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Royce Mohan, Ling Lei, Alexis Thompson, Camille Shaw, Kousuke Kasahara, Masaki Inagaki, Paola Bargagna-Mohan; Vimentin Phosphorylation Patterns Differentiate Corneal Fibroblasts from Myofibroblasts In Vitro and During Fibrosis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1302.
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© ARVO (1962-2015); The Authors (2016-present)
We investigated vimentin phosphorylation in corneal fibroblasts and myofibroblasts and response to vimentin drug withaferin A (WFA) in vitro and in corneal injury.
Rabbit corneal keratocytes were differentiated into fibroblasts and myofibroblasts. A cell spreading model was employed to investigate vimentin phosphorylation using immunohistochemistry and cell extracts were separated into soluble and insoluble fractions for western blotting. Wild type 129Svev and vimentin-deficient (Vim KO) mice were subjected to corneal alkali injury and treated with vehicle (DMSO) or WFA by intraperitoneal injection for 14 days post injury. Corneal tissue sections and tissues were analyzed for fibrotic markers and vimentin phosphorylation.
Corneal myofibroblasts, unlike fibroblasts, display slower cell spreading and cell polarization, which is associated with increased serine-38 phosphorylation of vimentin (pSer38Vim) but lower pSer71vim levels. Serine 72 and 82 phosphorylation was not changed. This pSer38Vim isoform is inefficiently incorporated into growing filaments of myofibroblasts, and as a result, myofibroblasts maintain higher soluble pSer38Vim levels compared to fibroblasts. WFA treatment caused a potent blockade of cell spreading selectively in myofibroblasts, increased expression of high molecular weight pSer38Vim (hyperphosphorylation). This hyperphosphorylated pSer38Vim species in WFA-treated myofibroblasts becomes complexed with adaptor protein filamin A (FlnA), and these complexes appear as short squiggles when displaced from focal adhesions. The extracellular-signal regulated kinase (ERK) is also phosphorylated (pERK) in response to WFA, but surprisingly, pERK does not enter the nucleus but remained bound to pSer38Vim in cytoplasmic complexes. In the model of corneal alkali injury, we found that fibrotic corneas of wild type mice possess high levels of pERK, whereas injured corneas of Vim KO mice that heal with significantly reduced fibrosis, have highly reduced pERK expression. Finally, WFA treatment caused a decrease in pERK and pSer38Vim expression in healing corneas of wild type mice demonstrating blockade of myofibroblasts.
Our findings identify a hereto-unappreciated role for serine phosphorylation in fibroblast to myofibroblast differentiation and illuminate also an important determinant of myofibroblast sensitivity to WFA.
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