Activation of nuclear protein binding to the antioxidant/electrophile response element in vascular smooth muscle cells by benzo(a)pyrene

This laboratory has previously shown that binding of nuclear proteins to the antioxidant/electrophile response element (ARE/EpRE) participates in deregulation of vascular gene expression by benzo(a)pyrene (BaP), a suspected atherogen. In the present study, oligonucleotides representing ARE/EpREs within the c-Ha-ras and glutathione-S-transferase (GST-Ya) promoters were employed to evaluate the role of flanking sequences in stabilizing protein:DNA interactions in BaP-treated vascular smooth muscle cells (vSMCs). We also wanted to define promoter-specific patterns of protein recognition to ARE/EpREs in this cell system. In electrophoretic mobility shift assays (EMSA), optimal protein binding to a human Ha-ras ARE/EpRE variant sequence fitted to match the extended mouse(m) GST-Ya ARE/EpRE core (5'-TMAnnRTGAYnnnGCR-3') was dependent on 5' nucleic acid sequence. Using immobilized DNA affinity chromatography (IDAC), we identified four nuclear proteins of M(r) 62, 60, 50, and 30 kDa that associated specifically with the mGSTYa ARE/EpRE. Photo crosslinking to a BrdU-substituted hHa-ras or mGST ARE/EpRE probe identified specific proteins of M(r) 80, 60, 55, 25, 23 kDa or 80, 60, 55, 27, 25, 23 kDa, respectively. Protein:DNA complexes detected using IDAC eluate overlapped with those observed in crude nuclear extracts. Chemical treatments known to modulate ARE/EpRE protein binding in vSMCs did not alter overall protein:DNA affinity and/or sequence recognition to either hHa-ras or mGST-Ya elements. We conclude that nucleotide sequences 5' to the core ARE/EpRE influence specific binding of nuclear proteins and that multiple proteins bind to ARE/EpREs in a promoter-specific manner in vSMCs.     

CCAAT/enhancer-binding protein beta isoforms and the regulation of alpha-smooth muscle actin gene expression by IL-1 beta

The role of IL-1beta in inflammation is amply documented, but its ability to inhibit myofibroblast differentiation and, in particular, the suppression of alpha-smooth muscle actin (alpha-SMA) gene expression is less well understood. Because IL-1beta can induce C/EBPbeta expression, the role of C/EBPbeta isoforms in IL-1beta regulation of alpha-SMA gene expression was investigated in rat lung myofibroblasts. The results showed that IL-1beta inhibited alpha-SMA expression in a dose-dependent manner, which was associated with stimulation of the expression of both C/EBPbeta isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP). However, a greater increase in LIP relative to LAP expression resulted in a reduced LAP/LIP ratio after IL-1beta treatment. Transfection with an LAP-expressing plasmid stimulated, whereas an LIP-expressing plasmid inhibited, alpha-SMA expression. Cells from C/EBPbeta-deficient mice had reduced levels of alpha-SMA expression and promoter activity, which failed to respond to IL-1beta treatment. Sequence analysis identified the presence of a C/EBPbeta consensus binding sequence in the alpha-SMA promoter, which, when mutated, resulted in diminished promoter activity and abolished its responsiveness to IL-1beta treatment. EMSA revealed binding of C/EBPbeta to this C/EBPbeta consensus binding sequence from the alpha-SMA promoter. Finally, IL-1beta enhanced the expression of eukaryotic initiation factor 4E, a stimulator of LIP expression, which may account for a mechanism by which IL-1beta could alter the LAP/LIP ratio. These data taken together suggest that C/EBPbeta isoforms regulate alpha-SMA gene expression, and that its inhibition by IL-1beta was due to preferential stimulation of LIP expression.     

DNA sequence determinants of nuclear protein binding to the c-Ha-ras antioxidant/electrophile response element in vascular smooth muscle cells: identification of Nrf2 and heat shock protein 90 beta as heterocomplex components

The antioxidant/electrophile response element (ARE/EpRE) is a cis-acting element involved in redox regulation of c-Ha-ras gene. Protein binding to the ARE/EpRE may be credited to deoxyribonucleic acid sequence; therefore, studies were conducted to evaluate the influence of internal and flanking regions to the 10-bp human c-Ha-ras ARE/EpRE core (hHaras10) on nuclear protein binding in oxidant-treated vascular smooth muscle cells. A protein doublet bound to an extended oligonucleotide comprising the ARE/EpRE core in genomic context (hHaras27), whereas a single complex bound to hHarasl0. Protein binding involved specific interactions of 25- and 23-kDa proteins with hHarasl0, and binding of 80-, 65-, and 55-kDa proteins to hHaras27. Competition assays with hNQO1 and rGSTA2 confirmed the specificity of deoxyribonucleic acid-protein interactions and indicated preferred binding of p25 and p23 to the c-Ha-ras ARE/EpRE. "NNN" sequences within the core afforded unique protein-binding profiles to the c-Ha-ras ARE/EpRE. In addition, Nrf2 and heat shock protein 90beta (p80) were identified as components of the c-Ha-ras ARE/EpRE heterocomplex. We conclude that both internal bases and flanking sequences regulate nuclear protein recruitment and complex assembly on the c-Ha-ras ARE/EpRE.     

Role of CREB in transcriptional regulation of CCAAT/enhancer-binding protein beta gene during adipogenesis

The proximal promoter of the C/EBPbeta gene possesses dual cis regulatory elements (TGA1 and TGA2), both of which contain core CREB binding sites. Comparison of the activities of C/EBPbeta promoter-reporter genes with 5'-truncations or site-directed mutations in the TGA elements showed that both are required for maximal promoter function. Electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) analyses with antibodies specific to CREB and ATF1 showed that these CREB family members associate with the proximal promoter both in vitro and ex vivo. Immunoblotting and ChIP analysis revealed that other CREB family members, CREM and ATF1, are up-regulated and associate with the proximal C/EBPbeta promoter in mouse embryonic fibroblasts (MEFs) from CREB(-/-) mice. ChIP analysis of wild-type MEFs and 3T3-L1 preadipocytes revealed that interaction of phospho-CREB, the active form of CREB, with the C/EBPbeta gene promoter occurs only after induction of differentiation of 3T3-L1 preadipocytes and MEFs. Consistent with the interaction of CREB and ATF1 at the TGA regulatory elements, expression of constitutively active CREB strongly activated C/EBPbeta promoter-reporter genes, induced expression of endogenous C/EBPbeta, and caused adipogenesis in the absence of the hormonal inducers normally required. Conversely, expression of a dominant-negative CREB blocked promoter-reporter activity, expression of C/EBPbeta, and adipogenesis. When subjected to the standard adipocyte differentiation protocol, wild-type MEFs differentiate into adipocytes at high frequency, whereas CREB(-/-) MEFs exhibit greatly reduced expression of C/EBPbeta and differentiation. The low level of expression of C/EBPbeta and differentiation in CREB(-/-) MEFs appears to be due to up-regulation of other CREB protein family members, i.e. ATF1 and CREM.     

Peroxisome proliferator-activated receptor-gamma activation inhibits interleukin-1beta -mediated platelet-derived growth factor-alpha receptor gene expression via CCAAT/enhancer-binding protein-delta in vascular smooth muscle cells

CCAAT/enhancer-binding protein (C/EBP)-binding motifs have been identified in the promoter regions of interleukin (IL)-6, tumor necrosis factor-alpha, and platelet-derived growth factor-alpha receptor (PDGFalphaR). Recently, peroxisome proliferator-activated receptors (PPARs) have been suggested to be important immunomodulatory mediators. Although many studies have demonstrated that the interaction between C/EBPs and PPARs plays a central role in lipid metabolism, expression and function of these factors are unknown in vascular smooth muscle cells (VSMCs). In the present study, we clarified a functional relationship between C/EBPs and PPARgamma in the regulation of IL-1beta-induced PDGFalphaR expression in VSMCs. PPARgamma activators, troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2), inhibited IL-1beta-induced PDGFalphaR expression and suppressed PDGF-induced proliferation activity of VSMCs. Electromobility shift and supershift assays for a C/EBP motif in the PDGFalphaR promoter region revealed that PPARgamma activators suppressed IL-1beta-induced DNA binding activity of C/EBPdelta and beta. PPARgamma activators also suppressed IL-1beta-induced C/EBPdelta expression. In contrast, overexpression of C/EBPdelta reversed the suppressive effect of PPARgamma activators on PDGFalphaR expression almost completely. From these results, we conclude that the inhibitory effect of PPARgamma activators on PDGFalphaR expression is mainly mediated by C/EBPdelta suppression. Regulation of C/EBPdelta by PPARgamma activators probably plays critical roles in modulating inflammatory responses in the arterial wall.     

A novel retinoid-response gene set in vascular smooth muscle cells

A modified suppression subtractive hybridization assay was performed to uncover genes induced by all-trans retinoic acid in cultured smooth muscle cells (SMC). Northern blotting studies confirmed the induction of 14 genes, many of which have heretofore been unrecognized as retinoid-inducible. Temporal expression and cycloheximide studies allowed us to categorize these genes as either immediate-early (LOX-1, endolyn, Stoned B/TFIIA alpha/beta-like factor, Src Suppressed C Kinase Substrate, and tissue transglutaminase) or delayed (cathepsin-L, ceruloplasmin, epithelin, importin alpha, alpha(8)-integrin, lactate dehydrogenase B, retinol dehydrogenase, spermidine/spermine N(1)-acetyltransferase, and VCAM-1) retinoid-response genes. A survey of rat tissues showed two of the genes (tissue transglutaminase and alpha(8)-integrin) to be highly restricted to vascular tissue. In situ hybridization verified expression of both tissue transglutaminase and alpha(8)-integrin to SMC in balloon-injured rat carotid artery. These findings unveil a new retinoid-response gene set that should be exploited to define molecular pathways involved in the antagonistic effects of retinoids on SMC growth and neointimal formation.