EXTRACELLULAR ATP ACTIVATES MAP KINASE CASCADES AND INCREASES INTESTINAL Caco-2 CELL PROLIFERATION

BUZZI, NATALIA; SCODELARO BILBAO, PAOLA GABRIELA; BOLAND, RICARDO; RUSSO DE BOLAND, ANA

Capital Federal

Congreso; XXVI Reunión Anual de la Asociación Argentina de Osteología y Metabolismo Mineral; 2009

Asociación Argentina de Osteología y Metabolismo Mineral

In the present work, we examined the role of ATP in the activation of the MAP kinases (MAPKs) ERK1/2, JNK1/2 and p38 and their involvement in the modulation of transcription factors and proliferation of human colon cancer Caco-2 cells. Our results showed that ATP induces the phosphorylation of MAPKs in a time and dose-dependent manner, peaking at 5 min at 10 mM ATP. Moreover, UTP and ATPgS but not ADP or ADPâS increased phosphorylation of MAPKs, indicating the involvement of, at least, P2Y2/P2Y4 purinergic receptor subtypes. RT-PCR studies and PCR product sequencing supported the expression of P2Y2 and P2Y4 receptors in this cell line. Spectrofluorimetric measurements showed that cell stimulation with ATP induced transient elevations in intracellular calcium concentration. In addition, western blot analysis revealed that ATP-induced phosphorylation of MAPKs in Caco-2 cells was dependent on calcium influx and intracellular calcium release, Src-family tyrosine kinases, and partially dependent on the cAMP/PKA and PKC pathways. The epidermal growth factor receptor (EGFR) specific inhibitor AG1478 decreased ERK1/2, JNK1/2 and p38 MAPK phosphorylation by ATP, suggesting that EGFR transactivation is important for ATP-mediated stimulation of MAPKs in this intestinal cell line. We demonstrate, by confocal microscopy, that stimulation of Caco-2 cells with ATP results in the translocation of active MAPKs to the nucleous where they induce the expression of c-Fos and Jun family proteins, the phosphorylation of ATF-1, ATF-2 and JunD transcription factors and stimulate Caco-2 cell proliferation. Moreover, MAPKs participate in the induction and phosphorylation of the dual phosphatase MKP-1, which is known to inactivate ERK, JNK and p38 MAPK by dephosphorylating two critical sites in the activation loop. These findings provide new molecular basis for further understanding the mechanisms involved in ATP functions, as a signal transducer and activator of MAP kinase cascades, in Caco-2 cells derived from human colon adenocarcinoma.