RESUMO
Several reports suggest putative interactions between valproic acid (VPA) treatment and the hypothalamus-pituitary-adrenal axis. Given that VPA alters mitochondrial functions, an action of this drug on a mitochondrial process such as steroid synthesis in adrenal cells should be expected. In order to disclose a putative action of VPA on the adrenocortical cell itself we evaluated VPA effects on regulatory steps of the acute stimulation of steroidogenesis in Y1 adrenocortical cells. This study demonstrates that VPA increases progesterone production in non-stimulated cells without inducing the levels of Steroidogenic Acute Regulatory (StAR) protein, which facilitates cholesterol transport. This result suggests that VPA increases mitochondrial cholesterol transport through a StAR-independent mechanism and is further supported by the fact that in isolated mitochondria VPA stimulates exogenous cholesterol metabolization to progesterone. VPA also reduces the cAMP-mediated increase of the StAR protein, mRNA levels, promoter activity and progesterone production. In summary, the present data show that VPA can alter steroid production in adrenal cells by a complex mechanism that mainly involves an action on cholesterol access to the inner mitochondrial membrane. The VPA-mediated increase of basal steroidogenesis could be linked to the increase of basal cortisolemia described in patients under VPA treatment.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Anticonvulsivantes/farmacologia , Colesterol/metabolismo , Mitocôndrias/efeitos dos fármacos , Ácido Valproico/farmacologia , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Córtex Suprarrenal/metabolismo , Animais , Anticonvulsivantes/toxicidade , Transporte Biológico , Linhagem Celular , Enzima de Clivagem da Cadeia Lateral do Colesterol/antagonistas & inibidores , AMP Cíclico/agonistas , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Camundongos , Mitocôndrias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ácido Valproico/toxicidadeRESUMO
Several stimuli, including stress conditions, promote the activation of MAP kinases family members (ERK1/2, JNK, p38). In turn, these enzymes regulate several cellular functions. Given that MAPK activation requires the phosphorylation of these proteins, their inactivation depends on the activity of specific phosphatases. MAPK phosphatase-1 (MKP-1), a phosphatase specifically involved in the inactivation of MAPK family members, is induced by mitogenic stimuli and stress conditions. Here we describe the effect of heat shock (HS), 10 min, 45 degrees C, on MAPKs activities and MKP-1 mRNA and protein levels in Y1 adrenocortical cells. Western blot analysis performed with antibodies against the phosphorylated forms of ERK1/2 and JNK revealed that HS produced the rapid activation of these kinases. Their inactivation was also a rapid event and occurred together with the increase of MKP-1 protein levels detected by Western blot analysis. In addition, the effect of HS on MKP-1 protein levels seems to be exerted at the transcriptional level, since the amount of its mRNA in heat shocked cells was higher than in nonheated cells. Comparison of the temporal profiles of MKP-1 protein induction and MAPKs phospho-dephosphorylation suggests that MKP-1 induction could contribute to ERK1/2 and JNK inactivation after HS.
Assuntos
Córtex Suprarrenal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transtornos de Estresse por Calor/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Córtex Suprarrenal/patologia , Animais , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular , Fosfatase 1 de Especificidade Dupla , Ativação Enzimática , Proteínas Imediatamente Precoces/genética , Camundongos , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/metabolismo , Choque/metabolismo , Fatores de TempoRESUMO
A key regulatory step in the steroidogenic hormones signaling pathway is the synthesis of steroidogenic acute regulatory protein (StAR). This protein facilitates the delivery of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. ACTH and LH pathway also includes tyrosine dephosphorylation processes. Indeed, our previous studies have demonstrated that both hormones increase protein tyrosine phosphatase (PTP) activity by a PKA-dependent mechanism and that the action of PTPs is required for the stimulation of steroid biosynthesis in adrenal and Leydig cells. In order to test the putative relationship between PTP activity and StAR protein induction in adrenocortical cells, in the present study we evaluated steroid production and StAR protein level in Y1 adrenocortical cells under PTP inhibition. Phenylarsine oxide (PAO), a powerful cell permeable PTP inhibitor, reduced ACTH-stimulated steroidogenesis in a concentration-dependent fashion. A concentration of 2.5 microM of this compound inhibited steroid synthesis in a 56% (ACTH = 318 +/- 30, ACTH + PAO = 145 +/- 18 ng progesterone/mL, P < 0.001) and also abrogated StAR protein induction. Phenylarsine oxide reduced the protein level after 60 min and this effect still remained at 120 min. A second PTP inhibitor, benzyl phosphonic acid, acting by a different mechanism, reproduced PAO effects on both steroidogenesis and StAR protein. Taken together, these results indicate that PTP activity participates in StAR protein induction and led us to attribute to the PKA-mediated PTP activation in steroidogenic systems a functional role, as mediator of StAR protein induction.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Esteroides/biossíntese , Animais , Arsenicais/administração & dosagem , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Camundongos , Organofosfonatos/farmacologia , Fosfoproteínas/antagonistas & inibidores , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Esteroides/antagonistas & inibidores , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Our recent reports indicate that protein tyrosine phosphorylation is an obligatory component of the mechanism of action of ACTH in its stimulatory action of corticosteroid production in adrenal zona fasciculata (ZF). The role of protein tyrosine phosphatase (PTP) activity in the regulation of steroidogenesis by LH/chorionic gonadotropin (CG) was tested using cell-permeable PTP inhibitors. Thus, PTP inhibition blocks LH- and 8-bromo-cAMP-stimulated testosterone production by Leydig cells without affecting 22(R)OH-cholesterol-supported steroidogenesis, similar results to those obtained in the adrenal ZF/ACTH system, leading us to propose that PTP action is an obligatory and common step in the cascade triggered by both hormones. Then, we continued the study testing whether LH modulates PTP activity in MA-10 cells, a Leydig cell line. In this regard, we observed by an in-gel PTP assay two PTPs of 110 and 50 kDa that are activated by hormone and 8-bromo-cAMP activation of the cells. Moreover, there is a transient increase by the second messenger in total PTP activity that correlates with the higher activity displayed by the 110 and 50 kDa proteins in the in-gel assay. In accordance with these results, analysis of tyrosine phosphorylated proteins showed the LH-induced dephosphorylation of proteins of 120, 68 and 50 kDa. The results of this study indicate that PTPs play an important role in the regulation of Leydig cell functions and that there exists a cross talk between serine/threonine phosphorylation and tyrosine dephosphorylation mediated by hormone-activated cAMP-dependent protein kinase and PTPs. These results are the first evidence of PTP having a role in LH/CG-stimulated steroidogenesis.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Gonadotropinas Hipofisárias/metabolismo , Células Intersticiais do Testículo/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Testosterona/biossíntese , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Análise de Variância , Animais , Linhagem Celular , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Gonadotropina Coriônica/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Hormônio Luteinizante/metabolismo , Masculino , Oxigenases/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Ratos , Ratos Wistar , Vanadatos/farmacologiaRESUMO
The present study investigated the effect of nitric oxide (NO) on megakaryocyte (Mk) proliferation induced by thrombopoietin (TPO). Low-density mononuclear cells (MNCs) and CD34+ cells from human bone marrow (BM) were cultured in liquid medium in the presence of sodium nitroprusside (SNP) or (Z)-1-[2-(aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA/NO) and then stimulated with TPO. Mk number decreased in both NO donors, as identified by flow cytometry 11 to 13 days after TPO stimulation. Nitrite, cyanide, or the carrier molecule DETA failed to reproduce the inhibition caused by NO donors. When CD34+ cells were treated with DETA/NO, the inhibition of Mk growth was even more pronounced than that in MNCs. Failure of the guanosine 3',5'-cyclic monophosphate (cGMP) analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) to inhibit Mk proliferation suggests that cGMP is not involved in Mk suppression mediated by NO. On the other hand, DNA analysis by flow cytometry showed that apoptosis of CD34+ cells and Mks seemed to be at least one of the mechanisms associated with the cytotoxic DETA/NO effect. Stimulation of MNCs or CD34+ cells with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) increased endogenous NO levels and suppressed Mk growth. Treatment with NO synthesis inhibitors such as L -N(G)-monomethyl arginine (L -NMMA) or L -N(G)-nitroarginine methyl ester hydrochloride (L -NAME) partially reversed Mk growth inhibition induced by TNF-alpha and IFN-gamma, although increased NO levels returned to normal values. The results presented here strongly indicate that NO regulates the growth of Mks induced by TPO by a direct effect on both progenitors and mature Mks.
Assuntos
Divisão Celular/fisiologia , Megacariócitos/citologia , Óxido Nítrico/fisiologia , Trombopoetina/farmacologia , Antígenos CD34/imunologia , Apoptose/fisiologia , Divisão Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Megacariócitos/imunologiaRESUMO
Thrombopoietin (TPO) and granulocyte colony-stimulating factor (G-CSF) may be administered together in aplastic patients. We evaluated the effect of both cytokines alone or combined on platelets and polymorphonuclear leukocytes (PMN) functional responses. TPO, G-CSF, or the combination of both cytokines, induced neither platelet nor PMN activation. TPO but not G-CSF synergized with threshold ADP concentrations to induce maximal aggregation and ATP release. The synergistic effect of TPO with ADP was not modified by the presence of G-CSF. Flow cytometry studies have shown that thrombin-induced loss of GPIb from platelet surface was significantly increased by pretreatment of platelets with TPO, G-CSF, or both cytokines. P-selectin expression induced by thrombin was augmented by TPO, but not by G-CSF. Coincubation of the cells with TPO and G-CSF did not modify the values obtained with TPO alone. Expression of CD11b on PMN surface was augmented by G-CSF or fMLP. G-CSF-treated PMN increased the effect of fMLP on CD11b expression. TPO did not modify either basal levels of CD11b or the increased expression induced by G-CSF or fMLP. Incubation of PMN with both cytokines showed no differences compared to G-CSF alone. Platelet-PMN aggregates induced by thrombin in whole blood were augmented by TPO. G-CSF alone neither synergized with thrombin nor changed the results observed with TPO. These data show that in vitro functional responses of platelets, or PMN induced by TPO or G-CSF alone, were neither further increased nor inhibited by treatment of the cells with both cytokines.
Assuntos
Plaquetas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Neutrófilos/efeitos dos fármacos , Trombopoetina/farmacologia , Difosfato de Adenosina/farmacologia , Humanos , Antígeno de Macrófago 1/biossíntese , Antígeno de Macrófago 1/efeitos dos fármacos , Ativação de Neutrófilo/efeitos dos fármacos , Selectina-P/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/efeitos dos fármacos , Trombina/farmacologiaRESUMO
In adrenal cortex, ACTH regulation of steroidogenesis depends on PKA-dependent serine/threonine phosphorylation and also on the activity of protein tyrosine phosphatases (PTPs). In addition, ACTH increases total PTPs involving at least three soluble PTPs (50, 82 and 115 kDa). Serine/threonine phosphorylation of these enzymes themselves could be a regulatory mechanism of their activity since the increase of total PTP activity is dependent on PKA-activation. In this report we analyzed the effect of in vitro phospho-dephosphorylation processes on the activity displayed by the ACTH-activated PTP of 115 kDa. Using an in-gel PTP assay we demonstrate that dephosphorylation catalyzed by potato acid phosphatase (PAP) reduces the activity of the 115 kDa PTP present in ZF from ACTH-treated animals and PKA-mediated phosphorylation reverses this effect.