RESUMO
Here we show that tyrosine phosphorylation of caveolin-2 (Cav-2) negatively regulates the anti-proliferative function of transforming growth factor beta (TGF-beta) in endothelial cells. In contrast to wild-type-Cav-2, retroviral re-expression of Y19/27F-Cav-2 in Cav-2 knockout endothelial cells did not affect anti-proliferative effect of TGF-beta compared to empty vector. Conversely, although less effective than wild-type, re-expression of S23/36A-Cav-2 reduced the effect of TGF-beta compared to empty vector. This differential effect of tyrosine and serine phosphorylation mutants of Cav-2 correlated with TGF-beta-induced Smad3 phosphorylation and transcriptional activation of plasminogen activator inhibitor-1. Thus tyrosine-phosphorylated Cav-2 counteracts anti-proliferative effect of TGF-beta in endothelial cells.
Assuntos
Caveolina 2/química , Caveolina 2/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Substituição de Aminoácidos , Animais , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Técnicas de Inativação de Genes , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serpina E2/genética , Proteína Smad3/metabolismo , Ativação Transcricional/efeitos dos fármacos , Tirosina/químicaRESUMO
Using a combination of wild-type (WT) and caveolin-2 (Cav-2) knockout along with retroviral reexpression approaches, we provide the evidence for the negative role of Cav-2 in regulating anti-proliferative function and signaling of transforming growth factor ß (TGF-ß) in endothelial cells (ECs). Although, TGF-ß had a modest inhibitory effect on WT ECs, it profoundly inhibited proliferation of Cav-2 knockout ECs. To confirm the specificity of the observed difference in response to TGF-ß, we have stably reexpressed Cav-2 in Cav-2 knockout ECs using a retroviral approach. Similar to WT ECs, the anti-proliferative effect of TGF-ß was dramatically reduced in the Cav-2 reexpressing ECs. The reduced anti-proliferative effect of TGF-ß in Cav-2-positive cells was evidenced by three independent proliferation assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell count, and bromodeoxyuridine incorporation and correlated with a loss of TGF-ß-mediated upregulation of cell cycle inhibitor p27 and subsequent reduction of the levels of hyperphosphorylated (inactive) form of the retinoblastoma protein in Cav-2 reexpressing ECs. Mechanistically, Cav-2 inhibits anti-proliferative action of TGF-ß by suppressing Alk5-Smad2/3 pathway manifested by reduced magnitude and length of TGF-ß-induced Smad2/3 phosphorylation as well as activation of activin receptor-like kinase-5 (Alk5)-Smad2/3 target genes plasminogen activator inhibitor-1 and collagen type I in Cav-2-positive ECs. Expression of Cav-2 does not appear to significantly change targeting of TGF-ß receptors I and Smad2/3 to caveolar and lipid raft microdomains as determined by sucrose fractionation gradient. Overall, the negative regulation of TGF-ß signaling and function by Cav-2 is independent of Cav-1 expression levels and is not because of changing targeting of Cav-1 protein to plasma membrane lipid raft/caveolar domains.
