Molecular Mechanism By HSP70 Knockdown Influencing LPS-induced Expression Of Inflammatory Cytokines

Severe trauma,burns,sepsis and various types of shock often leads to systemic inflammatory response syndrome(SIRS) occurred.SIRS is a very common clinical subjects,which can usually leads to death.Although the mechanism of SIRS has not entirely clear yet,but the important role of the expression of inflammatory and the activation of cellular signal transduction by LPS has been wide concerned.In order to understand the anti-inflammatory role of heat shock protein 70(HSP70),we investigated the effects of HSP70 knockdown on the lipopolysacchearide(LPS)-induced production of proinflammatory cytokines and the activation of NF-κB signaling pathways in RAW264.7 mouse macrophage cell line.Many studies have shown that elevation of HSP70 caused either by activation of the heat shock response(HSR) or through forced expression of the hsp70.1 gene down-regulated cytokine expression in RAW264.7 murine macrophages.Our data showed that inhibition of HSP70 by HSP70-siRNA could increase LPS-mediated expression of proinflammatory cytokines such as interleukin-1β(IL-1β) and interleukin-6(IL-6) at mRNA levels.Because NF-κB regulates the transcription of an exceptionally large number of genes,particularly those involved in inflammatory and acute stress response,which suggests a major role of NF-κB signaling pathway in critical diseases.We further determined the effects of HSP70 knockdown on NF-κB signaling pathway.Firstly western blot was used to analyze the subcellular distribution of NF-κB(p65),the transcriptionally active component of the NF-κB complex.It was shown that nuclear translocation of p65 induced by LPS was strongly inhibited in cells transfected with HSP70.But this effect was significantly abolished by HSP70 knockdown.Nuclear translocation of p65 occurs subsequently to the phos-phorylation and degradation of IκBα.Therefore we next determined the effects of HSP70 knockdown on the degradation of IκBα.It was shown that the degradation of IκBαinduced by LPS was significantly increased in the cells transfected with HSP70-siRNA,as compared with the cells transfected with HSP70.Because the phosphorylation and the consequent degradation of IκBαare also dependent on intracellular phosphatase activity apart from the effect of IκB kinase(IKK),we further investigated the effects of the inhibition of HSP70 on intracellular phosphatase 2A activity during LPS stimulation.HSP70 knockdown could down-regulate the phosphatase 2A activity in the cells at normal condition or stimulated with LPS for 30min.In conclusion,HSP70 could up-regulate the phosphatase 2A activity, which might inhibit the phosphorylation and degradation of IκBαand the subsequently nuclear translocation and activation of NF-κB.HSP70 could inhibit LPS-induced production of inflammatory cytokines by inactivating NF-κB pathway.