A New α5β1 Integrin-Dependent Survival Pathway Through GSK3β Activation in Leukemic Cells

Cell survival mediated by integrin engagement has been implicated in cell adhesion-mediated drug resistance. We have recently demonstrated that the activation of glycogen synthase kinase 3 beta (GSK3beta) is a new pathway supporting the chemoresistance of leukemic cells adhered to fibronectin.We show here that in conditions of serum starvation, the fibronectin receptor alpha(5)beta(1) integrin, but not alpha(4)beta(1), induced activation of GSK3beta through Ser-9 dephosphorylation in adherent U937 cells. The GSK3beta-dependent survival pathway occurred in adherent leukemic cells from patients but not in the HL-60 and KG1 cell lines. In adhesion, activated GSK3beta was found in the cytosol/plasma membrane compartment and was co-immunoprecipitated with alpha(5) integrin, the phosphatase PP2A and the scaffolding protein RACK1. PP2A and its regulatory subunit B' regulated the Ser-9 phosphorylation of GSK3beta. In adherent leukemic cells, alpha(5)beta(1) integrin but not alpha(4)beta(1) upregulated the resistance to TNFalpha-induced apoptosis. Both extrinsic and intrinsic apoptotic pathways were under the control of alpha(5)beta(1) and GSK3beta.Our data show that, upon serum starvation, alpha(5)beta(1) integrin engagement could regulate specific pro-survival functions through the activation of GSK3beta

α₅ integrin, PP2A and GSK3β are co-localized in adherent leukemic cells.

<p><b>A</b>- The Ser-9 phosphorylation of GSK3β was studied by Western blot in cytosolic (Ct) and membrane (Mb) compartments of U937 in adhesion or in suspension. <b>B</b>- The presence of GSK3β (and its Ser-9 phosphorylated form), PP2A (and its Tyr-307 phosphorylated form), the scaffolding protein RACK1 and the PI 3-kinase regulatory subunit p85 was checked by Western blot in α₅ immunoprecipitates from U937 in suspension (Susp.) or in adhesion (Adh.). The absence of flotillin in α₅ immunoprecipitate demonstrates specificity of the interactions between α₅ integrin, PP2A and GSK3β. Right panel (<i>input</i>) indicates the amounts of α₅ subunit and GSK3β proteins in the total lysates (Total) and in the supernatants (Sup.), before and after immunoprecipitation of α₅ respectively. Data are representative of three independent experiments.</p

GSK3β is differentially regulated by α₄β₁ and α₅β₁ integrins in leukemic cells.

<p><b>A</b>- Specificity and efficacy of siRNA integrins were checked by Western blot. <b>B</b>- Serum-starved U937 were allowed to adhere on fibronectin for 1 h and the Ser-9 phosphorylation state of GSK3β was compared to cells in suspension after downregulation of α₄ and α₅ expression by siRNA. SiC = non-targeting siRNA. Right panel shows the mean ± S.E.M. variations of the P(S9)GSK3β/GSK3β ratio analyzed by densitometry from three independent experiments. <b>C</b>- The phosphorylation state of GSK3β was studied in suspension or after adhesion of U937 on surfaces coated with fibronectin (Fn, 40 µg/ml), anti-α₄ (1 µg/ml) or -α₅ (clone P1D6 0.1 µg/ml; clone JBS5 0.2 µg/ml) antibodies in experimental conditions as for B. Adhesive capacities of U937 in each condition of coating were measured by colorimetry and are shown on right panel. Data are representative of three independent experiments.</p