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.     

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.     

Mitochondrial biogenesis and thyroid status maturation in brown fat require CCAAT/enhancer-binding protein alpha

Brown fat differentiation in mice is fully achieved in fetuses at term and entails the acquisition of not only adipogenic but also thermogenic and oxidative mitochondrial capacities. The present study of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein alpha (C/EBPalpha-null mice) demonstrates that C/EBPalpha is essential for all of these processes. Developing brown fat from C/EBPalpha-null mice showed a lack of uncoupling protein-1 expression, impaired adipogenesis, and reduced size and number of mitochondria per cell when compared with wild-type mice. Furthermore, immature mitochondrial morphology was found in brown fat, but not in liver or heart, from C/EBPalpha-null mice. Concordantly, expression of both nuclear and mitochondrial genome-encoded genes for mitochondrial proteins was reduced in C/EBPalpha-null brown fat, although expression of mitochondrial rRNA and mitochondrial DNA content were unaltered. Expression of nuclear respiratory factor-2, thyroid hormone nuclear receptors, and peroxisome proliferator-activated receptor gamma coactivator-1, was delayed in C/EBPalpha-null brown fat. Iodothyronine 5'-deiodinase activity and thyroid hormone content were also reduced in brown fat from C/EBPalpha-null mice, indicating for the first time a crucial role for C/EBPalpha in controlling thyroid status in developing brown fat, which may contribute to impaired mitochondrial biogenesis and cell differentiation. When survival of C/EBPalpha-null mice was achieved by transgenically expressing C/EBPalpha only in the liver, a substantial recovery in brown fat differentiation was found by day 7 of postnatal age, which is associated with a compensatory overexpression of C/EBPdelta and C/EBPbeta.