The Molecular Mechanism Of NF-κB Dependent Pro-inflammatroy Transcription

Atherosclerosis proceeds through a multi-step reaction that begins with endothelial injury caused by a host of stress signals including oxidative stress and inflammatory stimuli. The dysfunction of endothelium includes1) the aggregative adhesion of leukocyte to the vessel wall mediated by the up-regulation of cell adhesion molecules (CAM) expression; and2) the loss of vessel tone caused by decreased endothelial nitric oxide synthase (eNOS) expression leading to impaired availability of endothelium derived NO. However, the molecular mechanism of transcriptional regulation in endothelial dysfunction caused by stress stimuli remains poorly understood.As a transcriptional mediator, Myocardin-related transcription factor A (MRTF-A, also known as MKL1) have been to mediate transcriptional regulation of vascular smooth muscle cells (VSMC)-specific genes associated with contractile phenotype of SMC. Little is known regarding the role of MRTF-A in other cell types within the vasculature including vascualr endothelial cells (VECs). In the present study, we examined the role of of MRTF-A in ox-LDL induced endothelial injury. Expression levels as well as nuclear localization of were both increased in immortalized and primary endothelial cells in response to oxLDL treatment accompanying the upregulation of ICAM-1and downregualtion of eNOS. Overexpression of MRTF-A synergistically enhanced the stimulation of ICAM-1transcription and the repression of eNOS transcription by ox-LDL. In contrast, silencing MRTF-A by small interfering RNA or dominant negative mutation abolished the pathogenic program triggered by ox-LDL. Moreover, chromatin immunoprecipitation (ChIP) assays demonstrate that ox-LDL augmented MRTF-A recruitment to the ICAM-1and eNOS promoters. Importantly, the occupancy of MRTF-A on the ICAM-1promoter required NF-κB/p65whereas oxLDL promoted the formation of an MRTF-A/p65complex on the ICAM-1promoter. Finally, MRTF-A orchestrated key histone modifications on the ICAM-1and eNOS promoters. Taken together, we demonstrate that MRTF-A cooperates with p65to modulate the inflammatory response in ox-LDL induced endothelial dysfunction.Co-immunoprecipitations revealed a specific interaction between MRTF-A and p65, indicating that MRTF-A may play a potentially important role in p65dependent inflammatory response in macrophages. In response to TNF-a treatment, MRTF-A enhanced the transcription of p65target genes. On the contrary, MRTF-A knockdown attenuated the induction of a slew of pro-inflammatory mediators including IL1, IL6, MCP1and TNF-α in macrophages. These observations were corroborated by the microarray analysis of expression profiles in wild type and MRTF-A deficient mouse embroynic fibroblast (MEF) cells. Meanwhile, MRTF-A was enlisted to the proximal promoter regions of pro-inflammatory mediator genes in macrophages with a similar kinetics as p65. Knockdown of p65abrogated the occupancy of MRTF-A. Reciprocally, depletion of MRTF-A also destabilizes p65binding on its target promoters. More importantly, TNF-αcpromoted the formation of MRTF-A/p65complex on gene promoters. Mass spectrometry combined co-immunoprecipitation assay revealed that MRTF-A existed in a large histone H3K4methyltransferase complex that includes ASH2. In reporter assays, ASH2and other members of histone H3K4methyltransferase complex including SET1α and WDR5enhanced promoter activities of the pro-inflammatory genes induced by MRTF-A. Silencing MRTF-A blocked the recruitment of histone H3K4methyltransferases preventing the key epigenetic alterations. MRTF-A null mice exhibited diminished response to lipopolysaccharide (LPS) induced septic shock and dextran sulfate sodium (DSS) induced colitis,raising the possibility that MRTF-A might act as a key epigenetic coordinator determining the intensity and duration of p65-dependent pro-inflammatory transcription in macrophages. Therefore, therapeutic strategies targeting MRTF-A could serve as a potential treatment option to ameliorate inflammatory response.The transactivation of adhesion molecules including ICAM-1, VCAM-1and E-selection in endothelial injury in response to pro-inflammatory stimuli represents a key step in the pathogenesis of a host of cardiovascular diseases. The epigenetic regulation of this process remains largely undefined. Brahma related gene1(Brgl) and Brahma (Brm) are core components of the chromatin remodeling complex that utilizes ATP hydrolysis to alter the positioning of nucleosomes. Therefore, we tackled the role of Brgl/Brm in p65-dependent transactivation of CAM molecules in endothelial cells in response to pro-inflammatory stimuli as well as its implication in the pathogenesis of atherosclerosis. We find that pro-inflammatory stimuli augmented the expression of Brgl and Brm in vitro in cultured endothelial cells and in vivo in arteries isolated from rodents. Knockdown of Brgl and Brm abrogated transactivation of adhesion molecules and leukocyte adhesion induced by inflammatory signals. Compatible with these finding, over-expression of Brgl and Brm in SW-13cells in which there is no endogenous expression of Brgl and Brm restored the expression of adhesion molecules. Intriguingly, Brgl and Brm interacted with and were recruited to the CAM promoters by p65. Conversely, depletion of Brg1and Brm disturbed the kinetics of p65binding on CAM promoters and crippled CAM activation. Silencing of Brgl and Brm also altered key epigenetic changes associated with CAM transactivation. Most importantly, endothelial-targeted elimination of Brgl/Brm conferred atheroprotective effects to Apoe-/-mice on a Western diet. Therefore, our data suggest that Brgl and Brm integrate various pro-inflammatory cues into CAM transactivation and endothelial malfunction and as such may serve as potential therapeutic targets in treating inflammation related cardiovascular diseases.In conclusion, our studies have presented key evidence for the clarification of the molecular mechanism underlying p65induced inflammatory response. These results demonstrate that MRTF-A is transcriptional mediator while Brgl and Brm are epigenetic modulators in the regulation of NF-κB/p65targeted genes. As such,. MRFT-A and Brgl/Brm are potential therapeutic targets in the prevention and/or intervention of inflammatory diseases.