Transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene: GATA-4 and CCAAT/enhancer-binding protein beta confer synergistic responsiveness in hormone-treated rat granulosa and HEK293 cell models.

Steroidogenic acute regulatory protein (StAR) mediates translocation of cholesterol to the inner membranes of steroidogenic mitochondria, where it serves as a substrate for steroid synthesis. Transcription of StAR in the gonads and adrenal cells is upregulated by trophic hormones, involves downstream signaling pathways and a cohort of trans-factors acting as activators or suppressors of StAR transcription. This study suggests that a 21 basepair long sequence positioned at -81/-61 of the murine StAR promoter is sufficient to confer a robust hormonal activation of transcription in ovarian granulosa cells treated with FSH. We show that recombinant GATA-4 and CCAAT/enhancer-binding protein beta (C/EBPbeta) bind to the promoter at -66/-61 and -81/-70 and activate transcription of a reporter gene when co-expressed in heterologous human embryonic kidney 293 (HEK293) cells. In this cell model, C/EBPbeta and GATA-4 synergize in a sequence dependent manner and p300/CBP further maximizes their joint activities. Inhibitors of the transcriptional activators, such as liver-enriched inhibiting protein (C/EBPbeta-LIP), Friend of GATA-4 (FOG-2) protein and the viral E1A protein abolished the respective factor-dependent activities in HEK293 cells. Binding assays suggest that a dual binding of C/EBPbeta and GATA-4 to the promoter depends on the molar ratio of the factors present while demonstrating GATA-4 predominant association with the promoter DNA. This pattern may reflect on StAR expression at the time of corpus luteum formation when C/EBPbeta levels peak, as does StAR expression.

Activation of p90 Rsk1 is sufficient for differentiation of PC12 cells.

We investigated the role of Rsk proteins in the nerve growth factor (NGF) signaling pathway in PC12 cells. When rat Rsk1 or murine Rsk2 proteins were transiently expressed, NGF treatment (100 ng/ml for 3 days) caused three- and fivefold increases in Rsk1 and Rsk2 activities, respectively. Increased activation of both wild-type Rsk proteins could be achieved by coexpression of a constitutively active (CA) mitogen-activated protein kinase (MAPK) kinase, MEK1-DD, which is known to cause differentiation of PC12 cells even in the absence of NGF. Rsk1 and Rsk2 mutated in the PDK1-binding site were not activated by either NGF or MEK1-DD. Expression of constitutively active Rsk1 or Rsk2 in PC12 cells resulted in highly active proteins whose levels of activity did not change either with NGF treatment or after coexpression with MEK1-DD. Rsk2-CA expression had no detectable effect on the cells. However, expression of Rsk1-CA led to differentiation of PC12 cells even in the absence of NGF, as evidenced by neurite outgrowth. Differentiation was not observed with a nonactive Rsk1-CA that was mutated in the PDK1-binding site. Expression of Rsk1-CA did not lead to activation of the endogenous MAPK pathway, indicating that Rsk1 is sufficient to induce neurite outgrowth and is the only target of MAPK required for this effect. Collectively, our data demonstrate a key role for Rsk1 in the differentiation process of PC12 cells.

The life cycle of the steroidogenic acute regulatory (StAR) protein: from transcription through proteolysis.

The Steroidogenic Acute Regulatory (StAR) protein is a mitochondrial protein required for the transport of cholesterol substrate to the P450scc enzyme located in the inner mitochondrial membranes of steroid producing cells. This study suggests that the acute regulation of the rodent StAR gene in the ovary is mediated by two factors, C/EBPbeta and GATA-4. Once translated, the StAR precursor protein is either imported into the mitochondria, or it is rapidly degraded in the cytosol. We predicted that in order to perpetuate StAR activity cycles, imported StAR should turn over rapidly to avoid a potentially harmful accumulation of the protein in sub-mitochondrial compartments. Pulse-chase experiments in metabolically labeled cells showed that: (a) the turnover rate of mature mitochondrial StAR protein (30 kDa) is much faster (t(1/2) = 4-5 h) than that of other mitochondrial proteins; (b) dissipation of the inner membrane potential (-delta psi) by carbonyl cyanide m-chlorophenylhydrazone (mCCCP) accelerates the mitochondrial degradation of StAR; (c) unexpectedly, the mitochondrial degradation of StAR is inhibited by MG132 and lactacystin, but not by epoxomicin. Furthermore, StAR degradation becomes inhibitor-resistant two hours after import. Therefore, these studies suggest a bi-phasic route of StAR turnover in the mitochondria. Shortly after import, StAR is degraded by inhibitor-sensitive protease(s) (phase I), whereas at later times, StAR turnover proceeds to completion through an MG132-resistant proteolytic activity (phase II). Collectively, this study defines StAR as a unique protein that can authentically be used to probe multiple proteolytic activities in mammalian mitochondria.