RESUMO
For many years, research in the field of steroid synthesis has aimed to understand the regulation of the rate-limiting step of steroid synthesis, i.e. the transport of cholesterol from the outer to the inner mitochondrial membrane, and identify the protein involved in the conversion of cholesterol into pregnenolone. The extraordinary work by B Clark, J Wells, S R King, and D M Stocco eventually identified this protein and named it steroidogenic acute regulatory protein (StAR). The group's finding was also one of the milestones in understanding the mechanism of nonvesicular lipid transport between organelles. A notable feature of StAR is its high degree of phosphorylation. In fact, StAR phosphorylation in the acute phase is required for full steroid biosynthesis. As a contribution to this subject, our work has led to the characterization of StAR as a substrate of kinases and phosphatases and as an integral part of a mitochondrion-associated multiprotein complex, essential for StAR function and cholesterol binding and mitochondrial transport to yield maximum steroid production. Results allow us to postulate the existence of a specific cellular microenvironment where StAR protein synthesis and activation, along with steroid synthesis and secretion, are performed in a compartmentalized manner, at the site of hormone receptor stimulation, and involving the compartmentalized formation of the steroid molecule-synthesizing complex.
Assuntos
Fosfoproteínas , Esteroides , Fosfoproteínas/metabolismo , Colesterol/metabolismo , Microambiente CelularRESUMO
Hormone-receptor signal transduction has been extensively studied in adrenal gland. Zona glomerulosa and fasciculata cells are responsible for glucocorticoid and mineralocorticoid synthesis by adrenocorticotropin (ACTH) and angiotensin II (Ang II) stimulation, respectively. Since the rate-limiting step in steroidogenesis occurs in the mitochondria, these organelles are key players in the process. The maintenance of functional mitochondria depends on mitochondrial dynamics, which involves at least two opposite events, i.e., mitochondrial fusion and fission. This review presents state-of-the-art data on the role of mitochondrial fusion proteins, such as mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), in Ang II-stimulated steroidogenesis in adrenocortical cells. Both proteins are upregulated by Ang II, and Mfn2 is strictly necessary for adrenal steroid synthesis. The signaling cascades of steroidogenic hormones involve an increase in several lipidic metabolites such as arachidonic acid (AA). In turn, AA metabolization renders several eicosanoids released to the extracellular medium able to bind membrane receptors. This report discusses OXER1, an oxoeicosanoid receptor which has recently arisen as a novel participant in adrenocortical hormone-stimulated steroidogenesis through its activation by AA-derived 5-oxo-ETE. This work also intends to broaden knowledge of phospho/dephosphorylation relevance in adrenocortical cells, particularly MAP kinase phosphatases (MKPs) role in steroidogenesis. At least three MKPs participate in steroid production and processes such as the cellular cycle, either directly or by means of MAP kinase regulation. To sum up, this review discusses the emerging role of mitochondrial fusion proteins, OXER1 and MKPs in the regulation of steroid synthesis in adrenal cortex cells.
Assuntos
Dinâmica Mitocondrial , Hormônios Peptídicos , Humanos , Transdução de Sinais , Eicosanoides , Ácido Araquidônico , Hormônio Adrenocorticotrópico , Angiotensina IIRESUMO
The OXE receptor is a GPCR activated by eicosanoids produced by the action of 5-lipoxygenase. We previously found that this membrane receptor participates in the regulation of cAMP-dependent and -independent steroidogenesis in human H295R adrenocortical carcinoma cells. In this study we analyzed the effects of the OXE receptor physiological activator 5-oxo-ETE on the growth and migration of H259R cells. While 5-oxo-ETE did not affect the growth of H295R cells, overexpression of OXE receptor caused an increase in cell proliferation, which was further increased by 5-oxo-ETE and blocked by 5-lipoxygenase inhibition. 5-oxo-ETE increased the migratory capacity of H295R cells in wound healing assays, but it did not induce the production of metalloproteases MMP-1, MMP-2, MMP-9 and MMP-10. The pro-migratory effect of 5-oxo-ETE was reduced by pharmacological inhibition of the MEK/ERK1/2, p38 and PKC pathways. 5-oxo-ETE caused significant activation of ERK and p38. ERK activation by the eicosanoid was reduced by the "pan" PKC inhibitor GF109203X but not by the classical PKC inhibitor Gö6976, suggesting the involvement of novel PKCs in this effect. Although H295R cells display detectable phosphorylation of Ser299 in PKCδ, a readout for the activation of this novel PKC, treatment with 5-oxo-ETE per se was unable to induce additional PKCδ activation. Our results revealed signaling effectors activated by 5-oxo-ETE in H295R cells and may have significant implications for our understanding of OXE receptor in adrenocortical cell pathophysiology.
Assuntos
Córtex Suprarrenal/citologia , Ácidos Araquidônicos/farmacologia , Movimento Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Linhagem Celular , Citoproteção/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Metaloproteases/metabolismo , Receptores Eicosanoides/metabolismoRESUMO
In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the "classical" protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ACTH and discussed.
RESUMO
Hormone-regulated steroidogenesis and StAR protein induction involve the action of lipoxygenated products. The products of 5-lipoxygenase act on inflammation and immunity by stimulation of a membrane receptor called OxeR1. The presence of OxeR1 in other systems has not been described up to date and little is known about its mechanism of action and other functions. In this context, the aim of this study was the identification and characterization of OxeR1 as a mediator of cAMP-dependent and independent pathways. Overexpression of OxeR1 in MA-10 Leydig cells increased cAMP-dependent progesterone production. Angiotensin II and cAMP stimulation of adrenocortical human H295R cells produced an increase in StAR protein induction and steroidogenesis in cells overexpressing OxeR1 as compared to mock-transfected cells. Additionally, activation of OxeR1 caused a time-dependent increase in ERK1/2 phosphorylation. In summary, membrane receptor OxeR1 is involved in StAR protein induction and activation of steroidogenesis triggered by cAMP or angiotensin II, acting, at least in part, through ERK1/2 activation.
Assuntos
Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Angiotensina II/farmacologia , AMP Cíclico/farmacologia , Receptores Eicosanoides/metabolismo , Esteroides/biossíntese , Córtex Suprarrenal/efeitos dos fármacos , Animais , Ácidos Araquidônicos/farmacologia , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Plasmídeos/metabolismo , TransfecçãoRESUMO
Acyl-CoA synthetase 4 (Acsl4) is involved in several cellular functions including steroidogenesis, synaptic development and cancer metastasis. Although the expression of Acsl4 seems to be regulated by tissue- and cell-specific factors as well as pituitary hormones and growth factors, the transcriptional mechanisms involved remain unknown. We demonstrated hCG and cAMP regulation of Acsl4 mRNA in mouse steroidogenic MA-10 Leydig cells. We characterized the transcription initiation site and promoter of the Acsl4 mouse gene and identified three alternative splice variants present in MA-10 cells. Sequence analysis of a 1.5-kb fragment of the Acsl4 promoter revealed the absence of a TATA box and the presence of many putative binding sites for transcription factors including Sp1 and CREB. Functional characterization revealed that the specificity protein/Krüppel-like factor Sp1 binding site in the proximal promoter is involved in basal activity and that the cAMP response element-binding site is involved in cAMP stimulation of Acsl4 transcription.
Assuntos
Coenzima A Ligases/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fator de Transcrição Sp1/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Gonadotropina Coriônica/farmacologia , Clonagem Molecular , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , AMP Cíclico/farmacologia , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Transcrição Gênica/efeitos dos fármacosRESUMO
Hormonal regulation of steroidogenesis involves arachidonic acid (AA) metabolism through the 5-lipoxygenase pathway. One of the products, 5-hydroperoxy-eicosatetraenoic acid (5-HpETE), acts as a modulator of the activity of the steroidogenic acute regulatory (StAR) protein promoter. Besides, an oxoeicosanoid receptor of the leukotriene receptor family named OXE-R is a membrane protein with high affinity and response to 5-HpETE, among other AA derivatives. The aim of our work was to elucidate whether this receptor may be involved in steroidogenesis. RT-PCR and western blot analysis demonstrated the presence of the mRNA and protein of the receptor in human H295R adrenocortical cells. The treatment of H295R or MA-10 cells (murine Leydig cell line) with 8Br-cAMP together with docosahexaenoic acid (DHA, an antagonist of the receptor) partially reduced StAR induction and steroidogenesis. On the contrary, 5-oxo-ETE - the prototypical agonist, with higher affinity and potency on the receptor - increased cAMP-dependent steroid production, StAR mRNA and protein levels. These results lead us to conclude that AA might modulate StAR induction and steroidogenesis, at least in part, through 5-HpETE production and activation of a membrane receptor, such as the OXE-R.
Assuntos
Ácido Araquidônico/metabolismo , Fosfoproteínas/biossíntese , Fosfoproteínas/metabolismo , Receptores Eicosanoides , Esteroides/biossíntese , Córtex Suprarrenal/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Linhagem Celular , Ácidos Docosa-Hexaenoicos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Leucotrienos/metabolismo , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Fosfoproteínas/genética , RNA Mensageiro/biossíntese , Receptores Eicosanoides/agonistas , Receptores Eicosanoides/antagonistas & inibidores , Receptores Eicosanoides/biossíntese , Transcrição Gênica/efeitos dos fármacosRESUMO
Although the contribution of mitochondrial dynamics (a balance in fusion/fission events and changes in mitochondria subcellular distribution) to key biological process has been reported, the contribution of changes in mitochondrial fusion to achieve efficient steroid production has never been explored. The mitochondria are central during steroid synthesis and different enzymes are localized between the mitochondria and the endoplasmic reticulum to produce the final steroid hormone, thus suggesting that mitochondrial fusion might be relevant for this process. In the present study, we showed that the hormonal stimulation triggers mitochondrial fusion into tubular-shaped structures and we demonstrated that mitochondrial fusion does not only correlate-with but also is an essential step of steroid production, being both events depend on PKA activity. We also demonstrated that the hormone-stimulated relocalization of ERK1/2 in the mitochondrion, a critical step during steroidogenesis, depends on mitochondrial fusion. Additionally, we showed that the SHP2 phosphatase, which is required for full steroidogenesis, simultaneously modulates mitochondrial fusion and ERK1/2 localization in the mitochondrion. Strikingly, we found that mitofusin 2 (Mfn2) expression, a central protein for mitochondrial fusion, is upregulated immediately after hormone stimulation. Moreover, Mfn2 knockdown is sufficient to impair steroid biosynthesis. Together, our findings unveil an essential role for mitochondrial fusion during steroidogenesis. These discoveries highlight the importance of organelles' reorganization in specialized cells, prompting the exploration of the impact that organelle dynamics has on biological processes that include, but are not limited to, steroid synthesis.
Assuntos
Mitocôndrias/fisiologia , Dinâmica Mitocondrial , Progesterona/biossíntese , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Linhagem Celular , Gonadotropina Coriônica/farmacologia , Gonadotropina Coriônica/fisiologia , AMP Cíclico/farmacologia , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Retículo Endoplasmático/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/fisiologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Isoquinolinas/farmacologia , Cinética , Células Intersticiais do Testículo/metabolismo , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , Forma das Organelas , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Ionóforos de Próton/farmacologia , Interferência de RNA , Sulfonamidas/farmacologiaRESUMO
Acyl-CoA synthetase 4 (ACSL4) is implicated in fatty acid metabolism with marked preference for arachidonic acid (AA). ACSL4 plays crucial roles in physiological functions such as steroid synthesis and in pathological processes such as tumorigenesis. However, factors regulating ACSL4 mRNA and/or protein levels are not fully described. Because ACSL4 protein expression requires tyrosine phosphatase activity, in this study we aimed to identify the tyrosine phosphatase involved in ACSL4 expression. NSC87877, a specific inhibitor of the tyrosine phosphatase SHP2, reduced ACSL4 protein levels in ACSL4-rich breast cancer cells and steroidogenic cells. Indeed, overexpression of an active form of SHP2 increased ACSL4 protein levels in MA-10 Leydig steroidogenic cells. SHP2 has to be activated through a cAMP-dependent pathway to exert its effect on ACSL4. The effects could be specifically attributed to SHP2 because knockdown of the phosphatase reduced ACSL4 mRNA and protein levels. Through the action on ACSL4 protein levels, SHP2 affected AA-CoA production and metabolism and, finally, the steroidogenic capacity of MA-10 cells: overexpression (or knockdown) of SHP2 led to increased (or decreased) steroid production. We describe for the first time the involvement of SHP2 activity in the regulation of the expression of the fatty acid-metabolizing enzyme ACSL4.
Assuntos
Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Animais , Ácido Araquidônico/biossíntese , Ácido Araquidônico/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Fosfoproteínas/genética , Plasmídeos/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Quinolinas/farmacologia , Interferência de RNA , Ratos , Esteroides/biossínteseRESUMO
The phospho-dephosphorylation of intermediate proteins is a key event in the regulation of steroid biosynthesis. In this regard, it is well accepted that steroidogenic hormones act through the activation of serine/threonine (Ser/Thr) protein kinases. Although many cellular processes can be regulated by a crosstalk between different kinases and phosphatases, the relationship of Ser/Thr phosphorylation and tyrosine (Tyr)-dephosphorylation is a recently explored field in the regulation of steroid synthesis. Indeed in steroidogenic cells, one of the targets of hormone-induced Ser/Thr phosphorylation is a protein tyrosine phosphatase. Whereas protein tyrosine phosphatases were initially regarded as household enzymes with constitutive activity, dephosphorylating all the substrates they encountered, evidence is now accumulating that protein tyrosine phosphatases are tightly regulated by various mechanisms. Here, we will describe the role of protein tyrosine phosphatases in the regulation of steroid biosynthesis, relating them to steroidogenic acute regulatory protein, arachidonic acid metabolism and mitochondrial rearrangement.
Assuntos
Colesterol/metabolismo , Fosfoproteínas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Animais , Transporte Biológico , Hormônios/metabolismo , Humanos , Especificidade por SubstratoRESUMO
In order to achieve the goal of this article, as an example we will describe the strategies followed to analyze the presence of the multi-kinase complex at the mitochondria and the posttranslational modification of two key mitochondrial proteins, which participate in the regulation of cholesterol transport across the mitochondrial membranes and in the regulation of steroid biosynthesis. Hormones, ions or growth factors modulate steroid biosynthesis by the posttranslational phosphorylation of proteins. The question still remains on how phosphorylation events transmit a specific signal to its mitochondrial site of action. Cholesterol transport requires specific interactions in mitochondria between several proteins including a multi-kinase complex. The presence of this multi-kinase complex at the mitochondria reveals the importance of the posttranslational modification of mitochondrial proteins for its activity and functions. The activation of PKA triggers the posttranslational modification of the mitochondrial acyl-CoA thioesterase (Acot2), which releases arachidonic acid (AA) in the mitochondria, and the activation of a kinase cascade that leads to the phoshorylation of the steroidogenic acute regulatory (StAR) protein. The function of StAR is to facilitate the access of cholesterol to the first enzyme of the biosynthesis process and its induction is dependent on Acot2 and intramitochondrial AA release. Truncation of the StAR protein is associated with the steroid deficiency disease, congenital lipoid adrenal hyperplasia.
Assuntos
Mitocôndrias/enzimologia , Proteínas Quinases/análise , Proteínas Quinases/metabolismo , Esteroides/biossíntese , Tioléster Hidrolases/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/análise , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Mitocôndrias/química , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Tioléster Hidrolases/análise , Tioléster Hidrolases/genética , TransfecçãoRESUMO
It is known that ERK1/2 and MEK1/2 participate in the regulation of Star gene transcription. However, their role in StAR protein post-transcriptional regulation is not described yet. In this study we analyzed the relationship between the MAPK cascade and StAR protein phosphorylation and function. We have demonstrated that (a) steroidogenesis in MA-10 Leydig cells depends on the specific of ERK1/2 activation at the mitochondria; (b) ERK1/2 phosphorylation is driven by mitochondrial PKA and constitutive MEK1/2 in this organelle; (c) active ERK1/2 interacts with StAR protein, leads to StAR protein phosphorylation at Ser(232) only in the presence of cholesterol; (d) directed mutagenesis of Ser(232) (S232A) inhibited in vitro StAR protein phosphorylation by ERK1; (e) transient transfection of MA-10 cells with StAR S232A cDNA markedly reduced the yield of progesterone production. We show that StAR protein is a substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric complex that regulates cholesterol transport.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Mitocôndrias/metabolismo , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Dados de Sequência Molecular , Fosforilação , Alinhamento de Sequência , Esteroides/biossínteseRESUMO
The studies presented herein were designed to investigate the effect of mouse epidermal growth factor (mEGF) on arachidonic acid (AA) release in a clonal strain of cultured murine Leydig cells (designed MA-10). In MA-10 cells, mEGF promotes AA release and metabolism to lipoxygenated products to induce the steroidogenic acute regulatory (StAR) protein. However, the mechanism by which mEGF releases AA in these cells is not totally elucidated. We show that mEGF produces an increment in the mitochondrial AA content in a short-term incubation (30 min). This AA is released by the action of a mitochondrial acyl-CoA thioesterase (Acot2), as demonstrated in experiments in which Acot2 was down or overexpressed. This AA in turn regulates the StAR protein expression, indirect evidence of its metabolism to lipoxygenated products. We also show that mEGF induces the expression (mRNA and protein) of Acot2 and an acyl-CoA synthetase that provides the substrate, arachidonyl-CoA, to Acot2. This effect is also observed in another steroidogenic cell line, the adrenocortical Y1 cells. Taken together, our results show that: 1) mEGF can induce the generation of AA in a specific compartment of the cells, i.e. the mitochondria; 2) mEGF can up-regulate acyl-CoA synthetase and Acot2 mRNA and protein levels; and 3) mEGF-stimulated intramitochondrial AA release leads to StAR protein induction.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Animais , Ácido Araquidônico/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Células Clonais , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Tumor de Células de Leydig/genética , Tumor de Células de Leydig/metabolismo , Tumor de Células de Leydig/patologia , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfoproteínas/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismoRESUMO
ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Progesterona/biossíntese , Animais , Linhagem Celular , Colesterol/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Camundongos , Mitocôndrias/metabolismo , Fosfoproteínas/metabolismo , FosforilaçãoRESUMO
Recent studies demonstrated the importance of the mitochondrial ATP in the regulation of a novel long-chain fatty acid generation/export system in mitochondria of diabetic rat heart. In steroidogenic systems, mitochondrial ATP and intramitochondrial arachidonic acid (AA) generation are important for steroidogenesis. Here, we report that mitochondrial ATP is necessary for the generation and export of AA, steroid production and steroidogenic acute regulatory protein induction supported by cyclic 3'-5'-adenosine monophosphate in steroidogenic cells. These results demonstrate that ATP depletion affects AA export and provide new evidence of the existence of the fatty acid generation and export system involved in mitochondrial cholesterol transport.
Assuntos
Ácido Araquidônico/metabolismo , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Células Intersticiais do Testículo/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , AMP Cíclico/metabolismo , Células Intersticiais do Testículo/citologia , Masculino , Miocárdio/metabolismo , Fosfoproteínas/metabolismo , RatosRESUMO
In adrenocortical and Leydig cells PKA activation by trophic hormones increases the activity of protein tyrosine phosphatases and also induces the expression of MAP kinase phosphatase 1 (MKP-1), a dual activity protein phosphatase (serine/threonine and tyrosine). This work summarizes the knowledge on the regulation and the role played by cAMP-activated tyrosine phosphatases as well as MKP-1 in the hormonal activation of the acute and chronic phases of steroidogenesis.
Assuntos
Córtex Suprarrenal/metabolismo , Células Intersticiais do Testículo/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Esteroides/biossíntese , Córtex Suprarrenal/citologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/genética , Transcrição GênicaRESUMO
We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.
Assuntos
Ácido Araquidônico/metabolismo , Colesterol/metabolismo , AMP Cíclico/fisiologia , Células Intersticiais do Testículo/metabolismo , Mitocôndrias/metabolismo , Animais , Transporte Biológico , Ácidos Graxos/fisiologia , Masculino , Camundongos , Mitocôndrias/fisiologia , Organelas/metabolismo , Progesterona/biossíntese , Transfecção , Células Tumorais CultivadasRESUMO
A duplex polymerase chain reaction (PCR) assay was performed using a sense Leishmania genus-specific oligonucleotides to amplify the conserved region of 120 bp of minicircle kDNA and an additional antisense Leishmania donovani oligonucleotide that amplifies a complex-specific fragment of 90 bp. All 12 tested reference isolates of the L. donovani complex yielded a complex-specific amplification product of 90 bp concurrently with a genus-specific 120-bp product. All 17 tested reference isolates pertaining to other complexes presented only the genus-specific 120-bp product. Peripheral blood samples of twenty patients with visceral leishmaniasis positive by the genus-specific PCR presented both fragments with the duplex assay. This duplex PCR can be applied as a 1-step diagnostic tool where discrimination of L. donovani complex species is relevant for clinical and epidemiologic purposes.
Assuntos
Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Animais , Criança , Pré-Escolar , DNA de Cinetoplasto/genética , Feminino , Humanos , Lactente , Leishmania donovani/genética , Masculino , Pessoa de Meia-Idade , Oligonucleotídeos , Oligonucleotídeos Antissenso , Sensibilidade e Especificidade , TionucleotídeosRESUMO
The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.
Assuntos
Ácido Araquidônico/metabolismo , Coenzima A Ligases/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Proteínas Tirosina Fosfatases/metabolismo , Neoplasias do Córtex Suprarrenal , Hormônio Adrenocorticotrópico/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Tumor de Células de Leydig , Hormônio Luteinizante/farmacologia , Masculino , CamundongosRESUMO
We have described that, in adrenal and Leydig cells, the hormonal regulation of free arachidonic acid (AA) concentration is mediated by the concerted action of two enzymes: an acyl-CoA thioesterase (MTE-I or ARTISt) and an acyl-CoA synthetase (ACS4). In this study we analyzed the potential regulation of these proteins by hormonal action in steroidogenic cells. We demonstrated that ACS4 is rapidly induced by adrenocorticotropin (ACTH) and cAMP in Y1 adrenocortical cells. The hormone and its second messenger increased ACS4 protein levels in a time and concentration dependent way. Maximal concentration of ACTH (10 mIU/ml) produced a significant effect after 15 min of treatment and exerted the highest increase (3-fold) after 30 min. Moreover, (35)S-methionine incorporation showed that the increase in ACS4 protein levels is due to an increase in the de novo synthesis of the protein. On the contrary MTE-I protein levels in Y1 and MA-10 cells did not change after steroidogenic stimuli. In contrast with the effect observed on protein levels, stimulation of both cell lines did not change ACS4 RNA levels during the first hour of treatment, indicating that the effect of both stimuli is exerted at the level of ACS4 protein synthesis.StAR protein induction has a key role on the activation of steroidogenesis since this protein increases the rate of the limiting step of the whole process. In agreement with the fact that the inhibition of ACS4 activity by triacsin C blocks cAMP-stimulated progesterone production by MA-10 Leydig cells, here we demonstrated that ACS4 inhibition also reduces StAR protein levels. Moreover, exogenous AA was able to overcome the effect of triacsin C on both events, StAR induction and steroidogenesis. These results were confirmed by experiments using ACS4-targeted siRNA which result in a reduction in both ACS4 and StAR protein levels. The concomitant decrease in steroid production was overcome by the addition of AA to the knocked-out cells. In summary, this study suggests that in adrenal and Leydig cells the hormonal action prompts the synthesis of a labile protein, ACS4, which activity is involved in the regulation of AA release and is essential for steroidogenesis and StAR protein induction.