RESUMO
Interaction between signaling pathways regulates many cellular functions, including proliferation. The Galpha(s)/cAMP pathway is known to inhibit signal flow from receptor tyrosine kinases to mitogen-activated protein kinase (MAPK)-1,2 and, thus, inhibit proliferation. Elevation of cAMP or adenovirus-directed expression of mutant (Q227L)-Galpha(s) (alpha(s)*) inhibits the proliferation of rat vascular smooth muscle cells (VSMCs) in culture. Platelet-derived growth factor (PDGF) stimulated MAPK activation and DNA synthesis was also blocked by expression of alpha(s)*. However, it is not known whether such mechanisms are operative in vivo. Proliferation of vascular smooth muscle cells in vivo was induced by balloon injury of carotid arteries in the rat. Recombinant adenovirus encoding beta-galactosidase (beta-gal) or alpha(s)* was applied to arterial segments injured by the balloon catheters. The alpha(s)*-treated vessels showed decreased phospho-MAPK staining in the intima as compared with beta-gal-treated vessels. Application of alpha(s)*, but not beta-gal containing adenovirus, inhibited formation of neointima by 50%. No change was observed in total vessel diameter or in the media or adventitia. These results suggest that the interaction between the Galpha(s) and MAPK pathways can regulate proliferation in vivo and that targeted expression of activated Galpha(s) may have therapeutic potential for the treatment of vascular pathophysiologies that arise from intimal hyperplasia.
Assuntos
Angioplastia com Balão/efeitos adversos , Aorta/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Adenoviridae , Substituição de Aminoácidos , Animais , Aorta/citologia , Aorta/patologia , Células Cultivadas , AMP Cíclico/metabolismo , Replicação do DNA , Vetores Genéticos , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Hiperplasia , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Transfecção , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
The effects of expression of mutant (Q227L)-activated Galphas and elevation of cAMP on mitogen-activating protein kinase (MAPK) activity and the transformed phenotype were studied in the MCF-7 human mammary epithelial cell line. Elevation of cAMP partially inhibited the epidermal growth factor-stimulated DNA synthesis and the intrinsic MAPK (ERK-1 and ERK-2) of serum-starved MCF-7 cells. Addition of 8Br-cAMP or expression of mutant (Q227L)-activated Galphas in MCF-7 cells blocked the ability of these cells to grow in an anchorage-independent manner, as assessed by colony formation in soft agar. 8Br-cAMP in the culture medium also blocked estrogen stimulation of MCF-7 cell proliferation in vitro. MCF-7 cells expressing Q227L-Galphas grew very slowly in vitro, and when these cells were injected s.c. into athymic mice implanted with estrogen pellets, the frequency of tumor formation was reduced greatly and the sizes of the tumors formed were much smaller than those in mice injected with MCF-7 cells that had been transfected with the empty vector. These results indicate that the intracellular levels of cAMP in transformed mammary epithelial cells can be a crucial factor in determining the expression of the transformed phenotype. Interactions between the Gs/adenylyl cyclase and MAPK-1,2 signaling pathways could be one mechanism by which expression of the transformed phenotype in mammary epithelial cells are regulated.
