[Show abstract][Hide abstract] ABSTRACT: Activins are members of the transforming growth factor-β family of growth and differentiation factors. In this paper, we report
the results of a structure-function analysis of activin A. The primary targets for directed mutagenesis were charged, individual
amino acids located in accessible domains of the protein, concentrating on those that differ from transforming growth factor-β2,
the x-ray crystal structure of which is known. Based on the activities of the recombinant activin mutants in two bioassays,
4 out of 39 mutant proteins (D27K, K102A, K102E, and K102R) produced in a vaccinia virus system were selected for further
investigation. After production in insect cells and purification of these four mutants to homogeneity, they were studied in
bioassays and in cross-linking experiments involving transfected receptor combinations. Mutant D27K has a 2-fold higher specific
bio-activity and binding affinity to an ActRIIA/ALK-4 activin receptor complex than wild type activin, whereas mutant K102E
had no detectable biological activity and did not bind to any of the activin receptors. Mutant K102R and wild type activin
bound to all the activin receptor combinations tested and were equipotent in bioassays. Our results with the Lys-102 mutants
indicate that the positive charge of amino acid 102 is important for biological activity and type II receptor binding of activins.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A family of structurally related proteins homologous to the Drosophila mothers against dpp (MAD) gene product have been implicated in signal transduction by members of the TGF-beta superfamily. One of these MAD related proteins (DPC4) has been cloned as a candidate tumour suppressor in pancreas carcinomas, suggesting a role for DPC4 in growth regulation by TGF-beta related proteins. The involvement of DPC4 in TGF-beta1 induced growth inhibition and transcriptional response is demonstrated here, by the introduction of DPC4 in the TGF-beta and activin insensitive breast tumour cell line MDA-MB-468, from which the DPC4 gene is deleted. Transfection of DPC4 in this cell line restores both growth inhibition and the induction of a TGF-beta sensitive reporter construct (3TPlux) by TGF-beta1. In contrast, a DPC4 splice variant lacking amino acid residues 223-301 and cloned from another TGF-beta and activin resistant breast tumour cell line (MDA-MB-231), does not restore the induction of the 3TPlux reporter by TGF-beta1. We also show that in this latter cell line activin resistance is partly due to the absence of a functional activin type IB receptor. These results indicate that DPC4 is part of the TGF-beta signalling cascade and mediates TGF-beta induced growth inhibition. Together with the deletion of DPC4 from pancreas carcinomas these results suggest a role for DPC4 as a tumour suppressor.
[Show abstract][Hide abstract] ABSTRACT: Murine P19 embryonal carcinoma (EC) cells can be differentiated into various germ layer derivatives. The addition of retinoic acid (RA) to P19-EC cell aggregates results in a transient activation of receptor protein tyrosine phosphatase-alpha (RPTP alpha). Subsequent replating of these aggregates leads to neuronal differentiation. P19-EC cells expressing constitutively active RPTP alpha (P19-RPTP alpha) show extensive neuronal differentiation upon RA treatment in monolayer. P19-RPTP alpha cells thus provide a suitable in vitro model for studying neuronal differentiation. We used P19-RPTP alpha cells to study the effects of activin and basic fibroblast growth factor (bFGF) on neurogenesis. We show that P19-RPTP alpha cells express mRNA for types I and II activin receptors. RA addition causes an up-regulation of receptor type IIA expression. Complexes of type I and II receptors were detectable by cross-linking assays both before and after RA treatment. Receptor complexes were functional as determined by transient transfection assays with activin responsive reporter constructs. Undifferentiated as well as differentiated P19-RPTP alpha cells express also the FGF receptors (FGFRs) FGFR-1 and FGFR-2 but not FGFR-3 and FGFR-4. Their functionality was established by bFGF induced mitogen-activated protein kinase phosphorylation. Activin and bFGF appeared to exert differential actions on RA-induced neuronal differentiation. Although activin irreversibly changes the differentiation fate into nonneuronal directions, bFGF does not affect initial neurogenesis but regulates axonal outgrowth in a concentration-dependent way; low concentrations of bFGF enhance axonal outgrowth, whereas high concentrations inhibit this process. These results strengthen the notion that activin and bFGF are important regulators of neurogenesis in the mammalian embryo.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 01/1997; 7(12):1679-88.
[Show abstract][Hide abstract] ABSTRACT: Truncated activin type II receptors have been reported to inhibit activin receptor signaling in Xenopus embryos, although the mechanism of action for this effect has not been fully understood. In the present study we demonstrate that in P19 embryonal carcinoma cells both the induction of the activin responsive 3TP-lux reporter construct and the inhibition of retinoic acid-induced neuronal differentiation by activin are blocked by expression of a truncated activin receptor. To reveal the mechanism of action of truncated activin receptors, the interaction between different activin receptors has been investigated upon coexpression in COS cells followed by cross-linking of 125I-activin A and subsequent immunoprecipitation. Complexes between a truncated activin type IIA receptor and activin type IA and type IB receptors can be formed, as demonstrated by coimmunoprecipitation of these type I receptors with the truncated activin type IIA receptor. Other type I receptors known as ALK-1 and ALK-6 also coimmunoprecipitate with the truncated type IIA receptor, whereas ALK-3 and ALK-5 do not. Furthermore, the activin type IIB2 receptor does not coimmunoprecipitate with the truncated type IIA receptor, but decreases activin binding to the truncated type IIA receptor. In double immunoprecipitation experiments with cell lysates from COS cells, in which full-length activin type IIA and type IIB2 receptors were cotransfected, no interaction between these receptors was found. In contrast, homomeric complexes of full-length activin type IIA receptors were detected. These results implicate that truncated activin receptors can interfere with activin signaling by interacting with activin type I receptors. Additionally, truncated activin type IIB2 receptors might also interfere with type IIA receptor signaling by decreasing activin binding to the type IIA receptor and therefore might be more potent in inhibiting activin signal transduction. Furthermore, our data indicate that truncated type IIA receptors can interact with other type I receptors and as such might inhibit signal transduction by type I receptors other than activin type IA and type IB receptors.
Experimental Cell Research 06/1996; 224(2):323-34. DOI:10.1006/excr.1996.0142 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A solution-hybridization S1-nuclease protection assay was used to evaluate the expression of messenger RNAs for the activin beta A subunit and type II activin receptor in adult rat brain. Results indicate the presence of beta A subunit mRNA in both hypothalamus and brainstem, with approximately two-fold higher levels in brainstem. Levels of activin type II receptor mRNA were similar in the hypothalamus of young virgin and 15-day lactating females, and in females in which pups were removed after a 5-day lactation period. Male rats castrated prepubertally (30 days p.n.) had approximately 220% higher (P < 0.05) hypothalamic activin type II receptor mRNA levels than postpubertal, 3-month old age-matched sham controls. Two month treatment of castrate rats with estradiol (200 ng/g, i.p. every 2 days) reduced hypothalamic activin type II receptor mRNA expression to control levels; the same dose of testosterone had no effect. The expression of the hypothalamic activin type II receptor gene may be estrogen-regulated in vivo.
Journal of Neuroendocrinology 05/1996; 8(5):395-401. DOI:10.1046/j.1365-2826.1996.04618.x · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor beta (TGF-beta) is a potent growth inhibitor of non-malignant breast tissue, and TGF-beta resistance could play a role in tumorigenesis. Treatment of breast-tumor cells with anti-estrogens and progestins has been shown to correlate with an increase in the levels of secreted TGF-beta, suggesting that the growth inhibition observed with these (anti)hormones is mediated by this growth factor. In the present study we have investigated the effects of anti-estrogens and progestins on breast-tumor cell lines, which are either resistant or sensitive to TGF-beta. A hormone-independent variant of the MCF7 cell line is shown to have lost its sensitivity to TGF-beta during its progression towards an autonomous phenotype, but has preserved its sensitivity to anti-estrogens. In addition, evidence is presented showing that progestins and anti-estrogens inhibit proliferation, irrespective of the sensitivity to TGF-beta in variants of the T47D cell line. Therefore, we conclude that, although TGF-beta seems an important growth inhibitor for mammary epithelial cells, both progestins and anti-estrogens can inhibit cell proliferation independent of induced TGF-beta production.
International Journal of Cancer 03/1996; 65(5):682-7. DOI:10.1002/(SICI)1097-0215(19960301)65:5<682::AID-IJC20>3.0.CO;2-8 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Follistatin is an activin-binding protein, which inhibits activin bioactivity in several biological systems. In the present study it is demonstrated that preincubation of iodinated activin A with follistatin, purified from porcine follicular fluid, completely abolished the binding of activin to activin type IIA, IIB2 and IIB4 receptors, and consequently to activin type IB receptor, transiently transfected in COS cells. Binding of activin A to membrane proteins on the activin-responsive P19 embryonal carcinoma cells was also prevented by this follistatin preparation. The same results were obtained with a carboxy-terminally truncated form of follistatin (FS-288), which is only present in minor amounts in the purified follistatin preparation. Since FS-288 has a high affinity for heparan sulfate proteoglycans on the cell surface, we tested whether membrane-bound FS-288 presents activin A to the different activin receptors, thereby facilitating activin binding. FS-288 did bind to the cell surface of transfected COS cells, but inhibited the binding of activin A to its receptors IIA, IIB2 and IIB4. Furthermore, after addition of FS-288 to K562 erythroleukemia cells, the total binding of activin via cell surface-bound FS-288 was increased, whereas the binding of activin A to activin type II and type I receptors present on these cells was inhibited. These findings reveal that different forms of follistatin can neutralize activin bioactivity by interference with binding of activin to all known activin type II receptors, rather than that they inhibit the binding of the type I receptor to the activin/activin type II receptor complex. In addition, our studies indicate that cell surface-associated follistatin cannot present ligand to signalling receptors.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The expression and function of gonadotropin receptors, and the secretion of steroids, transferrin, and cytokines were investigated in three immortalized (single transfection with v-myc) mouse granulosa cell lines (GRM01, GRM01L, and GRM02). A dose-dependent increase in progesterone production was obtained in GRM01 and GRM02 cells after addition of LH, FSH, modulators of the adenylate cyclase enzyme system, and cAMP analogues. The LH-induced release of progesterone was already detectable in GRM02 cells after 8 h and was related to incubation time and cell number. Both epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) induced the secretion of progesterone in GRM02 cells, while no effect was obtained with TGF beta. LH receptor concentration was highest in the GRM02 cell line. FSH receptor mRNA was visualized in GRM01 and GRM02 cells. Aromatase activity in GRM02 cells was induced by androgens and inhibited by aromatase inhibitors. Whereas all cell lines were able to secrete transferrin, only in GRM01 cells was transferrin secretion increased significantly by LH. FSH did not affect transferrin secretion in the three cell lines, in contrast to forskolin or 8-bromo-cAMP. The immortalized mouse granulosa cell lines were able to express and release several growth factors. The expression and secretion of activin, inhibin, TGF beta, EGF, TGF alpha, insulin-like growth factor II, fibroblast growth factor (acidic and basic), platelet-derived growth factor, and interleukin-6 suggest an autocrine or paracrine role for these factors in follicular differentiation and function. In conclusion, these cells, derived from mural granulosa cells and immortalized in a preovulatory state, can be used to study granulosa cell physiology or to study the role of granulosa cells and their derivatives in the process of follicular maturation, fertilization, and early embryonic development.
Biology of Reproduction 06/1994; 50(5):1190-202. DOI:10.1095/biolreprod50.5.1190 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 müllerian 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 müllerian 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 müllerian 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)
Development 02/1994; 120(1):189-97. · 6.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The expression of mRNAs for inhibin subunits was studied in the human teratocarcinoma cell line Tera-2 clone 13 before and after differentiation with retinoic acid (RA). Both alpha- and beta B-subunits of inhibin were expressed. Subsequently, inhibin bio- and immunoactivity in the conditioned media of the Tera-2 cells were determined by studying the release of follicle-stimulating hormone from rat pituitary cells, by immunoassay and by immunoprecipitation using inhibin alpha- and beta B-subunit specific antibodies. Strikingly dissimilar high bio- and low immuno-activities were found. The ensuing hypothesis that the high bioactivity might be due to the presence of the activin-binding protein follistatin was confirmed by immunoprecipitation of 34 and 37 kDa labelled proteins, using a polyclonal anti-follistatin antiserum after culture of the Tera-2 cells with [35S]-methionine. Furthermore, expression of activin receptor types II and IIB mRNA was found in the cells. Addition of 5 microM RA to monolayer cultures of Tera-2 cells resulted in differentiation to flat endoderm-like cells and caused a slight suppression of the expression of the mRNA encoding the inhibin alpha- and beta B-subunits. The expression of follistatin and activin receptor type IIB was strongly suppressed, whereas the expression of the activin receptor type II was not affected. We conclude that Tera-2 cells secrete follistatin and express inhibin subunits and activin receptors differentially during RA-induced differentiation. The role of the decreased expression of follistatin and activin receptor IIB mRNA after addition of RA in the mechanism of RA-induced differentiation remains to be elucidated.
Biochemical and Biophysical Research Communications 06/1993; 192(3):1334-9. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The production of activin by Sertoli cells isolated from 21-day-old rats was studied using the mesoderm-inducing activity of activin on Xenopus laevis animal cap explants, immunoprecipitation and Western blotting. Furthermore, the effects of recombinant bovine activin-A on rat Sertoli cell aromatase activity and FSH and androgen receptor gene expression were examined. Animal cap explants from Xenopus laevis blastulas elongated after culture in conditioned medium of Sertoli cells cultured with or without ovine FSH or conditioned medium of the mouse Sertoli cell-derived TM4 cell line. Animal cap explants cultured in control medium remained spherical. This elongation was also found in the more than 10-kilodalton fraction of the conditioned medium and after heating for 10 min at 95 C, indicating that heat-stable activin-like bioactivity is present in the culture medium. Immunoprecipitation of [35S]methionine-labeled proteins and Western blotting of Sertoli cell-conditioned medium with polyclonal antisera against the inhibition beta-subunits indicated the presence of 24- to 25-kilodalton activin-like immunoreactive material. Sertoli cell aromatase activity was dose-dependently stimulated by ovine FSH after 72 h of culture. Recombinant bovine activin-A partly inhibited this stimulation in a dose-dependent way. This inhibition was also found after 24 h of culture. Furthermore, basal and FSH-stimulated androgen receptor mRNA expression in Sertoli cells and binding of the synthetic androgen R1881 to Sertoli cells were decreased after 24 h of culture in the presence of recombinant bovine activin-A. In the same experiments, FSH receptor mRNA expression was not significantly affected. These results indicate that activin can act as an autocrine regulator of Sertoli cell function.
[Show abstract][Hide abstract] ABSTRACT: The secretion of inhibin and inhibin-related proteins by testicular Leydig cells was studied by estimation of inhibin immunoreactivity and bioactivity in spent media of preparations of immature and mature rat Leydig cells and of tumor Leydig cells. Immature and mature rat Leydig cells expressed inhibin alpha-subunit mRNA and secreted immunoreactive inhibin. The immunoreactive material did not contain inhibin bioactivity as measured by an in vitro rat pituitary bioassay system. Results of pulse labeling with [35S]methionine followed by immunoprecipitation indicated that the inhibin-related proteins secreted by the immature Leydig cell preparations are 26 kDa and 44 kDa molecules. Mature rat Leydig cells only secreted the 44 kDa inhibin-related protein. Tumor Leydig cells (rat H540 and mouse MA10) secreted immunoreactive and bioactive inhibin, which could be immunoneutralized by an antibody against inhibin. In the culture medium of some H540 tumor Leydig cells 26 kDa and 42 kDa inhibin-related proteins and 30 kDa inhibin were detected. In culture medium of other H540 tumor Leydig cells, not secreting bioactive inhibin, only 26 kDa and 42 kDa inhibin-related proteins were found. No activin bioactivity was detected in culture media of immature rat Leydig cells, H540 and MA10 tumor Leydig cells. It is concluded that normal Leydig cells secrete inhibin alpha-subunits, while Leydig cell tumors can also secrete bioactive inhibin. Neither normal Leydig cells nor Leydig cell tumors produce activin.
[Show abstract][Hide abstract] ABSTRACT: cDNA encoding the extracellular domain of the rat activin receptor was cloned using the polymerase chain reaction (PCR). This cDNA is highly homologous to cDNA encoding the extracellular domain of the mouse activin receptor, whereas at the protein level the extracellular domains of both receptors are identical. Employing this cDNA as a probe in Northern blot analysis, expression of two activin receptor mRNAs (6 kb and 4 kb) was observed, in testes of immature and mature rats. Between day 21 and 28 of postnatal development, a large increase in testicular expression of the 4 kb mRNA was found, suggesting expression of this activin receptor mRNA in germ cells. The 4 kb mRNA was indeed present in isolated pachytene spermatocytes and round spermatids, but was absent in elongating spermatids. Sertoli cells obtained from immature and mature rats expressed both the 6 kb and 4 kb mRNAs, whereas the expression of these mRNAs in Leydig cell preparations was very low. These results may imply that activin has multiple actions in the control of testicular function.