of the most common SIRT1 haplotype, 1, had 1.5 times (95%CI 1.1-2.1) increased mortality risk compared to noncarriers. This risk further increased among smokers and those with low niacin intake. In the lowest tertile of niacin intake, mortality risk was increased 2.3 (95%CI 1.1-4.9) and 5.7 (95%CI 2.5-13.1) times for heterozygous and homozygous carriers of haplotype 1. Subjects with incident diabetes showed similar findings but only when they smoked. We conclude that in subjects with type 2 diabetes, SIRT1 genetic variation influences survival in interaction with dietary niacin and smoking. Correction of niacin deficiency and SIRT1 modulators may prolong the life span of patients with diabetes.

Bone resorption inhibitor alendronate normalizes the reduced bone thickness of TRPV5-/- mice (Article)

2008-11-01T00:00:00Z

TRPV5 is a Ca2+-selective channel involved in transcellular Ca2+absorption expressed in kidney and in the ruffled border of osteoclasts. Studies in hypercalciuric TRPV5 knockout (TRPV5-/-) mice, which display significantly increased vitamin D levels, showed that TRPV5 ablation increases number and size of osteoclasts but impairs osteoclast-mediated bone resorption. The latter is not in line with the observed decreased bone thickness in TRPV5-/-mice. Bisphosphonates also inhibit osteoclast-mediated bone resorption. The aim of this study was to evaluate the effect of alendronate on the expression of the Ca2+transporters in bone, kidney, and duodenum and, importantly, the bone phenotype in TRPV5-/-mice. Wildtype (TRPV5+/+) and TRPV5-/-mice were treated during 10 wk with 2 mg/kg alendronate or vehicle weekly and housed in metabolic cages at the end of treatment. Urine and blood samples were taken for biochemical analysis, and duodenum, kidney, and femur were sampled. Expression of Ca2+transporters and osteoclast ruffled border transporters in bone and cultured osteoclasts was determined by QPCR analysis. Femurs were scanned using μCT, and resorption pit assays were performed in bone marrow cultures isolated from TRPV5+/+and TRPV5-/-mice. Alendronate treatment enhanced bone thickness in TRPV5+/+mice but also normalized the disturbed bone morphometry parameters in TRPV5-/-mice. Bone TRPV5 expression was specifically enhanced by alendronate, whereas the expression of Ca2+transporters in kidney and intestine was not altered. The expression of the osteoclast ruffled border membrane proteins chloride channel 7 (CLC-7) and the vacuolar H+-ATPase did not differ between both genotypes, but alendronate significantly enhanced the expression and PTH levels in TRPV5-/-mice. The expression of TRPV5, CLC-7, and H+-ATPase in osteoclast cultures was not affected by alendronate. The number of resorption pits was reduced in TRPV5-/-bone marrow cultures, but the response to vitamin D was similar to that in TRPV5+/+cultures. The alendronate-induced upregulation of TRPV5 in bone together with the decreased resorptive capacity of TRPV5-/-osteoclasts in vitro suggests that TRPV5 has an important role in osteoclast function. However, our data indicate that significant bone resorption still occurs in TRPV5-/-mice, because alendronate treatment normalized bone thickness in these mice. Thus, TRPV5-/-mice are able to rescue the resulting defect in osteoclast-mediated bone resorption, possibly mediated by the long-term hypervitaminosis D or other (non)hormonal compensatory mechanisms.

Wnt signaling acts and is regulated in a human osteoblast differentiation dependent manner (Article)

2008-05-15T00:00:00Z

The Wnt signaling pathway is an important regulator of cellular differentiation in a variety of cell types including osteoblasts. In this study, we investigated the impact of Wnt signaling on the function of human osteoblasts in relation to the stage of differentiation. Differentiating osteoblasts were created upon glucocorticoid (GC) treatment, whereas nondifferentiating osteoblasts were created by excluding GCs from the culture medium. GC-induced differentiation suppressed endogenous β-catenin levels and transcriptional activity. During GC-induced osteoblast differentiation, activation of Wnt signaling slightly decreased alkaline phosphatase activity, but strongly suppressed matrix mineralization. In addition, mRNA expression of several Wnt signaling related genes was strongly regulated during GC-induced osteoblast differentiation, including frizzled homolog 8, dickkopf homolog 1, and secreted frizzled-related protein 1. In contrast, in the absence of GC-induced differentiation, Wnt signaling acted positively by stimulating basal alkaline phosphatase activity. Interestingly, pre-stimulation of Wnt signaling in early osteoblasts enhanced their differentiation capacity later on during the GC-induced differentiation process. In conclusion, we showed a differentiation-dependent effect of Wnt signaling on osteoblasts. Wnt signaling stimulated early osteoblasts in their capacity to differentiate, whereas mature osteoblasts were strongly inhibited in their capacity to induce mineralization. Moreover, osteoblast differentiation suppressed endogenous Wnt signaling and changed the expression of multiple Wnt signaling related genes.

Animal models for osteoarthritis: the effect of ovariectomy and estrogen treatment - a systematic approach (Article)

2008-05-01T00:00:00Z

OBJECTIVE: The prevalence of osteoarthritis (OA) increases dramatically in women after the age of 50. Animal models are used to study the effects of hormone depletion [by ovariectomy (OVX)] and estrogen treatment on OA. This review summarizes these animal studies, in order to get a better insight in the role of hormones on OA. METHOD: The literature was systematically reviewed until May 2007. The results were divided into two parts: the effect of OVX on cartilage, and the effect of estrogen treatment on cartilage. Only studies with an appropriate control group (e.g., sham-operated) were included. RESULTS AND DISCUSSION: Eleven out of 16 animal studies showed that OVX resulted in cartilage damage. When only studies using sexually mature animals were included, we saw that 11 out of 14 studies showed a detrimental effect, indicating considerable evidence for a relation between cartilage degeneration and OVX in mature animals. The effect of estrogen treatment was inconclusive with only 11 out of 22 animal studies reporting a beneficial effect on cartilage, whereas all six studies administering selective estrogen receptor modulators (SERMs) after OVX described protective effects. The discrepancy between the studies may be caused by the large variation in experimental set-up. We suggested a list of quality criteria for animal models since standardisation of design and outcome parameters of animal experiments may help to compare different studies and to gain better insight in the role of hormones in the osteoarthritic process.

Evidence for multiple peroxisome proliferator-activated receptor γ transcripts in bone: fine-tuning by hormonal regulation and mRNA stability (Article)

2008-05-01T00:00:00Z

The expression, regulation and functional significance of multiple Peroxisome proliferator-activated receptor γ transcript variants in bone were studied. PPARG transcripts giving rise to PPARg-1 protein were expressed in human osteoblasts, whereas PPARG-2 transcript and protein remained virtually absent. PPARG expression underwent homologous regulation, was upregulated during differentiation and directly induced by the osteogenic hormone dexamethasone, suggesting a role of PPARg-1 for osteogenesis. Differences between the stabilities of PPARG-1, -3 and -4 were observed. We hypothesize that cell-specific expression patterns of multiple PPARG transcript variants encoding for the same protein but differing in mRNA stabilities enable a fine-tuning of PPARG action, which eventually supports a well-adjusted signal transduction between the cell and its environment.

Evidence for multiple peroxisome proliferator-activated receptor γ transcripts in bone: fine-tuning by hormonal regulation and mRNA stability (Article)

2008-04-22T00:00:00Z

Cartilage damage pattern in relation to subchondral plate thickness in a collagenase-induced model of osteoarthritis (Article)

2008-04-01T00:00:00Z

OBJECTIVE: To see how initial differences in subchondral bone phenotype influence the development of cartilage damage and changes in subchondral bone architecture in an osteoarthritis (OA)-induced mouse model. METHOD: Intra-articular collagenase injections (right knee joint) and saline controls (left knee joint) were applied in the knees of two mouse strains known to have either a low or a high bone mass phenotype: the low bone mass C57Bl/6 mice with a thin subchondral bone plate and high bone mass C3H/HeJ mice with a thick subchondral bone plate. The ages of the mice were 16 and 30 weeks, with n=8 per group. The collagenase injection induced an osteoarthritic phenotype that was evaluated 4 weeks later in the tibia using histological analyses and micro-computed tomography (micro-CT). RESULTS: Both strains developed cartilage damage in the collagenase-injected right knee joints to a comparable extent, however, the spatial distribution of cartilage damage differed significantly: C57Bl/6 mice had most damage at the postero-lateral side, whereas in C3H/HeJ mice the postero-medial region was the most affected. Spontaneous cartilage damage was found in the saline-injected left control knees of C57Bl/6 mice, but in C3H/HeJ mice spontaneous cartilage damage was virtually absent. In both strains the subchondral bone plate of collagenase-injected joints became thinner, independent of the site of cartilage damage. TRAP-positive osteoclasts were observed underneath the subchondral bone plate, in line with the observed decreased thickness. No link was found between subchondral bone plate thickness and cartilage damage in the collagenase-injected joints. The subchondral trabecular architecture only changed in the high bone mass C3H/HeJ mice, with thinning of trabeculae and increased trabecular spacing. CONCLUSION: Thinning of the subchondral bone plate was found as a common observation 4 weeks after OA had been induced in two strains of mice having either a high or low bone phenotype, but no relation was found with the amount of cartilage damage. In addition, this study shows that different strains of mice can react differently to instability-induced OA with respect to the spatial arrangement of cartilage damage and changes in subchondral trabecular structure.

A role for subchondral bone changes in the process of osteoarthritis; a micro-CT study of two canine models (Article)

2008-02-01T00:00:00Z

BACKGROUND: This study evaluates changes in peri-articular bone in two canine models for osteoarthritis: the groove model and the anterior cruciate ligament transection (ACLT) model. METHODS: Evaluation was performed at 10 and 20 weeks post-surgery and in addition a 3-weeks time point was studied for the groove model. Cartilage was analysed, and architecture of the subchondral plate and trabecular bone of epiphyses was quantified using micro-CT. RESULTS: At 10 and 20 weeks cartilage histology and biochemistry demonstrated characteristic features of osteoarthritis in both models (very mild changes at 3 weeks). The groove model presented osteophytes only at 20 weeks, whereas the ACLT model showed osteophytes already at 10 weeks. Trabecular bone changes in the groove model were small and not consistent. This contrasts the ACLT model in which bone volume fraction was clearly reduced at 10 and 20 weeks (15-20%). However, changes in metaphyseal bone indicate unloading in the ACLT model, not in the groove model. For both models the subchondral plate thickness was strongly reduced (25-40%) and plate porosity was strongly increased (25-85%) at all time points studied. CONCLUSION: These findings show differential regulation of subchondral trabecular bone in the groove and ACLT model, with mild changes in the groove model and more severe changes in the ACLT model. In the ACLT model, part of these changes may be explained by unloading of the treated leg. In contrast, subchondral plate thinning and increased porosity were very consistent in both models, independent of loading conditions, indicating that this thinning is an early response in the osteoarthritis process.

ADAMTS5-/- mice have less subchondral bone changes after induction of osteoarthritis through surgical instability: implications for a link between cartilage and subchondral bone changes (Article)

2008-01-01T00:00:00Z

Objective: Osteoarthritis (OA) is characterized by damaged articular cartilage and changes in subchondral bone. Previous work demonstrated aggrecanase-2 deficient (ADAMTS5-/-) mice to be protected from cartilage damage induced by joint instability. This study analyzed whether this protective effect on cartilage is also reflected in the subchondral bone structure. Methods: Right knee joints from 10-week old male wild type (WT) and ADAMTS5-/- mice received transection of the medial meniscotibial ligament to induce OA, whereas left knees were left unoperated. After 8 weeks knee joints were scanned by micro-CT. The proximal tibia was selected for further analysis. Histology was performed to evaluate cartilage damage and osteoclast presence. Results: ADAMTS5-/- joints had a significantly thinner subchondral plate and less epiphyseal trabecular bone compared to WT joints. Histology confirmed previous findings that ADAMTS5-/- mice have significantly less cartilage damage than WT in the instability-induced OA model. Although the subchondral bone plate became significantly thicker at the medial tibial plateau in operated joints of both genotypes, the percentage increase was significantly smaller in ADAMTS5-/- mice (WT: 20.7 ± 4.7%, ADAMTS5-/-: 8.3 ± 1.2% compared to the left unoperated control joint). In ADAMTS5-/- animals a significant decrease was found in both Oc.N./BS and Oc.S./BS. Finally, in WT but not in ADAMTS5-/- mice a significant correlation was found between medial subchondral bone plate thickness and cartilage damage at the medial tibial plateau. Conclusion: ADAMTS5-/- joints that were protected from cartilage damage showed minor changes in the subchondral bone structure, in contrast to WT mice where substantial changes were found. This finding suggests links between the process of cartilage damage and subchondral bone changes in instability-induced OA.

Ghrelin and Bone (Article)

2007-11-06T00:00:00Z

A consequence of gastrectomy is loss of bone mass. Several mechanisms have been proposed, such as malabsorption of vitamins and minerals. Additionally, a peptide hormone produced in the stomach has been shown to mediate a calcitropic effect on bone. The identity of this peptide has not been elucidated, but ghrelin, produced by A-like cells in the fundus of the stomach, could be a good candidate. Ghrelin stimulates growth hormone (GH) secretion both in vivo and in vitro, and could by this means have a positive effect on bone. There is also evidence for direct effects of ghrelin on bone. We discuss here the role that ghrelin may play in bone metabolism, based on the most recent literature.

The activin A-follistatin system: Potent regulator of human extracellular matrix mineralization (Article)

2007-09-01T00:00:00Z

Bone quality is an important determinant of osteoporosis, and proper osteoblast differentiation plays an important role in the control and maintenance of bone quality. We investigated the impact of activin signaling on human osteoblast differentiation, extracellular matrix formation, and mineralization. Activins belong to the transforming growth factor-β superfamily and activin A treatment strongly inhibited mineralization in osteoblast cultures, whereas the activin antagonist follistatin increased mineralization. Osteoblasts produced activin A and follistatin in a differentiation-dependent manner, leading to autocrine regulation of extracellular matrix formation and mineralization. In addition, mineralization in a vascular smooth muscle cell-based model for pathological calcification was inhibited. Comparative activin A and follistatin gene expression profiling showed that activin signaling changes the expression of a specific range of extracellular matrix proteins prior to the onset of mineralization, leading to a matrix composition with reduced or no mineralizing capacity. These findings demonstrate the regulation of osteoblast differentiation and matrix mineralization by the activin A-follistatin system, providing the possibility to control bone quality as well as pathological calcifications such as atherosclerosis by using activin A, follistatin, or analogs thereof.

The catechol-O-methyltransferase Met158 low-activity allele and association with nonvertebral fracture risk in elderly men (Article)

2007-08-01T00:00:00Z

Context: Because sex steroids play an important role in bone development, variants in genes encoding proteins involved in estrogen synthesis and metabolism could contribute to interindividual variation in bone parameters and fracture risk. An example is catechol-O-methyltransferase (COMT), an estrogen-degrading enzyme involved in inactivation of catechol-estrogens. Its gene contains a functional valine to methionine substitution at codon 158. Objective: The aim of our study was to determine whether this polymorphism is associated with bone parameters and fracture risk in elderly subjects. Methods: COMT genotypes were determined using TaqMan allelic discrimination in 2515 men and 3554 women from the Rotterdam Study, a population-based cohort study of individuals aged 55 and older. Associations with bone mineral density (BMD) and bone loss were analyzed using ANOVA or analysis of covariance, whereas fracture risk was analyzed using Cox's proportional hazard regression analysis. COMT mRNA expression in three osteoblastic cell lines (SaOS, MG63, and SVHFO) was analyzed by RT-PCR. Results: Male carriers of the Met158allele had an increased risk for osteoporotic fractures (hazard ratio = 1.6; 95% confidence interval, 1.0 -2.4) and for fragility fractures (hazard ratio = 2.7; 95% confidence interval, 1.3-5.9), with evidence for a dominant effect. Adjustments for age, height, weight, and BMD did not change the risk estimates. In women, this association was weaker and not significant. BMD was not significantly associated with the variant in either men or women. COMT mRNA was expressed in all three osteoblastic cell lines tested. Conclusion: The COMT Val158Met polymorphism is associated with fracture risk in elderly men, through a mechanism independent of BMD. Copyright

Balans: buigen of barsten (Inaugural Lecture)

2007-06-01T00:00:00Z

Tijdens de voorbereiding op deze lezing kwam ik tot de conclusie dat er een duidelijke rode draad is, die loopt van mijn middelbare school, studiekeuze en keuzes binnen de studie naar mijn huidige werkzaamheden en leeropdracht. Centraal hierin staan de regelmechanismen die een organisme optimaal laten functioneren in zijn/haar omgeving en het organisme in balans doet zijn. Het organisme kan buigen; dat wil zeggen goed of voldoende reageren op veranderingen en het kan zich dan aanpassen aan veranderende omstandigheden. Het kan echter ook barsten bij foutief functioneren in geval van ziekte. Bij de handhaving van de balans zijn niet alleen interne (in het organisme) regelmechanismen betrokken, ook speelt de communicatie tussen het externe en interne milieu; de gen-omgevingsinteracties, een grote rol. In deze lezing zal ik de rode draad uiteenzetten en hoe deze verband houdt met mijn leeropdracht Calcium- en Botstofwisseling. rede In verkorte vorm uitgesproken ter gelegenheid van het aanvaarden van het ambt van bijzonder hoogleraar met als leeropdracht Calcium- en Botstofwisseling aan het Erasmus MC, faculteit van de Erasmus Universiteit Rotterdam op 1 juni 2007.

Long-term fluoxetine administration does not result in major changes in bone architecture and strength in growing rats (Article)

2007-05-01T00:00:00Z

Many studies have indicated that serotonin and its transporter play a role in bone metabolism. In this study we investigated the effect of selective serotonin re-uptake inhibitor (SSRI), fluoxetine (Prozac) on bone architecture and quality in growing female rats. We therefore administrated rats with clinically relevant doses of fluoxetine for a period of 6 months. DXA scans were performed during the treatment period in order to follow parameters as body weight, fat percentage and BMD. After 6 months of treatment, femurs were used to analyze bone architecture and bone strength, by means of microCT scans and three-point bending assays, respectively. We found a slightly diminished bone quality, reflected in a lower bone tissue strength, which was compensated by changes in bone geometry. As leptin and adiponectin could be possible factors in the serotonergic regulation of bone metabolism, we also determined the levels of these factors in plasma samples of all animals. Leptin and adiponectin levels were not different between the control group and fluoxetine-treated group, indicating that these factors were not involved in the observed changes in bone geometry and quality.

Vitamin D receptor gene haplotype is associated with body height and bone size (Article)

2007-04-01T00:00:00Z

Context: Adult stature is a complex genetic trait. The vitamin D endocrine system has pleiotropic effects on several physiological processes, especially on skeletal metabolism. We recently identified promoter and 3′-untranslated region (UTR) haplotype alleles that influence vitamin D receptor (VDR) mRNA expression. Objective: We studied whether VDR gene variants contribute to the genetic variation in height. Design and Subjects: We studied VDR haplotype alleles and body height in two independent populations (n = 7187). In a meta-analysis (n = 14,157 from 27 studies and our current data), we evaluated the effect of the Bsm I polymorphism. Results: Haplotypes of the linkage disequilibrium block 3 and block 5 were associated with body height differences with evidence for additive effects in the Rotterdam Study (P=0.00002) and the Longitudinal Aging Study Amsterdam study (P = 0.001). Height differences between the extreme genotypes were 1.4 and 2.7 cm, respectively. The relationship was independent of age, gender, presence of vertebral fractures, and age-related height loss. In the Rotterdam population, we found the combined genotype to be associated with decreased vertebral area (P = 0.03) and femoral narrow neck width (P = 0.002). In the meta-analysis, subjects with the "BB" genotype were 0.6 cm (95% confidence interval, 0.2-1.1 cm) taller than those with the "bb" genotype (P = 0.006). Conclusion: VDR gene variants are associated with differences in body height as evidenced by our study and by a meta-analysis. It remains for further studies to confirm whether the underlying mechanism of the association involves lower VDR expression in cells important for determining bone size. Copyright

Intrinsic differentiation potential of adolescent human tendon tissue: an in-vitro cell differentiation study (Article)

2007-02-23T00:00:00Z

Tendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential. METHODS: Cells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs). RESULTS: Tendon-derived cells stained D7-FIB (fibroblast-marker) positive, but alpha-SMA (marker for smooth muscle cells and pericytes) negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker), and 73% positive for CD105 (mesenchymal progenitor-cell marker). In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4) and PPARG (peroxisome proliferative activated receptor gamma). In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations. CONCLUSION: This study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis.

The novel vitamin D analog ZK191784 as an intestine-specific vitamin D antagonist (Article)

2006-10-01T00:00:00Z

Vitamin D [1,25(OH)2D3] plays a crucial role in Ca2+ homeostasis by stimulating Ca2+ (re)absorption and bone turnover. The 1,25(OH)2D3 analog ZK191784 was recently developed to dissociate the therapeutic immunomodulatory activity from the hypercalcemic side effects of 1,25(OH)2D3 and contains a structurally modified side chain characterized by a 22,23-double bond, 24R-hydroxy group, 25-cyclopropyl ring, and 5-butyloxazole unit. We investigated the effect of ZK191784 on Ca2+ homeostasis and the regulation of Ca2+ transport proteins in wild-type (WT) mice and mice lacking the renal epithelial Ca2+ channel TRPV5 (TRPV5-/-). The latter display hypercalciuria, hypervitaminosis D, increased intestinal expression of the epithelial Ca2+ channel TRPV6, the Ca2+-binding protein calbindin-D(9K), and intestinal Ca2+ hyperabsorption. ZK191784 normalized the Ca2+ hyperabsorption and the expression of intestinal Ca2+ transport proteins in TRPV5-/- mice. Furthermore, the compound decreased intestinal Ca2+ absorption in WT mice and reduced 1,25(OH)2D3-dependent 45Ca2+ uptake by Caco-2 cells, substantiating a 1,25(OH)2D3-antagonistic action of ZK191784 in the intestine. ZK191784 increased renal TRPV5 and calbindin-D(28K) expression and decreased urine Ca2+ excretion in WT mice. Both 1,25(OH)2D3 and ZK191784 enhanced transcellular Ca2+ transport in primary cultures of rabbit connecting tubules and cortical collecting ducts, indicating a 1,25(OH)2D3-agonistic effect in kidney. ZK191784 enhanced bone TRPV6 mRNA levels and 1,25(OH)2D3 as well as ZK191784 stimulated secretion of the bone formation marker osteocalcin in rat osteosarcoma cells, albeit to a different extent. In conclusion, ZK191784 is a synthetic 1,25(OH)2D3 ligand displaying a unique tissue-specific profile when administered in vivo. Because ZK191784 acts as an intestine-specific 1,25(OH)2D3 antagonist, this compound will be associated with less hypercalcemic side effects compared with the 1,25(OH)2D3 analogs currently used in clinical practice.

Stretch-induced modulation of matrix metalloproteinases in mineralizing osteoblasts via extracellular signal-regulated kinase-1/2 (Article)

2006-07-01T00:00:00Z

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) produced by osteoblasts play an essential role in bone remodeling. Hence, these proteins could provide an interesting means by which mechanical loading leads to adaptation of bone. Here, we examined the effect of stretch on MMP-1, -2, -3, -8, -9, -13, and -14, as well as TIMP-1 and -2 gene expression in differentiating, mineralizing, and nonmineralizing human SV-40 immortalized preosteoblast cells. In the mineralizing osteoblast culture, but not in the nonmineralizing cultures, cyclic stretch for only 15 min resulted in an increase of MMP-1 (fourfold) and -3 (depending on differentiation stage up to 25-fold) transcript abundance. No clear effect was observed for other MMPs, TIMP-1 or -2. The increase of MMP-1 and -3 was confirmed on the protein level. Stretching experiments performed in the presence of a specific inhibitor of extracellular signal-regulated kinase (ERK) showed a strong suppression of the stretch-induced increase in MMP-1 and -3. In conclusion, we show that MMP-1 and MMP-3 are mechanosensitive genes in mineralizing the human osteoblast, and that the mechano-induction of these genes is mediated via the ERK pathway. Our findings implicate that these MMPs are important factors in the mechanoregulation of bone turnover. With the ability to generate MMPs at highly stretched sites, osteoblasts can potantially direct osteoclasts to specific bone surface areas prepared for resorption.

Quantification of subchondral bone changes in a murine osteoarthritis model using micro-CT (Article)

2006-01-01T00:00:00Z

In the past few years there has been a considerable interest in the role of bone in osteoarthritis. Despite the increasing evidence of the involvement of bone in osteoarthritis, it remains very difficult to attribute the cause or effect of changes in subchondral bone to the process of osteoarthritis. Although osteoarthritis in mice provides a useful model to study changes in the subchondral bone, detailed quantification of these changes is lacking. Therefore, the goal of this study was to quantify subchondral bone changes in a murine osteoarthritis model by use of micro-computed tomography (micro-CT). We induced osteoarthritis-like characteristics in the knee joints of mice using collagenase injections, and after four weeks we calculated various 3D morphometric parameters in the epiphysis of the proximal tibia. The collagenase injections caused cartilage damage, visible in histological sections, particularly on the medial tibial plateau. Micro-CT analysis revealed that the thickness of the subchondral bone plate was decreased both at the lateral and the medial side. The trabecular compartment demonstrated a small but significant reduction in bone volume fraction compared to the contralateral control joints. Trabeculae in the collagenase-injected joints were thinner but their shape remained rod-like. Furthermore, the connectivity between trabeculae was reduced and the trabecular spacing was increased. In conclusion, four weeks after induction of osteoarthritis in the murine knee subtle but significant changes in subchondral bone architecture could be detected and quantified in 3D with micro-CT analysis.

The epithelial Ca2+ channel TRPV5 is essential for proper osteoclastic bone resorption. (Article)

2005-11-29T00:00:00Z

Bone remodeling involves the interplay of bone resorption and formation and is accurately controlled to maintain bone mass. Both processes require transcellular Ca(2+) transport, but the molecular mechanisms engaged remain largely elusive. The epithelial Ca(2+) channel TRPV5 is one of the most Ca(2+)-selective transient receptor potential (TRP) channels. In this study, the functional role of TRPV5 in bone was investigated. TRPV5 mRNA was expressed in human and murine bone samples and in osteoclasts along with other genes involved in transcellular Ca(2+) transport, including calbindin-D(9K) and calbindin-D(28K), Na(+)/Ca(2+) exchanger 1, and plasma membrane Ca(2+)-ATPase 1b. TRPV5 expression in murine osteoclasts was confirmed by immunostaining and showed predominant localization to the ruffled border membrane. However, TRPV5 was absent in osteoblasts. Analyses of femoral bone sections from TRPV5 knockout (TRPV5(-/-)) mice revealed increased osteoclast numbers and osteoclast area, whereas the urinary bone resorption marker deoxypyridinoline was reduced compared with WT (TRPV5(+/+)) mice. In an in vitro bone marrow culture system, the amount of osteoclasts and number of nuclei per osteoclast were significantly elevated in TRPV5(-/-) compared with TRPV5(+/+) mice. However, using a functional resorption pit assay, we found that bone resorption was nearly absent in osteoclast cultures from TRPV5(-/-) mice, supporting the impaired resorption observed in vivo. In conclusion, TRPV5 deficiency leads to an increase in osteoclast size and number, in which Ca(2+) resorption is nonfunctional. This report identifies TRPV5 as an epithelial Ca(2+) channel that is essential for osteoclastic bone resorption and demonstrates the significance of transcellular Ca(2+) transport in osteoclastic function.

Identification of acid-sensing ion channels in bone (Article)

2005-11-01T00:00:00Z

Bone balances serum pH variations and both osteoclasts and osteoblasts are regulated by subtle changes in pH. The aim of the current study was to identify molecules in bone that can sense pH. Interesting candidates are the acid-sensing ion channels (ASICs). In bone, ASIC2 and ASIC3 were most abundant, while in chondrocytes it was ASIC1. Isolated human monocytes expressed ASIC1, -2, and -3, which persisted after induction to osteoclast differentiation, albeit to a lower level. In human osteoblasts ASIC1, ASIC2, and ASIC3 mRNAs were shown. Western blot and immunostaining confirmed this at protein level. ASIC4 expression was always very low abundant. For the first time, we demonstrated ASICs in human skeleton, providing a means to sense and respond to differences in extracellular pH.

Hypervitaminosis D mediates compensatory Ca2+ hyperabsorption in TRPV5 knockout mice (Article)

2005-11-01T00:00:00Z

Vitamin D plays an important role in Ca(2+) homeostasis by controlling Ca(2+) (re)absorption in intestine, kidney, and bone. The epithelial Ca(2+) channel TRPV5 mediates the Ca(2+) entry step in active Ca(2+) reabsorption. TRPV5 knockout (TRPV5(-/-)) mice show impaired Ca(2+) reabsorption, hypercalciuria, hypervitaminosis D, and intestinal hyperabsorption of Ca(2+). Moreover, these mice demonstrate upregulation of intestinal TRPV6 and calbindin-D(9K) expression compared with wild-type mice. For addressing the role of the observed hypervitaminosis D in the maintenance of Ca(2+) homeostasis and the regulation of expression levels of the Ca(2+) transport proteins in kidney and intestine, TRPV5/25-hydroxyvitamin-D(3)-1alpha-hydroxylase double knockout (TRPV5(-/-)/1alpha-OHase(-/-)) mice, which show undetectable serum 1,25(OH)(2)D(3) levels, were generated. TRPV5(-/-)/1alpha-OHase(-/-) mice displayed a significant hypocalcemia compared with wild-type mice (1.10 +/- 0.02 and 2.54 +/- 0.01 mM, respectively; P < 0.05). mRNA levels of renal calbindin-D(28K) (7 +/- 2%), calbindin-D(9K) (32 +/- 4%), Na(+)/Ca(2+) exchanger (12 +/- 2%), and intestinal TRPV6 (40 +/- 8%) and calbindin-D(9K) (26 +/- 4%) expression levels were decreased compared with wild-type mice. Hyperparathyroidism and rickets were present in TRPV5(-/-)/1alpha-OHase(-/-) mice, more pronounced than observed in single TRPV5 or 1alpha-OHase knockout mice. It is interesting that a renal Ca(2+) leak, as demonstrated in TRPV5(-/-) mice, persisted in TRPV5(-/-)/1alpha-OHase(-/-) mice, but a compensatory upregulation of intestinal Ca(2+) transporters was abolished. In conclusion, the elevation of serum 1,25(OH)(2)D(3) levels in TRPV5(-/-) mice is responsible for the upregulation of intestinal Ca(2+) transporters and Ca(2+) hyperabsorption. Hypervitaminosis D, therefore, is of crucial importance to maintain normocalcemia in impaired Ca(2+) reabsorption in TRPV5(-/-) mice.

Bone mineral density and vertebral fracture history are associated with incident and progressive radiographic knee osteoarthritis in elderly men and women: the Rotterdam Study (Article)

2005-10-01T00:00:00Z

OBJECTIVE: To study the association between baseline femoral neck and lumbar spine bone mineral density (BMD), prevalent fractures and incident and progressive radiographic osteoarthritis (ROA) of the knee in men and women. METHODS: A sample of 1403 subjects (829 women and 574 men) was drawn from the Rotterdam Study, a prospective population-based cohort study of the elderly. Incidence and progression of ROA in quartiles of femoral neck (FN) and lumbar spine (LS) BMD were determined using the Kellgren score, and separate analyses were made for men and women. Furthermore, incidence and progression of ROA were compared in subjects with and without a prevalent vertebral or non-vertebral fracture at baseline. RESULTS: The incidence of knee ROA of subject in the highest FN BMD (10.5%) and LS BMD (14.3%) was significantly higher than of those in the lowest quartiles (3.4% and 3.3% respectively), with corresponding adjusted odds ratios (95% confidence interval) of 2.8 (1.2-6.8) and 4.7 (2.1-10.7). The same trend was seen in the association between LS BMD and the progression of knee ROA, but no association was found between FN BMD and progression of ROA. Separate analyses for men and women both showed significant increased risks in the presence of high baseline BMD, with higher odds ratios in men than in women but larger confidence limits due to lower number of cases in men. Combined incidence and progression of knee ROA in subjects with a prevalent vertebral but not with a prevalent non-vertebral fracture at baseline was 8 times lower than subject without a fracture, independent of baseline BMD. CONCLUSIONS: High systemic BMD at baseline is associated with increased incidence and progression of knee ROA in both men and women, while a prevalent vertebral fracture has a protective effect.

Estrogen receptor alpha gene polymorphisms are associated with estradiol levels in postmenopausal women. (Article)

2005-08-01T00:00:00Z

OBJECTIVE: Postmenopausal estradiol (E(2)) levels vary widely between individuals and this variation is an important determinant of diseases such as osteoporosis. It has been suggested that the estrogen receptor alpha (ESR1) gene may influence peripheral E(2) levels, but the role of common sequence variations in the ESR1 gene is unclear. METHODS: In 631 postmenopausal women and 528 men from the Rotterdam Study, a population-based, prospective cohort study of individuals aged 55 years and over, ESR1 PvuII-XbaI haplotypes were determined and correlated with plasma E2 levels. RESULTS: In women, haplotype 1 (T-A) was significantly associated with an allele-dose-dependent decrease in E(2). After adjusting for age, body mass index, years since menopause and testosterone levels, plasma E(2) levels decreased by 1.90 pmol/l per allele copy of this haplotype (P < 0.05). Extreme genotypes, representing 23 and 27% of the population, varied by 3.93 pmol/l. No association with plasma testosterone was observed. In a subset of 446 women, no association of genotype with plasma concentrations of dehydroepiandrosterone sulfate, androstenedione or estrone was seen. In men, none of the sex hormone levels was associated with the ESR1 PvuII-XbaI haplotypes. CONCLUSION: We have demonstrated a role for genetic variations in the ESR1 gene in determining post-menopausal E(2) levels in women.

11beta-Hydroxysteroid dehydrogenase expression and glucocorticoid synthesis are directed by a molecular switch during osteoblast differentiation. (Article)

2005-03-01T00:00:00Z

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays an important role in the prereceptor regulation of corticosteroids by locally converting cortisone into active cortisol. To investigate the impact of this mechanism on osteoblast development, we have characterized 11beta-HSD1 activity and regulation in a differentiating human osteoblast cell line (SV-HFO). Continuous treatment with the synthetic glucocorticoid dexamethasone induces differentiation of SV-HFO cells during 21 d of culture. Using this cell system, we showed an inverse relationship between 11beta-HSD1 activity and osteoblast differentiation. 11beta-HSD1 mRNA expression and activity were low and constant in differentiating osteoblasts. However, in the absence of differentiation (no dexamethasone), 11beta-HSD1 mRNA and activity increased strongly from d 12 of culture onward, with a peak around d 19. Promoter reporter studies provided evidence that specific regions of the 11beta-HSD1 gene are involved in this differentiation controlled regulation of the enzyme. Functional implication of these changes in 11beta-HSD1 is shown by the induction of osteoblast differentiation in the presence of cortisone. The current study demonstrates the presence of an intrinsic differentiation-driven molecular switch that controls expression and activity of 11beta-HSD1 and thereby cortisol production by human osteoblasts. This efficient mechanism by which osteoblasts generate cortisol in an autocrine fashion to ensure proper differentiation will help to understand the complex effects of cortisol on bone metabolism.

Promoter and 3'-untranslated-region haplotypes in the vitamin d receptor gene predispose to osteoporotic fracture: the rotterdam study (Article)

2005-01-01T00:00:00Z

Polymorphisms of the vitamin D receptor gene (VDR) have been shown to be associated with several complex diseases, including osteoporosis, but the mechanisms are unknown and study results have been inconsistent. We therefore determined sequence variation across the major relevant parts of VDR, including construction of linkage disequilibrium blocks and identification of haplotype alleles. We analyzed 15 haplotype-tagging SNPs in relation to 937 clinical fractures recorded in 6,148 elderly whites over a follow-up period of 7.4 years. Haplotype alleles of the 5' 1a/1e, 1b promoter region and of the 3' untranslated region (UTR) were strongly associated with increased fracture risk. For the 16% of subjects who had risk genotypes at both regions, their risk increased 48% for clinical fractures (P = .0002), independent of age, sex, height, weight, and bone mineral density. The population-attributable risk varied between 1% and 12% for each block and was 4% for the combined VDR risk genotypes. Functional analysis of the variants demonstrated 53% lower expression of a reporter construct with the 1e/1a promoter risk haplotype (P = 5 x 10(-7)) in two cell lines and 15% lower mRNA level of VDR expression constructs carrying 3'-UTR risk haplotype 1 in five cell lines (P = 2 x 10(-6)). In a further analysis, we showed 30% increased mRNA decay in an osteoblast cell line for the construct carrying the 3'-UTR risk haplotype (P = .02). This comprehensive candidate-gene analysis demonstrates that the risk allele of multiple VDR polymorphisms results in lower VDR mRNA levels. This could impact the vitamin D signaling efficiency and might contribute to the increased fracture risk we observed for these risk haplotype alleles.

Stretch-induced phosphorylation of ERK1/2 depends on differentiation stage of osteoblasts (Article)

2004-10-01T00:00:00Z

The goal of this study was to investigate the effect of mechanical loading on osteoblasts and extracellular signal-regulated kinase (ERK1/2) signaling in relation to osteoblast differentiation and mineralization. A human osteoblast cell line (SV-HFO) was triggered to differentiate to the advanced state of mineralization by addition of the osteogenic factors dexamethasone and beta-glycerophosphate. Osteoblasts were subjected to cyclic, equibiaxial stretch for 5, 15, or 60 min at different stages of differentiation (days 7, 14, and 21). Baseline (static) phosphorylated ERK1/2 and total ERK1/2 levels gradually increased during osteoblast differentiation. Cyclic stretch induced a rapid increase in ERK1/2 phosphorylation with a maximum between 5 and 15 min. Prolonged stretching for 60 min resulted in a decrease of phosphorylated ERK1/2 towards baseline level, suggesting a desensitization mechanism. The effect of stretch on ERK1/2 phosphorylation was strongest at later stages of differentiation (days 14 and 21). At day 21, the increase of ERK1/2 phosphorylation in response to stretch was significantly lower in non-differentiating than in differentiating osteoblasts. This could not be explained by differences in cell density, but did correlate with the formation of extracellular matrix, collagen fibrils. Mineralization of the extracellular matrix did not lead to a further increase of ERK1/2 phosphorylation. In conclusion, the current study demonstrates that the extent of activation of the ERK1/2 pathway is dependent on the differentiation or functional stage of the osteoblast. The presence of an extracellular matrix, but not per se mineralization, seems to be the predominant determinant of osteoblastic response to strain.

Fibroblast growth factor-2 in serum-free medium is a potent mitogen and reduces dedifferentiation of human ear chondrocytes in monolayer culture (Article)

2004-07-01T00:00:00Z

The loss of the differentiated phenotype (dedifferentiation) during the expansion culture of donor chondrocytes remains a large problem in cartilage tissue engineering. Dedifferentiated chondrocytes produce other matrix components and therefore the tissue produced will be of less suitable quality. Previously, the addition of fibroblast growth factor-2 (FGF2) to a serum-containing medium (SCM) during expansion culture was shown to have positive effects on the phenotype of articular chondrocytes. In the present study, we focused on a more defined, serum-free medium (SFM), to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. Adult human ear chondrocytes were expanded in serum-free medium supplemented with 100 ng/ml FGF2. Expansion culture in a conventional serum-containing medium (10% FCS) served as control. The cell yield during expansion culture in serum-free medium with FGF2 was significantly higher compared to serum-containing medium. In addition, chondrocytes expanded in the serum-free medium with FGF2 expressed a more differentiated phenotype at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I and aggrecan to versican. Also, a higher gene expression of Sox9 was found. Next, suspension in alginate and subsequent culture in vitro or subcutaneous implantation in nude mice was used to evaluate the capacity of the chondrocytes, expanded in either medium, to re-express the differentiated phenotype (redifferentiation) and to form cartilage. The observed beneficial effects of the serum-free medium with FGF2 on the chondrocyte phenotype at the end of monolayer culture were sustained on both transcriptional and extracellular level throughout both redifferentiation methods.

Estrogen receptor alpha gene polymorphisms and risk of myocardial infarction. (Article)

2004-06-23T00:00:00Z

CONTEXT: The role of estrogens in ischemic heart disease (IHD) is uncertain. Evidence suggests that genetic variations in the estrogen receptor alpha (ESR1) gene may influence IHD risk, but the role of common sequence variations in the ESR1 gene is unclear. OBJECTIVE: To determine whether the ESR1 haplotype created by the c.454-397T>C (PvuII) and c.454-351A>G (XbaI) polymorphisms is associated with myocardial infarction (MI) and IHD risk. DESIGN, SETTING, AND PARTICIPANTS: In 2617 men and 3791 postmenopausal women from The Rotterdam Study (enrollment between 1989-1993 and follow-up to January 2000), a population-based, prospective cohort study of participants aged 55 years and older, ESR1 c.454-397T>C and c.454-351A>G haplotypes were determined. Detailed interviews and physical examinations were performed, blood samples were obtained, and cardiovascular risk factors were assessed. MAIN OUTCOME MEASURE: The primary outcome was MI and IHD defined as MIs, revascularization procedures, and IHD mortality. RESULTS: Approximately 29% of women and 28.2% of men were homozygous carriers of the ESR1 haplotype 1 (-397 T and -351 A) allele, 49% of women and 50% of men were heterozygous carriers, and 22% of women and 21.4% of men were noncarriers. During a mean follow-up of 7.0 years, 285 participants (115 women; 170 men) had MI, and 440 (168 women; 272 men) had an IHD event, of which 97 were fatal. After adjustment for known cardiovascular risk factors, female heterozygous carriers of haplotype 1 had an increased risk of MI (event rate, 2.8%; relative risk [RR], 2.23; 95% confidence interval [CI], 1.13-4.43) compared with noncarriers (event rate, 1.3%), whereas homozygous carriers had an increased risk (event rate, 3.2%; RR, 2.48; 95% CI, 1.22-5.03). For IHD events, we observed a similar association. In women, the effect of haplotype 1 on fatal IHD was larger than on nonfatal IHD. In men, the ESR1 haplotypes were not associated with an increased risk of MI (event rate, 5.7%; RR, 0.93; 95% CI, 0.59-1.46 for heterozygous carriers; and event rate, 5.1%; RR, 0.82; 95% CI, 0.49-1.38 for homozygous carriers) compared with noncarriers (event rate, 5.8%) and were not associated with an increased risk of IHD. CONCLUSIONS: In this population-based, prospective cohort study, postmenopausal women who carry ESR1 haplotype 1 (c.454-397 T allele and c.454-351 A allele) have an increased risk of MI and IHD, independent of known cardiovascular risk factors. In men, no association was observed.

Height in pre- and postmenopausal women is influenced by estrogen receptor alpha gene polymorphisms (Article)

2004-01-01T00:00:00Z

The estrogen receptor alpha gene (ESR1) is known to be involved in metabolic pathways influencing growth. We have performed two population-based association studies using three common polymorphisms within this candidate gene to determine whether these are associated with variation in adult stature. In 607 women, aged 55-80 yr, from the Rotterdam Study, the ESR1 PvuII-XbaI haplotype 1 (px) and the L allele of the TA repeat polymorphism (<18 TA repeats) were significantly associated with an allele dose-dependent decrease in height. The per allele copy of ESR1 PvuII-XbaI haplotype 1 height was 0.9 cm shorter (P trend = 0.02) and 1.0 cm/allele copy of the TA repeat L allele (P trend = 0.003). These results were independent of age, age at menarche and menopause, and lumbar spine bone mineral density and remained significant after participants with vertebral fractures were excluded. In 483 men from the Rotterdam Study we found no association with height. In 1500 pre- and perimenopausal women from the Eindhoven Study a similar association was observed; women were 0.5 cm shorter per allele copy of the ESR1 haplotype 1 (P for trend = 0.03). In conclusion, we demonstrate a role for genetic variations in the estrogen receptor alpha gene in determining adult stature in women.

Homocysteine levels and the risk of osteoporotic fracture (Article)

2004-01-01T00:00:00Z

BACKGROUND: Very high plasma homocysteine levels are characteristic of homocystinuria, a rare autosomal recessive disease accompanied by the early onset of generalized osteoporosis. We therefore hypothesized that mildly elevated homocysteine levels might be related to age-related osteoporotic fractures. METHODS: We studied the association between circulating homocysteine levels and the risk of incident osteoporotic fracture in 2406 subjects, 55 years of age or older, who participated in two separate prospective, population-based studies. In the Rotterdam Study, there were two independent cohorts: 562 subjects in cohort 1, with a mean follow-up period of 8.1 years; and 553 subjects in cohort 2, with a mean follow-up period of 5.7 years. In the Longitudinal Aging Study Amsterdam, there was a single cohort of 1291 subjects, with a mean follow-up period of 2.7 years. Multivariate Cox proportional-hazards regression models were used for analysis of the risk of fracture, with adjustment for age, sex, body-mass index, and other characteristics that may be associated with the risk of fracture or with increased homocysteine levels. RESULTS: During 11,253 person-years of follow-up, osteoporotic fractures occurred in 191 subjects. The overall multivariable-adjusted relative risk of fracture was 1.4 (95 percent confidence interval, 1.2 to 1.6) for each increase of 1 SD in the natural-log-transformed homocysteine level. The risk was similar in all three cohorts studied, and it was also similar in men and women. A homocysteine level in the highest age-specific quartile was associated with an increase by a factor of 1.9 in the risk of fracture (95 percent confidence interval, 1.4 to 2.6). The associations between homocysteine levels and the risk of fracture appeared to be independent of bone mineral density and other potential risk factors for fracture. CONCLUSIONS: An increased homocysteine level appears to be a strong and independent risk factor for osteoporotic fractures in older men and women.

Cdx-2 polymorphism in the promoter region of the human vitamin D receptor gene determines susceptibility of fracture in the elderly (Article)

2003-09-01T00:00:00Z

A Cdx-2 binding site polymorphism (G to A) in the promoter region of the human vitamin D receptor gene was reported. In an ecological study in eight ethnic groups and an association study in 2848 elderly whites, we found the A-allele to be associated with decreased fracture risk. Our findings expand previous similar findings in a Japanese study to whites and show a relationship with fracture risk of this functional polymorphism. INTRODUCTION: A single nucleotide polymorphism (SNP) within a binding site of the intestinal-specific transcription factor Cdx-2 in the promoter region of the human vitamin D receptor (VDR) gene was previously reported. It was found to modulate the transcription of the hVDR gene and to be associated with decreased bone mineral density in a small group of postmenopausal Japanese women. In this study, we investigated the relationship between the VDR Cdx-2 genotype and risk of fracture. METHODS: We first determined the location of this SNP in the VDR gene by sequencing analysis, and we developed an allele-specific multiplex polymerase chain reaction test to determine the Cdx-2 genotype. We then performed an ecological study in eight ethnic groups and an association analysis in a large epidemiological cohort of 2848 Dutch white men and women, > or = 55 years old. RESULTS AND CONCLUSIONS: The location of the G to A substitution was found in the promoter region of exon le (le-G-1739A) of the VDR gene. By comparing the frequency of the A-allele in eight different ethnic groups, we observed a negative correlation between prevalence of the A-allele and published hip fracture incidence rates in these ethnic groups (p = 0.006 for men and p = 0.02 for women), suggesting a protective effect of this allele on fracture risk. Subsequently, in the association study, the A-allele (population frequency 19%) was observed to have a protective effect on occurrence of osteoporotic fractures, especially for nonvertebral fracture in women (relative risk of AA versus GG genotype is 0.2; 95% CI, 0.05-0.8). This effect remained after adjustment for age, weight, and bone mineral density. We conclude that the A-allele of the VDR Cdx-2 polymorphism is present in whites, albeit at low frequency, and show a protective effect of this allele on risk of fracture.

Association of 5' estrogen receptor alpha gene polymorphisms with bone mineral density, vertebral bone area and fracture risk (Article)

2003-07-15T00:00:00Z

This study investigates the influence of genetic variation of the estrogen receptor alpha (ESR1) gene locus on several bone parameters in 2042 individuals of The Rotterdam Study, a prospective population-based cohort study of elderly subjects. We analysed three polymorphic sites in the 5' region of the ESR1 gene; a (TA)(n)-repeat in the promoter region, and molecular haplotypes of the PvuII and XbaI RFLPs in intron 1, and inferred long-range haplotypes (LRH) thereof. We observed only three of the possible four PvuII-XbaI haplotypes in our population. A comparison with other Caucasian populations showed similar haplotype frequencies, while in Asian and African populations these were different. Linkage disequilibrium (LD) analysis between the PvuII-XbaI haplotype and the (TA)(n) repeat showed strong LD between the two sites. Reconstruction of long range haplotypes over the entire 5' region, revealed six frequent LRH. In men, we did not observe an association between the ESR1 polymorphisms studied and bone parameters. In women, we demonstrated an allele dose effect of haplotype "px" (P=0.003) and a low number of (TA)(n) repeats (P=0.008) with decreased lumbar spine bone mineral density (BMD) (4.8% lower BMD in women homozygous for haplotype "px", representing 28% of the population, compared with homozygous non-carriers) and decreased vertebral bone area (2.3% difference between extreme genotypes; P=0.016). Most importantly, we found an increased vertebral fracture risk with evidence for an allele dose effect with an odds ratio of 2.2 (95%CI 1.3-3.5) for haplotype "px", and 2.0 (1.5-3.2) for a low number of (TA)(n) repeats. The ESR1 genotype dependent fracture risk is largely independent of BMD and bone area. Combination of risk alleles at both loci by long-range haplotyping improved the associations slightly, but because of the strong LD between the two polymorphic sites, we were unable to determine if any particular polymorphic site is driving the associations found. We conclude that ESR1 polymorphism in the 5' (promoter) region is associated with vertebral fracture risk, lumbar spine BMD and vertebral bone area in postmenopausal women, but not in men. The molecular mechanism underlying this association needs further study.

Estrogen receptor and gene haplotype is associated with radiographic osteoarthritis of the knee in the elderly men and woman (Article)

2003-07-01T00:00:00Z

OBJECTIVE: Genetic influences have been shown to play an important role in the etiology of osteoarthritis (OA), but the genes involved are ill-defined. We studied the association between polymorphisms in the estrogen receptor alpha (ERalpha) gene and the prevalence of radiographic OA of the knee. METHODS: The study group comprised 1,483 men and women from the Rotterdam Study. Direct molecular haplotyping was used to determine the relationship between 2 polymorphisms in the ERalpha gene (the Pvu II and Xba I restriction fragment-length polymorphisms). Radiographs of the knee were evaluated according to the Kellgren/Lawrence (K/L) score, along with separate scores for osteophytosis and joint space narrowing. RESULTS: Three different haplotype alleles were identified: px (54%), PX (34%), and Px (12%). Allele PX was associated with an increased prevalence of radiographic knee OA (K/L score >/=2). The prevalence of radiographic OA was 22% among subjects without allele PX, 24% among those carrying 1 copy, and 35% among subjects carrying 2 copies. The corresponding odds ratios, after adjustment for confounding factors, were 1.3 (95% confidence interval [95% CI] 0.9-1.7) for heterozygotes and 2.2 (95% CI 1.5-3.4) for homozygotes. Separate analyses for men and women showed similar risk estimates. The association appeared to be driven by osteophytosis and is somewhat consistent with the association observed in previous studies of these polymorphisms in relation to OA. CONCLUSION: This study shows that polymorphisms in the ERalpha gene are associated with radiographic OA of the knee, and in particular with osteophytosis, in both elderly men and elderly women.

Mechanical control of human osteoblast apoptosis and proliferation in relation to differentiation (Article)

2003-04-01T00:00:00Z

Bone cells respond to mechanical stimulation. This is thought to be the mechanism by which bone adapts to mechanical loading. Reported responses of bone cells to mechanical stimuli vary widely and therefore there is no consensus on what mechanisms of mechanotransduction are physiologically relevant. We hypothesize that the differentiation stage of osteoblastic cells used to study responses to strain in vitro determines the outcome of applied loading. A human fetal osteoblast cell line was triggered to differentiate in culture to the advanced state of mineralization by addition of the osteogenic factors dexamethasone and b-glycerophosphate. Osteoblast cultures were subjected to increasing levels of cyclic, equibiaxial stretch at different stages of differentiation. We show that differentiation of human osteoblasts affects their responses to stretch in vitro. In 7-day osteoblast cultures, stretch results in decreased cell numbers as cells are triggered into apoptosis, independent of the stretch level (between 0.4-2.5%). In more mature cultures, apoptosis is not affected by the same treatment. Stretching differentiating cultures at day 14 actually increases proliferation. This is the first study reporting on differentiation-dependent mechanical control of osteoblast proliferation and apoptosis and is fundamental in understanding mechanotransduction processes in bone. The tight regulation of these responses by differentiation implies the significance of the differentiation stage of osteoblasts for the translation of mechanical signals and corroborates with the putative role of the osteoblastic lineage as mechanotransducer in bone.

Interaction between vitamin D receptor genotype and estrogen receptor alpha genotype influences vertebral fracture risk (Article)

2003-01-01T00:00:00Z

In view of the interactions of vitamin D and the estrogen endocrine system, we studied the combined influence of polymorphisms in the estrogen receptor (ER) alpha gene and the vitamin D receptor (VDR) gene on the susceptibility to osteoporotic vertebral fractures in 634 women aged 55 yr and older. Three VDR haplotypes (1, 2, and 3) of the BsmI, ApaI, and TaqI restriction fragment length polymorphisms and three ERalpha haplotypes (1, 2, and 3) of the PvuII and XbaI restriction fragment length polymorphisms were identified. We captured 131 nonvertebral and 85 vertebral fracture cases during a mean follow-up period of 7 yr. ERalpha haplotype 1 was dose-dependently associated with increased vertebral fracture risk (P < 0.001) corresponding to an odds ratio of 1.9 [95% confidence interval (CI), 0.9-4.1] per copy of the risk allele. VDR haplotype 1 was overrepresented in vertebral fracture cases. There was a significant interaction (P = 0.01) between ERalpha haplotype 1 and VDR haplotype 1 in determining vertebral fracture risk. The association of ERalpha haplotype 1 with vertebral fracture risk was only present in homozygous carriers of VDR haplotype 1. The risk of fracture was 2.5 (95% CI, 0.6-9.9) for heterozygous and 10.3 (95% CI, 2.7-40) for homozygous carriers of ERalpha haplotype 1. These associations were independent of bone mineral density. In conclusion, interaction between ERalpha and VDR gene polymorphisms leads to increased risk of osteoporotic vertebral fractures in women, largely independent of bone mineral density.

ERK activation and alpha v beta 3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts (Article)

2002-09-12T00:00:00Z

Osteoblast growth and differentiation are critical processes for bone development and maintenance, and are regulated by both humoral and mechanical factors. Humoral (hormonal) factors can affect gene transcription via MAPkinases, e.g., extracellular signal-regulated kinase (ERK). We studied whether the ERK pathway is also involved in processing mechanical inputs in human bone cells. Exposing MG63 cells to physiologically relevant levels of fluid flow resulted in ERK phosphorylation. Genistein blocked this response, indicating that it is dependent on tyrosine phosphorylation. Furthermore, alpha v beta 3 integrins were activated in response to fluid flow, as shown by recruitment of adaptor molecule Shc and clustering of alpha v beta 3 in focal adhesion-like structures. Antibodies blocking formation of beta 1 or beta 3 integrin-matrix interactions or RGD peptides could not inhibit fluid flow-induced ERK phosphorylation, suggesting that formation of new integrin-matrix interactions is not essential for this response and that other upstream mechanosensors regulate induction of ERK phosphorylation in response to fluid flow in human bone cells.

A central dinucleotide within vitamin D response elements modulates DNA binding and transactivation by the vitamin D receptor in cellular response to natural and synthetic ligands. (Article)

2002-04-26T00:00:00Z

There is considerable divergence in the sequences of steroid receptor response elements, including the vitamin D response elements (VDREs). Two major VDRE-containing and thus 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3))-regulated genes are the two non-collagenous, osteoblast-derived bone matrix proteins osteocalcin and osteopontin. We observed a stronger induction of osteopontin than osteocalcin mRNA expression by 1,25-(OH)(2)D(3). Subsequently, we have shown that vitamin D receptor/retinoid X receptor alpha (VDR/RXRalpha) heterodimers bind more tightly to the osteopontin VDRE than to the osteocalcin VDRE. Studies using point mutants revealed that the internal dinucleotide at positions 3 and 4 of the proximal steroid half-element are most important for modulating the strength of receptor binding. In addition, studies with VDRE-driven luciferase reporter gene constructs revealed that the central dinucleotide influences the transactivation potential of VDR/RXRalpha with the same order of magnitude as that observed in the DNA binding studies. The synthetic vitamin D analog KH1060 is a more potent stimulator of transcription and inducer of VDRE binding of VDR/RXR in the presence of nuclear factors isolated from ROS 17/2.8 osteoblast-like cells than the natural ligand 1,25-(OH)(2)D(3). Interestingly, however, KH1060 is comparable or even less potent than 1,25-(OH)(2)D(3) in stimulating VDRE binding of in vitro synthesized VDR/RXRalpha. Thus, the extent of 1,25-(OH)(2)D(3)- and KH1060-dependent binding of VDR/RXRalpha is specified by a central dinucleotide in the VDRE, and the ligand-induced effects on DNA binding are in part controlled by the cellular context of nuclear proteins.

Adjacent genes, for collagen type II?1 gene and the vitamin D receptor, are associated with separate features of the radiographic osteoarthritis at the knee (Article)

2000-01-01T00:00:00Z

OBJECTIVE: To study the association of the COL2A1 genotype, in relation to the vitamin D receptor (VDR) genotype, with features of radiographic osteoarthritis (ROA) in a population of elderly men and women. METHODS: In this cross-sectional study, we analyzed a population-based sample of 851 men and women ages 55-80 years from a large cohort study, the Rotterdam Study. We determined the prevalence of ROA of the knee according to the Kellgren/Lawrence (K/L) score and features of ROA (presence of osteophytes and narrowing of the joint space [JSN]) without considering clinical parameters of the disease. Genotypes were determined at a variable-number tandem repeats marker 1 kb downstream of the COL2A1 gene using a newly developed heteroduplexing method. The VDR genotype was previously determined by a direct molecular haplotyping polymerase chain reaction method to establish the phase of alleles at 3 adjacent restriction fragment length polymorphisms for Bsm I, Apa I, and Taq I. RESULTS: We found the COL2A1 genotype to be associated with a 2-fold increased risk for JSN, but not with osteophytes or the K/L score. We had previously found the VDR genotype to be associated with osteophytes and the K/L score, but not with JSN. When the COL2A1 genotype was analyzed in combination with the VDR genotype, we found evidence suggesting that the presence of haplotypes of the 2 genes was associated with increased risk for ROA. CONCLUSION: Our findings demonstrate that both the COL2A1 gene and the VDR gene are involved in ROA, but in separate features. The COL2A1 genotype is associated with JSN, while the VDR genotype is associated with osteophytes.

Estrogen receptor polymorphism predicts the onset of natural and surgical menopause (Article)

1999-01-01T00:00:00Z

Age at menopause and risk of hysterectomy have strong genetic components, but the genes involved remain ill defined. We investigated whether genetic variation at the estrogen receptor (ER) gene contributes to the variability in the onset of menopause in 900 postmenopausal women, aged 55-80 yr, of the Rotterdam Study, a population-based cohort study in The Netherlands. Gynecological information was obtained, and if women reported surgical menopause, validation of type and indication of surgery was accomplished by checking medical records. The ER genotypes (PP, Pp, and pp) were assessed by PCR using the PvuII endonuclease. Compared with women carrying the pp genotype, homozygous PP women had a 1.1-yr (P < 0.02) earlier onset of menopause. Furthermore, an allele dose effect was observed, corresponding to a 0.5-yr (P < 0.02) earlier onset of menopause per copy of the P allele. The risk of surgical menopause was 2.4 (95% confidence interval, 1.5-3.8) times higher for women carrying the PP genotype compared to those in the pp group, with the most prominent effect in women who underwent hysterectomy due to fibroids or menorrhagia. We conclude that genetic variations of the ER gene are related to the onset of natural menopause and the risk of surgical menopause, especially hysterectomy.

Relation of alleles of the collagen type Ialpha1 gene to bone density and the risk of osteoporotic fractures in postmenopausal women (Article)

1998-01-01T00:00:00Z

BACKGROUND: Osteoporosis is a common disorder with a strong genetic component. One way in which the genetic component could be expressed is through polymorphism of COLIA1, the gene for collagen type Ialpha1, a bone-matrix protein. METHODS: We determined the COLIA1 genotypes SS, Ss, and ss in a population-based sample of 1778 postmenopausal women using a polymerase-chain-reaction-based assay. We then related the genotypes to bone mineral density and the occurrence of osteoporotic fractures in these women. RESULTS: As compared with the 1194 women with the SS genotype, the 526 women with the Ss genotype had 2 percent lower bone mineral density at the femoral neck (P=0.003) and the lumbar spine (P=0.02); the 58 women with the ss genotype had reductions of 4 percent at the femoral neck (P= 0.05) and 6 percent at the lumbar spine (P=0.005). These differences increased with age (P=0.01 for modification by age of the effect of COLIA1 on femoral-neck bone density, and P=0.004 for modification of the effect on lumbar-spine bone density). Women with the Ss and ss genotypes were overrepresented among the 111 women who had incident nonvertebral fractures (relative risk per copy of the s allele, 1.5; 95 percent confidence interval, 1.1 to 2.1). CONCLUSIONS: The COLIA1 polymorphism is associated with reduced bone density and predisposes women to osteoporotic fractures.

Vitamin D receptor genotype is associated with osteoarthritis (Article)

1997-01-01T00:00:00Z

Osteoporosis and osteoarthritis are age-related disorders of the skeleton with genetic components. Low bone density is a risk factor for osteoporotic fracture while osteoarthritis is associated with increased bone density. The 1,25-dihydroxy-vitamin D3 receptor (VDR) gene locus was previously found to be associated with bone density. We therefore studied the relationship between radiographic osteoarthritis at the knee and VDR genotype in a population-based sample (n = 846), using molecular haplotyping of anonymous intragenic DNA polymorphisms. Radiographic osteoarthritis was defined using the Kellgren score, which is based on the assessment of osteophytes and joint space narrowing (JSN). We show that one VDR haplotype allele is significantly overrepresented in individuals with knee osteoarthritis and associated with a 2.27-fold increased relative risk (95% confidence interval 1.46, 3.52). Adjustment for bone density at the femoral neck did not change these results, indicating that the association is not mediated by bone density. The association appeared to be largely explained by the presence of osteophytes rather than JSN. Our results indicate a role of the VDR gene in the pathogenesis of osteophytes while linkage disequilibrium with another nearby gene, i.e., the collagen type IIa1 gene encoding the most abundant protein in cartilage, might contribute to the association.

Differential expression of estrogen receptors alpha and beta mRNA during differentiation of human osteoblast SV-HFO cells (Article)

1997-01-01T00:00:00Z

Estrogens have been shown to be essential for maintaining a sufficiently high bone mineral density and ER alpha expression has been demonstrated in bone cells. Recently, a novel estrogen receptor, estrogen receptor beta (ERbeta) has been identified. Here we demonstrate that also ERbeta is expressed in human osteoblasts, and that ER alpha and ERbeta are differentially expressed during human osteoblast differentiation. ERbeta mRNA expression increased gradually during osteoblast culture, resulting in an average increase of 9.9+/-5.3 fold (mean+/-S.D., n=3) at day 21 (mineralization phase) as compared to day 6 (proliferation phase). In contrast, ER alpha mRNA expression levels increased only slightly until day 10 (2.3+/-1.7 fold) and then remained constant. The observed differential regulation of ER alpha and beta is suggestive for an additional functional role of ERbeta to ER alpha in bone metabolism.

Conformational change and enhanced stabilization of the vitamin D receptor by the 1,25-dihydroxyvitamin D3 analog KH1060 (Article)

1996-10-01T00:00:00Z

The 1,25-dihydroxyvitamin D3[l,25(OH)2vitamin D3] analog KH1060 exerts very potent effects on cell proliferation and cell differentiation via the vitamin D receptor (VDR). However, the activities of KH1060 are not associated with an increased affinity for the VDR. We now show that increased stabilization of the VDR-KH1060 complex could be an explanation for its high potencies. VDR half-life studies performed with cycloheximide-translational blocked rat osteoblast-like ROS 17/2.8 cells demonstrated that, in the absence of ligand, VDR levels rapidly decreased. After 2 hr, less than 10% of the initial VDR level could be measured. In the presence of 1,25-(OH)2vitamin D3, the VDR half-life was 15 hr. After 24 hr, less than 20% of the initial VDR content was detectable, whereas, at this time-point, when the cells were incubated with KH1060 80% of the VDR was still present. Differences in 1,25-(OH)2vitamin D3- and KH1060-induced conformational changes of the VDR could underlie the increased VDR stability. As assessed by limited proteolytic digestion analysis, both 1,25-(OH)2vitamin D3and KH1060 caused a specific conformational change of the VDR. Compared with 1,25-(OH)2vitamin D3, KH1060 induced a conformational change that led to a far more dramatic protection of the VDR against proteolytic degradation. In conclusion, the altered VDR stability and the possibly underlying change in VDR conformation caused by KH1060 could be an explanation for its enhanced bioactivity.

A large-scale population-based study of the association of vitamin D receptor gene polymorphisms with bone mineral density. (Article)

1996-01-01T00:00:00Z

Conflicting results have been reported on the association between restriction fragment length polymorphisms (RFLPs) at the vitamin D receptor (VDR) gene locus (i.e., for BsmI, ApaI, and TaqI) and bone mineral density (BMD). We analyzed this association in a large population-based sample (n = 1782) of men and women aged 55-80 years using a novel direct haplotyping polymerase chain reaction (PCR) test to monitor the three polymorphic sites simultaneously. The direct haplotyping test we developed demonstrated a larger degree of genetic polymorphism at the VDR gene locus than described until now. None of the individual RFLPs were associated with BMD at the proximal femur. By analyzing allele dose effects, we identified a VDR haplotype allele weakly associated with low BMD. This allele, as one representative of the group of b alleles, is different from the BsmI allele previously reported by other groups to be associated with low BMD. This suggests allelic heterogeneity at the VDR locus in relation to BMD. Our results indicate at most a small effect of the VDR genotype on BMD in this elderly population. Since anonymous polymorphisms were analyzed, alternative explanations for our results include linkage to another nearby bone-metabolism related gene.

Antagonistic effects of transforming growth factor-beta on vitamin D3 enhancement of osteocalcin and osteopontin transcription: reduced interactions of vitamin D receptor/retinoid X receptor complexes with vitamin E response elements (Article)

1996-01-01T00:00:00Z

Osteocalcin and osteopontin are noncollagenous proteins secreted by osteoblasts and regulated by a complex interplay of systemic and locally produced factors, including growth factors and steroid hormones. We investigated the mechanism by which transforming growth factor-beta (TGF beta) inhibits 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-enhanced expression of the osteocalcin (OC) and osteopontin (OP) genes. ROS 17/2.8 cells, in which both genes are expressed, were transfected with reporter constructs driven by native (i.e. wild-type) rat OC and mouse OP promoters. TGF beta abrogated the 1,25-(OH)2D3 enhanced transcription of both the OC and OP genes. The inhibitory TGF beta response for each requires vitamin D response element (VDRE) sequences, although there are additional contributions from proximal basal regulatory elements. These transcriptional effects were further investigated for contribution of the trans-activating factors, which interact with OC and OP VDREs, involving the vitamin D receptor (VDR) and retinoid X receptor (RXR). Gel mobility shift assays show that TGF beta significantly reduces induction of the heterodimers VDR/RXR complexes in 1,25-(OH)2D3-treated ROS 17/2.8 cells. However, Western blot and ligand binding analysis reveal that TGF beta does not affect nuclear availability of the VDR. We also show that activator protein-1 activity is up-regulated by TGF beta; thus, activator protein-1 binding sites in the OC promoter may potentially contribute to inhibitory effects of TGF beta on basal transcription. Our studies demonstrate that the inhibitory action of TGF beta on the 1,25-(OH)2D3 enhancement of OC and OP transcription in osteoblastic cells results from modulations of protein-DNA interactions at the OC and OP VDRE, which cannot be accounted for by changes in VDR protein levels. As OC and OP participate in bone turnover, our results provide insight into the contributions of TGF beta and 1,25-(OH)2D3 to VDR-mediated gene regulatory mechanism operative in bone formation and/or resorption events.