Vimentin Phosphorylation Underlies Myofibroblast Sensitivity to Withaferin A In Vitro and during Corneal Fibrosis

PLoS One. 2015 Jul 17;10(7):e0133399. doi: 10.1371/journal.pone.0133399. eCollection 2015.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Movement / drug effects
  • Corneal Diseases / metabolism*
  • Corneal Diseases / pathology*
  • Enzyme Activation / drug effects
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Fibrosis
  • Filamins / metabolism
  • Focal Adhesions / drug effects
  • Focal Adhesions / metabolism
  • Intermediate Filaments / metabolism
  • MAP Kinase Signaling System / drug effects
  • Mice, Knockout
  • Myofibroblasts / drug effects
  • Myofibroblasts / metabolism*
  • Myofibroblasts / pathology*
  • Myosins / metabolism
  • Phosphorylation / drug effects
  • Phosphoserine / metabolism
  • Rabbits
  • Solubility
  • Vimentin / metabolism*
  • Withanolides / pharmacology*

Substances

  • Filamins
  • Vimentin
  • Withanolides
  • Phosphoserine
  • Extracellular Signal-Regulated MAP Kinases
  • Myosins
  • withaferin A