RESUMO
Activins are one of the three distinct subclasses within the greater Transforming growth factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.
Assuntos
Proteínas de Transporte , Fator de Crescimento Transformador beta , Camundongos , Animais , Fator de Crescimento Transformador beta/metabolismo , Ligantes , Receptores de Ativinas/genética , Receptores de Ativinas/metabolismo , Ativinas/metabolismoRESUMO
White adipose tissue (WAT) regulates energy balance through energy storage, adipokines secretion and the thermogenesis process. Beige adipocytes are responsible for WAT thermogenesis. They are generated by adipogenesis or transdifferentiation during cold or ß3-adrenergic agonist stimulus through a process called browning. Browning has gained significant interest for to its preventive effect on obesity. Glucocorticoids (GCs) have several functions in WAT biology; however, their role in beige adipocyte generation and WAT browning is not fully understood. The aim of our study was to determine the effect of dexamethasone (DXM) on WAT thermogenesis. For this purpose, rats were treated with DXM at room temperature (RT) or cold conditions to determine different thermogenic markers. Furthermore, the effects of DXM on the adipogenic potential of beige precursors and on mature beige adipocytes were evaluated in vitro. Our results showed that DXM decreased UCP-1 mRNA and protein levels, mainly after cold exposure. In vitro studies showed that DXM decreased the expression of a beige precursor marker (Ebf2), affecting their ability to differentiate into beige adipocytes, and inhibited the thermogenic response of mature beige adipocytes (Ucp-1, Dio2 and Pgc1α gene expressions and mitochondrial respiration). Overall, our data strongly suggest that DXM can inhibit the thermogenic program of both retroperitoneal and inguinal WAT depots, an effect that could be exerted, at least partially, by inhibiting de novo cell generation and the thermogenic response in beige adipocytes.
Assuntos
Tecido Adiposo Marrom , Tecido Adiposo Branco , Ratos , Animais , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Obesidade/metabolismo , Adipogenia , Dexametasona/farmacologia , TermogêneseRESUMO
Mammalian reproduction depends on the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone, which are secreted by pituitary gonadotrope cells. The zinc-finger transcription factor GATA2 was previously implicated in FSH production in male mice; however, its mechanisms of action and role in females were not determined. To directly address GATA2 function in gonadotropes, we generated and analyzed gonadotrope-specific Gata2 KO mice using the Cre-lox system. We found that while conditional KO (cKO) males exhibited â¼50% reductions in serum FSH levels and pituitary FSHß subunit (Fshb) expression relative to controls, FSH production was apparently normal in cKO females. In addition, RNA-seq analysis of purified gonadotropes from control and cKO males revealed a profound decrease in expression of gremlin (Grem1), a bone morphogenetic protein (BMP) antagonist. We show Grem1 was expressed in gonadotropes, but not other cell lineages, in the adult male mouse pituitary. Furthermore, Gata2, Grem1, and Fshb mRNA levels were significantly higher in the pituitaries of WT males relative to females but decreased in males treated with estradiol and increased following ovariectomy in control but not cKO females. Finally, we found that recombinant gremlin stimulated Fshb expression in pituitary cultures from WT mice. Collectively, the data suggest that GATA2 promotes Grem1 expression in gonadotropes and that the gremlin protein potentiates FSH production. The mechanisms of gremlin action have not yet been established but may involve attenuation of BMP binding to activin type II receptors in gonadotropes, facilitating induction of Fshb transcription by activins or related ligands.
Assuntos
Proteínas Morfogenéticas Ósseas , Hormônio Foliculoestimulante , Fator de Transcrição GATA2 , Gonadotrofos , Peptídeos e Proteínas de Sinalização Intercelular , Ativinas/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Feminino , Hormônio Foliculoestimulante/sangue , Subunidade beta do Hormônio Folículoestimulante/sangue , Fator de Transcrição GATA2/genética , Gonadotrofos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , CamundongosRESUMO
Activins selectively stimulate follicle-stimulating hormone (FSH) secretion by pituitary gonadotrope cells. More recently, other members of the TGFbeta superfamily, the bone morphogenetic proteins (BMPs), were reported to regulate FSH synthesis. Activins and BMPs independently and synergistically stimulate transcription of the FSHbeta subunit (Fshb) gene in immortalized gonadotrope-like cells. Both ligands can signal via the activin receptor type IIA (ACVR2A) to regulate FSH synthesis in vitro. In vivo, global Acvr2a knockout mice exhibit a 60% reduction in circulating FSH relative to wild-type animals, suggesting that activins, BMPs, or related ligands might signal through additional type II receptors to regulate FSH in vivo. Although the leading candidates are ACVR2B and the BMP type II receptor (BMPR2), only the latter mediates activin or BMP2 induction of Fshb transcription in vitro. Here, we generated mice carrying a loss of function mutation in Bmpr2 specifically in gonadotropes. Puberty onset, estrous cyclicity, and reproductive organ weights were similar between control and conditional knockout females. Serum FSH and luteinizing hormone (LH) and pituitary expression of Fshb and the LHbeta subunit (Lhb) were similarly unaffected by the gene deletion in both sexes. These results suggest that BMPR2 might not play a necessary role in FSH synthesis or secretion in vivo or that another type II receptor, such as ACVR2A, can fully compensate for its absence. These data also further contribute to the emerging concept that BMPs may not be physiological regulators of FSH in vivo.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Gonadotrofos/metabolismo , Reprodução/genética , Ativinas/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Camundongos Knockout , Hipófise/metabolismoRESUMO
OBJECTIVE: Loss of function mutations in the insulin receptor substrate 4 (IRS4) gene cause a rare form of X-linked congenital central hypothyroidism in boys and men. Affected individuals show decreased thyroid-stimulation hormone (TSH) secretion. Members of the IRS family canonically act as scaffold proteins between tyrosine kinase receptors and downstream effectors. How loss of IRS4 affects TSH synthesis or secretion is unresolved. We therefore assessed IRS4's role in the hypothalamic-pituitary-thyroid axis of Irs4 knockout mice. METHODS: We generated two global Irs4 knockout mouse lines harboring either two or four base-pair deletions that result in frameshifts and loss of most of the IRS4 protein. RESULTS: Under normal laboratory conditions, Irs4 knockout males did not exhibit impairments in pituitary expression of TSH subunit genes (Tshb or Cga) or in the thyrotropin-releasing hormone (TRH) receptor. Additionally, their serum thyroid hormone, T3 (triiodothyronine) and T4 (thyroxine), and hypothalamic Trh expression levels were normal. When Irs4 knockouts were rendered hypothyroid with a low-iodine diet supplemented with propylthiouracil (PTU) for 3 weeks, their serum TSH increased similarly to wild-type males. CONCLUSIONS: Overall, Irs4 knockout mice do not exhibit central hypothyroidism or otherwise appear to phenocopy IRS4 deficient patients. Compensation by another IRS protein may explain euthyroidism in these animals.
RESUMO
Activins are one of the three distinct subclasses within the greater Transforming Growth Factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, similar to ActC. Collectively, our results establish ActE as an ALK7 ligand, thereby providing a link between genetic and in vivo studies of ActE as a regulator of adipose tissue.
RESUMO
Inhibins are transforming growth factor-ß family heterodimers that suppress follicle-stimulating hormone (FSH) secretion by antagonizing activin class ligands. Inhibins share a common ß chain with activin ligands. Follistatin is another activin antagonist, known to bind the common ß chain of both activins and inhibins. In this study, we characterized the antagonist-antagonist complex of inhibin A and follistatin to determine if their interaction impacted activin A antagonism. We isolated the inhibin A:follistatin 288 complex, showing that it forms in a 1:1 stoichiometric ratio, different from previously reported homodimeric ligand:follistatin complexes, which bind in a 1:2 ratio. Small angle X-ray scattering coupled with modeling provided a low-resolution structure of inhibin A in complex with follistatin 288. Inhibin binds follistatin via the shared activin ß chain, leaving the α chain free and flexible. The inhibin A:follistatin 288 complex was also shown to bind heparin with lower affinity than follistatin 288 alone or in complex with activin A. Characterizing the inhibin A:follistatin 288 complex in an activin-responsive luciferase assay and by surface plasmon resonance indicated that the inhibitor complex readily dissociated upon binding type II receptor activin receptor type IIb, allowing both antagonists to inhibit activin signaling. Additionally, injection of the complex in ovariectomized female mice did not alter inhibin A suppression of FSH. Taken together, this study shows that while follistatin binds to inhibin A with a substochiometric ratio relative to the activin homodimer, the complex can dissociate readily, allowing both proteins to effectively antagonize activin signaling.
Assuntos
Folistatina , Glicoproteínas , Feminino , Camundongos , Animais , Glicoproteínas/metabolismo , Inibinas/metabolismo , Ativinas/metabolismo , Ligantes , Hormônio Foliculoestimulante/metabolismoRESUMO
Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cells, regulates spermatogenesis in males and ovarian follicle growth in females. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates FSHß subunit gene (Fshb) transcription, though the underlying mechanisms are poorly understood. To address this gap in knowledge, we examined changes in pituitary gene expression in GnRH-deficient mice (hpg) treated with a regimen of exogenous GnRH that increases pituitary Fshb but not luteinizing hormone ß (Lhb) messenger RNA levels. Activating transcription factor 3 (Atf3) was among the most upregulated genes. Activating transcription factor 3 (ATF3) can heterodimerize with members of the activator protein 1 family to regulate gene transcription. Co-expression of ATF3 with JunB stimulated murine Fshb, but not Lhb, promoter-reporter activity in homologous LßT2b cells. ATF3 also synergized with a constitutively active activin type I receptor to increase endogenous Fshb expression in these cells. Nevertheless, FSH production was intact in gonadotrope-specific Atf3 knockout [conditional knockout (cKO)] mice. Ovarian follicle development, ovulation, and litter sizes were equivalent between cKOs and controls. Testis weights and sperm counts did not differ between genotypes. Following gonadectomy, increases in LH secretion were enhanced in cKO animals. Though FSH levels did not differ between genotypes, post-gonadectomy increases in pituitary Fshb and gonadotropin α subunit expression were more pronounced in cKO than control mice. These data indicate that ATF3 can selectively stimulate Fshb expression in vitro but is not required for FSH production in vivo.
Assuntos
Fator 3 Ativador da Transcrição , Hormônio Foliculoestimulante , Feminino , Camundongos , Masculino , Animais , Hormônio Foliculoestimulante/metabolismo , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Regulação da Expressão Gênica , Sêmen/metabolismo , Gonadotropinas , Hormônio Liberador de Gonadotropina/metabolismo , Subunidade beta do Hormônio Folículoestimulante/genéticaRESUMO
Activin ligands are formed from two disulfide-linked inhibin ß (Inhß) subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; InhßA/InhßA) or activin C (ActC; InhßC/InhßC), or as heterodimers, as with activin AC (ActAC; InhßA:InhßC). While the biological functions of ActA and activin B (ActB) have been well characterized, little is known about the biological functions of ActC or ActAC. One thought is that the InhßC chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the InhßC chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature murine adipocytes, which exhibit high ALK7 expression, ActC elicited a SMAD2/3 response similar to ActB, which can also signal via ALK7. Collectively, these results establish that ActC and ActAC are active ligands that exhibit a distinct signaling receptor and antagonist profile compared to other activins.
Assuntos
Receptores de Ativinas Tipo I , Ativinas , Receptores de Ativinas/genética , Receptores de Ativinas/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Ativinas/metabolismo , Animais , Ligantes , Camundongos , Transdução de SinaisRESUMO
The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable regulator of adrenal and gonadal formation, playing roles in sex determination, hypothalamic development, and pituitary function. This study aimed to identify the roles of SF-1 in postnatal female reproductive function. Using a progesterone receptor-driven Cre recombinase, we developed a novel murine model, characterized by conditional depletion of SF-1 [PR-Cre;Nr5a1f/f; conditional knockout (cKO)] in the hypothalamic-pituitary-gonadal axis. Mature female cKO were infertile due to the absence of ovulation. Reduced gonadotropin concentrations in the pituitary gland that were nevertheless sufficient to maintain regular estrous cycles were observed in mature cKO females. The cKO ovaries showed abnormal lipid accumulation in the stroma, associated with an irregular expression of cholesterol homeostatic genes such as Star, Scp2, and Acat1. The depletion of SF-1 in granulosa cells prevented appropriate cumulus oöphorus expansion, characterized by reduced expression of Areg, Ereg, and Ptgs2. Exogenous delivery of gonadotropins to cKO females to induce ovulation did not restore fertility and was associated with impaired formation and function of corpora lutea accompanied by reduced expression of the steroidogenic genes Cyp11a1 and Cyp19a1 and attenuated progesterone production. Surgical transplantation of cKO ovaries to ovariectomized control animals (Nr5a1f/f) resulted in 2 separate phenotypes, either sterility or apparently normal fertility. The deletion of SF-1 in the pituitary and in granulosa cells near the moment of ovulation demonstrated that this nuclear receptor functions across the pituitary-gonadal axis and plays essential roles in gonadotropin synthesis, cumulus expansion, and luteinization.
Assuntos
Ovário , Fator Esteroidogênico 1 , Animais , Feminino , Células da Granulosa/fisiologia , Hipotálamo/fisiologia , Camundongos , Camundongos Knockout , Ovário/fisiologia , Ovulação/genética , Hipófise/fisiologia , Fator Esteroidogênico 1/genética , Fator Esteroidogênico 1/metabolismoRESUMO
A sex steroid-dependent modulation of the immune function in mammals is accepted, and evidence suggests that while estrogens enhance, androgens inhibit the immune response. The aim of this study was to explore in the adult male rat the effect of either neonatal flutamide (FTM) treatment or prepubertal orchidectomy (ODX) on endocrine markers in the basal condition and peripheral tumor necrosis factor alpha (TNFα) levels during inflammatory stress. For these purposes, (1) 5-day-old male rats were subcutaneously injected with either sterile vehicle alone or containing 1.75 mg FTM, and (2) 25-day-old male rats were sham operated or had ODX. Rats were sacrificed (at 100 days of age) in the basal condition for determination of peripheral metabolite levels. Additional rats were intravenously injected with bacterial lipopolysaccharide (LPS; 25 µg/kg body weight, i.v.) and bled for up to 4 h. Data indicate that (1) ODX increased peripheral glucocorticoid levels and reduced those of testosterone, whereas FTM-treated rats displayed low circulating leptin concentrations, and (2) LPS-induced TNFα secretion in plasma was significantly enhanced in the FTM and ODX groups. Our study supports that neonatal FTM treatment affected adiposity function, and adds data maintaining that androgens have a suppressive role in proinflammatory cytokine release in plasma during inflammation.
Assuntos
Reação de Fase Aguda/imunologia , Citocinas/metabolismo , Endotoxemia/imunologia , Endotoxemia/metabolismo , Neuroimunomodulação/fisiologia , Testosterona/imunologia , Reação de Fase Aguda/sangue , Animais , Animais Recém-Nascidos , Castração , Ensaio de Imunoadsorção Enzimática , Glucocorticoides/sangue , Leptina/sangue , Lipopolissacarídeos/toxicidade , Masculino , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Testosterona/sangue , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Follicle-stimulating hormone (FSH) regulates gonadal function and fertility. Measurement of FSH in bodily fluids and tissues is possible with radioimmunoassays and enzyme-linked immunosorbent assays (ELISAs). Recently, several novel assays were developed to measure pituitary hormones including growth hormone, prolactin, and luteinizing hormone in mice from small sample volumes. Here, we describe a novel and sensitive ELISA that enables the accurate measurement of FSH in serum, plasma, and whole blood from female and male mice. The assay can also be used to measure FSH in murine pituitary lysates and cell culture media. In summary, the new methodology described here will enable investigators to measure FSH from a variety of biological samples in mice accurately, at low cost, and in their own laboratories.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Hormônio Foliculoestimulante/sangue , Animais , Feminino , Masculino , Camundongos , Plasma/química , Sensibilidade e Especificidade , Soro/químicaRESUMO
Follicle-stimulating hormone (FSH), a key regulator of ovarian function, is often used in infertility treatment. Gonadal inhibins suppress FSH synthesis by pituitary gonadotrope cells. The TGFß type III receptor, betaglycan, is required for inhibin A suppression of FSH. The inhibin B co-receptor was previously unknown. Here, we report that the gonadotrope-restricted transmembrane protein, TGFBR3L, is the elusive inhibin B co-receptor. TGFBR3L binds inhibin B but not other TGFß family ligands. TGFBR3L knockdown or overexpression abrogates or confers inhibin B activity in cells. Female Tgfbr3l knockout mice exhibit increased FSH levels, ovarian follicle development, and litter sizes. In contrast, female mice lacking both TGFBR3L and betaglycan are infertile. TGFBR3L's function and cell-specific expression make it an attractive new target for the regulation of FSH and fertility.
RESUMO
To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation. We also identify transcriptional and chromatin accessibility programs distinguishing each major cell type. Regulons, which are co-regulated gene sets sharing binding sites for a common transcription factor driver, recapitulate cell type clustering. We identify both cell type-specific and sex-specific regulons that are highly correlated with promoter accessibility, but not with methylation state, supporting the centrality of chromatin accessibility in shaping cell-defining transcriptional programs. The sn multi-omics atlas is accessible at snpituitaryatlas.princeton.edu.
Assuntos
Cromatina/genética , Metilação de DNA , Redes Reguladoras de Genes , Hipófise/metabolismo , Regulon/genética , Transcriptoma/genética , Animais , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Modelos Genéticos , Hipófise/citologia , Regiões Promotoras Genéticas/genética , Fatores SexuaisRESUMO
Activins are selective regulators of FSH production by pituitary gonadotrope cells. In a gonadotrope-like cell line, LßT2, activins stimulate FSH via the activin type IIA receptor (ACVR2A) and/or bone morphogenetic protein type II receptor (BMPR2). Consistent with these observations, FSH is greatly reduced, though still present, in global Acvr2a knockout mice. In contrast, FSH production is unaltered in gonadotrope-specific Bmpr2 knockout mice. In light of these results, we questioned whether an additional type II receptor might mediate the actions of activins or related TGF-ß ligands in gonadotropes. We focused on the activin type IIB receptor (ACVR2B), even though it does not mediate activin actions in LßT2 cells. Using a Cre-lox strategy, we ablated Acvr2a and/or Acvr2b in murine gonadotropes. The resulting conditional knockout (cKO) animals were compared with littermate controls. Acvr2a cKO (cKO-A) females were subfertile (~70% reduced litter size), cKO-A males were hypogonadal, and both sexes showed marked decreases in serum FSH levels compared with controls. Acvr2b cKO (cKO-B) females were subfertile (~20% reduced litter size), cKO-B males had a moderate decrease in testicular weight, but only males showed a significant decrease in serum FSH levels relative to controls. Simultaneous deletion of both Acvr2a and Acvr2b in gonadotropes led to profound hypogonadism and FSH deficiency in both sexes; females were acyclic and sterile. Collectively, these data demonstrate that ACVR2A and ACVR2B are the critical type II receptors through which activins or related TGF-ß ligands induce FSH production in mice in vivo.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Hormônio Foliculoestimulante/biossíntese , Receptores de Activinas Tipo II/genética , Animais , Feminino , Hipogonadismo/genética , Masculino , Camundongos , Camundongos Knockout , Caracteres SexuaisRESUMO
Follicle-stimulating hormone (FSH), an essential regulator of mammalian fertility, is synthesized by pituitary gonadotrope cells in response to activins. In mice, activins signal via SMAD3, SMAD4, and FOXL2 to regulate transcription of the FSHß subunit (Fshb) gene. Gonadotrope-specific deletion of Foxl2, alone or in combination with Smad4, renders mice FSH-deficient. Whether human FSHB expression is similarly regulated is not known. Here, we used a combination of transgenic and conditional knockout mouse strains to assess the roles of activins, FOXL2, and SMAD4 in regulation of the human FSHB gene. First, we cultured pituitaries from mice harboring a human FSHB transgene (hFSHB mice) and measured both murine Fshb and human FSHB messenger ribonucleic acid (mRNA) expression in response to exogenous activins or two antagonists of endogenous activin-like signaling (follistatin-288 and SB431542). Both murine Fshb and human FSHB expression were stimulated by activins and reduced by the inhibitors. Next, we analyzed human FSHB expression in hFSHB mice carrying floxed Foxl2 and Smad4 alleles. Cre-mediated ablation of FOXL2 and SMAD4 strongly reduced basal and activin-stimulated murine Fshb and human FSHB expression in cultured pituitaries. Finally, the hFSHB transgene was previously shown to rescue FSH production and fertility in Fshb knockout mice. However, gonadotrope-specific Foxl2/Smad4 knockout females carrying the hFSHB transgene have significantly reduced murine Fshb and human FSHB pituitary mRNA levels and are hypogonadal. Collectively, these data suggest that similar to Fshb regulation in mice, FOXL2 and SMAD4 play essential roles in human FSHB expression.
Assuntos
Subunidade beta do Hormônio Folículoestimulante/genética , Proteína Forkhead Box L2/genética , Hipófise/metabolismo , Proteína Smad4/genética , Ativinas/farmacologia , Animais , Feminino , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Proteína Forkhead Box L2/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Smad4/metabolismo , Técnicas de Cultura de TecidosRESUMO
Bone morphogenetic proteins (BMPs) are pleiotropic ligands in the TGF-ß superfamily. In the early to mid-2000s, several BMPs, including BMP2, were shown to regulate FSH synthesis alone and in synergy with activins in immortalized gonadotrope-like cell lines and primary pituitary cultures. Activins are also TGF-ß family members, which were identified and named based on their abilities to stimulate FSH production selectively. Mechanistic analyses suggested that BMP2 promoted expression of the FSHß subunit gene (Fshb) via at least two nonmutually exclusive mechanisms. First, BMP2 stimulated the production of the inhibitor of DNA-binding proteins 1, 2, and 3 (Id1, Id2, and Id3), which potentiated the stimulatory actions of homolog of Drosophila mothers against decapentaplegic 3 (SMAD3) on the Fshb promoter. SMAD3 is an intracellular signaling protein that canonically mediates the actions of activins and is an essential regulator of Fshb production in vitro and in vivo. Second, BMP2 was shown to activate SMAD3-dependent signaling via its canonical type IA receptor, BMPR1A (also known as ALK3). This was a surprising result, as ALK3 conventionally activates distinct SMAD proteins. Although these initial results were compelling, they were challenged by contemporaneous and subsequent observations. For example, inhibitors of BMP signaling did not specifically impair FSH production in cultured pituitary cells. Of perhaps greater significance, mice lacking ALK3 in gonadotrope cells produced FSH normally. Therefore, the physiological role of BMPs in FSH synthesis in vivo is presently uncertain.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Hormônio Foliculoestimulante/biossíntese , Gonadotrofos/fisiologia , Ativinas/metabolismo , Animais , Inibinas/metabolismo , Reprodução , Proteínas Smad/metabolismoRESUMO
Inhibins are gonadal hormones that act on pituitary gonadotrope cells to suppress FSH synthesis and secretion. Inhibin A and B are heterodimers of the inhibin âº-subunit disulfide-linked to one of two inhibin ß-subunits. Homodimers or heterodimers of the inhibin ß-subunits form the activins, which stimulate FSH production. Activins signal through complexes of type I and II receptor serine/threonine kinases to increase transcription of the FSHß subunit gene. According to in vitro observations, inhibins impair FSH synthesis by competitively binding to activin type II receptors, particularly in the presence of the TGFß type III receptor (TGFBR3, or betaglycan). The role of TGFBR3 in inhibin action in vivo has not been determined. Here, we ablated Tgfbr3 specifically in murine gonadotropes. Conditional knockout females were supra-fertile, exhibiting enhanced folliculogenesis, numbers of ovulated eggs per cycle, and litter sizes relative to control mice. Despite these phenotypes, FSH levels appeared to be unaltered in knockout mice, and the mechanisms underlying their enhanced fertility remain unexplained. Inhibin B is the predominant form of the hormone in males and in females during most stages of the estrous cycle. Remarkably, inhibin A, but not inhibin B, suppression of FSH synthesis was impaired in cultured pituitaries of knockout mice, which may explain the absence of discernible changes in FSH levels in vivo. Collectively, these data challenge current dogma by demonstrating that TGFBR3 (betaglycan) functions as an inhibin A, but not an inhibin B, coreceptor in gonadotrope cells in vivo. Mechanisms of inhibin B action merit further investigation.
Assuntos
Gonadotrofos/metabolismo , Inibinas/metabolismo , Proteoglicanas/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Fertilidade/genética , Hormônio Foliculoestimulante/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Ligação Proteica , Multimerização Proteica , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/genéticaRESUMO
Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function expressed in thyrotropin (TSH)-producing thyrotrope cells of the anterior pituitary gland. The protein is cotranslationally cleaved, with only its C-terminal domain (CTD) being trafficked to the plasma membrane. Most intragenic IGSF1 mutations in humans map to the CTD. In this study, we used CRISPR-Cas9 to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele encodes a truncated protein that fails to traffic to the plasma membrane. Under standard laboratory conditions, Igsf1-deficient males exhibit normal serum TSH levels as well as normal numbers of TSH-expressing thyrotropes. However, pituitary expression of the TSH subunit genes and TSH protein content are reduced, as is expression of the receptor for thyrotropin-releasing hormone (TRH). When challenged with exogenous TRH, Igsf1-deficient males release TSH, but to a significantly lesser extent than do their wild-type littermates. The mice show similarly attenuated TSH secretion when rendered profoundly hypothyroid with a low iodine diet supplemented with propylthiouracil. Collectively, these results indicate that impairments in pituitary TRH receptor expression and/or downstream signaling underlie central hypothyroidism in IGSF1 deficiency syndrome.