Insulin suppresses transactivation by CAAT/Enhancer-binding proteins β (C/EBPβ). Signaling to p300/CREB-binding protein by protein kinase B disrupts interaction with the major activation domain of C/EBPβ
CAAT/enhancer-binding proteins (C/EBPs) play an important role in the regulation of gene expression in insulin-responsive tissues. We have found that a complex containing C/EBPbeta interacts with an insulin response sequence in the insulin-like growth factor-binding protein-1 (IGFBP-1) gene and that a C/EBP-binding site can mediate effects of insulin on promoter activity. Here, we examined mechanisms mediating this effect of insulin. The ability of insulin to suppress promoter activity via a C/EBP-binding site is blocked by LY294002, a phosphatidylinositol 3-kinase inhibitor, but not by rapamycin, which blocks activation of p70(S6 kinase). Dominant negative phosphatidylinositol 3-kinase and protein kinase B (PKB) block the effect of insulin, while activated PKB suppresses promoter function via a C/EBP-binding site, mimicking the effect of insulin. Coexpression studies indicate that insulin and PKB suppress transactivation by C/EBPbeta, but not C/EBPalpha, and that N-terminal transactivation domains in C/EBPbeta are required. Studies with Gal4 fusion proteins reveal that insulin and PKB suppress transactivation by the major activation domain in C/EBPbeta (AD II), located between amino acids 31 and 83. Studies with E1A protein indicate that interaction with p300/CBP is required for transactivation by AD II and the effect of insulin and PKB. Based on a consensus sequence, we identified a PKB phosphorylation site (Ser(1834)) within the region of p300/CBP known to bind C/EBPbeta. Mammalian two-hybrid studies indicate that insulin and PKB disrupt interactions between this region of p300 and AD II and that Ser(1834) is critical for this effect. Signaling by PKB and phosphorylation of Ser(1834) may play an important role in modulating interactions between p300/CBP and transcription factors and mediate effects of insulin and related growth factors on gene expression.
"Signaling via several pathways results in C/EBP phosphorylation and subsequent transport from the cytoplasm to the nucleus , –. Conversely, phosphorylation of other C/EBP residues by other signaling pathways can suppress the activation and DNA-binding potential of C/EBPβ , , , , . "
[Show abstract][Hide abstract] ABSTRACT: CCAAT-enhancer binding proteins are transcription factors that help to regulate a wide range of inflammatory mediators, as well as several key elements of energy metabolism. Because C/EBPs are expressed by rodent astrocytes and microglia, and because they are induced by pro-inflammatory cytokines that are chronically upregulated in the Alzheimer's disease (AD) cortex, we have investigated whether C/EBPs are expressed and upregulated in the AD cortex. Here, we demonstrate for the first time that C/EBPβ can be detected by Western blots in AD and nondemented elderly (ND) cortex, and that it is significantly increased in AD cortical samples. In situ, C/EBPβ localizes immunohistochemically to microglia. In microglia cultured from rapid autopsies of elderly patient's brains and in the BV-2 murine microglia cell line, we have shown that C/EBPβ can be upregulated by C/EBP-inducing cytokines or lipopolysaccharide and exhibits nuclear translocation possibly indicating functional activity. Given the known co-regulatory role of C/EBPs in pivotal inflammatory mechanisms, many of which are present in AD, we propose that upregulation of C/EBPs in the AD brain could be an important orchestrator of pathogenic changes.
PLoS ONE 01/2014; 9(1):e86617. DOI:10.1371/journal.pone.0086617 · 3.23 Impact Factor
"Consistent to this notion, it has been shown recently that exercise induces a reduction in C/EBPβ in cardiomyocytes, which mediates cardiomyocyte hypertrophy . In addition, the transactivation activity of C/EBPβ is suppressed by insulin, an anabolic hormone . Therefore, diverse signaling pathways that regulate protein homeostasis in striated muscles may converge upon C/EBPβ. "
[Show abstract][Hide abstract] ABSTRACT: Background
The p38 mitogen-activated protein kinases (MAPK) family plays pivotal roles in skeletal muscle metabolism. Recent evidence revealed that p38α and p38β exert paradoxical effects on muscle protein homeostasis. However, it is unknown why p38β, but not p38α, is capable of mediating muscle catabolism via selective activation of the C/EBPβ that upregulates atrogin1/MAFbx.
Tryptic phosphopeptide mapping was carried out to identify p38α- and p38β-mediated phosphorylation sites in C/EBPβ. Chromosome immunoprecipitation (ChIP) assay was used to evaluate p38α and p38β effect on C/EBPβ binding to the atrogin1/MAFbx promoter. Overexpression or siRNA-mediated gene knockdown of p38α and p38β, and site-directed mutagenesis or knockout of C/EBPβ, were used to analyze the roles of these kinases in muscle catabolism in C2C12 myotubes and mice.
Cellular expression of constitutively active p38α or p38β resulted in phosphorylation of C/EBPβ at multiple serine and threonine residues; however, only p38β phosphorylated Thr-188, which had been known to be critical to the DNA-binding activity of C/EBPβ. Only p38β, but not p38α, activated C/EBPβ-binding to the atrogin1/MAFbx promoter. A C/EBPβ mutant in which Thr-188 was replaced by alanine acted as a dominant-negative inhibitor of atrogin1/MAFbx upregulation induced by either p38β or Lewis lung carcinoma (LLC) cell-conditioned medium (LCM). In addition, knockdown of p38β specifically inhibited C/EBPβ activation and atrogin1/MAFbx upregulation induced by LCM. Finally, expression of active p38β in mouse tibialis anterior specifically induced C/EBPβ phosphorylation at Thr-188, atrogin1/MAFbx upregulation and muscle mass loss, which were blocked in C/EBPβ-null mice.
The α and β isoforms of p38 MAPK are capable of recognizing distinct phosphorylation sites in a substrate. The unique capacity of p38β in mediating muscle catabolism is due to its capability in phosphorylating Thr-188 of C/EBPβ.
"For instance, PKC-d-mediated p300 phosphorylation at serine 89 represses intrinsic acetyltransferase activity and transcriptional function (Yuan et al., 2002). In contrast, phosphorylation at serine 1835 by Akt/PKB enhances acetyltransferase activity (Huang and Chen, 2005), and also modulates p300 interaction with C/EBPb in response to insulin (Guo et al., 2001). Induction of phosphatidylinositol 3-kinase activity is associated with increased p300 stability and transcriptional activity (Chen et al., 2004). "
[Show abstract][Hide abstract] ABSTRACT: The transcriptional coactivator p300 is a ubiquitous nuclear phosphoprotein and transcriptional cofactor with intrinsic acetyltransferase activity. p300 controls the expression of numerous genes in cell-type and signal-specific manner, and plays a pivotal role in cellular proliferation, apoptosis, and embryogenesis. By catalyzing acetylation of histones and transcription factors, p300 plays a significant role in epigenetic regulation. Recent evidence suggests that abnormal p300 function is associated with deregulated target gene expression, and is implicated in inflammation, cancer, cardiac hypertrophy, and genetic disorders such as the Rubinstein-Taybi syndrome. The activity of p300 is regulated at multiple levels, including developmental stage-specific expression, post-translational modifications, subcellular localization, and cell-type and gene-specific interactions with transcription factors. Although p300 has been investigated extensively in epithelial and hematopoietic cells, its role in fibroblast biology and tissue repair has received little attention to date. Recent studies implicate p300 in the regulation of collagen synthesis by transforming growth factor-beta (TGF-beta). Both the acetyltransferase activity of p300 and its inducible interaction with Smad3 are essential for mediating TGF-beta-induced stimulation of collagen synthesis. As a signal integrator whose availability for intracellular interactions with transcription factors is strictly limiting, p300 mediates the antagonistic regulation of TGF-beta-induced collagen synthesis by IFN-gamma and TNF-alpha via intracellular competition for limiting amount of p300. Significantly, p300 is itself a direct transcriptional target of TGF-beta in normal fibroblasts, and its levels are significantly elevated in fibrotic lesions as well as in experimental models of fibrosis. The emerging appreciation of the importance of p300 in extracellular matrix (ECM) remodeling and fibrosis and novel insights concerning the regulation, mechanism of action, and significance of p300 in fibroblast biology are discussed in this minireview.
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