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J. L. Funderburgh, M. M. Mann, M. L. Funderburgh; Signaling Pathways Involved in the Keratocyte-Myofibroblast Transformation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4540.
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After corneal wounding some keratocytes become activated and express -smooth muscle actin. These myofibroblasts downregulate expression of normal corneal extracellular matrix and upregulate components of fibrotic scar tissue. Transforming growth factor ß (TGFß) plays a crucial role in this transformation, but other growth factors are also implicated. This study used chemical inhibitors of known signaling pathways to define the molecular mechanism by which cytokines participate in the production of corneal scar tissue.
Primary bovine keratocytes were cultured in serum-free conditions and stimulated with purified growth factors for 1-3 days in the presence of chemical inhibitors of known signaling pathways. mRNA abundance for matrix components was determined by qRT-PCR. Protein expression was determined by immunoblotting or immunostaining of cultures.
TGFß alone increased hyaluronan (HA) synthesis as well as the mRNA for its synthase, HAS2, in primary keratocytes. Other growth factors, IGF-1, insulin, PDGF, FGF2, and serum, also increased HAS2 mRNA and HA. TGFß in the presence of any of these caused a marked, non-additive stimulation of HAS2 and HA. A number of other TGFß-responsive genes, -smooth muscle actin, EDA-fibronectin, collagen 1, collagen 3, and biglycan, were induced in a similar synergistic manner. mRNA for keratocan and ALDH3 however were reduced by TGFß without a synergistic effect. All TGFß responses were reduced by inhibitors of TGFß receptor and of SMAD signaling. Several specific inhibitors of the PI3 kinase pathway completely blocked TGFß synergistic effects but not downregulation of keratocan or of nuclear SMAD translocation. Inhibition of AKT activation but not of mTOR, NF-kappa B, or MAP kinase signaling, was effective in blocking the synergistic effects. Inhibitors of EGF receptor phosphorylation were also effective in blocking synergistic activation of TGFß responses by several growth factors.
The myofibroblastic/fibrotic responses of keratocytes to TGFß require TGFß signaling via the SMAD pathway, but also require PI3 kinase signaling, which can be induced by a number of factors. Transactivation of the EGF receptor may be a common pathway by which this activation is mediated.
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