May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Gene Regulation of Type I Collagen by FGF–2 During Corneal Endothelial Mesenchymal Transformation
Author Affiliations & Notes
  • M.K. Ko
    Pathology,
    University of Southern California, Los Angeles, CA
  • E.P. Kay
    Ophthalmology,
    University of Southern California, Los Angeles, CA
    Doheny Eye Institute, Los Angeles, CA
  • Footnotes
    Commercial Relationships  M.K. Ko, None; E.P. Kay, None.
  • Footnotes
    Support  NIH grant EY06431
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 4513. doi:
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      M.K. Ko, E.P. Kay; Gene Regulation of Type I Collagen by FGF–2 During Corneal Endothelial Mesenchymal Transformation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4513.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: To investigate how FGF–2 regulates gene expression and its signaling pathway of type I collagen secretion during corneal endothelial mesenchymal transformation (EMT) Methods: Corneal endothelial cells (CECs) treated with FGF–2 from the primary culture to the third passage were transformed and designated as fCEC. Steady–state levels of both α1(I) and α2(I) collagen RNAs were measured using reverse transcriptional real–time PCR, and their half lives were determined in the presence of inhibitor of RNA synthesis. Limited proteolysis with pepsin was used to determine secretion of type I collagen. Protein–protein interaction was determined by co–immunoprecipitation, and co–localization of type I collagen with molecular chaperones was determined by immunofluorescence. LY294002 was used to block phosphatidylinositol 3–kinase (PI3K) pathways. Results: fCEC were characterized by increased cell proliferation, loss of contact inhibition, and multi–layers of fibroblastic cells. Of interest, the steady–state level of α1(I) collagen RNA was increased by FGF–2, while the steady–state level of α2(I) collagen RNA was decreased. fCEC secreted α1(I) collagen trimer as a major collagen species along with the heterotrimeric type I collagen as a minor collagen. The half–lives of α1(I) and α2(I) collagen RNAs of fCEC were much longer than those of normal CECs. FGF–2 also facilitated the subcellular localization of type I collagen to Golgi and reduced protein–protein interaction of the type I collagen with protein disulfide isomerase when compared to that in normal CECs. In fCEC, LY294002 reduced the steady state levels of both collagen RNAs, type I collagen secretion, and cell proliferation. But the inhibitor failed to alter the cell shape: fCEC maintained a fibroblastic morphology. When fCEC were switched to the growth media in the absence of FGF–2 and treated with LY294002 alone, there was a marked decrease of the steady–state levels of both collagen RNAs and type I collagen secretion. Conclusions: FGF–2 facilitated EMT of CECs through the action of PI3K. When CECs are fully transformed with FGF–2, there appear to be FGF–2–independent PI3K pathways to maintain EMT.

Keywords: cornea: endothelium • extracellular matrix • wound healing 
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