Studies On Molecular Mechanisms By Which HSP70 Attenuates Oxidative Stress-Mediated Nucleolar Impairment

Oxidative stress injury occurs in a variety of pathological processes, suchas infection, inflammation, toxication, trauma, hypoxia, ischemia,ischemia-repufusion, septic shock, systemic inflammatory response syndrome,multiple system organ failure and so on. Its basic mechanism is theimpairment of cells mediated by reactive oxygen species (ROS). ROSdirectly attack the cell membranes and organells, and induce aggregation ofproteins, injury of nucleic acid and apoptosis, etc.The nucleolus is a membrane-free organell within nucleus of eukaryoticcells and is formed around the ribosomal DNA (rDNA) repeats which bandedby multiple nucleolar proteins. The nucleolus is a "cellular factory" in whichribosomal RNA (rRNA) including 28S, 18S and 5.8S are transcribed,processed and assembled into ribosomal subunits, and it plays very importantroles in protein synthesis, growth, proliferation and differentiation ofeukaryotic cells. Many studies have shown that several stressors such as heat,TNF-αand cisplatin can induce nucleolar segregation or segmentation inseveral tumour cells. Our prior studies have shown that oxidative stress canalso result in nucleolar segregation in primarily cultured neonatal ratcardiomyocytes.However, it is still unclear what functional changes and molecularmechanisms are involved in the nucleolar segregation induced by oxidativestress.Recently, the roles of endogenous protective mechanism in the treatmentand prevention of diseases have attracted the attention of many investigatorsand most of which has primarily been focused on heat shock proteins (HSPs).HSPs are a family of molecular chaperons, which are indispensable inphysiological states and exert protective role in pathological processes. Theinducible expression of HSPs is regulated by heat shock transcription factor1(HSF1). Under unstressed conditions, HSF1 is maintained in the latent,non-DNA-binding state. Stresses, such as heat and ischemia, result inactivation of HSF1 monomers to form homotrimers, tanslocate into nucleus,and bind, with high affinity, to the conserved upstream heat shock elements(HSEs) contained in the promoters of all heat shock genes. HSP70 is the mostup-regulated and most important member among the HSPs. Some evidencesshowed that HSP70 could protect many kinds of cells from apoptosis inducedby a variety of stressors. We have demonstrated that over-expression ofHSP70 can attenuate oxidative stress-induced nucleolar segregation.But the mechanisms by which HSP70 attenuate oxidative stress-mediatednucleolar impairment are still uncertain.In present studies, nucleolar segregation of C2C12, RAW264.7 and K562cells was induced by exposure of the cells to 500 μM hydrogen peroxide(H2O2) for different durations, and the mechanisms of nucleolar segregationinduced by H2O2 and the mechanisms by which HSP70 inhibited nucleolarimpairment were investigated. The main results and findings are as follows:1 . Oxidative stress-induced cleavage and down-regulation ofnucleolar protein C23 were involved in nucleolar impairment.1) Exposure of the cells to 500 μM H2O2 for 3 hours resulted insignificant nucleolar segregation in C2C12, RAW264.7 and K562 cells. Andtotal protein synthesis decreased to about 40-50% after 6 h of H2O2 treatment.2) The activity of caspase -3 was significantly increased 4 h after H2O2treatment and reached peak at 8-12 hour;and apoptotic cells were observed12 h after H2O2 treatment.3) Cleavage and down-regulation of C23 were detected byimmunoblotting at the earlier stage of 30 min to 1 h after H2O2 treatment incardiomyocytes, C2C12, RAW264.7 and K562 cells.4) Antisense-mediated down-regulation of C23 resulted in remarkablenucleolar impairment, apoptosis and proliferation inhibition in C2C12 cells.2. Heat shock response inhibited H2O2–induced cleavage anddown-regulation of C23 and nucleolar injury by inducing the expressionof HSPs.The expression of HSP70 and αB-crystallin significantly increased inseveral kinds of cells 12 hour after heat shock response (HSR). And HSRcould inhibit both H2O2–induced cleavage and down-regulation of C23 andnucleolar impairment. Mouse embryonic fibroblasts (MEFs) were isolatedfrom HSF1 knock out (HSF1-/-) and wild type (HSF1+/+) mice andimmortalized by transfection of a eukaryotic expression vector containingSV40 large T antigen and screening with G418. It was showed that HSRcould induce the expression of HSP70 in HSF1+/+ MEFs but not in HSF1-/-MEFs, and alleviate nucleolar segregation and inhibition of total proteinsynthesis induced by H2O2 in HSF1+/+ MEFs but not in HSF1-/- MEFs. Theseresults suggested that HSR could suppress significantly H2O2-inducednucleolar impairment, which might be due to the inducible expression ofHSPs in HSF1+/+ MEFs and subsequent inhibition of the cleavage anddown-regulation of C23.3. HSP70 interacted with C23 and inhibited H2O2–induced cleavageand down-regulation of C23 and nucleolar injury.1) Eukaryotic expression plasmids pcDNA3.1 containing human fulllength cDNA of HSP70 (pcDNA3.1(-)-HSP70WT) and its deletion mutant innuclear localization sequence (NLS) at 246-273 amino acid residues (pcDNA3.1(-)-HSP70△NLS) and their relative enhanced GFP-(N1) recombinantplasmids were constructed and transfected into C2C12 cells. Their over-expression was confirmed by immunoblotting in cells stable transfection withpcDNA3.1(-)-HSP70WT and pcDNA3.1(-)-HSP70△NLS. It was showed thatH2O2 could induce the translocation of full length HSP70 but not its deletionmutant in NLS from cytoplasm to nucleus and nucleolus by fluorescentmicroscopy and immuno- blotting.2) HSP70 could inhibit H2O2-induced cleavage and down-regulation ofC23, and subsequently inhibit H2O2-mediated nucleolar impairment.3) Moreover, it was showed by co-immunoprecipitation (CO-IP) thatHSP70 interacted with C23 at 1 h after treatment with H2O2. In order tofurther clarify the domain by which HSP70 interacts with C23, two othereukaryotic expression plasmids encoding deletion mutants of HSP70 either atthe peptide binding domain (pcDNA3.1(-)-HSP70△PBD) or the ATP bindingdomain (pcDNA3.1(-)-HSP70△ATP-BD) were constructed apart frompcDNA3.1(-)- HSP70△NLS and pcDNA3.1(-)- HSP70WT, and HSP70△ATP-BDalso included the deletion mutation of NLS. These four plasmids weretransfected into C2C12 cells and their over-expression was confirmed 24 hafter transfection. Co-IP showed that H2O2 could only mediate the interactionbetween C23 and HSP70WT but not between C23 and HSP70△NLS, HSP70△PBDor HSP70△ATP-BD.4) Because both HSP70△NLS and HSP70△ATP-BD contained deletionmutation of NLS, during in vivo oxidative stress, the failure of these twomutants to translocate into nucleus would also affect their binding with C23.In order to verify this hypothesis, an in vitro protein overlay analysis wasperformed. First, HSP70WT and its three domain-deleted mutants wereconstructed into prokaryotic expression vectors (pQE31). After transfectionand inducible expression with IPTG, these four peptides were isolated andpurified by Ni++ affinity chromatography and cellulose DEAE-52 ionexchange chromatography. Immunoblotting showed that a goat anti-HSP70ployclonal antibody could recognize all the mutant proteins in thecarboxy-terminal end. Surprisingly, protein overlay analysis showed that C23interacted with HSP70WT, HSP70△NLS or HSP70△ATP-BD but not HSP70△PBD invitro.These results indicated that under oxidative stress, HSP70WT translocatedto nucleus and nucleolus from cytoplasm depending on its NLS included inATP-BD, and HSP70 interacted with C23 through its PBD. Subsequently,HSP70 executed its protective effects against H2O2-mediated nucleolarimpairment by inhibiting the cleavage and down-regulation of C23.In conclusion, oxidative stress could induce nucleolar impairment in allkinds of cells and its mechanism is related to H2O2-mediated cleavage anddown-regulation of C23. Heat shock response could inhibit H2O2-mediatednucleolar impairment by inducing the expression of HSPs and inhibitingcleavage and down-regulation of C23. HSP70 could suppress oxidativestress-mediated nucleolar impairment by its translocation into the nucleus andnucleolus, interaction with C23 through its peptide binding domain, theninhibiting the cleavage and down-regulation of C23.