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Mitchell A. Watsky, Zhaohong Yin; S1P-Induced Cl- Channel Activation, Block, and Smad Translocation in Human Keratocytes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1087.
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Sphingosine-1-phosphate (S1P) is a bioactive lipid that has been implicated in a number of fibrotic and autoimmune disorders. IClLPA is an S1P-activated anion current originally characterized in myofibroblasts isolated from wounded corneas that has been found in activated fibroblasts from several different tissue types. Importantly, regardless of the tissue of origin, IClLPA activity is observed almost exclusively in activated keratocytes and fibroblasts and not in their quiescent counterparts. Blocking this current or knocking down the associated ClC3 protein with shRNA or antisense prevents TGF beta-induced myofibroblast differentiation. The purpose of this study was to examine the signaling pathway leading to S1P-induced IClLPA activation and myofibroblast formation in corneal keratocytes.
We examined S1P and related LPA receptor subtypes in a human keratocyte cell line using real-time PCR. The involvement of Rho kinase (ROCK) in S1P-mediated IClLPA activation and examination of a non-typical drug to block IClLPA were studied using whole cell patch clamping with the ROCK inhibitor Y27632 and clomiphene, respectively. S1P-induced Smad 3 nuclear translocation was examined using immunostaining and confocal microscopy. The influence of IClLPA on this translocation was examined using the IClLPA blocker tamoxifen.
The primary bioactive lipid receptors present in the human keratocyte cell line are S1P3 and LPA1. Y27632 and clomiphene both blocked S1P-mediated IClLPA activation. S1P promoted nuclear translocation of Smad 3 in keratocytes, and this translocation was blocked by tamoxifen.
S1P-induced activation of IClLPA is likely proceeding through S1P3 via a ROCK-mediated pathway. IClLPA can be blocked using clomiphene. In addition, S1P promotes IClLPA-dependent Smad 3 nuclear translocation.
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