http://repub.eur.nl/res/aut/295/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/8700/ 1997-01-01T00:00:00Z To gain more insight in the mechanism of action of inhibin, we studied the effect of inhibin on activin signaling in Chinese hamster ovary cells. Inhibin specifically counteracted activin-induced expression of a plasminogen activator inhibitor 1 promoter element (3TP) and of the junB gene, but was ineffective when the responses were induced by transforming growth factor-beta. This indicates that inhibin acts only on the activin-specific part of these signaling cascades. Using a constitutively active activin type IB receptor we determined whether inhibin acted at the level of the activin-receptor complex or downstream of it. The mutant activin receptor stimulated the expression of the 3TP promoter in the absence of activin. This stimulation was insensitive to inhibin, indicating that inhibin acts exclusively at or upstream of this activin type I receptor. In addition, competition studies using labeled activin showed that inhibin displaced activin from the activin type II receptors, especially from the activin type IIB receptor, but not from the type I receptors. In conclusion, these data show that in Chinese hamster ovary cells inhibin acts directly at the activin receptor complex, most likely through displacement of activin from the activin type II receptor. http://repub.eur.nl/res/pub/23760/ 1994-06-08T00:00:00Z Inhibin and activin are gonadal protein hormones, which were originally defined by their negative and positive feedback action on the release of follicle stimulating hormone (FSH) from the pituitary gland. However, recent studies revealed that inhibin and activin do not only control FSH release but can also affect the functions of a large number of other cell types, as will be discussed in section 1.7. This section is preceded by short descriptions of the testicular cell types (section 1.2), the structures of inhibins, activins and other members of the TGF·-B family of growth and differentiation factors (sections 1.3 and 1.4), and of receptors and non-receptor binding proteins for inhibin and activin (sections 1.6 and 1.7). Finally, events occurring after binding of activin to its receptor are discussed in section 1.8. The aim of the experiments described in this thesis was the elucidation of intratesticular effects of activin. The expression of activin receptors, the secretion of activins and the effects of activins in the rat testis have been investigated. http://repub.eur.nl/res/pub/8584/ 1994-01-01T00:00:00Z The expression of activin type II and IIB receptors and inhibin alpha-, beta A-, and beta B-subunit messenger RNAs (mRNAs), and the secretion of immunoreactive and bioactive activin during culture of testicular peritubular myoid cells and peritubular myoid cell lines were studied. Cultured peritubular myoid cells and cell lines expressed high levels of inhibin beta A-subunit mRNA and some inhibin alpha- and beta B-subunit mRNA. Activin receptor type II mRNA was also detected, whereas activin receptor type IIB mRNA expression was not found. Expression of the beta A-subunit mRNA was present immediately after isolation of the cells and increased during culture in Eagle's Minimum Essential Medium containing 10% fetal calf serum. beta A-Subunit mRNA expression was not regulated by the synthetic androgen R1881. Western blotting of peritubular myoid cell- and peritubular cell line-conditioned media with a polyclonal antiserum against recombinant activin-A revealed the presence of 25-kilodalton activin-A, whereas activin bioactivity was detected using the animal cap assay. Because of the secretion of activin-A by peritubular myoid cells, the effects of recombinant activin-A on Sertoli cell inhibin and transferrin secretion were examined. Activin-A stimulated both basal and FSH-stimulated inhibin and transferrin production by Sertoli cells after 72 h of culture. These effects resemble the effects of the testicular paracrine factor PmodS on Sertoli cell function. It is concluded that activin-A is secreted by peritubular cells in vitro and that activin-A shares a number of effects on Sertoli cell function with PmodS. http://repub.eur.nl/res/pub/8588/ 1994-01-01T00:00:00Z The activin and TGF-beta type II receptors are members of a separate subfamily of transmembrane receptors with intrinsic protein kinase activity, which also includes the recently cloned TGF-beta type I receptor. We have isolated and characterized a cDNA clone (C14) encoding a new member of this subfamily. The domain structure of the C14-encoded protein corresponds with the structure of the other known transmembrane serine/threonine kinase receptors. It also contains the two inserts in the kinase domain that are characteristic for this subfamily. Using in situ hybridization, C14 mRNA was detected in the mesenchymal cells located adjacent to the mullerian ducts of males and females at day 15 (E15) of embryonic development. Marked C14 mRNA expression was also detected in the female gonads. In female E16 embryos, the C14 mRNA expression pattern remained similar to that in E15 embryos. However, in male E16 embryos C14 mRNA was detected in a circular area that includes the degenerating mullerian duct. The expression of C14 mRNA was also studied using RNase protection assays. At E15 and E16, C14 mRNA is expressed in the female as well as in the male urogenital ridge. However, at E19, a high C14 mRNA level in the female urogenital ridge contrasts with a lack of C14 mRNA in the male urogenital ridge. This correlates with the almost complete degeneration of the mullerian ducts in male embryos at E19. C14 mRNA expression was also detected in embryonic testes at E15, E16 and E19 using RNase protection assays, but at much lower levels than those found in the developing ovaries.(ABSTRACT TRUNCATED AT 250 WORDS) http://repub.eur.nl/res/pub/8881/ 1993-01-01T00:00:00Z Regulation of androgen receptor (AR) mRNA expression was studied in Sertoli cells and peritubular myoid cells isolated from immature rat testis, and in the lymph node carcinoma cell line derived from a human prostate (LNCaP). Addition of dibutyryl-cyclic AMP (dbcAMP) to Sertoli cell cultures resulted in a rapid transient decrease in AR mRNA expression (5 h), which was followed by a gradual increase in AR mRNA expression (24-72 h). This effect of dbcAMP mimicked follicle-stimulating hormone (FSH) action. In peritubular myoid cells, there was only a moderate but prolonged decrease during incubation in the presence of dbcAMP, and in LNCaP cells no effect of dbcAMP on AR mRNA expression was observed. When Sertoli cells or peritubular myoid cells were cultured in the presence of androgens, AR mRNA expression in these cell types did not change. This is in contrast to LNCaP cells, that showed a marked reduction of AR mRNA expression during androgen treatment. In the present experiments, transcriptional regulation of AR gene expression in Sertoli cells and LNCaP cells was also examined. Freshly isolated Sertoli cell clusters were transfected with a series of luciferase reporter gene constructs, driven by the AR promoter. It was found that addition of dbcAMP to the transfected Sertoli cells resulted in a small but consistent increase in reporter gene expression (which was interpreted as resulting from AR promoter activity); a construct that only contained the AR 5' untranslated region of the cDNA sequence did not show such a regulation. The same constructs, transfected into LNCaP cells, did not show any transcriptional down-regulation when the synthetic androgen R1881 was added to the cell cultures. A nuclear transcription elongation experiment (run-on), however, demonstrated that androgen-induced AR mRNA down-regulation in LNCaP cells resulted from an inhibition of AR gene transcription. The present results indicate that in Sertoli cells and LNCaP cells, hormonal effects on AR gene transcription play a role in regulation of AR expression. However, AR gene transcription in these cells is differentially regulated.