The Effects Of HSP70Inhibitor PFT-Μ On Induction Of Inducible Nitric Oxide Synthase And Its Stability

Backgroud: Nitric oxide (NO) is a fundamental signaling moleculeand effector involving in a variety of biological processes. In biologicalsystem, NO is produced by nitric oxide synthases (NOSs). Because of itsinducible expression and high-output features, iNOS is taken as aneffective tool to fight against microbe infection and eliminate tumor cells.Although many inflammatory factors can induce iNOS, the molecularmechanism is still largely unknown. Heat shock proteins (HSPs) are one ofthe most abundant protein families in the cytosolic, and they can be furtherinduced under heat shock stimuli. Our previous studies show that HSP90has great impact on iNOS induction, protein activity, and its stability. As arelevant protein, we evaluated whether HSP70has a similar effect on iNOS.George et al reported a small molecule, which strictly inhibits HSP70,called pifithrin-μ (PFT-μ), also called phenylacetylenylsulfonamide at thejournal Molecular Cell in2009. It gives us the possibility to study HSP70in vitro and in vivo. Up to now, many researchers have reported thisinhibitor can effectively at killing various types of tumor cells, but few reports are focused on inflammatory disorders or the role in iNOSinduction and protein quality control.Objective：To investigate the effects and mechanisms of the specificHSP70inhibtor, PFT-μ on LPS/IFN-γ-induced iNOS induction and nitricoxide production in murine RAW264.7cells; to study the effects of PFT-μon iNOS induction in endotoxemic mice model; to evaluate the effects ofPFT-μ on iNOS protein stability.Methods： To establish inflammatory cell line model by usingLPS/IFN-γ-stimulating murine RAW264.7cells. The level of iNOS proteinwas determined by Western-Blot, iNOS mRNA level was evaluated by realtime PCR, and nitric oxide production in media was determined by Griessreaction. HSP70siRNA was transfected to evulate the specific inhibition ofPFT-μ on RAW264.7cells. Western-Blot was used to determine thephosphoralation of STAT1, the induction and nuclear translocation of IRF-1.Chromation immunoprecipitaiton assay was used to evaluate the bindingefficiency of p-STAT1and IRF-1to its DNA elements. Western-Blot wasused to evaluate the soluable and insoluable iNOS after PFT-μ pretreatment.The endotoxemic mice model was conducted to evaluate the HSP70inhibition on iNOS induction in vivo.Results: Nitric oxide production, iNOS mRNA, and protein levelswere blunt after8hours in IFN-γ-stimulating RAW264.7cells bypretreatment with PFT-μ (P<0.05).Transfection with HSP70siRNA could inhibit iNOS protein expression. PFT-μ did not disturb thephosphoralation of STAT1, the induction and nuclear translocation ofIRF-1. In the presence of PFT-μ, IFN-γ-elicited STAT1and IRF-1bingdings to iNOS promoter were largerly abrogated (P<0.05). PFT-μ didnot change the stability of iNOS mRNA nor iNOS protein. HSP70function is essential for iNOS induction in endotoxemic mice model(P<0.05).Conclusion: PFT-μ prevents iNOS gene transcription, and proteinexpression, and nitric oxide production in LPS/IFN-γ-stimulating murineRAW264.7cell line. PFT-μ could inhibit STAT1and IRF-1bindings toiNOS promoter, and abrogate iNOS gene transcription. PFT-μ did notshorten the half-life of iNOS mRNA. PFT-μ pretreatment did not enhancethe insoluable portion of iNOS protein.