Mechanism And Significance Of Ubiquitination Enzyme BRCC36 In Regulating Smad3 Signaling Pathway In Cardiac Fibroblasts

Chronic stress load increased the most outstanding performance is high blood pressure. Long-term left ventricular high voltage can cause the heart of a series of pathophysiological reactions, eventually will take irreversible cardiac decompensation, lead to death.In addition heart valve disease and arrhythmia will cause the change of hemodynamic, resulting in increased ejection system pressure.Pressure overload mainly for increased myocardial fibrosis, extracellular matrix deposition, and myocardial compliance and shu function decline, etc.Cardiac fibroblasts proliferation, migration, into a fibroblast synthesis of extracellular matrix is an important pathophysiological process.If im can inhibit the proliferation and, therefore, reduce the synthesis of extracellular matrix, the outcome of cardiac function has an important positive significance.BRCC36is a newly protein which encoding by BRCC3gene, show the specificity of removing ubiquitin chains on Lys63loci, is an important member of JAB1/MPN/Mov34metal enzyme structure domain (JAMM) classes ubiquitin enzyme. Through our previous research, BRCC36can suppress TGF-β signaling pathway by ubiquitining Smad3, achieve the function of anti-inflammatory, anti-apoptotic and anti fibrosis. This significance of this research is showing the new negative regulatory mechanism of TGF-β by BRCC36, these results will provide new thought for research and development of new clinical treatment strategies.Objective:To observe the process of myocardial fibrosis, the effects and mechanism of BRCC36on Smad3,the influence on myocardial fibrosis formation induced by chronic stress load by regulate BRCC36expression level in CFs, in order to provides new thought and new molecular target for developing effective treatments strategies.Method:Using in vitro culture of the second generation of cardiac fibroblasts (using the specificity for α-SMA and anti-Vimentin to identification), using overexpression of BRCC36adenovirus and cut expression of BRCC36adenovirus, then infect CFs, make its produce the difference of BRCC36expression, marking to BRCC36overexpression group, cut expression group, blank control group,respectively. At the same time give cytokines TGF-β1stimulation respectively. In order to illustrate BRCC36regulation mechanism of Smad3signal pathways in CFs,we detection the expression level of BRCC36in CFs, the phosphorylation of Smad3by western blot, test the distribution of Smad3and nuclear ingression in CFs by immunofluorescence.Results1. When given TGF-β1to BRCC36expression group, smad3phosphorylation level significantly decreased compared with blank control group. In BRCC36cut expression group, smad3phosphorylation level not change compared with blank control group, suggesting BRCC36overexpression can inhibit TGF-β1pathway activation in CFs significantly, characterized by suppressed phosphorylation of smad3, the result is the fibrosis has been refrained in CFs.2. By immunofluorescence staining BRCC36, smad3protein in cells, it was found that in the control group1hour after given TGF-β1treated, smad3significant shift from whole cells scattered in the state transitions to the state of aggregation in the nucleus. Suggesting that the pathway is activated. The BRCC36over-expression group smad3nuclear transfer is significantly lower than the control group (P<0.01), BRCC36cut expression group smad3nuclear transfer is significantly increased than the control group. Showed that overexpression BRCC36prevent intracellular signaling molecules smad3into the nuclear pathway.3、Using in vitro cultured cardiac fibroblasts, when give TGF-β1stimulation, collected from different time points (0,0.5,1,6,12,24hours) protein to detect the expression of BRCC36level.We found BRCC36will occur spontaneously rise after give TGFb-β1stimulus, to12hours can reach the highest, increased2.45times(P<0.01), then began to decline, prompt BRCC36may be feedback as a negative feedback factors regulate TGF pathways activated, make its controlled, not excessive activation.Conclusion:BRCC36protein expression in cardiac fibroblasts, inhibits TGF-β1pathway activation.Its mechanism is as followed.①.Overexpress BRCC36can withhold Smad3in cytoplasm.②.Overexpressing BRCC36can inhibits the phosphorylation of smad3, preventing smad3convert to active model.③. Overexpression BRCC36can inhibits samd3into nuclear, stop signal transfer into the nucleus, thus inhibiting the target gene transcription.④. BRCC36as a kind of negative feedback factor, can inhibit TGF-β1signaling pathway over activation.