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.     

Age-specific CUGBP1-eIF2 complex increases translation of CCAAT/enhancer-binding protein beta in old liver

The RNA-binding protein CUGBP1 regulates translation of proteins in a variety of biological processes. In this study, we show that aging liver increases CUGBP1 translational activities by induction of a high molecular weight protein-protein complex of CUGBP1. The complex contains CUGBP1, subunits alpha, beta, and gamma of the initiation translation factor eIF2, and four proteins of the endoplasmic reticulum, eR90, CRT, eR60, and Grp78. The induction of the CUGBP1-eIF2 complex in old livers is associated with the elevation of protein levels of CUGBP1 and with the hyper-phosphorylation of CUGBP1 by a cyclin D3-cdk4 kinase, activity of which is increased with age. We have examined the role of the elevation of CUGBP1 and the role of cyclin D3-cdk4-mediated phosphorylation of CUGBP1 in the formation of the CUGBP1-eIF2 complex by using CUGBP1 transgenic mice and young animals expressing high levels of cyclin D3 after injection with cyclin D3 plasmid. These studies showed that both the increased levels of CUGBP1 and cdk4-mediated hyper-phosphorylation of CUGBP1 are involved in the age-associated induction of the CUGBP1-eIF2 complex. The CUGBP1-eIF2 complex is bound to C/EBPbeta mRNA in the liver of old animals, and this binding correlates with the increased amounts of liver-enriched activator protein and liver-enriched inhibitory protein. Consistent with these observations, the purified CUGBP1-eIF2 complex binds to the 5' region of C/EBPbeta mRNA and significantly increases translation of the three isoforms of C/EBPbeta in a cell-free translation system, in cultured cells, and in the liver. Thus, these studies demonstrated that age-mediated induction of the CUGBP1-eIF2 complex changes translation of C/EBPbeta in old livers.     

Surfactant protein D gene regulation. Interactions among the conserved CCAAT/enhancer-binding protein elements

Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following acute lung injury. Given the importance of CCAAT/enhancer-binding protein (C/EBP)-binding elements in the systemic acute-phase response and lung development and the expression of C/EBP isoforms by lung epithelial cells, we hypothesized that conserved C/EBP motifs in the near-distal and proximal promoters contribute to the regulation of SP-D expression by C/EBPs. Five SP-D motifs (-432, -340, -319, -140, and -90) homologous to the C/EBP consensus sequence specifically bound to C/EBPs in gel shift assays, and four of the five sites (-432, -340, -319, and -90) efficiently competed for the binding of C/EBPalpha, C/EBPbeta, or C/EBPdelta to consensus oligomers. Cotransfection of C/EBPalpha, C/EBPbeta, or C/EBPdelta cDNA in H441 lung adenocarcinoma cells significantly increased the luciferase activity of a wild-type SP-D promoter construct containing 698 bp of upstream sequence (SS698). Transfection of C/EBP also increased the level of endogenous SP-D mRNA in H441 cells. Transactivation of the reporter construct was abrogated by deletion of sequences upstream of -205. Independent site-directed mutagenesis of the sites at -432, -340, and -319 reduced C/EBP-mediated activation by approximately 50%, and mutagenesis of the site at -432 in combination with either of the tandem sites at -340 and -319 blocked activation. The conserved AP-1 element at -109 was required for maximal promoter activity, but not for the transactivation of SS698 by C/EBPs. Thus, interactions among C/EBP elements in the near-distal promoter can modulate the promoter activity of SP-D.     

Identification of new sites of binding and activation of the murine alpha1(I) collagen promoter by CCAAT/enhancer binding protein beta

The CCAAT/enhancer binding protein beta (C/EBPbeta) was previously shown to bind to the alpha(1)(I) collagen promoter at -365 to -335 (site 1) and to activate it. Acetaldehyde also activates the promoter, and this effect is mediated by an increase in stellate-cell C/EBPbeta protein and C/EBPbeta binding. The present study identified two additional distal sites (sites 2 and 3) of binding of C/EBPbeta, in the nuclear extracts of stellate cells, at -399 to -370 and -623 to -592 in the alpha(1)(I) collagen promoter. The C/EBPbeta protein activates the promoter at all three sites. Acetaldehyde increases C/EBPbeta binding to all three sites. Activation by acetaldehyde is abrogated in the transfected promoter mutated at either site 1 or site 3 but is not affected by mutation at site 2. Binding of the 20-kDa C/EBPbeta isoform (p20C/EBPbeta), which is eliminated by mutation at the distal site 3 of C/EBP binding, is necessary for the activation by acetaldehyde of the alpha(1)(I) collagen promoter.