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Paola Bargagna-Mohan, Royce Mohan; Role of Soluble Vimentin in Regulating the ERK Pathway during Corneal Fibrosis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3897. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Soluble vimentin (sVim) along with its cytoskeletal filamentous form drives corneal fibrosis via promotion of cell proliferation (Bargagna-Mohan JBC 2012). We have investigated whether sVim acts as a signaling chaperone that mediates extracellular-signal-regulated kinase 1/2 (ERK1/2) activity by controlling its nuclear expression since MAPK/ERK signaling is known to affect corneal repair. In this study, we employed genetic and chemical biology approaches to investigate the role of sVim-pERK axis in the healing cornea.
Primary cultures of rabbit corneal fibroblasts (RbCFs) were employed for cell proliferation studies. Cells were serum starved for 48h, trypsinized and then allowed to re-attach to culture plates for 2h in presence of 10% serum. To control the amount of sVim available in culture, cells were treated at the time of plating with different doses of Withaferin A (WFA). Cytoplasmic and nuclear extracts were collected and expression of phosphorylated-ERK (p-ERK) was analyzed at different time points by western blot analysis. Cells were also immunostained for p-ERK and phosphorylated-Vim (p-Vim). In vivo experiments were performed using a model of corneal alkali burn injury in Vim+/+ and Vim-/- mice. WFA was peritoneally injected (2 mg/kg/d) every day for 2 weeks and eyes were collected for immunohistochemistry and western blot analysis.
In proliferating RbCFs a time-dependent translocation of p-ERK from the cytoplasm into the nucleus occurred by 1h post-spreading and WFA treatment strongly inhibited this transition by maintaining cytoplasmic p-ERK levels 10-fold higher than its nuclear levels. At 24h post-plating, control cells displayed a well-extended cytoskeleton staining for p-Vim and strong nuclear p-ERK expression, whereas in WFA-treated cells p-ERK remained perinuclear and co-localized with p-Vim. Our in vivo data revealed that alkali injury induced p-ERK expression strongly in Vim+/+ corneas and WFA downregulated this at d14 post-injury. Interestingly, injured Vim-/- corneas displayed lower levels of p-ERK expression similar to that of Vim+/+- mice treated with WFA.
sVim engagement occurs with stimulation of the ERK1/2 pathway, which WFA potently antagonizes through reduction of nuclear pERK1/2 complexes. This mechanism may also explain lower pERK levels when vimentin is genetically abrogated in vivo.
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