The Regulation of Janus Kinase/signal Transducer and Activator of Transcription Pathway in High Mobility Group Box-1 Protein Expression and Inflammatory Response in Peritoneal Macrophages

Abstract: High mobility group box-1 protein (HMGB1) is a member of the highly reserved nonhistone DNA binding protein family, which is widely found in eukaryote nucleus and plays an important role in DNA repair, replication, transcription, and reconstruction. Recent studies have suggested that HMGB1 might be a potential "late" inflammatory mediator in sepsis. It was reported that serum HMGB1 levels delayedly increased during sepsis, maintained for a long time, and positively related to the late mortality in septic models. Moreover, we found that treatment with HMGB1 inhibitor could decrease the 1 – to 6-day mortality of septic animals. Therefore, HMGB 1 probably might become a potential target for therapeutic intervention against sepsis. Nevertheless, the possible role and effect of HMGB 1 in the development of sepsis remains to be further elucidated, especially for its signal regulation mechanism.It has been demonstrated that Janus kinase/signal transduction and transcription activator (JAK/STAT) pathway is critically involved in the signaltransduction of many important inflammatory cytokines, including IL-1, IL-6, IL-10, IL-4, and IFN-y etc. On the other hand, there is a close relationship between HMGB1 and these inflammatory cytokines, showing interaction among them. Based on the evidence above mentioned, JAK/STAT pathway seems to play a potential role in the signal transduction of HMGB 1 expression and inflammatory response. In addition, our animal experiments showed that inhibition of JAK/STAT pathway could reduce HMGB1 mRNA expression in vital organs. The present study was performed to clarify the relationship between the activation of JAK/STAT pathway and expression as well as inflammatory effect of HMGB1 through cultivation of rat peritoneal macrophages in vitro, thereby attempting to develope new therapy strategies for sepsis from the point of signal transduction.This experiment was conducted to investigat the influence of LPS on gene expression and protein release of HMGB1 by use of RT-PCR, IP, ELISA and Western blot. Also, we observed the effect of LPS and HMGB1 on activation of JAK/STAT pathway by EMSA and Western blot. Moreover, the potential signal transduction of JAK/STAT in HMGB1 expression and inflammatory response was investigated by using specific inhibitors for JAK/STAT pathway.The main conclusions are as follows:1. LPS can induce HMGB1 expression and release in rat peritoneal macrophages. HMGB1 is produced lately and decreased slowly, which might be identified as "a late inflammatory cytokine" during sepsis.2. LPS can early activate JAK2 and STAT1/STAT3 in rat peritoneal macrophages.3. JAK-STAT pathway is critically involved in HMGBl gene expression induced by LPS, but not in HMGBl protein release.4. HMGBl can induce TNF-α expression and release in rat macrophage. TNF-a expression and release are quick upon HMGB1 stimulation, showing that TNF-a serves as an early inflammatory cytokine.5. HMGBl can activate STAT1 and STAT3 within short time, but has no marked influence on JAK2 activity.6. JAK-STAT pathway plays an important role in TNF-α gene expression induced by HMGB1, but not in TNF-α protein release…
Key words: Macrophage; High mobility group box-1 protein; Signal transduction; Janus kinase; signal transducer and activator of transcription; Tumor necrosis factor-α; Sepsis; Systemic inflammatory response syndrome; Rats