Cardiac gap junctions, a target of growth factor signaling

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Doble, Bradley Wayne
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'Introduction'. We sought to: (a) examine the effects of fibroblast growth factor-2 (FGF-2) on cardiomyocyte gap junctions composed of connexin 43 (Cx43); (b) identify the kinase(s) responsible for mitogen induced Cx43 phosphorylation; (c) establish a cause and effect relationship between Cx43 phosphorylation and cardiomyocyte proliferation. 'Methods'. We used well coupled, primary neonatal rat cardiomyocytes as our model system. Cx43 protein and mRNA levels were determined by Western and Northern blotting, respectively. Labeling cells with [ 32P], followed by Cx43 immunoprecipitation, gel electrophoresis and autoradiography was used to assess Cx43 phosphorylation. The identity of phosphorylated Cx43 residues was examined by phospho-amino acid analysis. Scrape-loading and microinjection of fluorescent dye were used to assess gap junction-mediated coupling. Genistein, an inhibitor of tyrosine phosphorylation, PD98059, an inhibitor of mitogen ac ivated protein kinase (MAPK) kinase, and calphostin-C and chelerythrine, inhibitors of protein kinase C (PKC), were used to delineate FGF-2-initiated signals involved in regulating Cx43 phosphorylation and intercellular coupling. 'Results'. FGF-2 stimulated Cx43 phosphorylation on serine with a concomitant decrease in dye-coupling; FGF-2 had no effect on Cx43 accumulation or distribution, but elicited masking of epitope(s) contained within residues 260-270 of Cx43. Pathways requiring activation of tyrosine phosphorylation and PKC, but not MAPK, mediated the FGF-2 effects on Cx43 phosphorylation and dye-coupling. An interaction between Cx43 and PKC[epsilon] (but not PKC_), detected by co-immunoprecipitation, was strengthened by FGF-2 treatment. Also, more extensive co-localization of PKC[epsilon] and Cx43, upon FGF-2 or PMA treatment, was detected by immunofluorescence. Overexpression of dominant-negative PKC[epsilon] significantly reduced basal levels of Cx43 phosphorylation. 'Conclusions'. FGF-2 causes a reduction in dye coupling between neonatal rat cardiomyocytes through activation of its receptor tyrosine, kinase and a PKC pathway resulting in serine phosphorylation of Cx43. PKC[epsilon] is required for neonatal rat cardiomyocyte Cx43 phosphorylation and is directly responsible for PMA and FGF-2-induced phosphorylation of Cx43. S262 of Cx43 appears to be phosphorylated in the cellular milieu in response to PKC stimulation; its phosphorylation, triggered by mitogens, is likely required to cancel the growth-inhibitory effect exerted by Cx43. (Abstract shortened by UMI.)