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Padmanabhan P Pattabiraman, Vasanth Rao, Glaucoma; Regulation of Hic-5 Expression in Trabecular Meshwork Cells and Implications for Increased Resistance to Aqueous Humor Outflow. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5678.
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To investigate the hypothesis that Hic-5, a LIM-domain containing smooth muscle specific cell adhesive protein and the corticosteroid receptor co-activator which is upregulated by TGF-β and hydrogen peroxide, increases resistance to aqueous humor outflow via the trabecular pathway.
Regulation of Hic-5 expression and distribution profile by TGF-β2, lysophosphatidic acid (LPA), RhoA and dexamethasone was studied in primary cultures of human trabecular meshwork (HTM) cells using immunoblot and immunofluorescence analyses. Hic-5 induced changes in cell shape, focal adhesions, actin cytoskeletal reorganization and αV integrin distribution in HTM cells were evaluated by GFP-tagged Hic-5.
Hic-5 (50kDa) expression was detected in both human and porcine TM cells but not in lens extracts as assessed by immunoblotting. Hic-5 immunostaining distributed predominantly to focal adhesions and colocalized to the tips of actin stress fibers in HTM cells. Constitutively active RhoA (RhoAV14), LPA, TGF-β2 and dexamethasone stimulated significant increases in Hic-5 expression (greater than 2 fold, p<0.05) based on immunoblotting analyses. Expression of RhoAV14 or treatment with LPA induced a dramatic redistribution and clustering of Hic-5 to the leading edges in serum-starved HTM cells. In contrast TGF-β2 and dexamethasone elicited redistribution of Hic-5 to the cross linked actin network (CLAN). Hic-5 was also found to accumulate around the nucleus under the above described conditions. Additionally, expression of GFP-tagged recombinant human Hic-5 in HTM cells was noted to result in cell rounding, localization of Hic-5 to focal adhesions, and bundling of actin filament and CLANs. Interestingly, recombinant Hic-5 expression in HTM cells also induced localization of αV integrin to the focal adhesions as assayed by immunofluorescence.
Taken together, the upregulation of Hic-5 by TGF-β2, RhoA, LPA and dexamethasone, Hic-5-induced localization of αV integrin to the focal adhesions, actin cytoskeletal bundling and CLAN formation in HTM cells suggests the involvement of Hic-5 in increased resistance to aqueous humor outflow in glaucomatous eyes.
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