title: RhoA/Rho kinase (ROCK) alters fetuin-A uptake and regulates calcification in bovine vascular smooth muscle cells (BVSMC).
abstract: RhoA/Rho kinases (ROCK) play a critical role in vascular smooth muscle cell (VSMC) actin cytoskeleton organization, differentiation, and function and are implicated in the pathogenesis of cardiovascular disease. We have previously determined that an important step in the regulation of calcification is fetuin-A endocytosis, a process that is dependent on changes in the cytoskeleton, which, in turn, is known to be affected by the RhoA/ROCK signaling pathway. In the present study, bovine VSMC (BVSMC) were treated with the ROCK inhibitor Y-27632 or transfected with ROCK small interfering (si) RNA to knock down ROCK expression. Both conditions resulted in reduced actin stress fibers and increased Cy5-labeled fetuin-A uptake. Inhibition of ROCK by Y-27632 or siRNA also significantly increased BVSMC alkaline phosphatase (ALP) activity and calcification of BVSMC and rat aorta organ cultures. Cells were then incubated in calcification media in the presence or absence of Y-27632 and matrix vesicles (MV) isolated by collagenase digestion. These MV, isolated from BVSMC incubated with Y-27632, had increased ALP activity and increased ability of MV to subsequently calcify collagen by 66%. In contrast, activation of RhoA, which is upstream of ROCK, by transfecting plasmids encoding the dominant active Rho GTPase mutant (Rho-L63) led to decreased fetuin-A uptake and reduced calcification in BVSMC. These results demonstrate that the RhoA/ROCK signaling pathway is an important negative regulator of vascular calcification.
authors.affiliation: Indiana Univ. School of Medicine, Indianapolis, IN 46202, USA. xuechen@iupui.edu
authors.forename: Neal X
authors.initials: NX
authors.lastname: Chen
authors.forename: Xianming
authors.initials: X
authors.lastname: Chen
authors.forename: Kalisha D
authors.initials: KD
authors.lastname: O'Neill
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Atkinson
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Moe
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D002114:Calcinosis / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D023421:Models, Animal; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D000509:alpha-Fetoproteins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000577:Amides; D004791:Enzyme Inhibitors; D011725:Pyridines; D034741:RNA, Small Interfering; D000509:alpha-Fetoproteins; C108830:Y 27632; D054460:rho-Associated Kinases; D000469:Alkaline Phosphatase; D020742:rhoA GTP-Binding Protein