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PubMed1997

Presence of activin, inhibin, and follistatin in epithelial ovarian carcinoma.

Activin induces proliferation in epithelial ovarian carcinoma cell lines, whereas follistatin (FS), an activin binding protein, inhibits this action. To test the hypothesis that activin production, in excess of inhibin and FS, results in cell proliferation in epithelial ovarian tumors, messenger RNA (mRNA) expression of the activin family of proteins, FS, and activin type I and II receptors was examined in 25 primary epithelial ovarian tumors and tumor epithelium in culture (n = 7) using RT-PCR. Activin A was measured in the serum of ovarian cancer patients, and activin A, total inhibin, and FS protein secretion was measured from primary epithelial tumors in vitro. The effect of activin and FS on cell proliferation was assessed by measuring [3H]thymidine incorporation. All results were compared with normal ovarian epithelium. All epithelial ovarian tumors expressed mRNA for the alpha, beta A, and beta B subunits; FS 288 and 315; and the activin type IA, IB, II, and IIB receptors. beta A mRNA expression, as assessed using semiquantitative RT-PCR, was 3-fold greater in cultured tumor epithelium than in primary tumors (band density 0.86 +/- 0.17 vs. 0.28 +/- 0.09; P < 0.01). In addition, beta A mRNA was abundantly expressed in normal epithelium in culture (n = 2), whereas only trace amounts were seen in 2/9 primary epithelial samples. Activin protein was secreted by 24/25 primary epithelial ovarian tumors (range 0.2-155.8 ng/mL). In contrast, total inhibin was secreted by only 2/25 (range 0.01-0.92 ng/mL), whereas free FS was not detectable in the medium of any tumor (< 0.5 ng/mL). Treatment with activin or FS did not consistently affect cell growth. Measurement of serum activin A in a subset of subjects and in 27 additional subjects with epithelial ovarian carcinoma (n = 33) revealed preoperative activin A levels > 3 SD above the mean for pre- and postmenopausal women in 13/33 (39%) subjects. We conclude that in epithelial ovarian cancer: 1) beta A subunit mRNA is expressed, 2) activin protein is secreted more frequently than inhibin and in greater quantities than FS, 3) beta A subunit mRNA expression is greater in neoplastic and normal epithelium in culture than in the primary tissue, 4) the majority of tumors in culture do not respond to activin or FS treatment with proliferation, and 5) serum activin levels may reflect tumor secretion in some patients. Thus, activin A appears to be available as an autocrine/paracrine factor in epithelial ovarian tumors and may contribute to circulating levels, but its role in tumorigenesis has yet to be defined.

Activins↗
PubMed1995

Repression of glycoprotein hormone alpha-subunit gene expression and secretion by activin in alpha T3-1 cells.

The alpha T3-1 cell line, a GnRH-responsive gonadotroph cell line developed by targeted oncogenesis in transgenic mice, was used to study regulation of the glycoprotein hormone alpha-subunit by activin. Transient transfection assays established that activin suppressed transcription of both the human and mouse alpha-subunit genes. Initial studies demonstrated that activin decreased transcription of -846 and -180 human alpha-subunit-luciferase constructs by about 30%, but that inhibin and follistatin were without effect. Subsequent studies to localize sequences mediating responses to activin were carried out using a series of 5'-deletions (-507 to -133) of the mouse alpha-subunit promoter fused to luciferase. The luciferase activity of the -507-base pair construct was decreased by 60-70% in the presence of activin, and follistatin prevented this decrease. There were significant stepwise losses of activin responsiveness when sequences between -507 and -424, -424, and -288, and -288 and -205 base pairs were eliminated. Clustered point mutations of the mouse alpha-subunit gene, shown previously to reduce basal expression and GnRH responsiveness, were tested to further identify sequences mediating activin repression. Constructs containing a mutated (-337 to -330) pituitary glycoprotein hormone basal element (PGBE) showed significant loss of activin responsiveness in the context of both the native promoter (-507 to +46) and a minimal promoter downstream of the -507 to -205 region of the mouse alpha-subunit gene, whereas mutation of sequences (-406 to -399) in the GnRH-response element had no effect. Multimers of the PGBE element (-344 to -300) were insufficient to mediate a full activin response when linked to a minimal promoter. When added together with GnRH to transfected cells, activin abolished the stimulatory effect of GnRH on alpha-transcription. Secretion of free alpha-subunit by alpha T3-1 cells decreased 10-50% after exposure to activin for approximately 20 h, and steady state levels of alpha-subunit messenger RNA (mRNA) decreased by about 20-25% after 24-72 h. As changes in activin sensitivity could modulate its action, activin receptor II mRNA levels were measured by Northern blot hybridization at various times after activin (or inhibin) treatment. The three species of ActRII mRNA present in alpha T3-1 cells (approximately 6, 3, and 0.5 kilobases) were unaffected up to 72 h by these treatments. These observations provide the first demonstration that activin regulates a gonadotropin subunit gene at the level of transcription. Suppression of transcription of the mouse alpha-subunit gene by activin appears to involve several segments of the alpha-promoter, one of which is in the region of the PGBE. Thus, alpha T3-1 cells may provide a favorable system to further identify the DNA sequences and nuclear factors through which activin acts to alter transcription.

Activins↗
PubMed2002

Effect of activins AB and B on DNA synthesis stimulated by epidermal growth factor in primary cultured rat hepatocytes.

The effect of activins AB and B on DNA synthesis stimulated by epidermal growth factor (EGF) was studied in primary cultured rat hepatocytes and compared with the effect of activin A, a suppressor of DNA synthesis. Activin AB inhibited DNA synthesis as assessed by [3H]thymidine incorporation. The inhibition by activin AB was detected at 6 ng/ml, and the 12.5 ng/ml concentration produced almost maximal inhibition, approximately 40%, almost the same as that produced by activin A. Inhibition by activin A was detected at 3 ng/ml, and the 6 ng/ml concentration produced almost maximal inhibition. Activin B, on the other hand, had no effect on DNA synthesis up to 50 ng/ml. The increase in labeling index by EGF was also reduced to about 20% by 25 ng/ml activin A and activin AB, but not by activin B. Activin B, however, inhibited the binding of [125I]activin A to hepatocytes, but had no effect on the inhibition of DNA synthesis by activin A, even at 3-fold excess concentrations. These findings suggest that activin AB may act in the same manner as activin A does in terms EGF's inhibitory effect on DNA synthesis, although the effective concentration is higher than that of activin A. The findings also suggest that activin B receptors are present in hepatocytes but that they do not mediate signal transduction leading to the inhibition of DNA synthesis.

Animals↗
PubMed1996

Two distinct signaling pathways activated by activin A in glucose-responsive pancreatic beta-cell lines.

Activin A stimulates insulin secretion in pancreatic beta-cells by a calcium-dependent mechanism. The present study was conducted to further characterize the effects of activin A in two glucose-responsive insulinoma cell lines, MIN6 and HIT-T15 cells. In HIT-T15 cells, activin A evoked an increase in cytoplasmic free calcium concentration, stimulated insulin secretion, maintained glucose responsiveness of the cells and inhibited DNA synthesis. However, activin A did not have any effect in MIN6 cells. Measurement of 125I-labeled activin A binding in MIN6 cells revealed that the number of binding sites was markedly reduced, suggesting that the refractoriness was due, at least partly, to the reduced numbers of the activin receptor. Stable transfectants of MIN6 cells that overexpressed the type II activin receptor were then developed. The transfected cells (MIN6-ActR cells) expressed ten times more 125I-labeled activin A-binding sites than parental cells and the apparent Kd was 1.15 nM, which was nearly identical to that in parental cells. Affinity cross-linking in MIN6-ActR cells showed that a 90 kDa type II receptor as well as a 52 kDa protein, presumably follistatin, was markedly labeled with 125I-labeled activin A. Although MIN6-ActR cells expressed significant numbers of activin receptors, activin A did not induce immediate calcium-dependent responses in these cells. In contrast, activin A was capable of inducing long-term effects in MIN6-ActR cells; thus, reduction of the glucose concentration in culture medium from 25 to 5.5 mM for 4 days resulted in a remarkable loss of insulin response to glucose stimulation but this decline in response to glucose was prevented by the addition of activin A during culture. In addition, activin A inhibited DNA synthesis in MIN6-ActR cells. Hence, although activin A did not induce calcium-dependent responses, it evoked some calcium-independent effects in MIN6-ActR cells. Taken together, activin A elicits various effects in beta-cells by both calcium-dependent and -independent mechanisms.

Activin Receptors↗
PubMed2003

Activin signal transduction in the fetal rat adrenal gland and in human H295R cells.

The presence of activin A and its effects have previously been documented in the adrenal gland, particularly in the human fetal adrenal gland and the rat adrenal gland. The primary signaling pathway of activin involves interactions between receptor and intracellular (Smad) proteins that have not been completely described in the adrenal gland. In this study, we demonstrate that the components of the activin signaling cascade are present in two complementary models, the fetal rat adrenal gland and the human adrenocortical cell line, H295R, by means of RT-PCR, western analysis, and immunoprecipitation techniques. Using the cell line, activin signaling was analyzed using an activin-responsive reporter gene, p3TP-luc, and luciferase assays to assess transcriptional activity with co-expression of the different activin receptors and Smads to demonstrate the functionality of the signaling cascade. In the fetal rat adrenal gland, the relative amounts of mRNA of the type II receptors, RII and RIIB, were regulated by gestational age, such that the RIIB levels increased after birth while RII levels fell. Using immunodetection techniques, the activin receptors and the different Smad proteins were detected in the rat fetal adrenal glands. Notably, the presence of Smad4 protein is significantly increased after birth in the rat adrenal gland. RT-PCR established a similar profile in the H295R cells. Using p3TP-luc, the H295R cells show transcriptional activation of this activin-responsive reporter in the presence of activin A. Co-expression of type I and type II receptors as well as Smads, results in ligand-independent transcriptional activity in addition to an activin-stimulated response. In determining activin's effects on adrenal function, adrenal steroid production was evaluated by incubation of the H295R cells with increasing doses of activin A and inhibin A, resulting in a detectable increase in P450c17 expression. Co-incubation of activin A with follistatin diminishes this response. These results are consistent with a role for activin A in the adrenal gland by demonstrating that the elements of the activin signaling pathway are present, intact, and functional. This suggests that in the adrenal gland the components of the activin receptor/Smad pathway are dynamically changing in the transition from fetal to neonatal life, and are important to the function of this organ.

Activin Receptors, Type II↗
PubMed2006

The autocrine effect of activin A on human ovarian clear cell adenocarcinoma cells.

The functions of activin, a member of TGF-beta superfamily, in ovarian clear cell adenocarcinoma remain unsolved, although we recently found that inhibin betaA-subunit, activin A, activin receptor type IA, type IB, type IIA, type IIB, Smad2, Smad3 and Smad4 were localized in tumor cells of the ovarian clear cell adenocarcinoma tissue by immunohistochemistry. In the present study, in order to investigate the role of activin concerning cell growth in ovarian clear cell adenocarcinoma cells, we determined the production of activin A and inhibin A, and the expression of activin receptors and Smads using the human ovarian clear cell adenocarcinoma cell line JHOC-5. Moreover, we examined the effects of activin A on the activation of activin signaling pathway and on the proliferation in JHOC-5 cells. We detected a measurable amount of activin A in the culture medium of JHOC-5 cells, although inhibin A was not detected. The expression of activin receptor type IA, IB, IIA, IIB, Smad2, Smad3 and Smad4 was observed in JHOC-5 cells. Activin A induced a significant increase in proliferation of JHOC-5 cells compared with the untreated control. On the other hand, activin A did not affect the growth of JHOC-5 cells and no statistically significant difference was observed in the presence of follistatin which is a specific binding protein of activin. Phosphorylated Smad2, an activated form of Smad2, was detected both in treated JHOC-5 cells and in untreated cells by activin A. Activin A significantly increased the expression of phosphorylated Smad2 in JHOC-5 cells. Therefore, it is possible that activin has autocrine roles in tumor growth of ovarian clear cell adenocarcinoma cells.

Activin Receptors↗
PubMed1990

Activin-A modulates growth hormone secretion from cultures of rat anterior pituitary cells.

Activins, initially identified as FSH-releasing proteins, have now been shown to exert effects on other cell types of the anterior pituitary, including the somatotrophs. In the present study the inhibitory action of activin-A (beta A beta A) on GH secretion was characterized using primary cultures of rat anterior pituitary cells. Activin-A suppressed basal GH secretion for up to 72 h (the longest time tested). Immediately after the treatment period with activin-A, when the cells were thoroughly washed and further incubated with or without rat GH-releasing factor (rGRF), basal and stimulated GH secretion were partially inhibited as well. In parallel, activin-A pretreatment diminished rGRF-stimulated cAMP accumulation. The effects of activin-A were time- and concentration-dependent, with half-maximal inhibition occurring in the range of 20-30 pM activin-A. A minimum pretreatment time of 3 h was required for maximal effect, and when rGRF and activin-A were added simultaneously, no inhibition was evident. Secretory responses of activin-A-pretreated cells to rGRF were influenced by glucocorticoids. When cells were cultured in the presence of the synthetic glucocorticoid dexamethasone, pretreatment (72 h) with activin-A attenuated rGRF-stimulated GH secretion only during short (1-h), but not longer (3-h), exposure periods to the neuropeptide. In the absence of dexamethasone, rGRF-stimulated GH secretion was inhibited at all incubation times tested (up to 3 h). A 3-h exposure to the protein factor did not alter total (cellular plus secreted) immunoreactive GH levels, suggesting that the inhibition of secretion with the shorter treatment was not secondary to attenuated GH biosynthesis. However, longer (72-h) treatment with activin-A decreased total GH levels, indicating lower GH biosynthetic rates, as previously shown. Somatostatin is recognized as the primary negative modulator of GH secretion. Activin-A and SRIF inhibited GH secretion additively, suggesting distinct mechanisms of action for each. GH secretion in response to other secretagogues, such as 12-O-tetradecanoyl-phorbol-13-acetate, forskolin, cholera toxin, and 8-bromo-cAMP, was also suppressed after activin-A pretreatment. The presence of the RNA synthesis inhibitor actinomycin-D completely blocked the inhibitory effect of a 3-h activin-A pretreatment on subsequent rGRF-stimulated GH secretion. Pertussis toxin was only partially effective in preventing the inhibition by activin-A. The results of this study indicate that activin-A plays a crucial role as a modulator of somatotropic function, inhibiting GH secretion at the level of the secretory process and secondary to the inhibition of GH biosynthesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Alpha 2-macroglobulin is a binding protein of inhibin and activin.

The development of a two-site immunoassay for dimeric inhibin resulted in the detection of higher mol wt substances in follicular fluid that interfere with the measurement of dimeric inhibin by this assay, but not by a previously characterized inhibin alpha-subunit RIA. Therefore, we initiated a study to identify inhibin- and activin-binding proteins in biological fluids using 125I-labeled recombinant human inhibin-A and activin-A. By gel permeation chromatography, we found that [125I] inhibin A or [125I]activin-A incubated with either plasma or follicular fluid became associated with several higher mol wt proteins. The lowest mol wt complex eluted with a retention time similar to that of [125I] inhibin or [125I]activin bound to porcine follistatin, a known inhibin- and activin-binding protein. The largest mol wt complex eluted near the void volume, coincident with human alpha 2-macroglobulin (h alpha 2 M) incubated with [125I]inhibin or [125I]activin. Human plasma was incubated with [125I]inhibin or [125I]activin, and putative binding proteins were chemically cross-linked and analyzed by polyacrylamide gel electrophoresis. Cross-linked 125I-labeled inhibin or activin plasma binding protein complexes comigrated with [125I] inhibin or [125I]activin cross-linked to h alpha 2M. Antiserum to h alpha 2M precipitated 125I-labeled inhibin- and activin-binding proteins in human plasma that migrated identically in polyacrylamide gel electrophoresis to complexes formed between [125I]inhibin or [125I]activin cross-linked to h alpha 2M. Excess unlabeled inhibin or activin decreased the labeling of h alpha 2M and high mol wt human plasma binding proteins by [125I]inhibin or [125I]activin. These results demonstrate that in addition to follistatin, alpha 2M is a binding protein of both inhibin and activin. Although the physiological relevance of the inhibin-alpha 2M and activin-alpha 2M interaction is unknown, alpha 2M may play a role in the delivery or clearance of inhibin and activin similar to that proposed for transforming growth factor-beta and a number of other growth factors.

Activins↗
PubMed1995

Activin causes premature superovulation.

In the rat ovary, follicle cells produce and respond to activin, but as yet, the functional significance of the autocrine/paracrine effects of ovarian activin remains equivocal. To assess the effects of activin on folliculogenesis, normal cycling female rats were injected once every 8 h over a 40-h period with recombinant human activin A (120 micrograms/kg) beginning at 1300 h on estrus (day 1 of treatment). A total of 10 rats were injected with activin in 2 separate experiments. On days 3 and 4 of treatment, blood was obtained for hormone measurements, and the ovaries were removed for histology. Follicle counts were performed in 1 ovary from 3 representative animals in each treatment group. All antral (Graafian) follicles 300 microns or more in diameter were measured and classified as healthy or atretic based on the number of pyknotic nuclei in the largest cross-section. On day 3, all rats were in diestrus (diestrous day 2). After 3 days of activin administration, serum levels of estradiol were increased 200%, progesterone levels were decreased 67%, and FSH levels were unchanged compared with those in matched controls. By day 4 (i.e. 1 day after the last injection), no changes in the levels of these hormones were observed. Injection of activin for 3 days did not change the total number of antral follicles per ovary (control, 41.3 +/- 4.9; activin, 43.7 +/- 3.9); however, activin significantly increased the total number of atretic follicles (control, 69%; activin, 92%). Morphometric analysis of the ovaries removed on day 3 showed a marked increase (2-fold) in the number of large follicles, but most (89%) were atretic. Follicle counts suggested that the additional large follicles may have come from the pool of healthy small follicles. Histological studies showed that some of the day 3 activin-treated follicles had initiated ovulation. On day 4, control and activin-treated animals were at proestrus and estrus, respectively. Therefore, activin shortened the estrous cycle by 1 day. Little or n change in the follicle populations was observed in day 4 control ovaries. Interestingly, however, in 2 of the 3 activin-treated animals, 1 set of large follicles had ovulated (12 +/- 1 expanded egg cumulus complexes/oviduct), and another set (13 +/- 2) was just about to rupture. The activin-exposed oocytes (tubal and follicular) appeared arrested in metaphase I. After ovulation, activin-treated follicles developed into typical corpora lutea. No ovulations were found in control animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Activins↗
PubMed1994

Follistatin antagonizes the effects of activin-A on steroidogenesis in human luteinizing granulosa cells.

Activin-A decreases progesterone secretion and aromatase activity in cultured human luteinizing granulosa cells. Follistatin is a binding protein for activin and inhibin produced by granulosa cells and present in follicular fluid. The present study examines the hypothesis that follistatin reverses the actions of activin-A on human granulosa cells. Granulosa cells from women undergoing ovarian stimulation for in vitro fertilization were cultured in defined medium with recombinant human (rh) activin-A and purified porcine follistatin. Follistatin completely reversed the inhibition of basal progesterone production by rh-activin-A, but only when added in a greater than 2:1 molar ratio to activin-A. Although variable effects of activin-A on aromatase activity were observed in these studies, follistatin also antagonized these effects. Follistatin had no effect in the absence of added activin-A. rh-Inhibin-A did not alter steroidogenesis by granulosa cells either with or without added activin-A. Even when added in a 45-fold molar excess, inhibin-A did not displace sufficient activin-A from follistatin to inhibit progesterone secretion. This suggests that the affinity of inhibin-A for follistatin is much lower than that of activin-A, and/or that activin-A bound to follistatin dissociates slowly. alpha 2-Macroglobulin, another activin-binding protein, did not alter the inhibition of progesterone production by activin-A. We conclude that in human granulosa cells, follistatin, but not alpha 2-macroglobulin or inhibin-A, acts to modulate the actions of activin-A. Follistatin and activin may be viewed as components of an autocrine/paracrine system within the human follicle that regulate the differentiated functions of granulosa cells.

Activins↗
PubMed1992

Isolation and characterization of native activin B.

To examine whether activin binds to follistatin, an activin-binding protein, to form a complex in vivo, we attempted to purify activin-follistatin complex from porcine follicular fluid. Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B are present as a complex with follistatin in the follicular fluid. Reverse-phase high performance liquid chromatography of the purified complex yielded follistatin and activins A, AB, and B. The activity of the purified activin B was found to be significantly lower than those of other activins in various assay systems such as stimulation of follicle-stimulating hormone secretion, induction of erythrodifferentiation, and potentiation of expression of gonadotropin receptors on ovarian cells. Moreover, binding of 125I-activin A to erythroleukemic cells which are activin-responsive was competed by activin B with approximately 10-fold lower potency compared with other activins. In contrast to these results, activin B was proved to have a potent Xenopus mesoderm-inducing activity, comparable with that of other activins. This indicates that, unlike activins A and AB, activin B can only elicit mesoderm-inducing activity and cannot function in other biological systems, suggesting a specific role of activin B in early development and unknown biological functions.

Activins↗
PubMed2004

Activin isoforms signal through type I receptor serine/threonine kinase ALK7.

Activins play a fundamental role in cell differentiation and development. Activin A signaling is mediated through a combination of activin type II receptors (ActRIIs) and the activin type IB receptor, ALK4. Signaling receptors of other activin isoforms remain to be elucidated. Here, we found that activin AB and activin B are ligands for ALK7. ALK7 is an orphan receptor serine/threonine kinase expressed in neuroendocrine tissues including pancreatic islets. The combination of ActRIIA and ALK7, preferred by activin AB and activin B but not by activin A, is responsible for activin-mediated secretion of insulin from pancreatic beta cell line, MIN6. In contrast, all activins activate a combination of ActRIIA and ALK4 with various levels of potency. Thus, variation in activin signaling through type I receptors is dependent upon homo- and heterodimeric assembly of activin isoforms. Thus, the differential combination of receptor heterodimers mediates variation in activin isoform signaling.

Activin Receptors, Type I↗
PubMed2000

Differential regulation of activin A for hepatocyte growth and fibronectin synthesis in rat liver injury.

BACKGROUND/AIMS: Both hepatocyte growth and production of extracellular matrix such as fibronectin are essential for liver regeneration. Although activin A is reported to inhibit DNA replication in rat hepatocytes, the role of activin A for liver regeneration after acute injury has not been fully assessed. This study investigated the mechanism by which hepatocyte growth is regulated by activin A during liver regeneration and the effects of activin A on extracellular matrix production. METHODS: The mRNA for betaA subunit of activin A and activin receptors in hepatocytes and hepatic stellate cells after CCl4 administration were studied by Northern blotting. Binding of 125I-activin A was tested in these cells. Effects of activin A were examined by DNA, collagen and fibronectin synthesis. RESULTS: betaA mRNA was expressed in quiescent hepatocytes, and this expression peaked 12 h after CCl4 administration. Activin receptor mRNAs and cross-linked ligand/receptor complexes were expressed in hepatocytes and hepatic stellate cells However, these levels decreased specifically in hepatocytes at 24 h and had normalized by 72 h. The down-regulation of activin receptor was also observed after partial hepatectomy. Antiproliferative response to activin A decreased in hepatocytes at 24 h. Activin A stimulated production of fibronectin by hepatic stellate cells, but the synthesis of collagen was only slightly elevated in hepatic stellate cells following activin stimulation. CONCLUSIONS: The down-regulation of activin receptors in hepatocytes may be partly responsible for these cells becoming responsive to mitogenic stimuli. The increase of activin A at the early stage of liver injury has the potential to contribute to the regulation of fibronectin production in hepatic stellate cells.

Activin Receptors↗
PubMed2000

Hepatocyte growth factor induces branching tubulogenesis in MDCK cells by modulating the activin-follistatin system.

BACKGROUND: The activin-follistatin system is expressed in tubular cells of the kidney. The present study was conducted to examine the role of the activin-follistatin system in tubulogenesis using Madin-Darby canine kidney (MDCK) cells as a model system. METHODS: Tubulogenesis was assessed using MDCK cells cultured in collagen gel. The effect of recombinant human activin A on tubulogenesis was examined. Blockade of the action of endogenous activin was achieved by either adding follistatin or transfection of dominant-negative mutant of the type II activin receptor. The production of activin A was examined by Northern blotting, in situ hybridization, and Western blotting. RESULTS: MDCK cells expressed mRNA for the betaA subunit of activin. These cells formed spherical cysts when cultured in collagen gel. Hepatocyte growth factor (HGF) added to the spherical cysts induced branching tubulogenesis. When activin A was added together with HGF, activin A blocked the branching tubulogenesis induced by HGF, and the activin-treated cells were scattered. Conversely, follistatin, an antagonist of activin A, induced branching tubulogenesis qualitatively similar to that induced by HGF. Adenovirus vector-mediated transfer of the gene encoding truncated type II activin receptor, which acts as a dominant negative mutant, also induced branching tubulogenesis. Finally, HGF markedly inhibited the production of activin A in MDCK cells cultured in collagen gel. CONCLUSION: Activin A produced in MDCK cells tonically inhibits branching tubulogenesis, and HGF induced branching tubulogenesis mainly by blocking the production of activin A.

Activins↗
PubMed1993

Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes.

The present study was conducted to examine the effect of activin A on growth of rat hepatocytes. EGF induced a 10-fold increase in DNA synthesis as assessed by [3H]thymidine incorporation in cultured hepatocytes. When activin A was added together with EGF, DNA synthesis induced by EGF was markedly inhibited. Inhibition was detected at a concentration of 10(-10) M, and 5 x 10(-9) M activin A almost completely blocked EGF-mediated DNA synthesis. Similarly, activin A completely blocked DNA synthesis induced by hepatocyte growth factor/scatter factor. Activin A was capable of inhibiting EGF-mediated DNA synthesis, even when added 36 h after the addition of EGF. With the same time interval, TGF-beta also blocked EGF-induced DNA synthesis. Although both activin A and TGF-beta inhibited growth of hepatocytes in a similar manner, either activin A or TGF-beta did not compete with each other in their binding when assessed by competitive binding using an iodinated ligand. When hepatocytes were incubated with EGF, release of bioactivity of activin A into culture medium was detected after 48 h or later. Activity of activin A was released from parenchymal cells but not from nonparenchymal cells. mRNA for beta A subunit of activin was detected only slightly in unstimulated hepatocytes, but markedly increased at 48 h after the addition of EGF. To determine whether endogenously produced activin A affects DNA synthesis, we examined the effect of follistatin, an activin-binding protein that blocks the action of activin A. An addition of follistatin significantly enhanced EGF-induced DNA synthesis. Finally, in partial hepatectomized rat, expression of mRNA for beta A subunit in liver was markedly increased 24 h after the partial hepatectomy. These results indicate that activin A inhibits initiation of DNA synthesis in hepatocytes by acting on its own receptor and that activin A acts as an autocrine inhibitor of DNA synthesis in rat hepatocytes.

Activins↗
PubMed2002

Activins, inhibins, and follistatins: from endocrinology to signaling. A paradigm for the new millennium.

It has been 70 years since the name inhibin was used to describe a gonadal factor that negatively regulated pituitary hormone secretion. The majority of this period was required to achieve purification and definitive characterization of inhibin, an event closely followed by identification and characterization of activin and follistatin (FS). In contrast, the last 15-20 years saw a virtual explosion of information regarding the biochemistry, physiology, and biosynthesis of these proteins, as well as identification of activin receptors, and a unique mechanism for FS action-the nearly irreversible binding and neutralization of activin. Many of these discoveries have been previously summarized; therefore, this review will cover the period from the mid 1990s to present, with particular emphasis on emerging themes and recent advances. As the field has matured, recent efforts have focused more on human studies, so the endocrinology of inhibin, activin, and FS in the human is summarized first. Another area receiving significant recent attention is local actions of activin and its regulation by both FS and inhibin. Because activin and FS are produced in many tissues, we chose to focus on a few particular examples with the most extensive experimental support, the pituitary and the developing follicle, although nonreproductive actions of activin and FS are also discussed. At the cellular level, it now seems that activin acts largely as an autocrine and/or paracrine growth factor, similar to other members of the transforming growh factor beta superfamily. As we discuss in the next section, its actions are regulated extracellularly by both inhibin and FS. In the final section, intracellular mediators and modulators of activin signaling are reviewed in detail. Many of these are shared with other transforming growh factor beta superfamily members as well as unrelated molecules, and in a number of cases, their physiological relevance to activin signal propagation remains to be elucidated. Nevertheless, taken together, recent findings suggest that it may be more appropriate to consider a new paradigm for inhibin, activin, and FS in which activin signaling is regulated extracellularly by both inhibin and FS whereas a number of intracellular proteins act to modulate cellular responses to these activin signals. It is therefore the balance between activin and all of its modulators, rather than the actions of any one component, that determines the final biological outcome. As technology and model systems become more sophisticated in the next few years, it should become possible to test this concept directly to more clearly define the role of activin, inhibin, and FS in reproductive physiology.

Activins↗
PubMed1993

Perifusion of rat pituitary cells with gonadotropin-releasing hormone, activin, and inhibin reveals distinct effects on gonadotropin gene expression and secretion.

Gonadotropin biosynthesis and secretion are influenced by pulsatile GnRH derived from the hypothalamus as well as by paracrine factors. In the current studies, we compared the effects of inhibin, activin, and GnRH, alone and in combination, on gonadotropin subunit messenger RNA (mRNA) levels and gonadotropin secretion. A pituitary perifusion system was used to allow GnRH to be administered as pulses and to minimize paracrine effects. FSH beta mRNA levels were increased 25-fold by a maximal concentration of activin (3 ng/ml) and suppressed 83% by a maximal concentration of inhibin (30 ng/ml). When activin and inhibin were perifused together, inhibin attenuated the effects of maximal activin stimulation in a concentration-dependent manner, with a 10-fold excess of inhibin required to block the effects of activin entirely. Whole cell receptor assays using 125I-labeled activin confirmed that the inhibin used in the perifusion experiments competed for activin binding sites, although with a lower affinity. Direct competition at the activin receptor may thus account for part of the activin/inhibin antagonism observed at the level of FSH beta mRNA. Neither activin nor inhibin had a significant effect on levels of LH beta or alpha mRNAs. Hourly pulses of 10 nM GnRH elicited a 2- to 5-fold increase in FSH beta mRNA. This increment was maintained in the presence of activin and inhibin, suggesting separate, but dependent, mechanisms of action for GnRH vs. inhibin and activin. In studies of secretion, continuous activin stimulation (3 ng/ml) elicited only a small (approximately 30%) increase in basal FSH secretion. However, the response of FSH to pulses of GnRH was amplified 3-fold in the presence of activin. A similar enhancement of GnRH-induced, but not basal, LH release was also observed. Inhibin, in contrast, elicited no changes in basal or GnRH-stimulated release of FSH or LH. We conclude that activin and inhibin are the primary regulators of FSH beta mRNA levels, whereas GnRH appears to be the major effector for gonadotropin secretion. There is significant functional overlap, however, and the combined actions of activin, inhibin, and GnRH determine the final level of FSH beta mRNA and the pattern of gonadotropin secretion.

Activins↗
PubMed1999

A sensitive and specific in vitro bioassay for activin using a mouse plasmacytoma cell line, MPC-11.

A new in vitro bioassay for activin was developed using the mouse plasmacytoma cell line, MPC-11. Human recombinant (hr) activin A dose-dependently inhibited the proliferation of these cells, whereas a range of other factors, including inhibin, follistatin and transforming growth factor-beta1, -beta2 and -beta3 had no effect. Conditioned medium containing activin B induced an inhibition similar to hr-activin A. The inhibitory influence of activin A could be blocked by follistatin, but not by hr-inhibin A. This bioassay had a sensitivity for activin A of around 0.4 ng/ml, an ED50 response of 3.5 ng/ml, and an intra-assay coefficient of variation of <11%. It offers substantial advantages over existing in vitro activin bioassays in terms of ease of use, specificity and throughput. The utility of the MPC-11 bioassay was demonstrated in the purification of activin from amniotic fluid, where an almost identical profile of bioactive activin A was detected compared with the pituitary cell bioassay of activin. Bioactive activin could also be detected in unpurified ovine allantoic and amniotic fluids and bovine follicular fluid. Measuring activin in untreated and heat-treated human sera or seminal plasma was hampered by a non-specific inhibitory effect, so that several serum samples did not run parallel with the hr-activin A standard. This inhibitory effect by serum could not be overcome by addition of follistatin, suggesting it is not activin-like bioactivity. This new bioassay for activin demonstrates widespread applicability for monitoring of purified or partially purified samples during purification procedures, bioactivity measurements, receptor-binding studies and assays of cell culture medium.

Activins↗