Anti-inflammation Strategies Based On Thrombomodulin

Background: Inflammation has been recognized as an important process in the pathogenesis of many diseases. Thrombomodulin (TM) is a cell surface-expressed glycoprotein which has received much attention in recent years because of its anti-inflammatory effect. So the advancement of TM molecule will lead to the development of new possible therapies for atherosclerosis as well as other inflammatory diseases.Recent studies have identified TM as an antagonist of High mobility group box chromosomal protein 1(HMGB1), via the N-terminal lectin-like domain (PD1), binds HMGB1, thereby inhibiting a HMGB1-mediated inflammatory response. It has been demonstrated that the DNA-binding domain of HMGB1, the A box, could attenuate HMGB1-induced release of proinflammatory cytokines and significantly rescue mice from sepsis lethality. However, molecular weight of A box is just about 8 kDa, which makes it very difficult to express stably and biofunctionally in E.coli . Thus, any means that could overcome the problems described above could potentially make A box as a novel therapeutic approach in the clinical management of sepsis. Fusion protein technology is an effective way to achieve the goal by enhancing expression, decreasing the proteolytic degradation, and facilitating purification of the recombinant proteins. Accordingly, it is unclear whether is the existence of a fusion partner which can not only enhance expression level of the A box but also improve its anti- HMGB1 effect. So TMPD1 made of 155 amino acid residues can be a candidate of the fusion partner as described previously.Moreover, upregulation of TM provides an alternative to treat inflammatory diseases. The nuclear farnesoid X receptor (FXR) plays an important role in glucose metabolism, cholesterol metabolism, TG metabolism, so does the nuclear liver X receptor (LXR). The two nuclear receptors mention above also repress inflammatory responses, but their molecular mechanisms remain poorly understood. FXR,LXR and TM are highly expressed on vascular endothelial cells, then whether FXR or LXR could regulate TM to inhibit inflammation? The study on the effect of FXR or LXR on TM will provide useful information with regarding to the physiological and pathological roles TM play in anti-inflammatory metabolism, and supply a potential drug target. Objective: To prepare and select the best anti-HMGB1 fusion protein composed of TMPD1 and HMGB1 A box. To investigate the regulation of TM by FXR or LXR.Methods:1. Preparation and functional analysis of novel HMGB1 antagonists composed of HMGB1 A box and TMPD1The cDNA encoding murine or human TMPD1(hTMPD1) was amplified by PCR from the cDNA template, respectively. After being sequenced, the hTMPD1 cDNA (N-terminal or C-terminal placement)was coupled with the sequence encoding A box which was previously constructed by our laboratory directly or by a flexible linker sequence Gly4Ser, so was the murine TMPD1(mTMPD1). The fusion DNA fragments were ligated into the prokaryotic expression vector pQE-80L, and then transformed into Escherichia coli DH5α. The fusion proteins were purified by Ni-NTA chromatography and tested for their anti-HMGB1 activity in vitro. The anti-inflammation activity in vivo of the selected fusion protein consisting of mTMPD1 and A box was assessed by the survival rate of endotoxemia mouse.2. Upregulation of thrombomodulin by farnesoid X receptor and Liver X receptor in vascular endothelial cellsEa.hy926 cells (human vascular endothelial cell line) were stimulated with the given different concentrations of FXR agonists and LXR agonists, respectively. Using RT-PCR, Real-time PCR and western blot or flow cytometry detected the TM expression levels. TM activity was assessed by generation of activated protein C. Reporter assay detected the TM promoter activity with activation of FXR or LXR, by the assistant of bioinformatics analysis (http://www.nubiscan.unibas.ch/).Results: (1)The recombinant proteins named hTMPD1,A/hTMPD1,A/linker/hTMPD1,hTMPD1/A,hTMPD1/linker/A,mTMPD1,A/mTMPD1,A/linker/ mTMPD1,mTMPD1/A and mTMPD1/linker /A were successfully prepared.(2) hTMPD1/ A or A/mTMPD1 was the best anti-HMGB1 fusion protein in vitro. (3)hTMPD1/ A or A/mTMPD1 dose-dependently inhibited HMGB1 induced TNF-αrelease in vitro. (4)A/mTMPD1 increased the survival rate of endotoxemia mouse in vivo compared to DHFR/A or mTMPD1. (5)Treatment with FXR ligands resulted in upregulation of TM at the levels of both mRNA and protein. (6)The FXR ligand, GW4064, up-regulated activity of TM.(7) Reporter assays showed that FXR induced TM expression maybe by binding to a FXR element, an inverted repeat DNA motif, IR8(AGGTCCtcccaaagTGCCCT -503～-484). (8)Treatment with LXR ligand resulted in upregulation of TM at the levels of both mRNA and protein. (9)The LXR ligand, GW3965, up-regulated activity of TM. (10) Reporter assays showed that LXR had no effect on TM promoter(-2494～+160) activity.Conclusion: (1)The selected fusion protein hTMPD1/ A or A/mTMPD1 was a novel dual anti-HMGB1 molecule. (2) TMPD1 could be a candidate of the fusion partner which can not only enhance expression level of the A box but also improve its anti- HMGB1 effect. The result from this study maybe benefit on the development of new strategy for treating HMGB1-elevated sepsis. (3)The placement of the two partners from a hybrid could influence the activity of the fusion protein. (4)FXR induced TM expression maybe by binding to a FXR element, an inverted repeat DNA motif, IR8(AGGTCCtcccaaagTGCCCT -503～-484). (5)LXR induced TM expression, but the mechanisms is unkown. (6) FXR and LXR up-regulated activity of TM.