June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Role of Soluble Vimentin in Regulating the ERK Pathway during Corneal Fibrosis
Author Affiliations & Notes
  • Paola Bargagna-Mohan
    Neuroscience, Univ of Connecticut Health Center, Farmington, CT
  • Royce Mohan
    Neuroscience, Univ of Connecticut Health Center, Farmington, CT
  • Footnotes
    Commercial Relationships Paola Bargagna-Mohan, UKY Research Foundation, US8283323B2 (P); Royce Mohan, University of Kentucky Research Foundation (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3897. doi:https://doi.org/
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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)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 765 wound healing • 533 gene/expression • 480 cornea: basic science  
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