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1.
Endocrinology ; 151(6): 2788-99, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20332198

RESUMO

The injection of equine chorionic gonadotropin (eCG) in dairy goats induces the production of anti-eCG antibodies (Abs) in some females. We have previously shown that Abs negatively modulate the LH and FSH-like bioactivities of eCG, in most cases, compromising fertility in treated females. Surprisingly, we found out that some anti-eCG Abs improved fertility and prolificity of the treated females, in vivo. These Abs, when complexed with eCG, enhanced LH and FSH ability to induce steroidogenesis on specific target cells, in vitro. In the present study, we analyzed the impact of three eCG/anti-eCG Ab-enhancing complexes on two transduction mechanisms triggered by the FSH receptor: guanine nucleotide-binding protein alphaS-subunit/cAMP/protein kinase A (PKA) and beta-arrestin-dependent pathways, respectively. In all cases, significant enhancing effects were observed on ERK phosphorylation compared with eCG alone. However, cAMP production and PKA activation induced by eCG could be differently modulated by Abs. By using a pharmacological inhibitor of PKA and small interfering RNA-mediated knock-down of endogenous beta-arrestin 1 and 2, we demonstrated that signaling bias was induced and was clearly dependent on the complexed Ab. Together, our data show that eCG/anti-eCG Ab-enhancing complexes can differentially modulate cAMP/PKA and beta-arrestin pathways as a function of the complexed Ab. We hypothesize that enhancing Abs may change the eCG conformation, the immune complex acquiring new "biased" pharmacological properties ultimately leading to the physiological effects observed in vivo. The modulation of ligand pharmacological properties by Abs opens promising research avenues towards the optimization of glycoprotein hormone biological activities and, more generally, the development of new therapeutics.


Assuntos
Anticorpos/imunologia , Complexo Antígeno-Anticorpo/farmacologia , Gonadotropina Coriônica/imunologia , Hormônio Foliculoestimulante/metabolismo , Gonadotropinas Equinas/imunologia , Transdução de Sinais/efeitos dos fármacos , Complexo Antígeno-Anticorpo/imunologia , Arrestinas/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Isoquinolinas/farmacologia , Cinética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno , Sulfonamidas/farmacologia , beta-Arrestina 1 , beta-Arrestinas
2.
Mol Endocrinol ; 24(3): 561-73, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20107152

RESUMO

Deglycosylated FSH is known to trigger poor Galphas coupling while efficiently binding its receptor. In the present study, we tested the possibility that a deglycosylated equine LH (eLHdg) might be able to selectively activate beta-arrestin-dependent signaling. We compared native eLH to an eLH derivative [i.e. truncated eLHbeta (Delta121-149) combined with asparagine56-deglycosylated eLHalpha (eLHdg)] previously reported as an antagonist of cAMP accumulation at the FSH receptor (FSH-R). We confirmed that, when used in conjunction with FSH, eLHdg acted as an antagonist for cAMP accumulation in HEK-293 cells stably expressing the FSH-R. Furthermore, when used alone at concentrations up to 1 nM, eLHdg had no detectable agonistic activity on cAMP accumulation, protein kinase A activity or cAMP-responsive element-dependent transcriptional activity. At higher concentrations, however, a weak agonistic action was observed with eLHdg, whereas eLH led to robust responses whatever the concentration. Both eLH and eLHdg triggered receptor internalization and led to beta-arrestin recruitment. Both eLH and eLHdg triggered ERK and ribosomal protein (rp) S6 phosphorylation at 1 nM. The depletion of endogenous beta-arrestins had only a partial effect on eLH-induced ERK and rpS6 phosphorylation. In contrast, ERK and rpS6 phosphorylation was completely abolished at all time points in beta-arrestin-depleted cells. Together, these results show that eLHdg has the ability to preferentially activate beta-arrestin-dependent signaling at the FSH-R. This finding provides a new conceptual and experimental framework to revisit the physiological meaning of gonadotropin structural heterogeneity. Importantly, it also opens a field of possibilities for the development of selective modulators of gonadotropin receptors.


Assuntos
Arrestinas/metabolismo , Hormônio Luteinizante/análogos & derivados , Receptores do FSH/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Bovinos , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Cavalos , Humanos , Imunoprecipitação , Hormônio Luteinizante/química , Hormônio Luteinizante/metabolismo , Hormônio Luteinizante/farmacologia , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno , Receptores do FSH/agonistas , Receptores do FSH/antagonistas & inibidores , Proteína S6 Ribossômica/metabolismo , Suínos , beta-Arrestinas
3.
Mol Cell Endocrinol ; 315(1-2): 271-6, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-19778579

RESUMO

Follicle-stimulating hormone (FSH) controls the proliferation and differentiation of Sertoli cells of the testis. FSH binds a G protein-coupled receptor (GPCR) to stimulate downstream effectors of the phosphoinositide-3 kinase (PI3K)-dependent pathway, without enhancing PI3K activity. To clarify this paradox, we explored the activity of phosphatase and tensin homolog deleted in chromosome 10 (PTEN), the PI3K major regulator, in primary cultures of rat Sertoli cells. We show that, within minutes, FSH increases PTEN neo-synthesis, requiring the proteasomal degradation of an unidentified intermediate, as well as PTEN enzymatic activity. Importantly, introducing an antisense cDNA of PTEN into differentiating Sertoli cells restores FSH-dependent cell proliferation. In conclusion, these results provide a new mechanism of PTEN regulation, which could serve to block entry into S phase of Sertoli cells, while they are proceeding through differentiation in prepubertal animals.


Assuntos
Hormônio Foliculoestimulante/farmacologia , Mitose/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Puberdade/fisiologia , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/fisiologia , Animais , Células Cultivadas , Hormônio Foliculoestimulante/metabolismo , Humanos , Masculino , Mitose/fisiologia , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Ratos , Ratos Wistar , Células de Sertoli/citologia , Suínos , Transferrina/metabolismo
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