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X. Li, M. Funderburgh, M. Mann, Y. Du, D. Roh, J. Funderburgh; CD44 Is Involved in Smad Signaling by TGFß in Keratocytes. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4539.
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© ARVO (1962-2015); The Authors (2016-present)
: Keratocytes of the corneal stroma secrete a unique extracellular matrix essential for corneal transparency. In healing wounds, keratocytes exhibit a myofibroblastic phenotype expressing -smooth muscle actin (-SMA) and secrete hyaluronan (HA) and EDA-fibronectin (FN), components of a fibrotic extracellular matrix. Transforming growth factor beta (TGFβ) is a key inducer of this fibrotic response and the transformation to the myofibroblast phenotype. In corneal fibroblasts, TGFß binds cell-surface receptors causing rapid phosphorylation and nuclear translocation of SMAD proteins, but TGFß is involved in other signaling pathways as well. Our study investigated the requirement for SMAD pathway activation in upregulation of -SMA, FN, and HA by keratocytes and assessed the potential role of CD44, a cell surface HA receptor in regulating this SMAD mediated signaling.
Primary bovine keratocytes were treated with TGFß1 in the presence of inhibitors of SMAD3 phosphorylation (SIS3) and of TGF-β receptor I kinase (SB-43152, SB4). Expression of -SMA, FN, and CD44 were examined by qRT-PCR and immunoblotting. HA was measured by ELISA. Actin cytoskeleton organization and localization of SMAD4 in the nucleus were determined using imunofluorescence. Expression of CD44 was blocked using specific siRNA.
Inhibitors of SMAD3 phosphorylation (SIS3) and of TGF-β receptor I kinase (SB4) markedly inhibited the TGFβ-induction of HA in keratocytes in a dose-dependent manner. SIS3 and SB4 also inhibited TGFß upregualtion of -SMA, FN, and CD44, as well as the cytoskeletal reorganization of the keratocytes. SMAD4 nuclear translocation was inhibited by both SIS3 and SB4. Knockdown of CD44 expression reduced upregulation of HA, -SMA, and FN in response to TGFß treatment. Knockdown of CD44 also reduced cytoskeletal reorganization and SMAD nuclear translocation in response to TGFß.
Induction of fibrotic gene expression and -SMA in keratocytes by TGFß requires activation of SMAD signaling. The HA receptor CD44 appears to participate in SMAD signaling and reduction of CD44 expression reduces TGFß fibrotic/myofibroblastic responses.
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