title: Overexpression of a minimal domain of calpastatin suppresses IL-6 production and Th17 development via reduced NF-κB and increased STAT5 signals.
abstract: Calpain, a calcium-dependent cysteine protease, is reportedly involved in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA). In addition, autoantibodies against calpastatin, a natural and specific inhibitor of calpain, are widely observed in RA. We previously reported that E-64-d, a membrane-permeable cysteine protease inhibitor, is effective in treating experimental arthritis. However, the exact role of the calpastatin-calpain balance in primary inflammatory cells remains unclear. Here we investigated the effect of calpain-specific inhibition by overexpressing a minimal functional domain of calpastatin in primary helper T (Th) cells, primary fibroblasts from RA patients, and fibroblast cell lines. We found that the calpastatin-calpain balance varied during Th1, Th2, and Th17 development, and that overexpression of a minimal domain of calpastatin (by retroviral gene transduction) or the inhibition of calpain by E-64-d suppressed the production of IL-6 and IL-17 by Th cells and the production of IL-6 by fibroblasts. These suppressions were associated with reductions in RORγt expression and STAT3 phosphorylation. Furthermore, inhibiting calpain by silencing its small regulatory subunit (CPNS) suppressed Th17 development. We also confirmed that overexpressing a minimal domain of calpastatin suppressed IL-6 by reducing NF-κB signaling via the stabilization of IκBα, without affecting the upstream signal. Moreover, our findings indicated that calpastatin overexpression suppressed IL-17 production by Th cells by up-regulating the STAT5 signal. Finally, overexpression of a minimal domain of calpastatin suppressed IL-6 production efficiently in primary fibroblasts derived from the RA synovium. These findings suggest that inhibiting calpain by overexpressing a minimal domain of calpastatin could coordinately suppress proinflammatory activities, not only those of Th cells but also of synovial fibroblasts. Thus, this strategy may prove viable as a candidate treatment for inflammatory diseases such as RA.
authors: Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors: Mikiko
authors: M
authors: Iguchi-Hashimoto
authors: Takashi
authors: T
authors: Usui
authors: Hajime
authors: H
authors: Yoshifuji
authors: Masakazu
authors: M
authors: Shimizu
authors: Shio
authors: S
authors: Kobayashi
authors: Yoshinaga
authors: Y
authors: Ito
authors: Kosaku
authors: K
authors: Murakami
authors: Aoi
authors: A
authors: Shiomi
authors: Naoichiro
authors: N
authors: Yukawa
authors: Daisuke
authors: D
authors: Kawabata
authors: Takaki
authors: T
authors: Nojima
authors: Koichiro
authors: K
authors: Ohmura
authors: Takao
authors: T
authors: Fujii
authors: Tsuneyo
authors: T
authors: Mimori
mesh_terms: D001172:Arthritis, Rheumatoid / Q000473:pathology; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000473:pathology; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D007249:Inflammation; D020381:Interleukin-17 / Q000378:metabolism; D015850:Interleukin-6 / Q000096:biosynthesis*; D016328:NF-kappa B / Q000378:metabolism*; D011487:Protein Conformation; D050799:STAT5 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D058504:Th17 Cells / Q000166:cytology*
chemical_list: D002135:Calcium-Binding Proteins; C508600:IL6 protein, human; D020381:Interleukin-17; D015850:Interleukin-6; D016328:NF-kappa B; D050799:STAT5 Transcription Factor; C033668:calpastatin