Downstream effects of ROCK signaling in cultured human corneal stromal cells: microarray analysis of gene expression.
OBJECTIVE
Rho-associated coiled-coil-containing protein kinase (ROCK) is a downstream target of Rho GTPase signaling and regulates the assembly of stress fibers. Previous reports indicate that Rho/ROCK signaling is involved in the regulation of several cellular processes, some of which may be cell-type specific and are probably critical to corneal stromal cell activation. The present study identified ROCK-regulated gene expression in corneal stromal cells.


METHODS
Corneal stromal cells derived from eyes of three different donors were cultured to yield the following designated phenotypes: baseline fibroblasts (DMEM with 10% serum), activated fibroblasts (10% serum+bFGF+heparin), and myofibroblasts (1% serum+TGF-beta 1). Cells were exposed to the ROCK inhibitor Y-27632 or vehicle for 12 hours, and transcript levels altered by ROCK inhibition were identified with oligonucleotide microarrays (GeneChips; Affymetrix, Santa Clara, CA).


RESULTS
In these phenotypes, Y-27632 caused marked (twofold or more) increases or decreases in 14/4, 12/3, and 15/10 transcripts. In both fibroblast groups Y-27632-treatment increased expression of endothelin receptors and of parathyroid hormone-like hormone. The upregulation of alpha-smooth muscle actin in myofibroblasts was attenuated by Y-27632. Combining data from all groups identified ROCK-supported (Y-27632 inhibitable) expression of 10 transcripts, including ribonucleotide reductase M2, the cyclin B1-CDC2-CKS2 system, and four mitotic spindle-associated proteins.


CONCLUSIONS
ROCK inhibition causes broad inhibition of DNA synthesis and mitosis and causes changes that are different between (bFGF-activated) fibroblasts and (TGF-beta 1-induced) myofibroblasts. Thus, Rho/ROCK signaling regulates both common and distinct downstream events in corneal stromal cells activated (differentiated) to fibroblast or myofibroblast phenotype.
Harvey
Stephen A K
SA
Department of Ophthalmology, University of Pittsburgh, Pennsylvania 15213-2588, USA. harveysa@msx.upmc.edu
Anderson
Susan C
SC
SundarRaj
Nirmala
N
D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020411:Oligonucleotide Array Sequence Analysis; D044162:Parathyroid Hormone-Related Protein / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D017466:Receptors, Endothelin / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D015854:Up-Regulation; D054460:rho-Associated Kinases
D000199:Actins; D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D044162:Parathyroid Hormone-Related Protein; D011725:Pyridines; D017466:Receptors, Endothelin; C108830:Y 27632; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases