Identification and expression of the medaka inhibin ßE subunit.

Activin E, a member of the TGF-ß super family, is a protein dimer of mature inhibin ßE subunits. Recently, it is reported that hepatic activin E may act as a hepatokine that alter whole body energy/glucose metabolism in human. However, orthologues of the activin E gene have yet to be identified in lower vertebrates, including fish. Here, we cloned the medaka (Oryzias latipes) activin E cDNA from liver. Among all the mammalian inhibin ß subunits, the mature medaka activin E amino acid sequence shares the highest homology with mammalian activin E. Recombinant expression studies suggest that medaka activin E, the disulfide-bound mature form of mature inhibin ßE subunits, may exert its effects in a way similar to that in mammals. Although activin E mRNA is predominantly expressed in liver in mammals, it is ubiquitously expressed in medaka tissues. Since expression in the liver was enhanced after a high fat diet, medaka activin E may be associated with energy/glucose metabolism, as shown in mice and human.

Comparative analysis of activins A and B in the adult mouse epididymis and vas deferens.

Activin A regulates testicular and epididymal development, but the role of activin B in the epididymis and vas deferens is unknown. Mouse models with reduced activin A (Inhba+/- and InhbaBK/+), or its complete absence (InhbaBK/BK), were investigated to identify specific roles of activins in the male reproductive tract. In 8-week-old Inhba+/- mice, serum activin A decreased by 70%, with a 50% reduction of gene expression and protein in the testis, epididymis and vas deferens. Activin B and the activin-binding protein, follistatin, were similar to wild-type. Testis weights were slightly reduced in Inhba+/- mice, but the epididymis and vas deferens were normal, while the mice were fertile. Activin A was decreased by 70% in the serum, testis, epididymis and vas deferens of InhbaBK/+ mice and was undetectable in InhbaBK/BK mice, but activin B and follistatin levels were similar to wild-type. In 6-week-old InhbaBK/BK mice, testis weights were 60% lower and epididymal weights were 50% lower than in either InhbaBK/+ or wild-type mice. The cauda epididymal epithelium showed infoldings and less intra-luminal sperm, similar to 3.5-week-old wild-type mice, but at 8 weeks, no structural differences in the testis or epididymis were noted between InhbaBK/BK and wild-type mice. Thus, Inhbb can compensate for Inhba in regulating epididymal morphology, although testis and epididymal maturation is delayed in mice lacking Inhba Crucially, reduction or absence of activin A, at least in the presence of normal activin B levels, does not lead to major defects in the adult epididymis or vas deferens.

Endogenous protection derived from activin A/Smads transduction loop stimulated via ischemic injury in PC12 cells.

Activin A (ActA), a member of transforming growth factor-beta (TGF-b) super- family, affects many cellular processes, including ischemic stroke. Though the neuroprotective effects of exogenous ActA on oxygen-glucose deprivation (OGD) injury have already been reported by us, the endogenous role of ActA remains poorly understood. To further define the role and mechanism of endogenous ActA and its signaling in response to acute ischemic damage, we used an OGD model in PC12 cells to simulate ischemic injury on neurons in vitro. Cells were pre-treated by monoclonal antibody against activin receptor type IIA (ActRII-Ab). We found that ActRII-Ab augments ischemic injury in PC12 cells. Further, the extracellular secretion of ActA as well as phosphorylation of smad3 in PC12 cells was also up-regulated by OGD, but suppressed by ActRII-Ab. Taken together, our results show that ActRII-Ab may augment ischemic injury via blocking of transmembrane signal transduction of ActA, which confirmed the existence of endogenous neuroprotective effects derived from the ActA/Smads pathway. ActRIIA plays an important role in transferring neuronal protective signals inside. It is highly possible that ActA transmembrance signaling is a part of the positive feed-back loop for extracellular ActA secretion.

Human INHBB Gene Variant (c.1079T>C:p.Met360Thr) Alters Testis Germ Cell Content, but Does Not Impact Fertility in Mice.

Testicular-derived inhibin B (α/ß B dimers) acts in an endocrine manner to suppress pituitary production of follicle-stimulating hormone (FSH), by blocking the actions of activins (ß A/B/ß A/B dimers). Previously, we identified a homozygous genetic variant (c.1079T>C:p.Met360Thr) arising from uniparental disomy of chromosome 2 in the INHBB gene (ß B-subunit of inhibin B and activin B) in a man suffering from infertility (azoospermia). In this study, we aimed to test the causality of the p.Met360Thr variant in INHBB and testis function. Here, we used CRISPR/Cas9 technology to generate InhbbM364T/M364T mice, where mouse INHBB p.Met364 corresponds with human p.Met360. Surprisingly, we found that the testes of male InhbbM364T/M364T mutant mice were significantly larger compared with those of aged-matched wildtype littermates at 12 and 24 weeks of age. This was attributed to a significant increase in Sertoli cell and round spermatid number and, consequently, seminiferous tubule area in InhbbM364T/M364T males compared to wildtype males. Despite this testis phenotype, male InhbbM364T/M364T mutant mice retained normal fertility. Serum hormone analyses, however, indicated that the InhbbM364T variant resulted in reduced circulating levels of activin B but did not affect FSH production. We also examined the effect of this p.Met360Thr and an additional INHBB variant (c.314C>T: p.Thr105Met) found in another infertile man on inhibin B and activin B in vitro biosynthesis. We found that both INHBB variants resulted in a significant disruption to activin B in vitro biosynthesis. Together, this analysis supports that INHBB variants that limit activin B production have consequences for testis composition in males.

Activin B inhibits lipolysis in 3T3-L1 adipocytes.

Activin B, consisting of two inhibin betaB (INHBB) subunits, is a hormone known to affect gonadal function, reproduction and fetal development. We have reported that INHBB and activin B receptors are highly expressed in adipocytes suggesting that activin B may have local effects in adipose tissue. In this study, we investigate the effect of activin B on lipolysis, measured as release of non-esterified fatty acids and free glycerol. Recombinant activin B decreased lipolysis in a concentration-dependent manner and increased intracellular triglyceride content in 3T3-L1 adipocytes. siRNA-mediated knock-down of INHBB expression increased lipolysis, and this effect was abolished by addition of recombinant activin B. In line with its inhibitory effect on lipolysis, activin B caused a down regulation of the expression of adipose triglyceride lipase and hormone sensitive lipase, key genes involved in lipolysis. In summary, we suggest that activin B is a novel adipokine that inhibits lipolysis in a paracrine or autocrine manner.

Abnormalities in aggression and anxiety in transgenic mice overexpressing activin E.

To study the function of activin E, a TGF-beta superfamily member, in the regulation of affective behavior, we investigated the behavior of transgenic mice overexpressing activin E (TgActbetaE mice). Male TgActbetaE mice showed aggressive behavior in resident-intruder tests. In elevated plus-maze tests, the percentage of open arm entries was significantly increased in female TgActbetaE mice compared with that in wild-type mice. Furthermore, female TgActbetaE mice stayed in the central area for a significantly longer time than wild-type mice in open field tests. These results indicated that TgActbetaE mice had less anxiety-like behavior. The number of restraint-stress-evoked c-Fos-positive cells in the hypothalamic paraventricular nucleus in TgActbetaE mice was significantly decreased compared with that in wild-type mice. This suggests that synthesis of corticotrophin-releasing hormone induced by stress was decreased in TgActbetaE mice. Taking these results together, activin E may act as a regulator of the hypothalamic-pituitary-adrenal axis.

Effect of activin A on tubulointerstitial fibrosis in diabetic nephropathy.

AIM: The effect of activin A on tubulointerstitial fibrosis in diabetic nephropathy (DN) using streptozotocin (STZ)-induced diabetic rats and high glucose-cultured HK-2 cells was investigated. METHODS: Male Wistar rats were randomized into a normal control group (NC) and diabetes mellitus group (DM). Diabetes was induced by i.p. injection of STZ. Six rats were respectively killed 4, 8, 12 and 16 weeks after model establishment in each group. The changes of kidney weight/bodyweight (KW/BW), urine albumin excretion rate (AER) and creatinine clearance rate (Ccr) were determined. The morphology of tubulointerstitium was observed by light microscopy. Further biochemical analysis was provided using immunohistochemistry and real-time polymerase chain reaction. The different parameters in high glucose-cultured HK-2 cells were monitored by western blotting or enzyme-linked immunosorbent assay (ELISA) and the intervention of rh-follistatin on them was investigated. RESULTS: Compared with the NC group, there was marked enlargement in the levels of KW/BW, AER, Ccr and interstitial fibrosis index, and the production of P-Smad2/3 and fibronectin in the DM group from 8 to 16 weeks. Activin betaA, mainly located in tubular epithelial cells, was significantly higher in the DM group than that in the NC group throughout the study periods. Follistatin was abundant in the NC group, but was diminished gradually in the DM group. High glucose may facilitate the synthesis of activin betaA, transforming growth factor (TGF)-beta, P-Smad2/3 and fibronectin in HK-2 cells while rh-follistatin inhibited them except TGF-beta. CONCLUSION: Activin A is involved in tubulointerstitial fibrosis in DN by inducing the production of fibronectin through Smad signal pathway.

Interdependent fibroblast growth factor and activin A signaling promotes the expression of endodermal genes in differentiating mouse embryonic stem cells expressing Src Homology 2-domain inactive Shb.

The mechanisms controlling endodermal development during stem cell differentiation have been only partly elucidated, although previous studies have suggested the participation of fibroblast growth factor (FGF) and activin A in these processes. Shb is a Src homology 2 (SH2) domain-containing adapter protein that has been implicated in FGF receptor 1 (FGFR1) signaling. To study the putative crosstalk between activin A and Shb-dependent FGF signaling in the differentiation of endoderm from embryonic stem (ES) cells, embryoid bodies (EBs) derived from mouse ES cells overexpressing wild-type Shb or Shb with a mutated SH2 domain (R522K-Shb) were cultured in the presence of activin A. We show that expression of R522K-Shb results in up-regulation of FGFR1 and FGF2 in EBs. Addition of activin A to the cultures enhances the expression of endodermal genes primarily in EBs expressing mutant Shb. Inhibition of FGF signaling by the addition of the FGFR1 inhibitor SU5402 completely counteracts the synergistic effects of R522K-Shb and activin A. In conclusion, the present results suggest that expression of R522K-Shb enhances certain signaling pathways downstream of FGF and that an interplay between FGF and activin A participates in ES cell differentiation to endoderm.

Activin a causes cancer cell aggressiveness in esophageal squamous cell carcinoma cells.

BACKGROUND: Expression of activin A is associated with lymph node metastasis and clinical stage in esophageal cancer. METHODS: To clarify the aggressive behavior of tumors with high activin A expression, we used the beta subunit of activin A to establish stable activin betaA (Act-betaA)-transfected carcinoma cells in two human esophageal carcinoma cell lines, KYSE110 and KYSE140. The biological behavior of these cells was compared with that in mock-transfected cells from the same cell lines. We focused our attention on cell growth and tumorigenesis, and proliferation and apoptosis. RESULTS: Both Act-betaA-transfected carcinoma cell lines showed a higher growth rate than the mock-transfected carcinoma cells. In an in vitro invasion assay and a xenograft analysis, the Act-betaA-transfected carcinoma cells showed far higher proliferation in vitro and a higher potency for tumorigenesis in vivo, respectively. Moreover, in an analysis of apoptosis via Fas stimulation, the Act-betaA-transfected carcinoma cells showed a higher tolerance to apoptosis compared with the mock-transfected carcinoma cells. Moreover, anti-activin-neutralizing antibody-treated squamous cell cancer cell lines inhibited their migration. CONCLUSIONS: Collectively, these data indicate that continuous high expression of activin A in esophageal carcinoma cells is not related to tumor suppression, but rather to tumor progression in vitro and in vivo. The inhibition of activin might be one of the methods to attenuate tumor aggressiveness.

Intraovarian activins are required for female fertility.

Activins have diverse roles in multiple physiological processes including reproduction. Mutations and loss of heterozygosity at the human activin receptor ACVR1B and ACVR2 loci are observed in pituitary, pancreatic, and colorectal cancers. Functional studies support intraovarian roles for activins, although clarifying the in vivo roles has remained elusive due to the perinatal death of activin betaA knockout mice. To study the roles of activins in ovarian growth, differentiation, and cancer, a tissue-specific knockout system was designed to ablate ovarian production of activins. Mice lacking ovarian activin betaA were intercrossed to Inhbb homozygous null mice to produce double activin knockouts. Whereas ovarian betaA knockout females are subfertile, betaB/betaA double mutant females are infertile. Strikingly, the activin betaA and betaB/betaA-deficient ovaries contain increased numbers of functional corpora lutea but do not develop ovarian tumors. Microarray analysis of isolated granulosa cells identifies significant changes in expression for a number of genes with known reproductive roles, including Kitl, Taf4b, and Ghr, as well as loss of expression of the proto-oncogene, Myc. Thus, in contrast to the known tumor suppressor role of activins in some tissues, our data indicate that activin betaA and betaB function redundantly in a growth stimulatory pathway in the mammalian ovary.

Should activin betaC be more than a fading snapshot in the activin/TGFbeta family album?

The activin growth factors consist of dimeric proteins made up of activin beta subunits and have been shown to be essential regulators of diverse systems in physiology. Four subunits are known to be expressed in mammalian cells: betaA, betaB, betaC, and betaE. Surprisingly, deletion of activin betaC and betaE subunits in vivo does not affect embryonic development or adult physiology which has led to the activin betaC and betaE subunits being regarded as non-essential and unimportant. The steady accumulation of circumstantial evidence to the contrary has led this lab to reassess the role of the activin betaC subunit. Activin betaC protein is expressed more widely than indicated by mRNA localisation. Experiments overexpressing activin betaC subunit or adding exogenous Activin C in vitro are contradictory but suggest roles for activin betaC in regulating Activin A action in apoptosis and homeostasis. Sequestration of betaA subunits by dimerisation with betaC subunits to form Activin AC represents an intracellular regulator of Activin A bioactivity. Activins play a pivotal role in normal physiology and carcinogenesis, so any molecule, such as the activin betaC subunit, that can affect activin action is potentially significant. Advancing our understanding of the physiological role of the activin betaC subunit requires new tools and reagents. Direct detection of the Activin AC dimer will be essential and will necessitate the purification of heteromeric Activin AC protein. In addition, there is a need for the development of an in vivo model of activin betaC subunit overexpression. With development of these tools, research into activin action in development and physiology can expand to include the less well understood members of the activin family such as activin betaC.

Activin A mediates growth inhibition and cell cycle arrest through Smads in human breast cancer cells.

The transforming growth factor-beta (TGF-beta) superfamily of growth factors is responsible for a variety of physiologic actions, including cell cycle regulation. Activin is a member of the TGF-beta superfamily that inhibits the proliferation of breast cancer cells. Activin functions by interacting with its type I and type II receptors to induce phosphorylation of intracellular signaling molecules known as Smads. Smads regulate transcription of many genes in a cell- and tissue-specific manner. In this study, the role of activin A in growth regulation of breast cancer cells was investigated. Activin stimulated the Smad-responsive promoter, p3TP, 2-fold over control in T47D breast cancer cells. Activin inhibited cellular proliferation of T47D breast cancer cells after 72 hours, an effect that could be abrogated by incubation with the activin type I receptor inhibitor, SB431542. Activin arrested T47D cells in the G0-G1 cell cycle phase. Smad2 and Smad3 were phosphorylated in response to activin and accumulated in the nucleus of treated T47D cells. Infection of T47D cells with adenoviral Smad3 resulted in cell cycle arrest and activation of p3TP-luciferase, whereas a adenoviral dominant-negative Smad3 blocked activin-mediated cell cycle arrest and gene transcription. Activin maintained expression of p21 and p27 cyclin-dependent kinase inhibitors involved in cell cycle control, enhanced expression of p15, reduced cyclin A expression, and reduced phosphorylation of the retinoblastoma (Rb) protein. Smad3 overexpression recapitulated activin-induced p15 expression and repression of cyclin A and Rb phosphorylation. These data indicate that activin A inhibits breast cancer cellular proliferation and activates Smads responsible for initiating cell cycle arrest.

Activin A and inhibin A differentially regulate human uterine matrix metalloproteinases: potential interactions during decidualization and trophoblast invasion.

Embryo implantation and trophoblast invasion are tightly regulated processes, involving sophisticated communication between maternal decidual and fetal trophoblast cells. Decidualization is a prerequisite for successful implantation and is promoted by a number of paracrine agents, including activin A. To understand the downstream mechanisms of activin-promoted decidualization, the effects of activin on matrix metalloproteinases (MMPs) (important mediators of decidualization) were investigated. Activin A stimulated endometrial production of proMMPs-2, -3, -7, -9, and active MMP-2. In contrast, inhibin A was a potent inhibitor of proMMP-2, and antagonized the effect of activin on MMPs. Activin is up-regulated with decidualization, and MMPs-2, -3, and -9 increase in parallel. Furthermore, proMMP-2 production is stimulated when decidualization is accelerated with activin, and suppressed when activin is neutralized, attenuating decidualization. These data support that activin A promotes decidualization through up-regulating MMPs. Previous in vitro evidence proposes further roles for activin and MMPs in promoting trophoblast invasion; therefore, we examined their interrelationships in early human implantation sites. MMPs-7 and -9 were produced by static cytotrophoblast subpopulations, whereas MMP-2 was strikingly up-regulated in invasive extravillous cytotrophoblasts (EVT). Maternal decidua is the primary source of activin, where a role in stimulating MMP-2 in iEVTs can be envisaged. Inhibin was absent from cytotrophoblast populations, except for a dramatic up-regulation in endovascular EVT plugs, coinciding with a down-regulation of MMP-2. This suggests that inhibin may have a role in the cessation of vascular invasion. These data support that activin, via effects on MMPs, is an important factor in the maternal-fetal dialog regulating implantation.

Acute regulation of murine follicle-stimulating hormone beta subunit transcription by activin A.

In rodents, activins stimulate immediate-early increases in pituitary follicle-stimulating hormone beta (Fshb) subunit transcription. Here, we investigated the underlying signaling mechanisms using the mouse gonadotrope cell line, LbetaT2. Activin A increased mouse Fshb-luciferase reporter activity within 4 h through a Smad-dependent signaling pathway. The ligand rapidly stimulated formation of SMAD2/3/4 complexes that could interact with a consensus palindromic Smad binding element (SBE) in the proximal Fshb promoter. SMAD over-expression potently stimulated transcription, with the combination of SMADs 2, 3 and 4 producing the greatest synergistic activation. A mutation in the SBE that abolished Smad binding greatly impaired the effects of acute (4 h) activin A treatment and SMAD over-expression on promoter activity, but did not abolish the effects of chronic (24 h) activin A exposure. Within activated SMAD complexes, SMADs 3 and 4 appeared to bind the SBE simultaneously and the binding of both was required for maximal transcriptional activation. Interestingly, the human FSHB promoter, which lacks the consensus SBE, was neither rapidly stimulated by activin A nor by over-expressed SMADs, but was activated by 24 h activin A. Addition of the SBE to the human promoter increased both SMAD2/3/4-sensitivity and acute regulation by activin A, though not to levels observed in mouse. We postulate that short reproductive cycles in female rodents, particularly the brief interval between the primary and secondary FSH surges of the estrous cycle, require the Fshb promoter in these animals to be particularly sensitive to the rapid, Smad-dependent actions of activins on transcription. The human FSHB promoter, in contrast, is chronically regulated by activins seemingly through a SMAD-independent pathway.

Elevated levels of activin A in heart failure: potential role in myocardial remodeling.

BACKGROUND: Although modulation of inflammatory processes has been suggested as a new treatment modality in heart failure (HF), our knowledge about abnormalities in the cytokine network during HF is still limited. On the basis of a previous cDNA array study examining peripheral blood mononuclear cells from HF patients, we hypothesized a role for activin A, a member of the transforming growth factor (TGF)-beta superfamily, in the pathogenesis of HF. METHODS AND RESULTS: This study had 4 main and novel findings. First, serum levels of activin A were significantly elevated in patients with HF (n=86) compared with healthy control subjects (n=20), with increasing levels according to disease severity as assessed by clinical, hemodynamic, and neurohormonal parameters. Second, compared with control subjects, HF patients, as determined by real-time quantitative reverse transcriptase polymer chain reaction, also had markedly increased gene expression of the activin A subunit activin betaA in T cells but not in monocytes. Third, in a rat model of HF, we demonstrated a concerted induction of the gene expression of activin betaA and activin receptors IA, IB, IIA, and IIB after myocardial infarction. Immunohistochemical analysis localized activin A solely to cardiomyocytes. Finally, activin A markedly increased gene expression of mediators involved in infarction healing and myocardial remodeling (ie, atrial natriuretic peptide, brain natriuretic peptide, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta1, and monocyte chemoattractant protein-1) in neonatal rat cardiomyocytes. CONCLUSIONS: Together with our demonstration of activin A-induced gene expression in neonatal cardiomyocytes of mediators related to myocardial remodeling, the expression pattern of activin A during clinical and experimental HF suggests an involvement of this cytokine in the pathogenesis of HF.

Potential anti-inflammatory role of activin A in acute coronary syndromes.

OBJECTIVES: We sought to investigate whether activin A could be involved in the immunopathogenesis of acute coronary syndromes. BACKGROUND: Inflammatory mechanisms seem to play a pathogenic role in atherosclerosis and acute coronary syndromes, but the actual mediators have not been fully identified. Activin A, a pleiotropic member of the transforming growth factor-beta cytokine family, has recently been suggested to play a role in inflammation. METHODS: We examined the role of activin A and its endogenous inhibitor follistatin in patients with stable (n = 26) and unstable angina (n = 20) and healthy control subjects (n = 20) by different experimental approaches. RESULTS: 1) Patients with stable angina had raised activin A concentrations, as assessed by protein levels in serum and messenger ribonucleic acid levels in peripheral blood mononuclear cells (PBMCs). 2) Although several activin A-related mediators were upregulated in PBMCs from patients with stable angina compared with controls (i.e., activin A and Smad3), no changes or even downregulation (i.e., Smad2) were seen in unstable disease. 3) The activin type II receptors, representing the primary ligand-binding proteins, were downregulated in unstable compared with stable angina. 4) Percutaneous coronary intervention induced a decrease in the activin A/follistatin ratio, suggesting downregulatory effects on activin A activity. 5) Although activin A dose-dependently suppressed the release of inflammatory cytokines from PBMCs in angina patients, an opposite effect was found in healthy controls. CONCLUSIONS: Our findings suggest an anti-inflammatory potential of activin A in angina patients, and such effects may be of particular relevance in unstable angina in which several of the activin parameters were downregulated.

The secretion and effect of inhibin A, activin A and follistatin on first-trimester trophoblasts in vitro.

OBJECTIVE: The objectives of this study were to investigate the effect of activin A and follistatin on first-trimester cytotrophoblast invasion in culture and to study the secretion of inhibin A, activin A and follistatin by these cells in vitro. DESIGN AND METHODS: Cytotrophoblasts were isolated from human placental chorionic villous tissue obtained from 6-8, 8-10 and 10-12 weeks gestation. Cells were cultured for 3 days on cell-culture inserts coated with gelatine for invasion studies and in 24-well culture plates for secretion studies. The effects of activin A (10 ng/ml), follistatin (100 ng/ml), interleukin 1beta (IL-1beta; 10 ng/ml) and epidermal growth factor (EGF; 10 ng/ml) on cytotrophoblast invasion were investigated using a non-radioactive invasion assay. Secretion of inhibin A, activin A and follistatin in the presence of EGF, IL-1beta, activin A and follistatin were measured using in-house ELISAs. RESULTS AND CONCLUSION: Activin A, follistatin and EGF had a significant stimulatory effect on cytotrophoblast invasion from 6-10 weeks gestation. IL-1beta had a significant stimulatory effect at 8-10 weeks and a significant inhibitory effect on invasion at 10-12 weeks gestation. Follistatin also had a significant inhibitory effect on invasion at 10-12 weeks gestation. In the secretion study, activin A secretion at 8-10 weeks was significantly stimulated by IL-1beta and EGF. At 10-12 weeks, follistatin and EGF had a significant inhibitory effect on activin A secretion. Follistatin secretion was significantly increased in the presence of IL-1beta at 6-8 weeks gestation. Inhibin A secretion was not significantly altered by EGF, IL-1beta, activin A and follistatin. These results show that activin A promotes invasion of first-trimester cytotrophoblasts until 10 weeks gestation. There is a difference in the control of secretion of these proteins dependent on the gestation, suggesting that there is a tight regulation in the function of first-trimester trophoblasts depending on the gestational age.

Differential regulation of follicle stimulating hormone by activin A and TGFB1 in murine gonadotropes.

BACKGROUND: Activins stimulate the synthesis of follicle stimulating hormone (FSH) in pituitary gonadotropes, at least in part, by inducing transcription of its beta subunit (Fshb). Evidence from several laboratories studying transformed murine LbetaT2 gonadotropes indicates that activins signal through Smad-dependent and/or Smad-independent pathways, similar to those used by transforming growth factor beta-1 (TGFB1) in other cell types. Therefore, given common intracellular signaling mechanisms of these two ligands, we examined whether TGFBs can also induce transcription of Fshb in LbetaT2 cells as well as in purified primary murine gonadotropes. METHODS: Murine Fshb promoter-reporter (-1990/+1 mFshb-luc) activity was measured in LbetaT2 cells treated with activin A or TGFB1, and in cells transfected with either activin or TGFB receptors. The ability of the ligands to stimulate phosphorylation of Smads 2 and 3 in LbetaT2 cells was measured by western blot analysis, and expression of TGFB type I and II receptors was assessed by reverse transcriptase polymerase chain reaction in both LbetaT2 cells and primary gonadotropes purified from male mice of different ages. Finally, regulation of endogenous murine Fshb mRNA levels by activin A and TGFB1 in purified gonadotropes and whole pituitary cultures was measured using quantitative RT-PCR. RESULTS: Activin A dose-dependently stimulated -1990/+1 mFshb-luc activity in LbetaT2 cells, but TGFB1 had no effect at doses up to 5 nM. Similarly, activin A, but not TGFB1, stimulated Smad 2 and 3 phosphorylation in these cells. Constitutively active forms of the activin (Acvr1b-T206D) and TGFB (TGFBR1-T204D) type I receptors strongly stimulated -1990/+1 mFshb-luc activity, showing that mechanisms down stream of Tgfbr1 seem to be intact in LbetaT2 cells. RT-PCR analysis of LbetaT2 cells and whole adult murine pituitaries indicated that both expressed Tgfbr1 mRNA, but that Tgfbr2 was not detected in LbetaT2 cells. When cells were transfected with a human TGFBR2 expression construct, TGFB1 acquired the ability to significantly stimulate -1990/+1 mFshb-luc activity. In contrast to LbetaT2 cells, primary murine gonadotropes from young mice (8-10 weeks) contained low, but detectable levels of Tgfbr2 mRNA and these levels increased in older mice (1 yr). A second surprise was the finding that treatment of purified primary gonadotropes with TGFB1 decreased murine Fshb mRNA expression by 95% whereas activin A stimulated expression by 31-fold. CONCLUSION: These data indicate that TGFB1-insensitivity in LbetaT2 cells results from a deficiency in Tgfbr2 expression. In primary gonadotropes, however, expression of Tgfbr2 does occur, and its presence permits TGFB1 to inhibit Fshb transcription, whereas activin A stimulates it. These divergent actions of activin A and TGFB1 were unexpected and show that the two ligands may act through distinct pathways to cause opposing biological effects in primary murine gonadotropes.

Control of pelage hair follicle development and cycling by complex interactions between follistatin and activin.

Members of the transforming growth factor beta/bone morphogenetic protein (TGF-beta/BMP) family are involved in the control of hair follicle (HF) morphogenesis and cycling. The activities of several members of this family activins and BMP-2, -4, -7, and -11) are controlled by antagonists such as follistatin. Because follistatin-deficient mice show abnormalities in vibrissae development, we explored the role of follistatin and activin in pelage HF development and cycling. We show here that during HF development follistatin mRNA was prominently expressed by hair matrix and outer root sheath keratinocytes as well as by interfollicular epidermal cells, whereas activin betaA mRNA was mainly expressed in dermal papilla cells. Compared with age-matched wild-type controls, both follistatin knockout mice and activin betaA transgenic mice showed a significant retardation of HF morphogenesis. Treatment of wild-type embryonic skin explants with follistatin protein stimulated HF development. This effect was inhibited by addition of recombinant activin A protein. Activin betaA transgenic mice demonstrated retardation of catagen entry, down-regulation of BMP-2, and up-regulation of expression of its antagonist matrix GLA protein. These observations suggest that follistatin and activin interaction plays an important role in both HF development and cycling, possibly in part by regulating expression of BMP-2 and its antagonist.

Role of TGF-beta family in osteoclastogenesis induced by RANKL.

Recent studies have revealed that both transforming growth factor-beta (TGF-beta) and activin A play pivotal roles in osteoclastogenesis. In this report, we show that the effect of TGF-beta family members, TGF-beta1 and activin A, but not BMP-2, enhance multinucleated osteoclast-like cell (OCL) formation induced by receptor activator of NF-kappaB ligand (RANKL) in isolated bone marrow macrophages and monocytic cell line, RAW264.7. TGF-beta1 and activin A caused the growth suppression and concomitant expression of tartrate-resistant acid phosphatase (TRAP) and c-Src, without inducing syncytium formation or increasing the survival rate in RAW264.7 cells. Although TGF-beta1 and activin A had no effect on NF-kappaB and JNK activities, these factors enhanced the expression of JunB, a component of the AP-1 transcriptional complex. These results suggest that TGF-beta1 and activin A may function as commitment factors in osteoclastic differentiation, not as a crucial component for terminal differentiation to form multinucleated OCLs nor in OCL survival.