Platelet-derived growth factor-BB activates calcium/calmodulin-dependent and -independent mechanisms that mediate Akt phosphorylation in the neurofibromin-deficient human Schwann cell line ST88-14

Abstract

Neurofibromatosis type 1-derived Schwann cells isolated from malignant peripheral nerve sheath tumors (MPNSTs) overexpress PDGF receptor-β and generate an aberrant intracellular calcium increase in response to PDGF-BB. Using the human MPNST Schwann cell line ST88-14, we demonstrate that, in addition to a transient phosphorylation of Akt, PDGF-BB stimulation produces an atypical sustained phosphorylation of Akt that is dependent on calcium and calmodulin (CaM). The sustained Akt phosphorylation did not occur in PDGF-BB-stimulated normal human Schwann cells or ST88-14 cells stimulated with stem cell factor, whose receptor is also overexpressed in ST88-14 cells. The sustained Akt phosphorylation induced by PDGF-BB was inhibited by pretreatment of the cells with either the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA-AM) or the CaM antagonist W7, whereas the transient portion was not inhibited. Akt also co-immunoprecipitated with CaM in a PDGF-BB-dependent manner, suggesting that direct interaction between Akt and CaM is involved in the sustained phosphorylation of Akt. Furthermore, we provide evidence that anti-apoptotic effects of PDGF-BB on serum-deprived ST88-14 cells can be inhibited by W7, implicating the PDGF-BB-induced activation of calcium/CaM in promoting cell survival, presumably through sustained Akt activation. We conclude that the activation of the calcium/CaM/Akt pathway resulting from stimulation of overexpressed PDGF receptor-β may contribute to the survival and tumorigenicity of MPNST cells.

FIGURE 1.

Comparison of basal levels of Akt and ERK1/2 phosphorylation and expression in ST88-14 cells and nhSc. Total cell lysates were prepared from cells that were serum-starved for 4 h prior to harvesting and analyzed by Western blotting. The difference in neurofibromin expression between the two cell types was demonstrated by immunostaining with a polyclonal antibody whose epitope maps within the C terminus of human neurofibromin. Immunostaining for pERK1/2 and pAkt^Ser-473 was done using appropriate phosphospecific antibodies. Peroxidase-conjugated secondary antibodies were used for visualization, after which the blots were stripped and reprobed with anti-Akt and anti-ERK antibodies. GAPDH was used as a loading control. Notice that there are similar levels of total Akt and pAkt^Ser-473 for the two cell types but markedly different amounts of total ERK and pERK. The results shown are representative of three or more independent experiments.

FIGURE 2.

Time course of Akt Ser-473 and ERK1/2 phosphorylation after growth factor stimulation. Cells were plated at a density of 1.5 × 10⁵ cells/35-mm tissue culture dish for ST88-14 cells or type I collagen-coated dish for nhSc, and the following day, they were serum-starved for 4 h prior to being treated with growth factors (20 ng/ml for both SCF and PDGF-BB) for the indicated times. Total cell lysates were prepared and subjected to Western blot analysis for pAkt, pERK1/2, and GAPDH (loading control). Phosphorylation of Akt and ERK1/2 over a 2-h time period is compared in SCF-stimulated (A) and PDGF-BB-stimulated (B) ST88-14 cells and in PDGF-BB-stimulated nhSc (C) and PDGF-BB-stimulated ST88-14 cells (D). The results shown are representative of three independent experiments.

FIGURE 3.

Effect of intracellular calcium chelator BAPTA-AM and CaM antagonist W7 on PDGF-BB-induced transient and sustained phosphorylation of Akt at Ser-473. ST88-14 cells were serum-starved for 4 h and then pretreated with vehicle, 25 μm BAPTA-AM (A), 10 μm W7 (B), or 10 μm W5 (B) for 30 min prior to PDGF-BB stimulation. Cells were harvested at 0 min (untreated), 30 min (to represent transient phosphorylation), and 120 min (to represent sustained phosphorylation), and total cellular protein was subjected to Western blot analysis. Blots were immunostained for pAkt^Ser-473 and GAPDH (loading control), followed by peroxidase-conjugated secondary antibodies. Results are mean ± S.E. from three (A) or four (B) independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

Effect of PI3K inhibition with LY294002 on PDGF-BB-stimulated transient and sustained Akt Ser-473 phosphorylation. ST88-14 cells were serum-starved for 4 h and then pretreated with vehicle or 50 μm LY294002 for 30 min prior to PDGF-BB (20 ng/ml) stimulation. Cells were harvested at 0 min (untreated), 15 min (to represent transient phosphorylation), and 120 min (to represent sustained phosphorylation), and total cellular protein was subjected to Western blot analysis. Blots were immunostained for pAkt^Ser-473, pERK, and GAPDH (loading control), followed by peroxidase-conjugated secondary antibodies. Results are representative of three independent experiments.

FIGURE 5.

PDGF-BB-dependent co-immunoprecipitation of Akt with CaM. ST88-14 cells (1.5 × 10⁶/time point) were serum-starved for 4 h and then treated with 20 ng/ml PDGF-BB for 0 (untreated), 30, and 120 min. Cells were solubilized in lysis buffer, and the lysates were incubated overnight with anti-CaM antibody and precipitated with protein A/G-agarose. Immunoprecipitated Akt was detected by Western blotting (WB) using an antibody to total Akt. Results are mean ± S.E. from five independent experiments. *, p < 0.05. IP, immunoprecipitation.

FIGURE 6.

Effect of CaM antagonist W7 on PDGF-BB-induced cell survival. Shown is the morphology of ST88-14 cells incubated for 24 h in normal medium (DMEM and 5% FBS) (A), serum-free/low glucose MEM (B), MEM + 20 ng/ml PDGF-BB (C), MEM + 20 ng/ml PDGF-BB + 10 μm W7 (D), MEM + 20 ng/ml PDGF-BB + 10 μm W5 (E), or MEM + 20 ng/ml SCF (F). W7 or W5 was added to the medium 30 min prior to PDGF-BB treatment. Arrowheads in B and D indicate cells with morphologies consistent with dying cells. Scale bar = 100 μm. G and H, after incubating ST88-14 cells for 24 h under the conditions indicated in A–F, cells were harvested, and total cellular protein was subjected to Western blot analysis. Blots were immunostained for cleaved PARP, PARP, and GAPDH (loading control), followed by peroxidase-conjugated secondary antibodies. Results are mean ± S.E. from four independent experiments. *, p < 0.05; **, p < 0.01.