Molecular Mechanism Of Heat Shock Protein 27 Regulating CBP And Inhibiting Inflammatory Overreaction

Inflammation is a defensive response to harmful stimuli.In general,adaptive immune response is usually beneficial.However,when the body’s regulatory dysfunction is uncontrolled,the immune inflammatory response is out of control and immune dysfunction,leading to the development of systemic inflammatory response syndrome(SIRS)and sepsis.Heat-shock protein 27(Hsp27)is a member of the small heat shock protein family that has been reported to protect cells against pro-inflammatory stresses.High mobility group box 1(HMGB1)is a proinflammatory cytokine associated with death from sepsis and other inflammatory diseases,and how to effectively control the nucleocytoplasmic transport and extracellular release of HMGB1,especially the production of inflammatory factors caused by HMGB1 is the key to control mortality.CREB binding protein(CBP)is a transcriptional coactivator and acetyltransferase that has been reported to be involved in the pathogenesis of inflammation-related diseases.However,the studies on how CBP is regulated in excessive inflammation and its role in the pathogenesis of septic inflammatory diseases are not systematic and complete.In the present study,we report the mechanisms of Hsp27 inhibits HMGB1 translocation by regulating CBP acetyltransferase activity and ubiquitination,and that CBP bromodomain inhibitor prevents sepsis-related HMGB1 secretion and blocks HMGB1 pro-inflammatory activity.Firstly,we have studied the inhibitory effect of Hsp27 on the release of HMGB1 by regulating CBP acetyltransferase activity and ubiquitination.After being acetylated by CREB-binding protein(CBP),the transcriptional adaptor and acetyltransferase,HMGB1 translocates from the nucleus to the cytoplasm.In the present study,we investigated the effects of Hsp27 on HMGB1 translocation from the nucleus to the cytoplasm in THP-1 cells.We found that Hsp27 phosphorylation decreased LPS-induced HMGB1 acetylation and translocation from the nucleus to the cytoplasm,as well as its release from THP-1 cells.The study further showed that cytosolic non-phosphorylated Hsp27 enhanced CBP ubiquitination and degradation in LPS-unstimulated cells,which suggested that Hsp27 maintained suitable CBP levels under normal physiological conditions.After LPS stimulation,Hsp27 was phosphorylated at serine residues 15/78 and translocated from the cytoplasm into the nucleus.Consequently,LPS stimulation increased CBP levels and promoted its translocation into the nucleus.In the nucleus,Hsp27 bound to CBP and suppressed CBP acetyltransferase activity and the subsequent CBP-dependent acetylation of HMGB1.If the phosphorylation of Hsp27 is blocked after LPS stimulation,Hsp27 will not enter the nucleus and interact with CBP in the nucleus,which will lead to the increase of acetylation modification of HMGB1 and the increase of extracellular release of HMGB1.Secondly,the blocking effect of the CBP bromodomain potency inhibitor SGC-CBP30 on HMGB1-mediated inflammatory response was investigated in vivo and in vitro.In sepsis mice model,treatment with SGC-CBP30 at 8 hours after modeling could significantly increased the survival rate,improved tissue damage,and even reduced the level of HMGB1 and TNF-α in blood circulation.Data from THP-1 and mouse primary peritoneal macrophages showed that CBP bromodomain inhibition suppresses LPS-induced expression and release of HMGB1.Moreover,CBP restraint attenuated pro-inflammatory activity of HMGB1.Furthermore,our findings provide evidence that SGC-CBP30 down-regulated rh HMGB1-induced activation of MAPKs and NF-κB signaling by triggering the reactivation of PP2 A and the stabilization of MKP-1.In conclusion,we demonstrate that cytosolic non-phosphorylated Hsp27 enhanced the ubiquitin-mediated degradation of CBP,while phosphorylated Hsp27 inhibited CBP acetyltransferase activity in the nucleus.By regulating CBP,Hsp27 maintained cell homeostasis and inhibited excessive inflammatory response.At the same time,our work reveals that CBP potent inhibitor SGC-CBP30 has a good therapeutic effect on sepsis mice,and its mechanism of action is to reduce the expression and release of HMGB1 in sepsis,and to inhibit the pro-inflammatory activity of HMGB1 by saving the activity of PP2 A and the protein level of MKP-1.This provides a comprehensive explanation of the effectiveness of CBP as a therapeutic target for sepsis.It explores a useful strategy to control the occurrence and development of sepsis after the release of late inflammation from a new perspective,providing an important basis for the creation of anti-sepsis drugs.