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A. M. Bernstein, A. O. Imas, L. E. Kang, B. S. Pedroja; Fibrotic Wound Healing: The Accumulation of Cell Surface TGFβRII and vβ3 Promotes Enhanced TGFβ Signaling. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4817.
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© ARVO (1962-2015); The Authors (2016-present)
PAI-1 (plasminogen activator inhibitor) is a multifunctional molecule that promotes the internalization of both uPA/uPAR (urokinase plasmingen activator and its receptor) and integrin αvβ3 from the cell surface. In previous studies we found that TGFβ1-treatment over 7 days caused increased cleavage of uPAR as the fibroblasts differentiated into myofibroblasts. Since cleaved cell-surface uPAR cannot bind uPA and PAI-1, we now propose that 1) uPAR cleavage results in the inhibition of αvβ3 internalization from the cell-surface and 2) the resultant increase in cell-surface αvβ3 leads to amplified TGFβ signaling through the prolonged interaction between αvβ3 with TGFβRII.
To promote the accumulation of αvβ3 and TGFβRII on the cell surface, we used MEFs that are null for PAI-1 (PAI-1 KO) and therefore lack the ability for PAI-1 to induce the internalization of αvβ3. These findings were extended in a primary human corneal fibroblast (HCF) wound-healing model. PAI-1 WT and KO MEFs, and HCFs were cultured on collagen or vitronectin in supplemented serum-free media for 1-7 days. HCFs were treated with 1ng/ml TGFβ1 to induce myofibroblast differentiation. We investigated TGFβ signaling as detected by SMAD2/3 nuclear localization, and TGFβRII and αvβ3 localization and expression by western blot, cell-surface biotinylation followed by immunoprecipitation, and immunofluoresence.
In PAI-1 KO MEFs but not WT, TGFβ signaling through SMAD 2/3 was constitutively active on both collagen and vitronectin, and was sustained on vitronectin. Expression of TGFβRII and αvβ3 was dramatically enhanced in PAI-1 KO cells compared to WT. In PAI-1 KO cells αvβ3 was detected in large, elongated focal adhesions, which aligned with the terminations of the actin cytoskeleton in contrast to more dispersed and smaller focal points in WT. In HCFs, 7-day TGFβ treatment increased cell-surface expression of β3 proportionally with time.
These data suggest that the accumulation of αvβ3 on the cell-surface promotes the interaction between αvβ3 and TGFβRII. This enhanced TGFβ signaling could promote sustained myofibroblast differentiation and fibrotic wound healing.
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