No abstract

Sterol regulatory element-binding proteins (SREBPs) 3 are transcription factors that regulate expression of genes controlling cholesterol homeostasis and de novo fatty acid synthesis (1-7). SREBP-1a and SREBP-1c, which differ only in their first exon, are derived from a single gene through the use of alternative promoters, whereas SREBP-2 is encoded by a separate gene (8). Although there is clearly some functional overlap among the three SREBP isoforms (5), these proteins regulate different metabolic pathways. SREBP-1c preferentially affects transcription of genes that regulate de novo lipid synthesis, whereas SREBP-2 regulates genes involved in cholesterol biosynthesis and metabolism. The SREBP-1a isoform transactivates both lipogenic and cholesterogenic genes (9). In addition, the three SREBP isoforms exhibit differential tissue-specific expression. In replicating tumor cell lines, SREBP-1a constitutes greater than 90% of the SREBP-1 pool; conversely, SREBP-1c is the predominant isoform in liver and adipose tissue (9). Increased hepatic levels of nuclear SREBP-1c are thought to mediate the development of hyperlipidemia in type II diabetes and hyperinsulinemia (10 -12). Nutritional and hormonal factors have been shown to regulate expression of SREBP-1c and its downstream regulatory targets (10,(13)(14)(15). Insulin induces the expression of SREBP-1c mRNA and nascent precursor protein (10,16,17). Glucagon opposes this effect of insulin via its second messenger cAMP (18). Newly synthesized SREBPs contain two transmembrane domains that are embedded in the endoplasmic reticulum (ER) with the NH 2 -and COOH-terminal sequences exposed to the cytoplasm. Following transport from ER to Golgi, the transcriptionally active NH 2 -terminal segments of SREBPs are liberated by two successive cleavages; the first cleavage in the loop extending into the vesicular lumen is carried out by site 1 protease (S1P), and the second cleavage is executed within the NH 2 -proximal transmembrane domain by site 2 protease (S2P).Regulation of post-translational proteolysis has been studied most extensively in the case of SREBP-2 and SREBP-1a, both of which are regulated primarily by sterols. Within the ER, the

The redox state plays an important role in gene regulation. Thiols maintain the intracellular redox homeostasis. To understand the role of thiols in redox signaling, we have studied the effect of thiol alkylation on platelet-derived growth factor-BB (PDGF-BB)-induced cell survival events in vascular smooth muscle cells. PDGF-BB stimulated Akt phosphorylation predominantly at Ser-473. N-Ethylmaleimide (NEM), a thiol alkylating agent, blocked PDGF-BB-induced Akt phosphorylation without affecting its upstream phosphatidylinositol 3-kinase (PI3K). On the other hand, LY294002 and wortmannin, specific inhibitors of PI3K, prevented PDGF-BB-induced phosphorylation of Akt and its downstream effector molecules, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. NEM also abrogated the phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E induced by PDGF-BB, suggesting that thiol alkylation interferes with the PI3K/Akt pathway at the level of Akt. In addition, NEM blocked PDGF-BBinduced phosphorylation of BAD and forkhead transcription factor FKHR-L1, and these events correlated with increased apoptosis. NEM alone and in concert with PDGF-BB increased reactive oxygen species (ROS) production and protein phosphatase 2A (PP2A) activity in VSMC. The inhibition of PDGF-BB-induced Akt phosphorylation by NEM was completely reversed by PP2A inhibitors fostriecin and okadaic acid, ceramide synthase inhibitor fumonisin B1, and ROS scavenger Nacetylcysteine (NAC). NAC also attenuated the apoptosis induced by NEM, alone or in combination with PDGF-BB. Together, these findings demonstrate for the first time that PP2A mediates thiol alkylation-dependent redox regulation of Akt and cell survival.The cellular redox state plays an important role in the regulation of gene expression in prokaryotes and eukaryotes (1-4). The following observations support this notion: 1) Oxidants regulate the activities of several transcription factors, including activator protein-1, nuclear factor kappa B, and p53 (5-7); 2) Oxidants are capable of activating several early response events, including stimulation of protein tyrosine phosphorylation, activation of mitogen-activated protein kinases and induction of expression of proto-oncogenes (8 -11); 3) Oxidants are produced acutely in response to various agents, including growth factors and cytokines in several cell types (12, 13), and a requirement for their production in the mitogenic effects of receptor tyrosine kinase and G protein-coupled receptor agonists has been demonstrated (14, 15); and 4) In addition to producing oxidants, cells also possess enzymatic and non-enzymatic mechanisms for their removal (16 -18), and this feature attests to the role of oxidants as second messenger molecules (19). Despite the growing body of information on the role of oxidants in the regulation of gene expression, the mechanisms by which these molecules transmit the extracellular signals from the plasma membrane to the nucleus are less clear. Thiols play a critical role in the reduction/oxidation reactions as w...

The regulation of lipid homeostasis by insulin is mediated in part by the enhanced transcription of the gene encoding sterol regulatory element-binding protein-1c (SREBP-1c). The nascent SREBP-1c is embedded in the endoplasmic reticulum (ER) and must be transported to the Golgi where two sequential cleavages generate its NH(2)-terminal fragment, nSREBP-1c. We have shown recently that in primary cultures of rat hepatocytes, insulin rapidly and selectively stimulates proteolytic processing of the nascent SREBP-1c by enhancing the affinity of the SREBP cleavage-activating protein (SCAP).SREBP-1c complex for coatomer protein complex II (COPII) vesicles. The SCAP.SREBP complex is retained in the ER by Insig proteins. We report here that insulin persistently stimulates controlled proteolysis of the nascent SREBP-1c by selectively reducing the level of Insig-2a protein via accelerated degradation of its cognate mRNA. Insulin enhanced the rate of turnover of Insig-2a mRNA via its 3'-untranslated region. Insulin-induced depletion of Insig-2a promotes association of the SCAP.SREBP-1c complex with COPII vesicles and subsequent migration to the Golgi where site-1 and site-2 proteases process the nascent SREBP-1c. Consistent with this mechanism, experimental knockdown of Insig-2a expression with small interfering RNA mimicked insulin-induced proteolysis of the nascent SREBP-1c, whereas exogenous expression of Insig-2a in hepatocytes led to reduced intramembrane proteolysis of the newly synthesized SREBP-1c. The action of insulin on the processing of the nascent SREBP-1c via Insig-2a was highly selective, as proteolysis of the newly synthesized SREBP-2 remained unchanged under identical conditions. On the basis of these data, we propose that the stimulation of SREBP-1c processing by insulin is mediated by a selective depletion of Insig-2a protein by promoting decay of its cognate mRNA. Thus, insulin-induced reduction in Insig-2a protein leads to an enhanced export of the SCAP.SREBP-1c complex from ER to the Golgi.

We have studied the role of nuclear factor of activated T-cells (NFAT) transcription factors in the induction of vascular smooth muscle cell (VSMC) growth by platelet-derived growth factor-BB (PDGF-BB) and thrombin, the receptor tyrosine kinase (RTK) and G-protein-coupled receptor (GPCR) agonists, respectively. NFATc1 but not NFATc2 or NFATc3 was translocated from the cytoplasm to the nucleus upon treatment of VSMCs with PDGF-BB or thrombin. Translocation of NFATc1 was followed by an increase in NFAT-DNA binding activity and NFAT-dependent reporter gene expression. Cyclosporin A (CsA), a potent and specific inhibitor of calcineurin, a calcium/calmodulin-dependent serine phosphatase involved in the dephosphorylation and activation of NFATs, blocked NFAT-DNA binding activity and NFAT-dependent reporter gene expression induced by PDGF-BB and thrombin. CsA also completely inhibited PDGF-BB- and thrombin-induced VSMC growth, as measured by DNA synthesis and cell number. In addition, forced expression of the NFAT-competing peptide VIVIT for calcineurin binding significantly attenuated the DNA synthesis induced by PDGF-BB and thrombin in VSMCs. Together, these findings for the first time demonstrate a role for NFATs in RTK and GPCR agonist-induced growth in VSMCs.