Blockade of connexin 43 expression by stable transfection of antisense cDNA in cultured vascular smooth muscle cells.
Gap junctional communication is involved in embryogenesis, cell growth control, and coordinated contraction of cardiac myocytes. It has been hypothesized that gap junctions coordinate responses of vascular cells to constrictor or dilator stimulation. Three connexin (Cx) proteins, 37, 40, and 43, are found in the vasculature. Cx43 gap junctions are widely distributed along the vascular tree, although a precise physiologic role in vascular function is unknown because of a lack of specific functional inhibitors and of suitable animal models. To investigate the role of Cx43 in intercellular communication among vascular smooth muscle (VSM) cells, we selectively modified the expression of the Cx43 gene using antisense cDNA stable transfections in culture. Results show that in cells stably transfected with antisense Cx43 cDNA, gene expression of Cx43 could be reduced to 20% of that observed in vector-transfected cells. In spite of the mRNA and protein reduction, the antisense Cx43 cDNA-transfected cells did not show a significant reduction in dye transfer or a difference in cell growth rate as compared with control. These results suggest either that the residual amount of Cx43 protein is sufficient for dye transfer and growth control or that the dye transfer in these cells can be mediated by Cx40 or other connexin proteins. Therefore, more potent approaches, such as dominant negative and gene knockout, are required to fully block gap junctional communication in VSM cells.
Liao
Y
Y
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22906-0011, USA.
Duling
B R
BR
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002450:Cell Communication; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D016373:DNA, Antisense / Q000235:genetics* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000502:physiology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014162:Transfection
D018031:Connexin 43; D016373:DNA, Antisense; D005456:Fluorescent Dyes