Activation of the bone morphogenetic protein signaling pathway induces inhibin beta(B)-subunit mRNA and secreted inhibin B levels in cultured human granulosa-luteal cells

During the human menstrual cycle the circulating levels of inhibin B, a dimer of inhibin alpha- and beta(B)-subunits, fluctuate in a fashion distinct from that of inhibin A, the alpha-beta(A)-subunit dimer. This suggests that human inhibin subunits are each regulated in a distinct manner in human ovarian granulosa cells by endocrine and local factors. We have previously shown using cultures of human granulosa-luteal (hGL) cells that gonadotropins stimulate the steady state mRNA levels of inhibin alpha- and beta(A)-subunits, but not those of the beta(B)-subunit, which, on the other hand, are up-regulated by, for instance, activin and TGF beta. We recently identified the TGF beta gene family member bone morphogenetic protein-3 (BMP-3) as a granulosa cell-derived growth factor, but whether BMP-3 or other structurally related BMPs regulate human granulosa cell inhibin production is not known. We show here that hGL cells express mRNAs for distinct serine/threonine kinase receptors (BMP-RIA and BMP-RII) and Smad signaling proteins (Smad1, Smad4, and Smad5) involved in the mediation of cellular effects of BMPs. Subsequently, we determined in hGL cell cultures the effects of distinct members of the BMP family previously found to be expressed in mammalian ovaries. Recombinant BMP-2 induces potently in a time- and concentration-dependent manner the expression of the inhibin beta(B)-subunit mRNAs in hGL cells without affecting the levels of alpha- or beta(A)-subunit mRNAs. BMP-6 has a similar, but weaker, effect than BMP-2, whereas BMP-3 and its close homolog, BMP-3b (also known as growth differentiation factor-10) had no effect on inhibin subunit mRNA expression. hCG treatment of hGL cells was previously shown to abolish the stimulatory effect of activin on beta(B)-subunit mRNA levels, and here hCG is also shown to suppress the effect of BMP-2. Furthermore, BMP-2 stimulates hGL cell secreted dimeric inhibin B levels in a concentration-dependent manner. Depending on the experiment, maximal increases in inhibin B levels of 6- to 28-fold above basal levels were detected during a 72-h culture period. We conclude that activation of the BMP-signaling pathway in hGL cells stimulates inhibin beta(B)-subunit mRNA levels and leads at the protein level to a dramatic stimulation of secreted inhibin B dimers. Our results are consistent with the suggestion that in addition to the distinct activin- and TGF beta-activated signaling pathways, the BMP-activated pathway is likely to be implicated in the complex regulation of inhibins in the human ovary.     

Interaction between activin and follicle-stimulating hormone-suppressing protein/follistatin in the regulation of basal inhibin production by cultured rat granulosa cells.

There is evidence that FSH-suppressing protein (FSP) antagonizes the action of activin on the differentiation of rat granulosa cells by binding activin in vitro. We tested the interaction of activin and FSP in this in vitro system by examining the effects of FSP on activin dose-related stimulation of immunoreactive inhibin release by rat granulosa cells. Granulosa cells (2 x 10(5) viable cells/well) from diethylstilbestrol-treated immature rats were cultured for 48 h in McCoy's 5a serum-free medium with additives and increasing doses of bovine FSP (0-30 nM) and human recombinant activin (0-20 nM). Inhibin was measured in the medium by RIA. Activin caused a dose-related increase in basal inhibin production, which was maximal between 4-10 nM activin (ED50, 0.6 nM). With the addition of FSP, an apparent increase in the ED50 of the activin dose-response curves was observed, but there were no changes in the maximum response. This pattern closely resembled that of chemical antagonism of an agonist by an agent that binds with relatively high affinity to form a biologically inactive complex. Based on this premise, apparent high affinity activin binding to FSP was determined by Scatchard analysis to have a Kd of 0.13 +/- 0.07 nM (mean +/- SD) and to occur in a 2:1 or greater FSP/activin molar ratio. These data support the proposition that the antagonistic effect of FSP on activin is due to the formation of an inactive complex.     

Effect of activin A and inhibin A on expression of the inhibin/activin beta-B-subunit and gonadotropin receptors in granulosa cells of the hen

Activin A has been shown to be abundant in the theca layer of the large pre-ovulatory follicles of the hen whereas inhibin A is produced in the granulosa layer. The purpose of this study was to investigate the effects of activin A and inhibin A on granulosa cell expression of inhibin beta-B-subunit, FSH receptor (FSHR), and LH receptor (LHR). Granulosa cells were isolated from the F1, F3+F4, and small yellow follicles (SYF; 6-12 mm diameter) of laying hens and pooled according to size. The cells were dispersed and plated in the presence of 0, 10, or 50 ng/ml recombinant human activin A (n=5 replicate cultures). RNA was subsequently extracted from the cells and Northern blots performed. Cell proliferation was determined for all treatments. An identical set of experiments was performed in which the granulosa cells were treated with recombinant human inhibin A (n=4 replicate cultures). Treatment with activin A at 50 ng/ml significantly (p<0.05) increased expression of beta-B-subunit for granulosa cells from all follicles. This dose also significantly increased expression of FSHR in granulosa cells from all follicles (p<0.05) and increased expression of LHR in cells from F1 and F3+F4 follicles (p<0.01) with no significant effect on cells from the SYF. Overall, activin A treatment significantly (p<0.05) decreased cell proliferation at the 50 ng/ml dose. Inhibin A had no significant effect on expression of beta-B-subunit, FSHR or LHR at any dose. There was a moderate stimulatory effect of inhibin A on granulosa cell proliferation. These results suggest that activin A may have an important role in regulating granulosa cell responsiveness to gonadotropins while also modulating follicle development by attenuating cell proliferation.     

Regeneration of axons in injured spinal cord by activation of bone morphogenetic protein/Smad1 signaling pathway in adult neurons

Axon growth potential is highest in young neurons but diminishes with age, thus becoming a significant obstacle to axonal regeneration after injury in maturity. The mechanism for the decline is incompletely understood, and no effective clinical treatment is available to rekindle innate growth capability. Here, we show that Smad1-dependent bone morphogenetic protein (BMP) signaling is developmentally regulated and governs axonal growth in dorsal root ganglion (DRG) neurons. Down-regulation of the pathway contributes to the age-related decline of the axon growth potential. Reactivating Smad1 selectively in adult DRG neurons results in sensory axon regeneration in a mouse model of spinal cord injury (SCI). Smad1 signaling can be effectively manipulated by an adeno-associated virus (AAV) vector encoding BMP4 delivered by a clinically applicable and minimally invasive technique, an approach devoid of unwanted abnormalities in mechanosensation or pain perception. Importantly, transected axons are able to regenerate even when the AAV treatment is delivered after SCI, thus mimicking a clinically relevant scenario. Together, our results identify a therapeutic target to promote axonal regeneration after SCI.     

Retinoic acid stimulates chondrocyte differentiation and enhances bone morphogenetic protein effects through induction of Smad1 and Smad5

Whereas bone morphogenetic protein (BMP)-signaling events induce maturational characteristics in vitro, recent evidence suggests that the effects of other regulators might be mediated through BMP-signaling events. The present study examines the mechanism through which retinoic acid (RA) stimulates differentiation in chicken embryonic caudal sternal chondrocyte cultures. Both RA and BMP-2 induced expression of the chondrocyte maturational marker, colX, in chondrocyte cultures by 8 d. Though the RA effect was small, it synergistically enhanced the effect of BMP-2 on colX and phosphatase activity. Inhibition of either RA or BMP signaling, with selective inhibitors, interfered with the inductive effects of these agents but also inhibited the complementary pathway, demonstrating a codependence of RA and BMP signaling during chondrocyte maturation. BMP-2 did not enhance the effects of RA on an RA-responsive reporter construct, but RA enhanced basal activity and synergistically enhanced BMP-2 stimulation of the BMP-responsive chicken type X collagen reporter. A similar synergistic interaction between RA and BMP-2 was observed on colX expression. RA did not increase the expression of the type IA BMP receptor but did markedly up-regulate the expression of Smad1 and Smad5 proteins, important participants in the BMP pathway. Inhibition of RA signaling, with the selective inhibitor AGN 193109, blocked RA-mediated induction of the Smad proteins and chondrocyte differentiation. These findings demonstrate that RA induces the expression of BMP-signaling molecules and enhances BMP effects in chondrocytes.     

Activin signaling through type IB activin receptor stimulates aromatase activity in the ovarian granulosa cell-like human granulosa (KGN) cells

In addition to a stimulatory effect on FSH production by the pituitary gland, activin is thought to have a paracrine or autocrine role in follicular development in the ovary, where it is produced. Recently, we established a human ovarian granulosa tumor cell line, KGN, which possesses in vivo characteristics of granulosa cells, namely the expression of functional FSH receptors and cytochrome P-450 aromatase. Here, we have demonstrated the activin signaling pathway and its role in KGN cells. A series of transient transfection experiments revealed that activin type IB receptor (ActRIB) is an essential component of the activin signaling pathway in KGN cells. Smad2 was found to act downstream of ActRIB as an intracellular signal transmitter. Smad7, but not Smad6, was an inhibitory Smad in the pathway. Finally, we show that FSH receptor expression and cytochrome P-450 (P-450) aromatase activity was up-regulated by activin stimulation through ActRIB in KGN cells. These results show that we have clarified the signaling mechanisms and the roles of activin in the human granulosa cell line, KGN. Activin signaling mediated by ActRIB-Smad2 system in the ovary may thus be essential for the regulation of follicular differentiation.     

Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells.

Ovarian granulosa cells produce inhibin and activin, structurally related proteins with potentials to directly modulate follicular steroidogenesis. The aim of the present study was to compare development-related effects of inhibin-A and activin-A on steroidogenesis in marmoset monkey (Callithrix jacchus) granulosa cells. Granulosa cells from "immature" (< 1.0 mm diameter) and "mature" (> 2 mm diameter) follicles were incubated in serum-free culture medium for 96 h with and without peptide (1-100 ng/mL), in the presence and absence of gonadotropins [human (h) FSH or hLH] (10 ng/mL). Spent medium was collected and stored frozen for progesterone assay. Aromatase activity was determined by incubating cells for a further 6 h in the presence of 1 mumol testosterone and assaying accumulation of oestradiol. Granulosa cells from immature follicles showed characteristically low basal rates of steroid synthesis that were unaffected by treatment alone with either inhibin or activin. Treatment with hFSH stimulated both progesterone production and aromatase activity. Cotreatment with activin and hFSH further enhanced aromatase activity by up to 4-fold. The progesterone response to activin plus hFSH was related to the effect of hFSH in the absence of activin: high-level responsiveness to hFSH was suppressed by activin while low-level responsiveness was enhanced. Inhibin had no significant effect on FSH-responsive progesterone production, but at high concentrations (> 10 ng/mL) it caused slight (up to 30%) reduction in FSH-induced aromatase activity. Granulosa cells from mature follicles showed relatively high basal rates of steroidogenesis, and treatment with inhibin did not influence either basal or gonadotropin responsive steroidogenesis. Treatment with activin had divergent effects on aromatase activity and progesterone synthesis in that it increased both basal and hLH-responsive aromatase activity (up to 11-fold), had no effect on basal progesterone production, and markedly suppressed (by more than 50%) the progesterone response to hLH. These data reveal development-dependent effects of inhibin and activin on granulosa cell steroidogenesis that are likely to have physiological relevance to ovarian function in vivo.     

Ovarian follicle development in the laying hen is accompanied by divergent changes in inhibin A,inhibin B,activin A and follistatin production in granulosa and theca layers

To study the potential involvement of inhibin A (inhA), inhibin B (inhB), activin A (actA) and follistatin (FS) in the recruitment of follicles into the preovulatory hierarchy, growing follicles (ranging from 1 mm to the largest designated F1) and the three most recent postovulatory follicles (POFs) were recovered from laying hens (n=11). With the exception of <4 mm follicles and POFs, follicle walls were dissected into separate granulosa (G) and theca (T) layers before extraction. Contents of inhA, inhB, actA and FS in tissue extracts were assayed using specific two-site ELISAs and results are expressed per mg DNA. InhB content of both G and T followed a similar developmental pattern, although the content was >4-fold higher in G than in T at all stages. InhB content was very low in follicles <4 mm but increased ~50-fold (P<0.0001) to peak in 7-9 mm follicles, before falling steadily as follicles entered and moved up the follicular hierarchy (40-fold; 8 mm vs F2). In stark contrast, inhA remained very low in prehierarchical follicles (< or =9 mm) but then increased progressively as follicles moved up the preovulatory hierarchy to peak in F1 (approximately 100-fold increase; P<0.0001); In F1 >97% of inhA was confined to the G layer whereas in 5-9 mm follicles inhA was only detected in the T layer. Both inhA and inhB contents of POFs were significantly reduced compared with F1. Follicular actA was mainly confined to the T layer although detectable levels were present in G from 9 mm; actA was low between 1 and 9 mm but increased sharply as follicles entered the preovulatory hierarchy (approximately 6-fold higher in F4; P<0.0001); levels then fell approximately 2-fold as the follicle progressed to F1. Like actA, FS predominated in the T although significant amounts were also present in the G of prehierarchical follicles (4-9 mm), in contrast to actA, which was absent from the G. The FS content of T rose approximately 3-fold from 6 mm to a plateau which was sustained until F1. In contrast, the FS content of G was greatest in prehierarchical follicles and fell approximately 4-fold in F4-F1 follicles. ActA and FS contents of POFs were reduced compared with F1. In vitro studies on follicle wall explants confirmed the striking divergence in the secretion of inhA and inhB during follicle development. These findings of marked stage-dependent differences in the expression of inhA, inhB, actA and FS proteins imply a significant functional role for these peptides in the recruitment and ordered progression of follicles within the avian ovary.     

Expression of activin/inhibin receptor and binding protein genes and regulation of activin/inhibin peptide secretion in human adrenocortical cells

Activins and inhibins are glycoprotein hormones produced mainly in gonads but also in other organs. They are believed to be important para/autocrine regulators of various cell functions. We investigated activin/inhibin receptor and binding protein gene expression and the regulation of activin/inhibin secretion in human adrenal cells. RT-PCR revealed inhibin/activin alpha-, betaA/B-subunit, follistatin, activin type I/II receptor, and inhibin receptor (betaglycan and inhibin-binding protein) mRNA expression in fetal and adult adrenals and cultured adrenocortical cells. Cultured cells secreted activin A and inhibin A/B as determined by specific ELISAs. ACTH stimulated inhibin A/B secretion in fetal (1.8- and 1.8-fold of control, respectively) and in adult cells (3.4- and 1.7-fold of control, respectively) without significant effect on activin A. 8-bromoadenosine cAMP (protein kinase A activator) increased activin A and inhibin A/B secretion in the human adrenocortical NCI-H295R cell line (32-, 17-, and 3-fold of control, respectively). 12-O-tetradecanoyl phorbol-13-acetate (protein kinase C activator) stimulated both activin A and inhibin A secretion (764- and 32-fold of control, respectively), and activin treatment increased inhibin B secretion in these cells (25-fold of control). In conclusion, human adrenocortical cells produce dimeric activins and inhibins. ACTH stimulates inhibin secretion and decreases activin/inhibin secretion ratio, probably via the protein kinase A signal transduction pathway. This, together with the adrenocortical activin/ inhibin receptor and binding protein expression, suggests a physiological role for activins and inhibins in the human adrenal gland.