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S.A. Harvey, S.C. Anderson, N. SundarRaj; Downstream effects of ROCK signaling in cultured human corneal stromal cells: microarray analysis of gene expression. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4548.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: ROCK (Rho–associated coiled–coil containing protein kinase) is a downstream target of Rho GTPase signaling, and regulates the assembly of actin–containing stress fibers. The present study identifies ROCK–regulated gene expression in human corneal stromal cells (HCSCs). Methods: HCSCs from each of three different donor eyes were cultured for 48h in DMEM with additions to yield the following designated phenotypes: baseline fibroblasts (10% serum), bFGF–activated fibroblasts (10% serum plus bFGF plus heparin) and myofibroblasts (1 % serum plus TGF–ß1). Cells were then exposed to the ROCK inhibitor Y–27632 or vehicle for 12 hours. Total RNA was extracted, processed and analyzed using either Affymetrix HU95Av2 or HU133A GeneChips. ROCK–supported (i.e., Y–27632–inhibited) and ROCK–suppressed (i.e., Y–27632–enhanced) changes used the following criteria: within each phenotype, minimum ≥ 1.5 fold and mean ≥ 2–fold. For changes across all phenotypes, minimum ≥ 1.25 fold and mean ≥ 1.5 –fold. Results: ROCK supported 4, 3, and 10 transcripts, in the respective phenotypes. In bFGF–activated fibroblasts, ROCK supported ASPM and CENPF (two mitotic–spindle associated products) and ENC1, an actin–associated protein regulating cytoskeletal reorganization. In myofibroblasts, ROCK supported both α– (smooth muscle) and γ– (enteric) isoforms of actin. Combining data for all phenotypes identified ROCK–supported expression of RRM2 (DNA synthesis), the CCNB1–CDC2–CKS2 triad (cell cycle control), ASPM, and three other mitotic spindle associated proteins. ROCK suppressed 14, 12 and 15 transcripts in the respective phenotypes. In myofibroblasts, ROCK suppressed a subset of extracellular matrix (ECM) transcripts: biglycan, perlecan, agrin and adlican. Combining data from all groups identified global suppression of stromal cell derived factor 1, cytosolic phospholipase A2, phosphatidic acid phosphatase 2A, and three other transcripts. In myofibroblasts ROCK supported the expression of TGF–ß2 (the predominant stromal isoform of TGF–ß) but in baseline fibroblasts ROCK suppressed TGF–ß2 expression. Conclusions: These data suggest that ROCK plays an important role in corneal wounding, panphenotypically supporting cell proliferation (DNA synthesis/cell cycle control/mitosis) while modulating both actin and ECM levels in myofibroblasts. By suppressing TGF–ß2 in baseline fibroblasts ROCK will delay the autacoid TGF–ß2 –mediated conversion to myofibroblasts: however once conversion has started, ROCK will accelerate it by supporting myofibroblast output of TGF–ß2.
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