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X. Li, M. Funderburgh, M. Mann, D. Roh, Y. Du, J. Funderburgh; CD44 Mediates Keratocyte Response to TGF-beta. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4821. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
During healing, keratocytes become motile, mitotically active, and begin secretion of an array of fibrotic extracellular matrix components responsible for reducing corneal transparency. Transforming growth factor beta (TGFβ) is a key inducer of the fibrotic response during wound healing in many tissues including cornea. TGFβ also induces mitotic activation and alpha-smooth muscle actin, an expression identified with the myofibroblast phenotype. Previously we showed that TGFβ induces keratocytes expression of several matrix components characteristic of corneal scars, including the EDA-fibronectin, collagen III, and biglycan. Hyaluronan (HA) secretion is induced by mitotic activation of keratocytes and HA is upregulated synergistically by TGFβ. We reported (ARVO 2007) that HA secretion appears to play a direct role in mediating keratocyte response to TGFβ. The purpose of this study was to test the role of HA receptor CD44 in the keratocyte response to TGFβ.
Primary bovine keratocytes were treated with TGFbeta1 in the presence of inhibitors of CD44: CD44 antibody (IM7), and/or siRNA to CD44. CD44 expression at protein and mRNA levels was measured by western blot and qRT-PCR. Smooth muscle actin, EDA-fibronectin, collagen III, and HAS2 mRNA and protein levels were examined by qRT-PCR, western blot, and immunohistochemistry. Cell growth was measured by Alamar blue and by incorporation of BrdU.
CD44 mRNA and protein in keratocytes are upregulated in response to TGFβ peaking at 24-48h after induction. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) blocked the TGFβ-induced upregulation of CD44. Reduction of CD44 expression using siRNA to CD44 or neutralizing antibody IM7 reduced TGFß-induction of fibronectin, smooth muscle actin, and HA. Inhibition of CD44 also reduced cell division in response to TGFβ, as well as cytoskeletal reorganization and matrix organization by cultured cells.
HA, not present in normal corneal matrix, appears to act as a potent enhancer of the keratocyte response to TGFß. Upregulation of CD44, a HA cell-surface trans-membrane receptor, closely correlates with fibrotic response. Inhibition of CD44 blocked a variety of TGFβ-induced responses, suggesting that HA signaling via CD44 mediates keratocyte response to TGF during wound healing.
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