Identification Of A Novel Autoantibody Against HSF1 In Myositis Patients And HSF1 Expression In Myogenesis

Part I.Identification of a novel autoantibody against heat shock transcription factor 1 in idiopathic inflammatory myopathyObjective:Myositis autoantibodies show great utility in the diagnosis and clinic-serological phenotyping of idiopathic inflammatory myopathy(IIM).We identified a novel autoantibody against heat shock transcription factor 1(HSF1)and further evaluated its disease specificity and clinical significance in IIM patients.Methods:A human protein microarray was used to identify autoantibodies in myositis sera.ELISA,immunoblot and dot blot assays were applied to examine anti-HSF1 autoantibodies in IIM patients and controls.Immunofluorescence was used to detect HSF1 expression in muscle tissues.Results:Anti-HSF1 was identified as a novel autoantibody by protein microarray and the seroreactivity was confirmed by immunoprecipitation,ELISA,immunoblot and dot blot assays.Anti-HSF1 autoantibodies were present in 64/581(11.0%)?M,4/37(10.8%)rheumatoid arthritis,5/40(12.5%)primary Sjogren's syndrome,2/40(5%)systemic lupus erythematosus,while largely negative in healthy controls.Anti-HSF1 autoantibodies were significantly associated with pruritus,hypergammaglobulinemia,and elevated erythrocyte sedimentation rate in IIM patients.Anti-HSFl autoantibodies were more prevalent in cancer-associated myositis(CAM)compared to non-CAM patients(17.2%vs,7.5%,p=0.009),nevertheless were undetectable in cancer controls.Meanwhile,cross-sectional and longitudinal analyses revealed positive correlations between anti-HSF1 levels and disease activity in IIM patients without cancer.Additionally,increased expression of HSF 1 was found in regenerating muscle cells of myositis muscle tissues.Conclusion:These data reveal anti-HSF1 as a new autoantibody associated with CAM in IIM.The autoimmunity against HSF1 may be involved in the immunopathogenesis of myositis.Part ?.Expression of heat shock transcription factor 1 in myogenesisObjective:We previously identified anti-heat shock factor 1(HSF1)autoantibodies in patients with idiopathic inflammatory myopathy(?M),and found HSF1 was highly expressed in regenerating muscle cells of ?M muscle.We undertook this study to further investigate HSF1 expression kinetics during muscle regeneration,and explore the physiological function of HSF1 in myogenesis.Methods:We detected the expression of HSF1 in IIM muscle tissues by Immunofluorescence.The expression of HSF1 in proliferating or differentiating muscle cells(C2C1G and primary human CD56+ muscle cells)was analyzed by immunofluorescence and Western blot.The effect of HSF1 on C2C12 differentiation was studied by premature HSF1 silencing using RNA interfering technology(RNAi).Results:In IIM muscle tissues,HSF1 expression was observed in the cytoplasm of regenerating muscle cells,while was negative in normal muscle cells.We induced the differentiation of C2C12 cells and primary human CD56+muscle cells in vitro,and found the percentage of HSF1 expressing muscle cells were increased as differentiation induction.Uneven HSF1 expression was found in the cytoplasm of differentiating muscle cells,but was not observed in proliferating muscle cells.Western blot analysis revealed that HSF1 was highly expressed in the early stage of C2C12 differentiation.Premature silencing of HSF1 using siHSF1 in C2C12 cells under differentiation induction resulted in lower expression of myogenin,which is a transcription factor regulating muscle cell differentiation.Conclusion:These results revealed that HSF1 expression was markedly up-regulated during muscle regeneration.Premature silencing of HSF1 expression in muscle cell may retard its differentiation.