MinHee K Ko1, EunDuck P Kay. 1. Department of Pathology, Keck School of Medicine of University of Southern California, Los Angeles, USA.
Abstract
PURPOSE: To investigate the regulatory role of FGF-2 on type I collagen expression during 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 fibroblastic CECs (fCECs). Steady state levels of both type I collagen RNAs were measured using reverse transcription-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 coimmunoprecipitation, and subcellular localization was studied by immunofluorescence. RESULTS: fCECs were characterized by greatly stimulated proliferative potential, loss of contact inhibition, and multilayer fibroblastic cells. The steady state level of alpha1(I) collagen RNA was greatly upregulated through stabilization of the message in fCECs, whereas steady state level and half-life of the alpha2(I) collagen RNA were slightly increased compared with the corresponding levels in normal CECs. Of interest, fCECs predominantly secreted homotrimeric type I collagen, [alpha1(I)](3), with heterotrimeric type I collagen as a minor species. Type I collagen in fCECs was preferentially associated and colocalized with Hsp47 at Golgi apparatus as opposed to its association with protein disulfide isomerase in CECs. LY294002 (a specific PI 3-kinase inhibitor) greatly reduced the steady state levels and stability of alpha1(I) and alpha2(I) collagen RNAs and the secretion of type I collagen. CONCLUSIONS: FGF-2 directly mediates corneal EMT through the action of PI 3-kinase, which acts on posttranscriptional regulation by affecting the stability of type I collagen RNA.
PURPOSE: To investigate the regulatory role of FGF-2 on type I collagen expression during 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 fibroblastic CECs (fCECs). Steady state levels of both type I collagen RNAs were measured using reverse transcription-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 coimmunoprecipitation, and subcellular localization was studied by immunofluorescence. RESULTS: fCECs were characterized by greatly stimulated proliferative potential, loss of contact inhibition, and multilayer fibroblastic cells. The steady state level of alpha1(I) collagen RNA was greatly upregulated through stabilization of the message in fCECs, whereas steady state level and half-life of the alpha2(I) collagen RNA were slightly increased compared with the corresponding levels in normal CECs. Of interest, fCECs predominantly secreted homotrimeric type I collagen, [alpha1(I)](3), with heterotrimeric type I collagen as a minor species. Type I collagen in fCECs was preferentially associated and colocalized with Hsp47 at Golgi apparatus as opposed to its association with protein disulfide isomerase in CECs. LY294002 (a specific PI 3-kinase inhibitor) greatly reduced the steady state levels and stability of alpha1(I) and alpha2(I) collagen RNAs and the secretion of type I collagen. CONCLUSIONS:FGF-2 directly mediates corneal EMT through the action of PI 3-kinase, which acts on posttranscriptional regulation by affecting the stability of type I collagen RNA.
Authors: Li-Fong Seet; Roseline Su; V A Barathi; Wing Sum Lee; Rebekah Poh; Yee Meng Heng; Ed Manser; Eranga N Vithana; Tin Aung; Matt Weaver; E Helene Sage; Tina T Wong Journal: PLoS One Date: 2010-02-25 Impact factor: 3.240