Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells

Background

We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β _B-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.

Methodology

hGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin β _B-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey''s test.

Principal Findings

GDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin β _B-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin β _B-subunit mRNA and inhibin B production were attenuated by adding FST.

Conclusion

GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin β _B-subunit mRNA expression and inhibin B production in hGL cells.     

Differential Modulation of Integrin Receptors and Extracellular Matrix Laminin by Transforming Growth Factor-β1 in Rat Alveolar Epithelial Cells

The transforming growth factors-β (TGFs-β) family of genes plays important roles in cell growth and differentiation in many cell types. TGFβ modulates the synthesis and accumulation of extracellular matrix (ECM) components and the expression of cell surface receptors for ECM components. TGFβ is increased in alveolar lining fluid during inflammatory reactions of the lung and has been identified in alveolar epithelial cells of developing lungs and hyperplastic type II cells during repair. However, little is known about how TGFβ may regulate expression of extracellular matrix proteins and ECM receptors in lung alveolar epithelial cells. Laminin, a major glycoprotein component of epithelial basement membrane, is synthesized and secreted by alveolar epithelial cells. To study the effects of TGFβ on modulation of laminin and its integrin receptors α₆β₁ and α₃β₁ in lung alveolar epithelial cells, a rat alveolar type II cell-derived cell line, LM5, was incubated with TGFβ₁ (0-100 pg/ml) in serum-free medium for 0-16 h. We examined the expression of integrin subunits and laminin β2 chain (s-laminin) mRNAs and protein expression. By Northern blot analysis, TGFβ₁ induced dose-dependent increases in α₆ and β₁ mRNA levels. TGFβ₁ also increased the expression of laminin β2 chain mRNA at 12-16 h poststimulation. In contrast, TGFβ decreased α₃ mRNA expression. Immunoprecipitation studies of TGFβ₁-treated cells showed increased surface expression of both α₆ and β₁ protein while surface expression of the α₃ integrin subunit was decreased. The same treatment resulted in increased laminin protein expression. These data suggest that TGFβ₁ may regulate alveolar epithelial cell differentiation in part through its modulation of integrins and laminin chains.     

Differentiation of aggregated murine P19 embryonal carcinoma cells is induced by a novel visceral endoderm-specific FGF-like factor and inhibited by activin A

Aggregation of P19 embryonal carcinoma cells in the presence of a factor, secreted by the visceral endoderm-like cell line END-2, induces differentiation to cell types including visceral endoderm, mesoderm-derived muscle tissue and neurons. This factor is different from activin A, type beta transforming growth factors (TGF beta) and fibroblast growth factors (FGF) although its acid- and heat-lability and its stability in the presence of reducing agents resemble the properties of the FGFs. The END-2 factor is completely inhibited in its action by activin A. This inhibitory effect of activin A is not specific for the END-2 factor as retinoic acid (RA)-induced differentiation of aggregated P19 EC cells into neurons (10(-8) M RA) or mesoderm-derived muscle tissue (10(-9) M RA) is also completely inhibited by activin A. The results of this study suggest that the END-2 activity and activin A are intimately involved in the induction and regulation, respectively, of early differentiation processes in vertebrate embryogenesis.     

Opposing Effects of Activin A and Follistatin on Developing Skeletal Muscle Cells

Activin and the activin-binding protein follistatin modulate a variety of biological processes and are abundant at sites of muscle development. Activin and follistatin were expressed in developing chick pectoral musclein vivoand in primary cell culture. Addition of recombinant activin inhibited muscle development in a dose-dependent manner as measured by the number of nuclei in myosin heavy chain positive cells and creatine phosphokinase activity. Conversely, follistatin potentiated muscle development. The effects of activin were found to be distinct from those of the related protein transforming growth factor (TGF) β1. Muscle development was repressed by activin at all time points investigated and did not recover with the removal of activin following a limited exposure. In contrast, while myogenic differentiation in TGFβ1 was initially repressed, muscle marker expression recovered to control levels—even in the continued presence of TGFβ1. Fibroblast growth factor (FGF) had little effect on inhibiton of muscle development caused by activin A. However, inhibition of development produced by TGFβ increased with increasing concentrations of FGF. Finally, early expression of myoD and myf5 mRNA by muscle cultures in the presence of activin and follistatin was analyzed. Activin-treated cultures expressed reduced myoD and myf5 levels at 1.5 days after plating. Myf5 levels in follistatin-treated cultures were elevated, but, surprisingly, these cultures showed a reduction in myoD levels. These data suggest that endogenously expressed activin and follistatin are important modulators of muscle development.     

Phylogenomic Analyses Reveal the Evolutionary Origin of the Inhibin α-Subunit,a Unique TGFβ Superfamily Antagonist

Transforming growth factor-beta (TGFβ) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFβ superfamily that share a common β-subunit. The evolution of the inhibin α-subunit created the only antagonist within the TGFβ superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin α-subunit appear to reflect specific insertion-deletion changes within the inhibin β-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFβ superfamily.     

The complexity of TGFβ/activin signaling in regeneration

The role of transforming growth factor β TGFβ/activin signaling in wound repair and regeneration is highly conserved in the animal kingdom. Various studies have shown that TGF-β/activin signaling can either promote or inhibit different aspects of the regeneration process (i.e., proliferation, differentiation, and re-epithelialization). It has been demonstrated in several biological systems that some of the different cellular responses promoted by TGFβ/activin signaling depend on the activation of Smad-dependent or Smad-independent signal transduction pathways. In the context of regeneration and wound healing, it has been shown that the type of R-Smad stimulated determines the different effects that can be obtained. However, neither the possible roles of Smad-independent pathways nor the interaction of the TGFβ/activin pathway with other complex signaling networks involved in the regenerative process has been studied extensively. Here, we review the important aspects concerning the TGFβ/activin signaling pathway in the regeneration process. We discuss data regarding the role of TGF-β/activin in the most common animal regenerative models to demonstrate how this signaling promotes or inhibits regeneration, depending on the cellular context.     

Adult Human Glia,Pericytes and Meningeal Fibroblasts Respond Similarly to IFNy but Not to TGFβ1 or M-CSF

The chemokine Interferon gamma-induced protein 10 (IP-10) and human leukocyte antigen (HLA) are widely used indicators of glial activation and neuroinflammation and are up-regulated in many brain disorders. These inflammatory mediators have been widely studied in rodent models of brain disorders, but less work has been undertaken using human brain cells. In this study we investigate the regulation of HLA and IP-10, as well as other cytokines and chemokines, in microglia, astrocytes, pericytes, and meningeal fibroblasts derived from biopsy and autopsy adult human brain, using immunocytochemistry and a Cytometric Bead Array. Interferonγ (IFNγ) increased microglial HLA expression, but contrary to data in rodents, the anti-inflammatory cytokine transforming growth factor β1 (TGFβ₁) did not inhibit this increase in HLA, nor did TGFβ₁ affect basal microglial HLA expression or IFNγ-induced astrocytic HLA expression. In contrast, IFNγ-induced and basal microglial HLA expression, but not IFNγ-induced astrocytic HLA expression, were strongly inhibited by macrophage colony stimulating factor (M-CSF). IFNγ also strongly induced HLA expression in pericytes and meningeal fibroblasts, which do not basally express HLA, and this induction was completely blocked by TGFβ₁, but not affected by M-CSF. In contrast, TGFβ₁ did not block the IFNγ-induced increase in IP-10 in pericytes and meningeal fibroblasts. These results show that IFNγ, TGFβ₁ and M-CSF have species- and cell type-specific effects on human brain cells that may have implications for their roles in adult human brain inflammation.     

Expression of Inhibin/activin Subunits alpha (-α), beta A (-β A) and beta B (-β B) in Placental Tissue of Normal and Intrauterine Growth Restricted (IUGR) Pregnancies

During human pregnancy the placenta produces a variety of proteins like steroid hormones and their receptors that are responsible for the establishment and ongoing of the feto-placental unit. Inhibins are dimeric glycoproteins, composed of an α-subunit and one of two possible β-subunits (β _A or β _B). Aims of the present study were the determination of the frequency and tissue distribution patterns of the inhibin/activin subunits in human placental tissue of normal pregnancies and pregnancies complicated with fetal growth restriction (IUGR). Slides of paraffin embedded placental tissue were obtained after delivery from patients diagnosed with IUGR (n = 6) and normal term placentas (n = 8). Tissue samples were fixed and incubated with monoclonal antibodies inhibin/activin-subunits -α, -β _A, -β _B. Intensity of immunohistochemical reaction on the slides was analysed using a semi-quantitative score and statistical analysis was performed (P<0.05). A significant lower expression of the inhibin-α subunit in IUGR extravillous trophoblast compared to normal pregnancies was observed, while the inhibin-α immunostaining was significantly upregulated in syncytiotrophoblast. Additionally, a significant down-regulation of inhibin-β _B subunit in extravillous trophoblast cells in IUGR syncytiotrophoblast cells was demonstrated. A co-localisation of inhibin-α and the β-subunits was also observed, suggesting a production and secretion of intact inhibin A and inhibin B. Although the precise role of these inhibin/activin subunits in human placenta and IUGR pregnancies is still unclear, they could be involved in autocrine/paracrine signalling, contributing to several aspects like angiogenesis and tissue remodelling.     

Transforming growth factor type β can act as a potent competence factor for AKR-2B cells

Transforming growth factor type β (TGFβ) is a pleiotropic regulator of cell growth with specific high-affinity cell-surface receptors on a large number of cells; its mechanism of action, however, is poorly defined. In this report, we utilized the mouse fibroblast line AKR-2B to explore the question of the temporal requirements during the cell cycle in regard to both the growth inhibitory and the growth stimulatory action of TGFβ. The results indicate that AKR-2B cells are most sensitive to the inhibitory action of TGFβ during early to mid-G₁. In addition, TGFβ need be present only briefly (as little as l min) in order to exert its inhibitory effect on EGF-induced DNA synthesis. Likewise, the stimulatory effect of TGFβ in the absence of EGF requires only an equally brief exposure to TGFβ. Use of homogeneous ¹²⁵I-labeled TGFβ in a cell-binding assay demonstrates that TGFβ bound to cell-surface receptors can readily exchange into the culture medium T_1/2 = 120 min), helping to rule out the possibility that persistent receptor-bound TGFβ is the source of a continuous stimulus. The data indicate that TGFβ exposure induces a stable state in the cell (T_1/2 = 20 h) similar to but distinct from the state of “competence” induced by platelet-derived growth factor (PDGF).     

Pluripotency Gene Expression and Growth Control in Cultures of Peripheral Blood Monocytes during Their Conversion into Programmable Cells of Monocytic Origin (PCMO): Evidence for a Regulatory Role of Autocrine Activin and TGF-β

Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-β(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21^WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TβRII, ALK5 as well as TGF-β1 and the β_A subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-β1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-β1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-β antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.     

Suppression of Transforming Growth Factor β Receptor 2 and Smad5 Is Associated with High Levels of MicroRNA miR-155 in the Oral Mucosa during Chronic Simian Immunodeficiency Virus Infection

Chronic human immunodeficiency virus and simian immunodeficiency virus (HIV and SIV) infections are characterized by mucosal inflammation in the presence of anti-inflammatory cytokines such as transforming growth factor β (TGFβ). The mechanisms for refractiveness to TGFβ are not clear. Here we show that the expression of microRNA miR-155 was significantly upregulated in the oropharyngeal mucosa during chronic SIV infection and was coincident with downregulation of TGFβ receptor 2 (TGFβ-R2) and SMAD5, key TGFβ signaling genes that harbor putative target sites for miR-155. Ectopic expression of miR-155 in vitro was found to significantly downregulate TGFβ-R2 and Smad5 expression, suggesting a role for miR-155 in the suppression of TGFβ-R2 and SMAD5 genes in vivo. The downregulation of TGFβ signaling genes by miR-155 likely contributes to the nonresponsiveness to TGFβ during SIV infection and may inadvertently aid in increased immune activation during HIV and SIV infections.     

The Role of SnoN in Transforming Growth Factor β1-induced Expression of Metalloprotease-Disintegrin ADAM12

Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor β1 (TGFβ1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFβ1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFβ signaling pathway, is a master regulator of ADAM12 expression in response to TGFβ1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFβ1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFβ1-induced expression of ADAM12. In a panel of TGFβ1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFβ1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFβ1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies.     

Structural basis for a functional antagonist in the transforming growth factor beta superfamily

Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.     

Hypoxia-Induced Changes in DNA Methylation Alter RASAL1 and TGFβ1 Expression in Human Trabecular Meshwork Cells

PurposeFibrosis and a hypoxic environment are associated with the trabecular meshwork (TM) region in the blinding disease glaucoma. Hypoxia has been shown to alter DNA methylation, an epigenetic mechanism involved in regulating gene expression such as the pro-fibrotic transforming growth factor (TGF) β1 and the anti-fibrotic Ras protein activator like 1 (RASAL1). The purpose of this study was to compare DNA methylation levels, and the expression of TGFβ1 and RASAL1 in primary human normal (NTM) with glaucomatous (GTM) cells and in NTM cells under hypoxic conditions.MethodsGlobal DNA methylation was assessed by ELISA in cultured age-matched NTM and GTM cells. qPCR was conducted for TGFβ1, collagen 1α1 (COL1A1), and RASAL1 expression. Western immunoblotting was used to determine protein expression. For hypoxia experiments, NTM cells were cultured in a 1%O₂, 5%CO₂ and 37°C environment. NTM and GTM cells were treated with TGFβ1 (10ng/ml) and the methylation inhibitor 5-azacytidine (5-aza) (0.5μM) respectively to determine their effects on DNA Methyltransferase 1 (DNMT1) and RASAL1 expression.ResultsWe found increased DNA methylation, increased TGFβ1 expression and decreased RASAL1 expression in GTM cells compared to NTM cells. Similar results were obtained in NTM cells under hypoxic conditions. TGFβ1 treatment increased DNMT1 and COL1A1, and decreased RASAL1 expression in NTM cells. 5-aza treatment decreased DNMT1, TGFβ1 and COL1A1 expression, and increased RASAL1 expression in GTM cells.ConclusionsTGFβ1 and RASAL1 expression, global DNA methylation, and expression of associated methylation enzymes were altered between NTM and GTM cells. We found that hypoxia in NTM cells induced similar results to the GTM cells. Furthermore, DNA methylation, TGFβ1 and RASAL1 appear to have an interacting relationship that may play a role in driving pro-fibrotic disease progression in the glaucomatous TM.     

The role of activins and follistatins in skin and hair follicle development and function

Investigations of the signalling between epithelial and mesenchymal compartments of skin during hair follicle initiation in utero and hair cycling have revealed the importance of the TGFβ superfamily in ectodermal organogenesis and morphogenesis. In particular the activins, their receptors and binding proteins such as follistatin, have been shown to be important regulators of cell proliferation, differentiation and apoptosis in hair follicle initiation, hair cycling, normal skin homeostasis and wound healing. Transgenic mice lacking various components of the activin signalling pathways display varying ectodermal pathologies including altered pelage hair follicle initiation. This review summarises the activin signal transduction pathways and the interactions between activins and other TGFβ signalling systems during hair follicle formation, hair growth cycling, skin function and wound healing.     

Rapamycin induces the TGFβ1/Smad signaling cascade in renal mesangial cells upstream of mTOR

The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGFβ/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly, rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGFβ₁ as shown by ELISA and by the inhibitory effects of a neutralizing TGFβ antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGFβ type II receptor (TGFβ-RII) we furthermore demonstrate a functional involvement of both types of TGFβ receptors. However, rapamycin did not compete with TGFβ for TGFβ-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGFβ/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin.