The anti-mitogenic effect of TGFbeta1 in pituitary tumor cells is enhanced by the inhibition of PI3K/Akt and MEK/ERK1/2 pathways.

PETITI JP, ; SABATINO E,; GUTIÉRREZ S, ; SOSA L, ; VACA A, ; DE PAUL A, ; TORRES AI.

Bariloche

Simposio; Second South American Spring Symposium in Signal Transduction and Molecular Medicine (SISTAM); 2012

TGFbeta1 is a tumor suppressor that induces cell cycle arrest through the activation of Smads, which in turn can be regulated by other signal pathways. Our objective was to determine the contribution of the PI3K/Akt and MEK/ERK1/2 pathways as modulators of TGFbeta1-induced anti-proliferative response in tumoral adenohypophysial cells. GH3B6 cells were treated with TGFbeta1 (4ng/ml, 30 min or 24h) in presence or absence of the inhibitors: LY294002 (PI3K 10µM) and PD98059 (MEK1/2; 50µM). Cell cycle was analyzed by flow cytometry and the PRL secretion by RIA. The interaction of Smad2/3 with Akt or ERK1/2 was determined by immunoprecipitation and subsequent Western blot. The translocation of Smad2/3 and ERK1/3 was observed by confocal microscopy. Statistics: ANOVA-Fisher. The TGFbeta1 treatment induced Smad2/3 phosphorylation and cell arrest in G0/G1 phase (p lower tha 0.01). Both effects were potentiated when the cells were pre-incubated with PI3K and MEK1/2 inhibitors (p lower than 0.01). TGFbeta1 decreased the PRL secretion, reaching lowest values when cells were pretreated with PD98059 (p lower than 0.01). No changes were detected after incubation with LY294002. The immunoprecipitation assays showed interaction of Smad2/3 with ERK1/2 and Akt. These associations were increased after TGFbeta1 treatment, effect that was blocked when the cells were pre-incubated with the PI3K or MEK1/2 inhibitors (p lower than 0.01). The confocal microscopy showed Smad2/3 translocation from cytosol to the nucleus after TGFbeta1 incubation and co-localization of Smad2/3 with ERK1/2 was also observed. The PI3K and ERK1/2 inhibitors increased Smad2/3 phosphorylation potentiating the anti-mitogenic effect of TGFbeta1 in GH3B6 cells. These results may contribute to the knowledge of intracellular mechanisms involved in the resistance to TGFbeta1 growth–inhibitory effect in tumoral pituitary cells.