Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK‐mediated MLC phosphorylation

Abstract

The retinal pigment epithelium (RPE) forms the outer blood-retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial-mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR-1 induces the time- and dose-dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho-MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y-27632, whereas MLCK inhibitor ML-7 and PLC-β inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC-β-mediated calcium increase. Additionally, thrombin-induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP(3)/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin-induced RPE cell transformation subsequent to BRB dysfunction.

HIF-3α Promotes Metastatic Phenotypes in Pancreatic Cancer by Transcriptional Regulation of the RhoC–ROCK1 Signaling Pathway

“…Inhibition of ROCK1 expression blocks LIMK2/cofilin phosphorylation and subsequent stress fiber formation (38,39). MLC stimulates the formation and stabilization of actin stress fibers (40). In this study, we found that HIF-3a transcriptionally regulates RhoC and ROCK1 and enhances phospho-cofilin, and phospho-MLC2 levels, demonstrating activation of RhoC-ROCK1 signaling.…”

Shared Triggering Mechanisms of Retinal Regeneration in Lower Vertebrates and Retinal Rescue in Higher Ones

“…It was found that RPE cells lost their initial phenotype, dedifferentiated and acquired mesenchymal migratory morphology and cytoskeleton proteins (Casaroli-Marano et al, 1999). Recently thrombin (see above) pretends to play a promoting role in actin stress fiber formation, an important determinant in eye diseases involving transformation and migration of RPE cells (Ruiz-Loredo et al, 2011 Earlier we showed a decrease of fibronectin in Bruch membrane and its redistribution in RPE after NR detachment in the newt (Grigoryan et al, 1990). Similar results were obtained by Ortiz and co-authors (1992): at the beginning of RPE cell transdifferentiation in the eye of the adult newt, fibronectin was the first to appea r i n t h e c e l l b o r d e r o f t h e n e w f o r m i n g neuroepithelium.…”

Estrogen regulates Hippo signaling via GPER in breast cancer

“…Collectively, these observations suggest a model in which Rho and ROCK serve as important mediators downstream of GPER and are required for GPER to activate TAZ. The Rho/ROCK pathway potently modulates cellular actin dynamics, particularly stress fiber formation, in response to various GPCR agonists such as LPA or thrombin (42,43). We therefore examined whether cytoskeletal reorganization contributes to TAZ activation in response to G1 stimulation.…”