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Molecular mechanism of transglutaminase-2 in corneal epithelial migration and adhesion.

Biochimica et biophysica acta (2013-03-08)
Louis Tong, Evelyn Png, Hou Aihua, Siew Sian Yong, Hui Ling Yeo, Andri Riau, Earnest Mendoz, Shyam S Chaurasia, Chwee Teck Lim, Ting Wai Yiu, Siiri E Iismaa
ABSTRACT

Migration of cells in the ocular surface underpins physiological wound healing as well as many human diseases. Transglutaminase (TG)-2 is a multifunctional cross-linking enzyme involved in the migration of skin fibroblasts and wound healing, however, its functional role in epithelial migration has not been evaluated. This study investigated the importance of TG-2 in a murine corneal wound healing model as well as the mechanistic role of TG-2 in the regulation of related biological processes such as cell adhesion and migration of cultured human corneal epithelial (HCE-T) cells. Corneal wound closure was delayed in homozygous TG-2 deleted mice compared to wild type mice. HCE-T cells that were knocked-down for TG-2 expression through stable expression of a short-hairpin (sh) RNA targeting TG-2, were delayed in closure of scratch wounds (48 compared to 12h in control cells expressing scrambled shRNA). TG-2 knockdown did not influence epithelial cell cycle progression or proliferation, rather, it led to reduced epithelial cell adhesion, spreading and velocity of migration. At the molecular level, TG-2 knockdown reduced phosphorylation of β-3 integrin at Tyr747, paxillin at Ser178, vinculin at Tyr822 and focal adhesion kinase at Tyr925 simultaneous with reduced activation of Rac and CDC42. Phosphorylation of paxillin at Ser178A has been shown to be indispensable for the migration of corneal epithelial cells (Kimura et al., 2008) [18]. TG-2 dependent β-3 integrin activation, serine-phosphorylation of paxillin, and Rac and CDC42 activation may thus play a key functional role in enhancing corneal epithelial cell adhesion and migration during wound healing.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Mouse IgG (whole molecule)–Peroxidase antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Transglutaminase from guinea pig liver, lyophilized powder, ≥1.5 units/mg protein
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Monoclonal Anti-Vinculin antibody produced in mouse, clone VIN-11-5, ascites fluid
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Monoclonal Anti-Talin antibody produced in mouse, clone 8d4, ascites fluid
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Anti-Mouse IgG (whole molecule)–FITC antibody produced in goat, affinity isolated antibody, buffered aqueous solution
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Monoclonal Anti-Paxillin antibody produced in mouse, clone PXC-10, ascites fluid
Sigma-Aldrich
Anti-Integrin β1 Antibody, a.a. 82-87, clone JB1A (a.k.a. J10), ascites fluid, clone JB1A (J10), Chemicon®