Activation of liver resident macrophages and increased pro-inflammatory cytokine production is a hallmark in the pathogenesis of alcoholic liver disease (ALD). Alcohol-induced oxidative stress plays an important role in macrophage activation. Stress induced heat shock proteins (hsps) function as molecular chaperones of signaling molecules important in macrophage activation. Heat shock protein 90 (hsp90) and its ER form, Grp94/gp96 are crucial chaperones of signaling molecules in the TLR4 pathway and thus regulate inflammatory cytokines. Together, hsp90 and gp96 can link stress and inflammatory pathways and play an important role in development of ALD. Studies during the last funding period of this project established that: 1) hsp90? (cytoplasmic) and gp96/grp94 (endoplasmic reticulum (ER) form of hsp90), but not hsp70 is increased in hepatic macrophages and whole livers of chronic alcohol-fed mice and in human alcoholic hepatitis livers, 2)Hsp90? chaperones IKK? and IRF3 and facilitates TLR4 signaling 2) Inhibition of hsp90 in vivo using pharmacological agent 17-DMAG protects mice from alcoholic liver injury, 3) Myeloid-specific gp96 deficiency alleviates alcoholic liver injury 4) HSF1, although activated by alcohol is not sufficient for induction of hsp90?. On the other hand, HSF1 and hsp70 induced by hsp90 inhibitors during decreased injury is crucial for reduction of liver inflammatory responses. Here we hypothesize that cytoplasmic hsp90? and ER gp96/grp94 contribute to hepatic macrophage activation and are plausible therapeutic targets in ALD. Increased hsp90? promotes macrophage activation and gp96 (unfolded protein response (UPR) gene) induced toll-like receptor 4 surface expression and hyperresponsiveness contributes to macrophage sensitization in alcoholic liver disease. Finally we propose that HSF1 and hsp70 exert anti-inflammatory effects during hsp90 inhibition in ALD. Use of hsp90 inhibitors will help unravel innovative pathways of cross-talk between stress proteins and inflammatory responses and establish hsp90 as a new therapeutic target in ALD.
The Specific Aims are as follows: 1) To study the effect of hsp90? inhibition on macrophage activation in ALD by: A) Using hsp90? siRNA to target liver macrophages and 17-DMAG packaged nanoparticles in ALD. B) Examining the effect of hsp90? inhibition on macrophage polarization in the liver. C) Analysis of paracrine effect of macrophage specific hsp90? inhibition on alcoholic steatosis. 2) To investigate the function of myeloid gp96, ER paralog of hsp90, in alcohol-induced sensitization of macrophages by: A) Determine the regulation of gp96 expression during alcoholic liver injury. B) Study importance of gp96/TLR4 interaction and TLR4 hyperresponsiveness during ALD. C) Characterize the role of gp96 in macrophage UPR activation during ALD. 3) To assess anti-inflammatory effects of HSF1 and hsp70 during hsp90 inhibition in ALD: A) Evaluate the role of myeloid-specific HSF1 in macrophage activation and ALD. B) Investigate the role of HSF1 during 17-DMAG treatment and inhibition of ALD. C) Determine the role of hsp70, in anti-inflammatory responses during 17- DMAG treatment.
Alcoholic Liver Disease (ALD) continues to be a global health problem with high morbidity and mortality and limited treatment options. This RO1 renewal resubmission application builds on results from previous funding period showing hsp90 as a plausible target in ALD. This application proposes novel pre-clinical approaches including liver and cell-specific siRNA therapeutics and nanoemulsion approaches to inhibit macrophage pro-inflammatory activation in the liver during ALD. Macrophage activation is central to the pathogenesis of liver injury by alcohol. Targeting pathways important in macrophage activation will provide novel mechanisms and thus extend our horizons to identify drug targets for alcoholic liver disease.
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