RESUMO
Ovarian hyperstimulation syndrome (OHSS) is a common complication of ovarian stimulation associated with the administration of human chorionic gonadotropin (hCG) during assisted reproduction. We have determined the expression of luteinizing hormone receptor (Lhcgr) mRNA, vascular endothelial growth factor (VEGF), and its transcription factor, HIF1α, during the periovulatory period in a rodent model of OHSS and compared these results with normal ovulatory periods. These results showed that the downregulation of Lhcgr mRNA in response to conditions that mimic preovulatory LH surge was significantly impaired in the OHSS group compared to the complete downregulation seen in the control group. Most importantly, the downregulation of luteinizing hormone receptor mRNA expression following hCG administration was sustained in the control group up to 48 h, whereas it remained at significantly higher levels in the OHSS group. This impairment of hCG-induced Lhcgr downregulation in the OHSS group was accompanied by significantly elevated levels of VEGF and its transcription factor, HIF1α. Furthermore, the downregulation of Lhcgr that occurs in response to a preovulatory LH surge in normal cycles was accompanied by low levels of VEGF. This study shows that, while downregulation of Lhcgr as well as low VEGF levels are seen in response to a preovulatory LH surge in normal ovarian cycle, impaired Lhcgr downregulation and elevated VEGF levels were found in the OHSS group.
Assuntos
Gonadotropina Coriônica/farmacologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Síndrome de Hiperestimulação Ovariana/patologia , Indução da Ovulação/métodos , Receptores do LH/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Feminino , Síndrome de Hiperestimulação Ovariana/tratamento farmacológico , Síndrome de Hiperestimulação Ovariana/genética , Síndrome de Hiperestimulação Ovariana/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores do LH/genética , Substâncias para o Controle da Reprodução/farmacologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
CONTEXT: LH receptor (LHR) expression has been shown to be regulated posttranscriptionally by LHR mRNA binding protein (LRBP) in rodent and human ovaries. LRBP was characterized as mevalonate kinase. The gene that encodes mevalonate kinase is a member of a family of genes that encode enzymes involved in lipid synthesis and are regulated by the transcription factor sterol regulatory element binding proteins (SREBPs). OBJECTIVE: The current study examined the regulation of LHR mRNA expression in human granulosa-lutein cells in response to alterations in cholesterol metabolism. DESIGN: Using atorvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase to inhibit cholesterol biosynthesis, we examined its effect on LHR mRNA expression. The effect of atorvastatin on SREBP and mRNA expression as well as LHR mRNA binding protein expression was examined. Finally, the effect of atorvastatin on human chorionic gonadotropin (hCG)-stimulated progesterone production and the expression of key steroidogenic enzymes was also examined. RESULTS: Statin treatment reduced LHR mRNA expression by increasing the levels of SREBP1a and SREBP2, leading to an increase in LRBP. RNA gel shift assay showed that increased binding of LHR mRNA to LRBP occurred in response to atorvastatin, leading to LHR mRNA degradation. The granulosa-lutein cells pretreated with atorvastatin also showed decreased responsiveness to hCG by decreasing the mRNA and protein expression of steroidogenic enzymes. Atorvastatin also attenuated LH/hCG-induced progesterone production. CONCLUSION: These results imply that LHR mRNA expression by the human granulosa-lutein cells is regulated by cholesterol, through a mechanism involving SREBP and SREBP cleavage activating protein serving as the cholesterol sensor.
Assuntos
Células Lúteas/metabolismo , Receptores do LH/genética , Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Atorvastatina/farmacologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Lúteas/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/fisiologiaRESUMO
Luteinizing hormone/chorionic gonadotropin receptor (LHR) expression in the ovary is regulated by a messenger RNA (mRNA) binding protein, which specifically binds to the coding region of LHR mRNA. We have shown that miR-122, a short noncoding RNA, mediates LHR mRNA levels by modulating the expression of LHR mRNA-binding protein (LRBP) through the regulation of sterol regulatory element binding protein (SREBP) activation. The present results show that miR-122 regulates LRBP levels by increasing the processing of SREBP through the degradation of Insig1, the anchoring protein of SREBP. We present evidence showing that mRNA and protein levels of Insig1 undergo a time-dependent increase following the treatment of rat granulosa cells with follicle-stimulating hormone (FSH), which leads to a decrease in LRBP levels. Furthermore, overexpression of miR-122 using an adenoviral vector (AdmiR-122) abolished FSH-induced increases in Insig1 mRNA and protein. We further confirmed the role of Insig1 by showing that inhibition of Insig1 using a specific small interfering RNA prior to FSH treatment resulted in the abrogation of LHR upregulation. Silencing of Insig1 also reversed FSH-mediated decreases in SREBP and LRBP activation. These results show that decreased levels of miR-122 increase Insig1 and suppress SREBP processing in response to FSH stimulation of rat granulosa cells.
Assuntos
Células da Granulosa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , MicroRNAs/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Receptores do LH/genética , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Animais , Feminino , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica , Hormônios/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , RNA Interferente Pequeno , Proteínas de Ligação a RNA/metabolismo , Ratos , Receptores do LH/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismoRESUMO
The expression of luteinizing hormone receptor (LHR) in the mammalian ovary is regulated in response to changes in the secretion of follicle-stimulating hormone and luteinizing hormone by the anterior pituitary, at least in part, through posttranscriptional mechanisms. The steady-state levels of LHR mRNA are maintained by controlling its rate of degradation by an RNA-binding protein designated as LHR mRNA-binding protein (LRBP). LRBP forms a complex with LHR mRNA and targets it for degradation in the p bodies. miR-122, an 18 nucleotide noncoding RNA, regulates the expression of LRBP. Thus, the levels of miR-122 determine the cellular levels of LHR mRNA expression. This phenomenon has been examined during the induction of LHR mRNA expression that occurs during follicle maturation in response to rising levels of FSH. In this situation, miR-122 and LRBP levels decrease as LHR mRNA expression undergoes downregulation in response to preovulatory LH surge. miR-122 expression as well as LRBP levels show robust increases. The mechanism of induction of LRBP by miR-122 has also been discussed.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Ciclo Menstrual , MicroRNAs/metabolismo , Modelos Biológicos , Ovário/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores do LH/metabolismo , Animais , Feminino , Fármacos para a Fertilidade Feminina/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Ciclo Menstrual/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Interferência de RNA , Estabilidade de RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/agonistas , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Receptores do LH/agonistas , Receptores do LH/antagonistas & inibidores , Receptores do LH/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
We have shown that the transient changes in the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) messenger RNA (mRNA) during the ovarian cycle occurs, at least in part, through a posttranscriptional mechanism involving an LHCGR mRNA-binding protein (LRBP). Eukaryotic initiation factor 5A (eIF5A), an LRBP-interacting protein, participates in this process. eIF5A undergoes hypusination, a unique posttranslational modification that is necessary for its functions. This study examined the role of eIF5A in follicle-stimulating hormone (FSH)-induced LHCGR expression during follicular growth. Treatment of primary cultures of rat granulosa cells with FSH and 17ß-estradiol (E2) showed a time-dependent increase in LHCGR mRNA expression. Conversely, inhibition of endogenous hypusination of eIF5A using N1-guanyl-1,7-diaminoheptane (GC7), a hypusination inhibitor, showed a greater increase in LHCGR mRNA expression over that produced by FSH and E2 alone. Further studies were carried out to determine the mechanism by which inhibition of hypusination of eIF5A causes an increase in LHCGR mRNA expression. Because LHCGR expression is negatively regulated by LRBP, the effect of inhibiting hypusination of eIF5A on LRBP expression was examined. The results showed a decrease in the expression of LRBP mRNA and protein when hypusination of eIF5A was inhibited by GC7. Because LRBP promotes LHCGR mRNA degradation, the results of this study support the notion that by inhibiting eIF5A hypusination, FSH reduces the expression of LRBP. This increases LHCGR mRNA expression by abrogating the inhibitory action of LRBP.
Assuntos
Hormônio Foliculoestimulante/farmacologia , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores do LH/metabolismo , Animais , Feminino , Regulação da Expressão Gênica/fisiologia , Células da Granulosa/fisiologia , Fatores de Iniciação de Peptídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Receptores do LH/genética , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
We have previously reported that LHCGR expression in the ovary is regulated through a post-transcriptional mechanism involving an mRNA binding protein designated as LRBP, which is regulated, at least in part, by a non-coding RNA, miR-122. Our present study examined the regulatory role of miR-122 in FSH-induced LHCGR expression during follicle development. Treatment of rat granulosa cells concurrently with FSH and 17ß estradiol showed, as expected, a time-dependent increase in LHCGR mRNA levels as well as hCG-induced progesterone production. However, miR-122 expression was decreased during the early time periods, which preceded the increased expression of LHCGR mRNA. The role of miR-122 in FSH-induced LHCGR mRNA expression was then examined by overexpressing miR-122 prior to FSH stimulation by infecting granulosa cells with an adenoviral vector containing a miR-122 insert (AdmiR-122). Pretreatment with AdmiR-122 resulted in complete abrogation of FSH- mediated upregulation of LHCGR. AdmiR-122 also blocked FSH-induced decrease in LRBP expression and increased the binding of LHCGR mRNA to LRBP. Based on these results, we conclude that miR-122 plays a regulatory role in LHCGR expression by modulating LRBP levels during FSH-induced follicle growth.
Assuntos
MicroRNAs/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Receptores do LH/metabolismo , Animais , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/metabolismo , Progesterona/metabolismo , RNA Mensageiro/metabolismo , Ratos , Regulação para Cima/fisiologiaRESUMO
LH/human chorionic gonadotropin receptor (LHR) undergoes down-regulation during preovulatory LH surge or in response to exposure to a supraphysiological concentration of its ligands through a posttranscriptional mechanism involving an RNA binding protein designated as LHR mRNA binding protein (LRBP). miR-122, a short noncoding RNA, has been shown to mediate the up-regulation of LRBP. In the present study, we show that inhibition of miR-122 using a locked nucleic acid (LNA)-conjugated antagomir suppressed human chorionic gonadotropin (hCG)-induced up-regulation of LRBP as well as its association with LHR mRNA, as analyzed by RNA EMSA. Most importantly, inhibition of miR-122 resulted in the abolishment of hCG-mediated LHR mRNA down-regulation. We also show that the transcription factor, sterol regulatory element binding protein (SREBP) (SREBP-1a and SREBP-2 isoforms), is an intermediate in miR-122-mediated LHR mRNA regulation. HCG-stimulated increase in the activation of both SREBP-1a and SREBP-2 was inhibited by pretreatment with the miR-122 antagomir. The inhibition of cAMP/protein kinase A (PKA) and ERK pathways, upstream activators of miR-122, abolished SREBP activation after hCG treatment. SREBP-mediated regulation of LRBP expression is mediated by recruitment of LRBP promoter element to SREBP-1a, because chromatin immunoprecipitation assay revealed that association of LRBP promoter to SREBP was increased by hCG treatment. Pretreatment with miR-122 antagomir suppressed this response. Inhibition of SREBP activation by pretreating the rats with a chemical compound, fatostatin abrogated hCG-induced up-regulation of LRBP mRNA and protein. Fatostatin also inhibited LHR-LRBP mRNA-protein complex formation and LHR down-regulation. These results conclusively show that miR-122 plays a regulatory role in LH/hCG-induced LHR mRNA down-regulation by increasing LRBP expression through the activation of SREBP pathway.
Assuntos
MicroRNAs/metabolismo , Ovário/metabolismo , Receptores do LH/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Animais , Antagomirs , Gonadotropina Coriônica , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Oligonucleotídeos , Fosfotransferases (Aceptor do Grupo Álcool) , Piridinas , Proteínas de Ligação a RNA/metabolismo , Ratos Sprague-Dawley , Ribonucleoproteínas/metabolismo , TiazóisRESUMO
Luteinizing hormone receptor (LHR) mRNA expression in the ovary is regulated post-transcriptionally by an LH receptor mRNA binding protein (LRBP). Eukaryotic initiation factor 5A (EIF5A), identified as an LRBP-interacting protein plays a crucial role in LHR mRNA expression. In this study, we have demonstrated that during hCG-induced LHR downregulation, a significant upregulation of eIF5A mRNA expression and hypusination of eIF5A protein occurs in a time dependent manner. Pretreatment with H89, a specific inhibitor of PKA, and U0126, a specific inhibitor of ERK1/2 significantly inhibited both hCG-induced eIF5A mRNA expression and hypusination of eIF5A protein. Pretreatment with GC7, a specific inhibitor of eIF5A hypusination significantly abolished hCG-induced LRBP mRNA and protein expression. Furthermore, GC7 pretreatment significantly inhibited hCG-induced interaction of LRBP with LHR mRNA as assessed by RNA electrophoretic mobility gel shift assay (REMSA). GC7 treatment also reversed LHR mRNA downregulation. Taken together, these results suggest that hCG-induced LHR mRNA downregulation is mediated by cAMP-PKA-ERK1/2 signaling leading to activation of eIF5A hypusination.
Assuntos
AMP Cíclico/metabolismo , Regulação da Expressão Gênica/fisiologia , Lisina/análogos & derivados , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ovário/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Receptores do LH/biossíntese , Animais , Proteínas Quinases Dependentes de AMP Cíclico , Feminino , Hormônio Luteinizante/metabolismo , Lisina/metabolismo , Fatores de Iniciação de Peptídeos , Fosfotransferases (Aceptor do Grupo Álcool) , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Down-regulation of LH receptor (LHR) in the ovary by its ligand is mediated by a specific RNA-binding protein, designated LH receptor mRNA-binding protein (LRBP), through translational suppression and mRNA degradation. Using yeast 2-hybrid screens, we previously identified eukaryotic initiation factor 5A (eIF5A) as one of the proteins that interacts with LRBP during LHR mRNA down-regulation. The present study examined the role of eIF5A and its hypusination in the context of LHR mRNA down-regulation. The association of eIF5A with LRBP or LHR mRNA was determined using immunoprecipitation and RNA immunoprecipitation assays. The results showed that the association of eIF5A with the LHR mRNA-LRBP complex increased significantly during down-regulation. Furthermore, gel fractionation and the hypusination activity assay both showed increased hypusination of eIF5A during LHR mRNA down-regulation. Abolishment of hypusination by pretreatment with the chemical inhibitor GC7 prevented the association of eIF5A with LHR mRNA and LRBP. Inhibition of hypusination also reduced the extent of ligand-induced down-regulation of LHR mRNA as well as the expression of functional LHRs assessed by real-time PCR and (125)I-human chorionic gonadotropin (hCG) binding assays, respectively. The loss of human chorionic gonadotropin-mediated downstream signaling during LHR down-regulation was also restored by inhibition of hypusination of eIF5A. Thus, the present study, for the first time, reveals the crucial role of eIF5A and its hypusination in the regulation of LHR expression in the ovary.
Assuntos
Hormônio Luteinizante/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores do LH/metabolismo , Animais , Gonadotropina Coriônica/metabolismo , Regulação para Baixo/genética , Feminino , Humanos , Hormônio Luteinizante/genética , Ovário/metabolismo , Fatores de Iniciação de Peptídeos/genética , RNA/genética , RNA/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Receptores do LH/genética , Transdução de Sinais/genética , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
A specific luteinizing hormone receptor (LHR) mRNA binding protein (LRBP) has been identified and purified. This LH receptor mRNA binding protein selectively binds to the polypyrimidine rich bipartite sequence in the coding region of the LHR mRNA and accelerates its degradation. In response to preovulatory LH surge, the LH receptor expression in the ovary undergoes downregulation by accelerated degradation of LH receptor mRNA through the involvement of this RNA binding protein. Here we describe the intracellular mechanism triggered by LH/hCG (human chorionic gonadotropin) that leads to the regulated degradation of LH receptor mRNA. Downregulation of LH receptor mRNA was induced by treatment of cultured human granulosa cells with 10 IU of hCG. Activation of downstream target, extracellular signal-regulated protein kinase 1 and 2 (ERK 1/2) showed an increase within five min and sustained up to 1 h. Confocal analysis showed that ERK1/2 translocates to the nucleus after 15 min of hCG treatment. This leads to an increase in LRBP expression which then causes downregulation of LH receptor mRNA by accelerating its degradation. Treatment with UO126 or transfection with ERK specific siRNA (small interfering RNA) resulted in the abolishment of ERK activation as well as LHR mRNA downregulation. RNA electrophoretic mobility gel shift assay of the cytosolic fractions showed that hCG-induced increase in the LH receptor mRNA binding activity was also abrogated by these treatments. These results show that LH/hCG-induced LH receptor mRNA downregulation is initiated by the activation of ERK1/2 pathway by regulating the expression and activity of LH receptor mRNA binding activity.
Assuntos
Hormônio Luteinizante/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Ovário/metabolismo , Proteólise , Proteínas de Ligação a RNA/metabolismo , Receptores do LH/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Northern Blotting , Butadienos , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Células da Granulosa/metabolismo , Humanos , Nitrilas , Ovário/citologia , RNA Interferente Pequeno/farmacologiaRESUMO
LH receptor (LHR) expression in the ovary is regulated by the RNA binding protein, (LHR mRNA binding protein [LRBP]), which has been identified as being mevalonate kinase. This study examined the role of microRNA miR-122 in LRBP-mediated LHR mRNA expression. Real-time PCR analysis of ovaries from pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-primed female rats treated with hCG to down-regulate LHR expression showed that an increase in miR-122 expression preceded LHR mRNA down-regulation. The expression of miR-122 and its regulation was confirmed using fluorescent in situ hybridization of the frozen ovary sections using 5'-fluorescein isothiocyanate-labeled miR-122 locked nucleic acid probe. The increased expression of miR-122 preceded increased expression of LRBP mRNA and protein, and these increases were followed by LHR mRNA down-regulation. Inhibition of protein kinase A (PKA) and ERK1/2 signaling pathways by H89 and UO126, respectively, attenuated the hCG-mediated up-regulation of miR-122 levels. This was also confirmed in vitro using human granulosa cells. These results suggest the possibility that hCG-mediated miR-122 expression is mediated by the activation of cAMP/PKA/ERK signaling pathways. Inhibition of miR-122 by injection of the locked nucleic acid-conjugated antagomir of miR-122 abrogated the hCG-mediated increases in LRBP protein expression. Because it has been previously shown that miR-122 regulates sterol regulatory element-binding proteins (SREBPs) and SREBPs, in turn, regulate LRBP expression, the role of SREBPs in miR-122-mediated increase in LRBP expression was then examined. The levels of active forms of both SREBP-1a and SREBP-2 were increased in response to hCG treatment, and the stimulatory effect was sustained up to 4 hours. Taken together, our results suggest that hCG-induced down-regulation of LHR mRNA expression is mediated by activation of cAMP/PKA/ERK pathways to increase miR-122 expression, which then increases LRBP expression through the activation of SREBPs.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Ovário/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Células Cultivadas , Gonadotropina Coriônica/farmacologia , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Hormônio Luteinizante/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , MicroRNAs/genética , Ovário/citologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ratos , Ratos Sprague-Dawley , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Regulação para CimaRESUMO
Luteinizing hormone receptor undergoes downregulation during preovulatory Luteinizing hormone surge through a post-transcriptional mechanism involving an RNA binding protein designated as LRBP. The present study examined the mechanism by which LRBP induces the degradation of Luteinizing hormone receptor mRNA, specifically the role of decapping of Luteinizing hormone receptor mRNA and the translocation of LRBP-bound Luteinizing hormone receptor mRNA to degradative machinery. Immunoprecipitation of the complex with the 5'cap structure antibody followed by real time PCR analysis showed progressive loss of capped Luteinizing hormone receptor mRNA during downregulation suggesting that Luteinizing hormone receptor mRNA undergoes decapping prior to degradation. RNA immunoprecipitation analysis confirmed dissociation of eukaryotic initiation factor 4E from the cap structure, a step required for decapping. Furthermore, RNA immunoprecipitation analysis using antibody against the p body marker protein, DCP1A showed that Luteinizing hormone receptor mRNA was associated with the p bodies, the cytoplasmic foci that contain RNA degradative enzymes and decapping complex. Immunohistochemical studies using antibodies against LRBP and DCP1A followed by confocal analysis showed colocalization of LRBP with DCP1A during downregulation. This was further confirmed by co-immunoprecipitation of LRBP with DCP1A. The association of LRBP and Luteinizing hormone receptor mRNA in the p bodies during downregulation was further confirmed by examining the association of a second p body component, rck/p54, using immunoprecipitation and RNA immunoprecipitation respectively. These data suggest that the association of LRBP with Luteinizing hormone receptor mRNA results in the translocation of the messenger ribonucleoprotein complex to the p bodies leading to decapping and degradation.
Assuntos
Hormônio Luteinizante/metabolismo , RNA Mensageiro , Proteínas de Ligação a RNA/metabolismo , Receptores do LH/metabolismo , Animais , Citoplasma/genética , Citoplasma/metabolismo , Regulação para Baixo , Feminino , Humanos , Ovário/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Transcrição GênicaRESUMO
The present study examined the effect of insulin-mediated activation of the mammalian target of rapamycin complex 1 (MTORC1) signaling network on the proliferation of primary culture of theca-interstitial (T-I) cells. Our results show that insulin treatment increased proliferation of the T-I cells through the MTORC1-dependent signaling pathway by increasing cell cycle regulatory proteins. Inhibition of ERK1/2 signaling caused partial reduction of insulin-induced phosphorylation of RPS6KB1 and RPS6 whereas inhibition of PI3-kinase signaling completely blocked the insulin response. Pharmacological inhibition of MTORC1 with rapamycin abrogated the insulin-induced phosphorylation of EIF4EBP1, RPS6KB1 and its downstream effector, RPS6. These results were further confirmed by demonstrating that knockdown of Mtor using siRNA reduced the insulin-stimulated MTORC1 signaling. Furthermore, insulin-stimulated T-I cell proliferation and the expression of cell cycle regulatory proteins CDK4, CCND3 and PCNA were also blocked by rapamycin. Taken together, the present studies show that insulin stimulates cell proliferation and cell cycle regulatory proteins in T-I cells via activation of the MTORC1 signaling pathway.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Insulina/farmacologia , Complexos Multiproteicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Células Tecais/citologia , Animais , Proteínas de Transporte/metabolismo , Bovinos , Proliferação de Células/efeitos dos fármacos , Feminino , Inativação Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Alvo Mecanístico do Complexo 1 de Rapamicina , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Complexos Multiproteicos/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína S6 Ribossômica/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fatores de TempoRESUMO
LH triggers the biosynthesis of androgens in the theca-interstitial (T-I) cells of ovary through the activation of a cAMP-dependent pathway. We have previously shown that LH/human chorionic gonadotropin (hCG) activates mammalian target of rapamycin complex 1 (mTORC1) signaling network, leading to cell proliferation. In the present study, we provide evidence that the LH/hCG-mediated activation of the mTORC1 signaling cascade is involved in the regulation of steroidogenic enzymes in androgen biosynthesis. Treatment with LH/hCG increased the expression of downstream targets of mTORC1, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E as well as steroidogenic enzymes. LH/hCG-mediated stimulation of the steroidogenic enzyme mRNA was blocked by the mTORC1 inhibitor, rapamycin. This inhibitory effect was selective because rapamycin failed to block hCG-mediated increase in the expression of Star mRNA levels. Furthermore, pharmacological targeting of mTORC1 with rapamycin also blocked LH/hCG- or forskolin-induced expression of cAMP response element-binding protein (CREB) and steroidogenic enzymes (P450 side-chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase type 1, and 17α-hydroxylase/17,20 lyase) but produced no effect on steroidogenic acute regulatory protein levels. These results were further confirmed by demonstrating that the knockdown of mTOR using small interfering RNA selectively abrogated the LH/hCG-induced increase in steroidogenic enzyme expression, without affecting steroidogenic acute regulatory protein expression. LH/hCG-stimulated androgen production was also blocked by rapamycin. Furthermore, the pharmacological inhibition of mTORC1 or ribosomal protein S6 kinase 1 signaling prevented the LH/hCG-induced phosphorylation of CREB. Chromatin immunoprecipitation assays revealed the association of CREB with the proximal promoter of the Cyp17a1 gene in response to hCG, and this association was reduced by rapamycin treatment. Taken together, our findings show for the first time that LH/hCG-mediated activation of androgen biosynthesis is regulated by the mTORC1 signaling pathway in T-I cells.
Assuntos
Androgênios/biossíntese , Gonadotropina Coriônica/fisiologia , Hormônio Luteinizante/fisiologia , Proteínas/metabolismo , Células Tecais/metabolismo , Androstenodiona/biossíntese , Animais , Células Cultivadas , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Gonadotropina Coriônica/farmacologia , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Indução Enzimática , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Hormônio Luteinizante/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas/antagonistas & inibidores , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
OBJECTIVE: To determine whether metformin has direct effects on ovarian theca-interstitial (T-I) cell proliferation through activation of adenosine monophosphate-activated protein kinase (AMPK). DESIGN: In vitro experimental study. SETTING: Academic medical center laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Ovarian T-I cells were isolated, purified, and cultured in the absence (control) or presence of insulin (1 µg/mL) with or without metformin or other activators/inhibitors of AMPK (AICAR, compound C). MAIN OUTCOME MEASURE(S): Proliferation assessed by determination of expression levels of proteins involved in cell cycle progression, cyclin D3, and cyclin-dependent kinase 4 (CDK4) with Western blot analysis, and determination of DNA synthesis with bromodeoxyuridine (BrdU) incorporation assay; activation of AMPK, Erk1/2, and S6K1 determined by Western blot analysis with the use of antibodies specific for the phosphorylated (activated) forms. RESULT(S): Metformin inhibited insulin-induced ovarian T-I cell proliferation and the up-regulation of the cell cycle regulatory proteins, cyclin D3 and CDK4. Metformin independently activated AMPK in a dose-dependent manner. Treatment with metformin inhibited insulin-induced activation of Erk1/2 and S6K1. This effect was reversed with the addition of compound C, a known AMPK inhibitor. CONCLUSION(S): Metformin directly inhibits proliferation of ovarian T-I cells via an AMPK-dependent mechanism. These findings further validate the potential benefits of metformin in the treatment of conditions associated with hyperinsulinemia and excessive growth of ovarian T-I cells (such as polycystic ovary syndrome).
Assuntos
Proliferação de Células/efeitos dos fármacos , Metformina/farmacologia , Ovário/efeitos dos fármacos , Células Tecais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/fisiologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Feminino , Hipoglicemiantes/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ovário/citologia , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/metabolismo , Células Tecais/citologia , Células Tecais/fisiologiaRESUMO
We have previously reported that 5α-dihydrotestosterone (DHT) inhibits FSH-mediated granulosa cell proliferation by reducing cyclin D2 mRNA expression and blocking cell cycle progression at G1/S phase. The present study investigated the role of AMP activated protein kinase (AMPK) in DHT-mediated inhibition of granulosa cell proliferation. Granulosa cells harvested from 3-d estradiol primed immature rats were exposed to different concentrations of DHT (0, 45, and 90 ng/ml) for 24 h. Western blot analysis of immunoprecipitated AMPK showed a dose-dependent activation (P < 0.05) as evidenced by the increased phosphorylation at thr 172. In addition, time-courses studies (0, 6, 12, and 24 h) using DHT (90 ng/ml) showed a time-dependent increase in AMPK activation with maximum effect at 24 h. FSH inhibited AMPK phosphorylation and promoted granulosa cell proliferation, but pretreatment with DHT (90 ng/ml) for 24 h prior to FSH treatment reduced this effect. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1-ß4-ribofuranoside abolished FSH-mediated ERK phosphorylation, indicating that AMPK is a negative upstream regulator of ERK. Furthermore, inhibition of AMPK activation by compound C reversed the DHT-mediated reduction in positive cell cycle regulator, cyclin D2, and 5-bromo-2'-deoxyuridine incorporation. These results suggest that elevated levels of DHT activate AMPK, which in turn inhibits ERK phosphorylation. Thus, inhibition of ERK phosphorylation by activated AMPK in response to DHT might contribute to decreased granulosa cell mitogenesis and ovulatory dysfunction seen in hyperandrogenic states.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proliferação de Células/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Androgênios/farmacologia , Animais , Western Blotting , Células Cultivadas , Ciclina D2/genética , Ciclina D2/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Hormônios/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Luteinizing hormone receptor and follicle stimulating hormone receptor play a crucial role in female and male reproduction. Significant new information has emerged about the structure, mechanism of activation, and regulation of expression of these receptors. Here we provide an overview of the current information on those aspects with an in-depth discussion of the recent developments in the post-transcriptional mechanism of LH receptor expression mediated by a specific LH receptor mRNA binding protein, designated as LRBP. LRBP was identified by electrophoretic gel mobility shift assay using cytosolic fractions from ovaries in the down regulated state. LRBP was purified, its binding site on LH receptor mRNA was identified and characterized. During ligand-induced down regulation, LRBP expression is increased through the cAMP/PKA and ERK signaling pathway, is translocated to translating ribosomes, binds LH receptor mRNA and forms an untranslatable ribonucleoprotein complex. This complex is then routed to the mRNA degradation machinery resulting in diminished levels of both LHR mRNA and cell surface expression of LH receptor. The studies leading to these conclusions are presented.
Assuntos
Receptores do FSH/fisiologia , Receptores do LH/fisiologia , Animais , Regulação da Expressão Gênica , Humanos , Mutação , Especificidade de Órgãos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Processamento de Proteína Pós-Traducional , Receptores do FSH/genética , Receptores do FSH/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Transdução de SinaisRESUMO
The ligand-induced down-regulation of LH receptor (LHR) expression in the ovaries, at least in part, is regulated by a posttranscriptional process mediated by a specific LH receptor mRNA binding protein (LRBP). The LH-mediated signaling pathways involved in this process were examined in primary cultures of human granulosa cells. Treatment with 10 IU human chorionic gonadotropin (hCG) for 12 h resulted in the down-regulation of LHR mRNA expression while producing an increase in LHR mRNA binding to LRBP as well as a 2-fold increase in LRBP levels. The activation of ERK1/2 pathway in LH-mediated LHR mRNA down-regulation was also established by demonstrating the translocation of ERK1/2 from the cytosol to the nucleus using confocal microcopy. Inhibition of protein kinase A using H-89 or ERK1/2 by U0126 abolished the LH-induced LHR mRNA down-regulation. These treatments also abrogated both the increases in LRBP levels as well as the LHR mRNA binding activity. The abolishment of the hCG-induced increase in LRBP levels and LHR mRNA binding activity was further confirmed by transfecting granulosa cells with ERK1/2 specific small interfering RNA. This treatment also reversed the hCG-induced down-regulation of LHR mRNA. These data show that LH-regulated ERK1/2 signaling is required for the LRBP-mediated down-regulation of LHR mRNA.
Assuntos
Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Mensageiro/genética , Receptores do LH/genética , Western Blotting , Butadienos/farmacologia , Linhagem Celular , Núcleo Celular/metabolismo , Gonadotropina Coriônica/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Imunofluorescência , Células da Granulosa/metabolismo , Humanos , Isoquinolinas/farmacologia , Microscopia Confocal , Nitrilas/farmacologia , Ovário , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Reação em Cadeia da Polimerase , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/farmacologia , Receptores do LH/metabolismo , Transdução de Sinais , Sulfonamidas/farmacologiaRESUMO
This study examined the role of Rab5a GTPase in regulating hCG-induced internalization and trafficking of the hCG-LH receptor complex in transfected 293T cells. Coexpression of wild-type Rab5a (WT) or constitutively active Rab5a (Q79L) with LHR significantly increased hCG-induced LHR internalization. Conversely, coexpression of dominant negative Rab5a (S34N) with LHR reduced internalization. Confocal microscopy showed LHR colocalizing with Rab5a (WT) and Rab5a (Q79L) in punctuate structures. Coexpression of Rab5a (WT) and Rab5a (Q79L) with LHR significantly increased colocalization of LHR in early endosomes. Conversely, dominant negative Rab5a (S34N) decreased this colocalization. While Rab5a stimulated internalization of LHR, it significantly decreased LHR recycling to the cell surface and increased degradation. Dominant negative Rab5a (S34N) increased LHR recycling and decreased degradation. These results suggest that Rab5a plays a role in LHR trafficking by facilitating internalization and fusion to early endosomes, increasing the degradation of internalized receptor resulting in a reduction in LHR recycling.
Assuntos
Gonadotropina Coriônica/metabolismo , Endocitose , Receptores do LH/metabolismo , Proteínas rab5 de Ligação ao GTP/fisiologia , Linhagem Celular , Gonadotropina Coriônica/análise , Endossomos/metabolismo , Humanos , Microscopia Confocal , Receptores do LH/análise , Transfecção , Proteínas rab5 de Ligação ao GTP/análise , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
The objective of the study was to evaluate the effect of valproic acid (VPA) on ovarian androgen biosynthesis in primary cultures of theca-interstitial (T-I) cells isolated from rat ovaries. Ovarian T-I cells were cultured with VPA in the presence or absence of hCG. VPA did not increase basal or hCG-stimulated androgen synthesis when added to primary cultures of T-I cells. However, the addition of VPA caused a marked concentration-dependent inhibitory effect on hCG-stimulated androstendione synthesis. Treatment of T-I cells with 8-Bromo-cAMP resulted in a marked increase in the production of androstenedione, and VPA inhibited this stimulatory effect, suggesting that the mechanism of VPA's inhibitory effect on androstenedione production occurs at a step after second messenger activation. Treatment of T-I cells with hCG resulted in a significant increase in the mRNA expression of steroidogenic enzymes CYP17A1 and 17ß-hydroxysteroid dehydrogenase. Addition of VPA sharply blunted the stimulatory effect of hCG, reducing the mRNA expression of the steroidogenic enzymes to basal levels. In conclusion, VPA exerts an inhibitory effect on hCG-stimulated androgen synthesis in rat T-I cells.