RESUMEN
The endogenous phospholipid l-alpha-lysophosphatidylinositol (LPI) was recently identified as a novel ligand for the orphan G protein-coupled receptor 55 (GPR55). In this study we define the downstream signaling pathways activated by LPI in a human embryonic kidney (HEK) 293 cell line engineered to stably express recombinant human GPR55. We find that treatment with LPI induces marked GPR55 internalization and stimulates a sustained, oscillatory Ca(2+) release pathway, which is dependent on Galpha13 and requires RhoA activation. We then establish that this signaling cascade leads to the efficient activation of NFAT (nuclear factor of activated T cells) family transcription factors and their nuclear translocation. Analysis of cannabinoid ligand activity at GPR55 revealed no clear effect of the endocannabinoids anandamide and 2-arachidonoylglycerol; however, the classical CB(1) antagonist AM251 evoked GPR55-mediated Ca(2+) signaling. Thus, LPI is a potent and efficacious ligand at GPR55, which is likely to be a key plasma membrane mediator of LPI-mediated signaling events and changes in gene expression.
Asunto(s)
Señalización del Calcio/fisiología , Lisofosfolípidos/farmacología , Factores de Transcripción NFATC/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Línea Celular , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Regulación de la Expresión Génica , Humanos , Transporte de Proteínas , Receptores de Cannabinoides , Proteína de Unión al GTP rhoA/genéticaRESUMEN
BACKGROUND AND PURPOSE: Increased circulating levels of L-alpha-lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G-protein-coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55. EXPERIMENTAL APPROACH: Quantitative RT-PCR was used to measure GPR55 expression in breast cancer cell lines. Cell migration and invasion were measured using a Boyden chamber chemotaxis assay and Cultrex invasion assay, respectively. Cell polarization and orientation in response to the microenvironment were measured using slides containing nanometric grooves. KEY RESULTS: GPR55 expression was detected in the highly metastatic MDA-MB-231 breast cancer cell line. In these cells, LPI stimulated binding of [(35)S]GTPgammaS to cell membranes (pEC(50) 6.47 +/- 0.45) and significantly enhanced cell chemotaxis towards serum. MCF-7 cells expressed low levels of GPR55 and did not migrate or invade towards serum factors. When GPR55 was over-expressed in MCF-7 cells, serum induced a robust migratory and invasive response, which was further enhanced by LPI and prevented by siRNA to GPR55. The physical microenvironment has been identified as a key factor in determining breast tumour cell metastatic fate. LPI endowed MDA-MB-231 cells with the capacity to detect shallow (40 nm deep) grooved slides and induced marked cancer cell polarization on both flat and grooved surfaces. CONCLUSIONS AND IMPLICATIONS: LPI and GPR55 play a role in the modulation of migration, orientation and polarization of breast cancer cells in response to the tumour microenvironment.