Pankaj Lamba

Mechanisms of FSH synthesis: what we know, what we don't, and why you should care

The pituitary gonadotropin hormones, FSH and LH, are key regulators of reproductive physiology. Though the two hormones are produced by the same cell type, often in response to the same endocrine and paracrine regulators, they sub-serve different biological functions and their synthesis and secretion are differentially regulated. This stems largely from differences in transcriptional, post-transcriptional, and post-translational regulation of their unique beta subunits. That is, both hormones are dimeric glycoproteins and share a common alpha subunit. Their unique beta subunits, however, derive from different genes encoding distinct proteins. Past and recent research indicates synthesis and release of the two hormones are subject to extensive and independent regulation. LH appears to be secreted predominantly via the regulated secretory pathway, whereas FSH release is largely constitutive. As such, investigations of FSH-beta subunit synthesis may lend direct insight into mechanisms underlying patterns of secreted FSH, more so than investigations of the LHbeta subunit. Here, we review recent investigations of transcriptional regulation of the FSH-beta subunit gene from different mammalian species, including humans. The results reveal both conserved and species-specific regulatory mechanisms that might contribute to inter-species variation in FSH release.

Activator Protein-1 and Smad Proteins Synergistically Regulate Human Follicle-Stimulating Hormone β-Promoter Activity

GnRH1 stimulates the synthesis and secretion of FSH and LH from the anterior pituitary gland. The molecular mechanisms through which GnRH1 produces these effects in humans have not been determined. Here, we examined transcriptional regulation of the human FSHbeta (FSHB) subunit using reporter assays in immortalized murine gonadotrope cells. GnRH1 dose and time dependently stimulated FSHB promoter activity, with peak stimulation occurring at 8 h. GnRH1 rapidly stimulated various MAPK cascades, though the ERK1/2 and p38 pathways appeared to be most critical for FSHB induction. Indeed, constitutively active forms of both Raf1 kinase and MAP2K6 (MKK6) were sufficient to stimulate reporter activity. GnRH1 stimulated activator protein-1 (AP-1) (FosB, c-fos, JunB, and cJun) synthesis and complex formation, the latter of which bound to a conserved cis-element within -120 bp of the transcription start site. A second, lower affinity, site was mapped more proximally. Mutations of both cis-elements diminished GnRH1-stimulated promoter activity, though disruption of the higher affinity site had a more dramatic effect. A dominant-negative Fos protein dose dependently inhibited GnRH1-stimulated FSHB transcription, confirming a role for endogenous AP-1 proteins. MAPK kinase 1 (MEK1) and p38 inhibitors significantly attenuated GnRH1-stimulated c-fos, FosB, and JunB synthesis, suggesting a mechanism whereby the ERK1/2 and p38 signaling pathways regulate FSHB transcription. Activins and inhibins potently regulate FSH synthesis in rodents, but their roles in FSH regulation in humans are less clear. Activin A, though weak on its own, synergized with GnRH1 to stimulate human FSHB promoter activity. In contrast, activin A partially inhibited GnRH1-stimulated LHbeta subunit (LHB) transcription. The GnRH1 and activin A signaling pathways appear to converge at the level of the high-affinity AP-1 site. Fos and Jun proteins synergistically regulate reporter activity through this element, and their effects are potentiated by coexpression of either Smad2 or Smad3, effectors in the activin signaling cascade. In summary, GnRH1 and activin A synergistically regulate human FSHB subunit transcription. The combined actions of AP-1 and Smad proteins acting through a conserved AP-1 element provide a candidate mechanism for this effect. The ability of activins to potentiate selectively the effects of GnRH1 on FSHB expression suggests a model for preferential increases in FSH secretion at the luteal-follicular transition of the menstrual cycle.

A Novel Role for the Forkhead Transcription Factor FOXL2 in Activin A-Regulated Follicle-Stimulating Hormone β Subunit Transcription

Selective synthesis and release of FSH from pituitary gonadotropes is regulated by activins. Activins directly stimulate murine FSHbeta (Fshb) subunit gene transcription through a consensus 8-bp Sma- and Mad-related protein-binding element (SBE) in the proximal promoter. In contrast, the human FSHB promoter is relatively insensitive to the direct effects of activins and lacks this SBE. The proximal porcine Fshb promoter, which is highly conserved with human, similarly lacks the 8-bp SBE, but is nonetheless highly sensitive to activins. We used a comparative approach to determine mechanisms mediating differential activin induction of human, porcine, and murine Fshb/FSHB promoters. We mapped an activin response element in the proximal porcine promoter and identified interspecies variation in a single base pair in close proximity that conferred strong binding of the forkhead transcription factor FOXL2 to the porcine, but not human or murine, promoters. Introduction of the human base pair into the porcine promoter abolished FOXL2 binding and activin A induction. FOXL2 conferred activin A induction to the porcine promoter in heterologous cells, whereas knockdown of the endogenous protein in gonadotropes inhibited the activin A response. The murine Fshb promoter lacks the high-affinity FOXL2-binding site, but its activin induction is FOXL2 sensitive. We identified a more proximal FOXL2-binding element in the murine promoter, which is conserved across species. Mutation of this site attenuated activin A induction of both the porcine and murine promoters. Collectively, the data indicate a novel role for FOXL2 in activin A-regulated Fshb transcription.

SMADs and FOXL2 Synergistically Regulate Murine FSHβ Transcription Via a Conserved Proximal Promoter Element

Pituitary FSH regulates ovarian and testicular function. Activins stimulate FSHβ subunit (Fshb) gene transcription in gonadotrope cells, the rate-limiting step in mature FSH synthesis. Activin A-induced murine Fshb gene transcription in immortalized gonadotropes is dependent on homolog of Drosophila mothers against decapentaplegic (SMAD) proteins as well as the forkhead transcription factor FOXL2 (FOXL2). Here, we demonstrate that FOXL2 synergizes with SMAD2, SMAD3, and SMAD4 to stimulate murine Fshb promoter-reporter activity in heterologous cells. Moreover, SMAD3-induction of Fshb promoter activity or endogenous mRNA expression is dependent upon endogenous FOXL2 in homologous cells. FOXL2/SMAD synergy requires binding of both FOXL2 and SMAD3 or SMAD4 to DNA. Of three putative forkhead-binding elements identified in the murine Fshb promoter, only the most proximal is absolutely required for activin A induction of reporter activity in homologous cells. Additionally, mutations to the minimal SMAD-binding element adjacent to the proximal forkhead-binding element abrogate activin A or FOXL2/SMAD3 induction of reporter activity. In contrast, a mutation that impairs an adjacent PBX1/PREP1 (pre-B cell leukemia transcription factor 1-PBX/knotted-1 homeobox-1) binding site does not alter activin A-stimulated promoter activity in homologous cells. Collectively, these and previous data suggest a model in which activins stimulate formation of FOXL2-SMAD2/3/4 complexes, which bind to the proximal murine Fshb promoter to stimulate its transcription. Within these complexes, FOXL2 and SMAD3 or SMAD4 bind to adjacent cis-elements, with SMAD3 brokering the physical interaction with FOXL2. Because this composite response element is highly conserved, this suggests a general mechanism whereby activins may regulate and/or modulate Fshb transcription in mammals.

Cloning of Gonadotropin-Releasing Hormone I Complementary DNAs in Songbirds Facilitates Dissection of Mechanisms Mediating Seasonal Changes in Reproduction

Temperate zone animals exhibit seasonal variation in reproductive physiology. In most cases, seasonal changes in reproductive states are regulated by changes in GnRH1 secretion, rather than synthesis, from the preoptic area (POA)/anterior hypothalamus. An important exception occurs in some songbirds that become photorefractory to the stimulatory effects of long days and show profound decreases in brain GnRH1 protein content. Whether this decline reflects changes in gene expression is unknown because of past failures to measure GNRH1 mRNA levels, due in large part to the absence of available GNRH1 gene sequence in this taxon. Here, we report the first cloning of GNRH1 cDNAs in two songbirds: European starlings and zebra finches. Consistent with the size of the prepro-hormone in other avian and non-avian species, the open-reading frames predict proteins of 91 and 92 amino acids, respectively. Whereas the decapeptide in both species is perfectly conserved with chicken GnRH1, the amino acid identity in the signal peptide and GNRH associated peptide subdomains are significantly less well conserved. At the nucleotide level, the starling and zebra finch coding sequences are approximately 88% identical to each other but only approximately 70% identical to chicken GNRH1. In situ hybridization using radiolabeled cRNA probes demonstrated GNRH1 mRNA expression primarily in the POA, consistent with previous studies on the distribution of the GnRH1-immunoreactive cell bodies. Furthermore, we provide evidence for photoperiod-dependent regulation of GNRH1 mRNA in male starlings. Declines in GNRH1 mRNA levels occur in parallel with testicular involution. Thus, photorefractoriness is associated with decreases in GNRH1 gene expression in the medial POA.

Paired-Like Homeodomain Transcription Factors 1 and 2 Regulate Follicle-Stimulating Hormone β-Subunit Transcription through a Conserved cis-Element

Paired-like homeodomain transcription factors (PITX) regulate the activity of pituitary hormone-encoding genes. Here, we examined mechanisms through which the family of PITX proteins control murine FSH beta-subunit (Fshb) transcription. We observed that endogenous PITX1 and PITX2 isoforms from murine LbetaT2 gonadotrope cells could bind a highly conserved proximal cis-element. Transfection of PITX1 or PITX2C in heterologous cells stimulated both murine and human Fshb/FSHB promoter-reporter activities, and in both cases, mutation of the critical cis-element abrogated these effects. In homologous LbetaT2 cells, the same mutation decreased basal reporter activity and greatly reduced activin A-stimulated transcription from murine and human promoter-reporters. Transfecting dominant-negative forms of PITX1 or PITX2C or knocking down PITX1 or -2 expression by RNA interference in LbetaT2 cells inhibited murine Fshb transcription, confirming roles for endogenous PITX proteins. Both PITX1 and PITX2C interacted with Smad3 (an effector of the activin signaling cascade in these cells) in coprecipitation experiments, and the PITX binding site mutation greatly inhibited Smad2/3/4-stimulated Fshb transcription. In summary, both PITX1 and PITX2C regulate murine and human Fshb/FSHB transcription through a conserved cis-element in the proximal promoter. Furthermore, the data indicate both common and distinct mechanisms of PITX1 and PITX2C action.

Activin A Regulates Porcine Follicle-Stimulating Hormone β-Subunit Transcription via Cooperative Actions of SMADs and FOXL2

Activins stimulate FSH synthesis and secretion by pituitary gonadotrope cells. Activin A induction of porcine and murine FSHβ (Fshb) gene transcription in immortalized gonadotropes is dependent on homolog of Drosophila mothers against decapentaplegic (SMAD) proteins as well as the forkhead transcription factor L2 (FOXL2). Using both heterologous and homologous cell models, we demonstrate that FOXL2 functionally synergizes with SMAD3/4 to stimulate porcine Fshb promoter-reporter activity. We further show that endogenous FOXL2 and SMAD2/3 physically interact in homologous cells. We identify two composite cis-elements of adjacent FOXL2 and SMAD binding sites in the proximal porcine Fshb promoter that mediate activin A, FOXL2, and SMAD3 actions. FOXL2 can bind these elements independently of SMADs, whereas SMAD3/4 binding requires high-affinity FOXL2 binding. Conversely, FOXL2 alone is insufficient to regulate Fshb transcription and requires SMADs to induce promoter activity. Collectively, our data suggest a model in which activins stimulate formation and nuclear accumulation of SMAD3/4 complexes, which interact with FOXL2 bound to at least two proximal promoter elements. This association stabilizes SMAD3/4 binding to adjacent SMAD binding elements. SMAD-FOXL2 complexes then mediate activation of transcription through a currently unknown mechanism. Conservation of one of the two composite cis-elements suggests that this may form part of a general mechanism whereby activins regulate Fshb subunit transcription and FSH synthesis.

Conservation of mechanisms mediating gonadotrophin-releasing hormone 1 stimulation of human luteinizing hormone β subunit transcription

Gonadotrophin-releasing hormone (GNRH1) regulates pituitary luteinizing hormone (LH). Previous studies have delineated a mechanism for GNRH1-induced LHbeta subunit gene (Lhb) transcription, the rate-limiting step in LH production. GNRH1 induces expression of early growth response 1 (EGR1), which interacts with steroidogenic factor 1 (SF1) and paired-like homeodomain transcription factor 1 (PITX1) to regulate Lhb promoter activity. Though the cis-elements for these factors are conserved across species, regulation of human LHB transcription has not been thoroughly investigated. We therefore characterized LHB transcriptional regulation by GNRH1 using promoter-reporter analyses in LbetaT2 cells. GNRH1 stimulated LHB transcription via an extracellular signal-regulated kinase 1/2 pathway. EGR1 bound to two binding sites on the LHB promoter and this binding was increased by GNRH1. Mutation of either site or knockdown of endogenous EGR1 decreased basal and/or GNRH1-regulated promoter activity. The human LHB promoter also contains low and high affinity SF1 binding sites. Mutation of these elements or depletion of endogenous SF1 impaired basal and ligand-induced transcription. Knockdown of PITX1 or PITX2 isoforms impaired GNRH1 induction, and endogenous PITX1 bound to the candidate PITX binding site on the LHB promoter. Thus, the mechanism described for GNRH1 regulation of Lhb in other species is largely conserved for human LHB. We also uncover a previously unappreciated role for PITX2 isoforms in this process.

Mono-(2-ethylhexyl) phthalate (MEHP) regulates glucocorticoid metabolism through 11β-hydroxysteroid dehydrogenase 2 in murine gonadotrope cells

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolite mono-(2-ethylhexyl) phthalate (MEHP) have been classified as toxicants to the reproductive system at the testis level and DEHP may also impair reproductive axis function at the pituitary levels. However, MEHP is 10-fold more potent than DEHP in toxicity and little is known about the toxicological effect of MEHP on pituitary. In this study, we demonstrated that 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), not 11beta-HSD1, is strongly expressed in murine gonadotrope LbetaT2 cells. Interestingly, MEHP inhibited Hsd11b2 mRNA level and 11beta-HSD2 enzyme activity in LbetaT2 cells at as low as 10(-7)M. Corticosterone (CORT) at a concentration of 10(-6)M significantly inhibited LbetaT2 cell proliferation after 2-day culture, and 10(-6)M RU486, an antagonist of glucocorticoid receptor (GR), reversed this inhibition. However, in the presence of 10(-5) or 10(-4)M MEHP, the minimal concentration of CORT to inhibit the proliferation of LbetaT2 cells was lowered to 10(-7)M, and 10(-6)M RU486 was not able to completely reverse the CORT effect. In conclusion, along with the regulation of GR, 11beta-HSD2 may have a key role in glucocorticoid metabolism in LbetaT2 cells. MEHP may participate in the glucocorticoid metabolism in LbetaT2 cells through inhibition of 11beta-HSD2 enzyme activity. Such perturbation may be of pathological significance as MEHP may interfere with the reproductive system at pituitary level through regulation of glucocorticoid metabolism, especially in neonates with higher risk of phthalates exposure.

Minireview: Activin Signaling in Gonadotropes: What Does the FOX say… to the SMAD?

The activins were discovered and named based on their abilities to stimulate FSH secretion and FSHβ (Fshb) subunit expression by pituitary gonadotrope cells. According to subsequent in vitro observations, activins also stimulate the transcription of the GnRH receptor (Gnrhr) and the activin antagonist, follistatin (Fst). Thus, not only do activins stimulate FSH directly, they have the potential to regulate both FSH and LH indirectly by modulating gonadotrope sensitivity to hypothalamic GnRH. Moreover, activins may negatively regulate their own actions by stimulating the production of one of their principal antagonists. Here, we describe our current understanding of the mechanisms through which activins regulate Fshb, Gnrhr, and Fst transcription in vitro. The activin signaling molecules SMAD3 and SMAD4 appear to partner with the winged-helix/forkhead transcription factor, forkhead box L2 (FOXL2), to regulate expression of all 3 genes. However, in vivo data paint a different picture. Although conditional deletion of Foxl2 and/or Smad4 in murine gonadotropes produces impairments in FSH synthesis and secretion as well as in pituitary Fst expression, Gnrhr mRNA levels are either unperturbed or increased in these animals. Surprisingly, gonadotrope-specific deletion of Smad3 alone or with Smad2 does not impair FSH production or fertility; however, mice harboring these mutations may express a DNA binding-deficient, but otherwise functional, SMAD3 protein. Collectively, the available data firmly establish roles for FOXL2 and SMAD4 in Fshb and Fst expression in gonadotrope cells, whereas SMAD3s role requires further investigation. Gnrhr expression, in contrast, appears to be FOXL2, SMAD4, and, perhaps, activin independent in vivo.