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Identification and characterization of the unique guanine nucleotide exchange factor, SmgGDS, in vascular smooth muscle cells
Author(s) -
Thill Rebecca,
Campbell William B.,
Williams Carol L.
Publication year - 2008
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.21740
Subject(s) - rhoa , guanine nucleotide exchange factor , transfection , vascular smooth muscle , microbiology and biotechnology , myosin light chain phosphatase , myosin , rho associated protein kinase , biology , myosin light chain kinase , cell culture , gtpase , phosphorylation , signal transduction , endocrinology , smooth muscle , genetics
The guanine nucleotide exchange factor (GEF), SmgGDS, promotes nucleotide exchange by several GTPases in both the Ras and Rho families, especially by RhoA. Because RhoA plays an important role in regulating the contraction of vascular smooth muscle cells (VSMC), we examined the expression and function of SmgGDS in VSMC. SmgGDS is expressed in primary rat aortic smooth muscle (ASM) cells, primary bovine coronary artery smooth muscle (BCASM) cells, and the immortalized A7r5 line of rat ASM cells. Down regulation of SmgGDS expression by siRNA transfection resulted in a decrease of RhoA‐GTP levels, enhanced cell spreading, and loss of the characteristic elongated morphology of VSMC. A similar morphology was also observed following treatment with the Rho‐kinase inhibitor, Y27632. In contrast, cells with reduced RhoA expression exhibit an elongated shape. Subsequent immunofluorescent staining revealed a disruption of the myosin filament organization in the cells with reduced SmgGDS expression. Further studies analyzed the effect of SmgGDS siRNA transfection on the contraction of A7r5 cells and BCASM cells, which is also a Rho‐regulated pathway. Transfection of SmgGDS siRNA or RhoA siRNA resulted in an impaired ability of the A7r5 and BCASM cells to undergo contraction in a collagen gel matrix. However, phosphorylation of the myosin‐binding subunit of myosin phosphatase (MYPT1) or the light chain of myosin II (MLC) was not altered by downregulating expression of either SmgGDS or RhoA GTPase. Taken together these results identify SmgGDS as a novel regulator of myosin organization and contraction in VSMC. J. Cell. Biochem. 104: 1760–1770, 2008. © 2008 Wiley‐Liss, Inc.