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Vimentin Phosphorylation Underlies Corneal Myofibroblast Sensitivity to Withaferin A
Author(s) -
BargagnaMohan Paola,
Lei Ling,
Thompson Alexis,
Shaw Camille,
Mohan Royce
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.721.28
Subject(s) - vimentin , myofibroblast , withaferin a , phosphorylation , microbiology and biotechnology , intermediate filament , chemistry , pathology , cancer research , cell , biology , cytoskeleton , medicine , immunohistochemistry , fibrosis , biochemistry , alternative medicine , withania somnifera
Objective To compare vimentin phosphorylation in corneal fibroblasts with myofibroblasts and determine their sensitivity to withaferin A. Methods: Primary rabbit corneal fibroblasts and myofibroblasts were subjected to cell spreading assays. Wild type and vimentin‐deficient mice were subjected to corneal alkali injury and treated with withaferin A (WFA). Corneas were subjected to immunohistochemistry and western blot analyses. Results: We show that myofibroblasts produce increased serine‐38 phosphorylated vimentin (pSer38Vim) and display impaired cell spreading and cell polarization compared to fibroblasts. The pSer38Vim isoform is inefficiently incorporated into growing vimentin IFs of myofibroblasts during cell spreading as myofibroblasts maintain higher levels of soluble pSer38Vim compared to fibroblasts. WFA treatment causes a potent blockade of cell spreading selectively in myofibroblasts, targeting pSer38Vim for hyperphosphorylation, which is not induced in fibroblasts. Hyperphosphorylated pSer38Vim in myofibroblasts complexes with adaptor protein filamin A (FlnA), and appears as short squiggles displaced from focal adhesions upon WFA treatment. The extracellular‐signal regulated kinase (ERK) is also phosphorylated (pERK) in response to WFA, but pERK does not enter the nucleus because it remains bound to pSer38Vim in cytoplasmic complexes. Fibrotic corneas of injured wild type mice possess high levels of pERK compared to injured vimentin‐deficient mice. WFA treatment causes a decrease in pERK and downregulates pSer38Vim expressing myofibroblasts in corneas of wild type mice. Conclusions We have identified pSer38Vim as a major determinant of myofibroblast sensitivity to WFA.
Support: R01 EY016782