RESUMEN
The heterotrimeric G-protein subunits Galpha and Gbetagamma are involved in cellular transformation and tumor development. Here, we report the expression of Galpha(olf) in human digestive and urogenital epithelial cells using RT-PCR and Western blot. When the constitutively activated form of Galpha(olf)Q214L (AGalpha(olf)) was stably transfected in canine kidney MDCKts.src and human colonic HCT-8/S11 epithelial cells, it induced cellular invasion in collagen gels. AGalpha(olf)-mediated invasion was abrogated by agonists of platelet activating factor receptors (PAF-R) and protease-activated receptors -1 (PAR-1), pharmacological inhibitors of PI3'-Kinase (wortmannin), protein kinase C (Gö6976 and GF109203X), Rho GTPase (C3T exoenzyme), but was independent of protein kinase A. Accordingly, the invasive phenotype induced by AGalpha(olf) in HCT-8/S11 cells was reversed by the RhoA antagonist RhoD (G26V). Although AGalpha(olf) protected MDCKts.src cells against serum starvation-mediated apoptosis via a Rho-independent pathway, both AGalpha(olf) and Rho inhibition by C3T induced neuroendocrine-like differentiation linked to extensive neurite outgrowth and parathyroid hormone-related protein expression in human prostatic LNCaP-AGalpha(olf) cells. Since prostate tumors with a larger neuroendocrine cell population display increased invasiveness, persistent activation of the G-protein alpha(olf) may exert convergent adverse effects on cellular invasion and survival in solid tumors during the neoplastic progression towards metastasis. doi:10.1038/sj.onc.1205498
Asunto(s)
Células Epiteliales/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Invasividad Neoplásica/patología , Proteínas de Unión al GTP rho , Apoptosis/fisiología , Western Blotting , Diferenciación Celular , Supervivencia Celular , Colágeno/metabolismo , Colon/citología , Colon/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cartilla de ADN/química , Subunidades alfa de la Proteína de Unión al GTP , Sustancias de Crecimiento/metabolismo , Proteínas de Unión al GTP Heterotriméricas/genética , Humanos , Riñón/citología , Riñón/metabolismo , Masculino , Mutación , Neoplasias Hormono-Dependientes/metabolismo , Sistemas Neurosecretores/fisiología , Mucosa Olfatoria , Próstata/citología , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Because we recently identified the G(alpha)olf subunit in rat pancreatic beta-cells, we investigated the downstream effectors and the biological functions of this G protein in HEK-293T cells and the insulin-secreting mouse betaTC-3 cell line. With the use of transient transfection of HEK-293T cells with constitutively activated G(alpha)olf (G(alpha)olfQ214L, i.e., AG(alpha)olf), together with expression vectors encoding the adenylyl cyclase (AC) isoforms (AC-I to -VIII and soluble AC), compared with cotransfections using AG(alphas) (G(alphas)R201C), we observed that AG(alpha)olf preferentially activates AC-I and -VIII, which are also expressed in beta-cells. Stable overexpression of wild-type or AG(alpha)olf in betaTC-3 cells resulted in partial attenuation of insulin secretion and biosynthesis, suggesting that chronic activation of the G(alpha)olf-signaling pathway is associated with beta-cell desensitization. In agreement, transfected betaTC-3 cells present a decreased insulin content with respect to parental cells, whereas the proinsulin convertases PC-1 and PC-2 were unaffected. Furthermore, betaTC-3-AG(alpha)olf cells are resistant to serum starvation-induced apoptosis. Our findings suggest that G(alpha)olf is involved in insulin status, cell survival, and regeneration of the insulin-secreting beta-cells during development and diabetes.