Vimentin is a newly recognized target for corneal
fibrosis. Using primary rabbit corneal fibroblasts and myofibroblasts we show that myofibroblasts, unlike fibroblasts, display impaired cell spreading and cell polarization, which is associated with increased levels of soluble serine-38 phosphorylated
vimentin (pSer38Vim). This pSer38Vim
isoform is inefficiently incorporated into growing
vimentin intermediate filaments (IFs) of myofibroblasts during cell spreading, and as a result, myofibroblasts maintain higher soluble pSer38Vim levels compared to fibroblasts. Moreover, the soluble
vimentin-targeting small molecule and fibrotic inhibitor
withaferin A (WFA) causes a potent blockade of cell spreading selectively in myofibroblasts by targeting soluble pSer38Vim for hyperphosphorylation. WFA treatment does not induce
vimentin hyperphosphorylation in fibroblasts. 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 remains bound to pSer38Vim in cytoplasmic complexes. Using a model of corneal
alkali injury, we show that fibrotic corneas of wild type mice possess high levels of pERK, whereas injured corneas of
vimentin-deficient (Vim KO) mice that heal with reduced
fibrosis have highly reduced pERK expression. Finally, WFA treatment causes a decrease in pERK and pSer38Vim expression in healing corneas of wild type mice. Taken together, these findings identify a hereto-unappreciated role for pSer38Vim as an important determinant of myofibroblast sensitivity to WFA.