Communication between NF-kappa B and Sp1 controls histone acetylation within the proximal promoter of the monocyte chemoattractant protein 1 gene

The induction of the monocyte chemoattractant protein 1 gene (MCP-1) by TNF occurs through an NF-kappaB-dependent distal regulatory region and an Sp1-dependent proximal regulatory region that are separated by 2.2 kb of sequence. To investigate how these regions coordinate activation of MCP-1 in response to TNF, experiments were performed to examine the role of coactivators, changes in local chromatin structure, and the acetylation of histones at the MCP-1 regulatory regions. An E1a-sensitive coactivator was found to be required for expression. In vivo nuclease sensitivity assays identified changes in response to TNF at both the proximal and distal regions that were dependent on the p65 subunit of NF-kappaB and Sp1. Chromatin immunoprecipitations used to analyze factor assembly and histone acetylation at the distal and proximal regions showed that Sp1 binding to and histone acetylation of the proximal region was dependent on NF-kappaB p65. Conversely, Sp1 assembly at the proximal region was required for p65 binding to and acetylation of the distal region, suggesting communication between the two regions during gene activation. These data and the NF-kappaB p65-dependent histone acetylation of a middle region sequence suggest a potential order for the assembly, acetylation and accessibility of the MCP-1 regulatory regions in response to TNF.     

Functional role of the cis-acting elements in human monocyte chemotactic protein-1 gene in the regulation of its expression by phorbol ester in human glioblastoma cells

The monocyte chemotactic protein-1 (MCP-1) is a 76 amino acid protein that specifically attracts monocytes. The expression of MCP-1 gene can be induced by lipopolysaccharides (LPS), phorbol esters (TPA) and several cytokines. However, how they regulate MCP-1 gene expression is not known. We tested whether the two putative TPA-responsive elements (TREs) and one B enhancer-like region found in the MCP-1 promoter region, are involved in this regulation of MCP-1 gene expression. The 5 untranslated region of MCP-1 gene was linked to chloramphenicol acetyl transferase (CAT) reporter gene and transfected into human glioblastoma cells in which endogenous MCP gene expression was found to be stimulated by TPA and tumor necrosis factor- (TNF-). The 128 bp 5-flanking region containing one TRE was adequate for basal promoter activity but the presence of both TREs in the MCP-1 promoter region were needed to give TPA responsive enhancement (2.5 fold) of expression of the marker gene. Mutations in either of the TRE's could abolish the TPA induction of CAT expression. Replacement of the B enhancer-like element with a TRE-like sequence caused a 10-fold enhancement of CAT expression by TPA treatment. Random mutation of B enhancer-like element did not affect CAT expression or its TPA induction. None of the MCP promoter constructs showed significant increase in CAT expression by treatment with tumor necrosis factor- (TNF-). This result suggested that the TNF regulation of MCP-1 gene involves other parts of the gene besides the proximal 5 flanking region. (Mol Cell Biochem141: 121–128, 1994)     

Retinoic acid-mediated down-regulation of Oct3/4 coincides with the loss of promoter occupancy in vivo.

Oct3/4, a hallmark of the earliest stages of embryogenesis, is expressed in undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cells. Oct3/4 gene expression is dependent on the promoter region, the proximal enhancer and the newly identified distal enhancer. We have analysed in vivo occupancy of these elements. In undifferentiated EC and ES cells, strong footprints were detected at specific sites of all three regulatory elements. These were promptly lost upon RA treatment in ES cells and in P19 EC cells, in parallel with sharply reduced Oct3/4 mRNA levels. Thus, the occupancy of regulatory elements is coupled with Oct3/4 expression, and RA treatment causes coordinated factor displacement, leading to extinction of gene activity. In F9 EC cells, footprint was first abolished at the proximal enhancer. However, this loss of binding site occupancy did not result in a decrease in Oct3/4 mRNA levels. The partial factor displacement seen in F9 EC cells, combined with the observation that EC and ES cells utilize the proximal and distal enhancers in differential manner, indicate the complex pattern of Oct3/4 gene regulation, which could reflect a cell type- and lineage-specific expression of the gene in vivo.     

Activation of PKC-epsilon and ERK1/2 participates in shear-induced endothelial MCP-1 expression that is repressed by nitric oxide

Vascular endothelial cells (ECs) continuously experience hemodynamic shear stress generated from blood flow. Previous studies have demonstrated that shear stress modulates monocyte chemotactic protein-1 (MCP-1) expression in ECs. This study explored the roles of protein kinase C (PKC), extracellular signal-regulated protein kinase (ERK1/2), and nitric oxide (NO) in sheared-induced MCP-1 expression in ECs. The activation of PKC-alpha and PKC-epsilon isoforms was observed in ECs exposed to shear stress. The use of an inhibitor (calphostin C) to PKC-alpha and PKC-epsilon decreased ERK1/2 activation and MCP-1 induction by shear, whereas an inhibitor (Go6976) to PKC-alpha did not affect ERK1/2 activation or MCP-1 induction. Inhibition of ERK1/2 activation by PD98059 blocked MCP-1 induction. Transfection of ECs with an antisense to PKC-epsilon abolished the shear inducibility of MCP-1 promoter. These results demonstrate that PKC-epsilon and ERK1/2 participate in shear-induced MCP-1 expression. We also examined the regulatory role of NO in MCP-1 expression. An NO donor (NOC18) suppressed shear-induced activation of PKC-epsilon and ERK1/2, and also repressed MCP-1 induction. Consistently, overexpression of endothelial nitric oxide synthase (eNOS) to enhance the endogenous generation of NO in ECs decreased the activation of PKC-epsilon and ERK1/2, and also inhibited MCP-1 expression. Taken together, these findings suggest that PKC-epsilon and ERK1/2 are critical in the signaling pathway(s) leading to the MCP-1 expression induced by shear stress. Additionally, this study indicates that NO, by repressing PKC-epsilon activity and ERK pathway activation, attenuates shear-induced MCP-1 expression.     

Angiotensin II induces monocyte chemoattractant protein-1 expression via a nuclear factor-kappaB-dependent pathway in rat preadipocytes

Both monocyte chemoattractant protein-1 (MCP-1), a member of chemokine family, and angiotensinogen, a precursor of angiotensin (ANG) II, are produced by adipose tissue and increased in obese state. MCP-1 has been shown to decrease insulin-stimulated glucose uptake and several adipogenic genes expression in adipocytes in vitro, suggesting its pathophysiological significance in obesity. However, the pathophysiological interaction between MCP-1 and ANG II in adipose tissue remains unknown. The present study was undertaken to investigate the potential mechanisms by which ANG II affects MCP-1 gene expression in rat primary cultured preadipocytes and adipose tissue in vivo. ANG II significantly increased steady-state MCP-1 mRNA levels in a time- and dose-dependent manner. The ANG II-induced MCP-1 mRNA and protein expression was completely abolished by ANG II type 1 (AT1)-receptor antagonist (valsartan). An antioxidant/NF-kappaB inhibitor (pyrrolidine dithiocarbamate) and an inhibitor of 1kappaB-alpha phosphorylation (Bay 11-7085) also blocked ANG II-induced MCP-1 mRNA expression. ANG II induced translocation of NF-kappaB p65 subunit from cytoplasm to nucleus by immunocytochemical study. Luciferase assay using reporter constructs containing MCP-1 promoter region revealed that two NF-kappaB binding sites in its enhancer region were essential for the ANG II-induced promoter activities. Furthermore, basal mRNA and protein of MCP-1 during preadipocyte differentiation were significantly greater in preadipocytes than in differentiated adipocytes, whose effect was more pronounced in the presence of ANG II. Exogenous administration of ANG II to rats led to increased MCP-1 expression in epididymal, subcutaneous, and mesenteric adipose tissue. In conclusion, our present study demonstrates that ANG II increases MCP-1 gene expression via ANG II type 1 receptor-mediated and NF-kappaB-dependent pathway in rat preadipocytes as well as adipose MCP-1 expression in vivo. Thus the augmented MCP-1 expression by ANG II in preadipocytes may provide a new link between obesity and cardiovascular disease.