The anti-mitogenic effect of TGFâ1 in pituitary tumor cells is enhanced by the inhibition of PI3K/Akt and MEK/ERK1/2 pathways

PETITI JUAN PABLO; SABATINO MARíA EUGENIA; GUTIéRREZ SILVINA; SOSA LILIANA; VACA ALICIA MALDRE; DE PAUL ANA LUCíA; TORRES ALICIA

Bailoche

Simposio; Second South American Spring Symposium in Signal Transduction and Molecular Medicine”; 2012

Petiti JP, Sabatino E, Gutiérrez S, Sosa L, Vaca A, De Paul A, Torres AI.

TGFâ1 is a tumor suppressor that induces cell cycle arrest through the activation of Smads, which 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 the anti-proliferative response induced by TGFâ1 in tumor adenohypophysial cells. GH3B6 cells were treated with TGFâ1 (4ng/ml, 30 min or 24h) in the presence or absence of the inhibitors added 30 min before: LY294002 (PI3K 10ìM) and PD98059 (MEK1/2; 50ìM). The 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 WB. The translocation of Smad2/3 and ERK1/3 was observed using confocal microscopy. Statistics: ANOVA-Fisher. The treatment with TGFâ1 induced phosphorylation of Smad2/3 and cell arrest in G0/G1 phase (p<0.01). These effects were potentiated when cells were pre-incubated with the inhibitors of PI3K and MEK1/2 (p<0.01). With respect to hormone release, TGFâ1 decreased the PRL secretion, reaching lower values when cells were pretreated with PD98059 and not with LY294002 (p<0.01). The immunoprecipitation assays showed interaction between Smad2/3 and ERK1/2 and/or Akt. These associations were increased after TGFâ1 treatment, effect that was blocked when the cells were pre-incubated with the inhibitors of PI3K or MEK1/2 (p<0.01). The confocal microscopy showed translocation of Smad2/3 from cytosolic to nucleus after TGFâ1 incubation and co-localization of Smad2/3 with ERK1/2. These results demonstrate that the anti-mitogenic effect of TGFâ1 in tumoral pituitary cells is regulated by PI3K and ERK1/2 which inhibit the activation of Smad2/3 and consequently the anti-proliferative actions of TGFâ1. GH3B6 cells were treated with TGFâ1 (4ng/ml, 30 min or 24h) in the presence or absence of the inhibitors added 30 min before: LY294002 (PI3K 10ìM) and PD98059 (MEK1/2; 50ìM). The 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 WB. The translocation of Smad2/3 and ERK1/3 was observed using confocal microscopy. Statistics: ANOVA-Fisher. The treatment with TGFâ1 induced phosphorylation of Smad2/3 and cell arrest in G0/G1 phase (p<0.01). These effects were potentiated when cells were pre-incubated with the inhibitors of PI3K and MEK1/2 (p<0.01). With respect to hormone release, TGFâ1 decreased the PRL secretion, reaching lower values when cells were pretreated with PD98059 and not with LY294002 (p<0.01). The immunoprecipitation assays showed interaction between Smad2/3 and ERK1/2 and/or Akt. These associations were increased after TGFâ1 treatment, effect that was blocked when the cells were pre-incubated with the inhibitors of PI3K or MEK1/2 (p<0.01). The confocal microscopy showed translocation of Smad2/3 from cytosolic to nucleus after TGFâ1 incubation and co-localization of Smad2/3 with ERK1/2. These results demonstrate that the anti-mitogenic effect of TGFâ1 in tumoral pituitary cells is regulated by PI3K and ERK1/2 which inhibit the activation of Smad2/3 and consequently the anti-proliferative actions of TGFâ1. The treatment with TGFâ1 induced phosphorylation of Smad2/3 and cell arrest in G0/G1 phase (p<0.01). These effects were potentiated when cells were pre-incubated with the inhibitors of PI3K and MEK1/2 (p<0.01). With respect to hormone release, TGFâ1 decreased the PRL secretion, reaching lower values when cells were pretreated with PD98059 and not with LY294002 (p<0.01). The immunoprecipitation assays showed interaction between Smad2/3 and ERK1/2 and/or Akt. These associations were increased after TGFâ1 treatment, effect that was blocked when the cells were pre-incubated with the inhibitors of PI3K or MEK1/2 (p<0.01). The confocal microscopy showed translocation of Smad2/3 from cytosolic to nucleus after TGFâ1 incubation and co-localization of Smad2/3 with ERK1/2. These results demonstrate that the anti-mitogenic effect of TGFâ1 in tumoral pituitary cells is regulated by PI3K and ERK1/2 which inhibit the activation of Smad2/3 and consequently the anti-proliferative actions of TGFâ1. These results demonstrate that the anti-mitogenic effect of TGFâ1 in tumoral pituitary cells is regulated by PI3K and ERK1/2 which inhibit the activation of Smad2/3 and consequently the anti-proliferative actions of TGFâ1.