Research Service, Edward Hines, Jr. Veterans Administration Hospital, Hines, Illinois 60141, USA.
J Biol Chem. 2013 Apr 19;288(16):11066-73. doi: 10.1074/jbc.M112.442244. Epub 2013 Mar 1.
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.
神经纤维瘤病 1 型衍生的施万细胞从恶性外周神经鞘瘤(MPNST)中分离出来,过表达 PDGF 受体-β,并对 PDGF-BB 产生异常的细胞内钙增加。使用人 MPNST 施万细胞系 ST88-14,我们证明,除了 Akt 的短暂磷酸化外,PDGF-BB 刺激还产生依赖于钙和钙调蛋白(CaM)的非典型持续 Akt 磷酸化。这种持续的 Akt 磷酸化不会发生在 PDGF-BB 刺激的正常人类施万细胞中,也不会发生在 ST88-14 细胞中刺激干细胞因子时发生,其受体也在 ST88-14 细胞中过度表达。PDGF-BB 诱导的持续 Akt 磷酸化可通过用细胞内钙螯合剂 1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸四(乙酰氧甲基)酯(BAPTA-AM)或 CaM 拮抗剂 W7 预处理细胞来抑制,而瞬时部分则不受抑制。Akt 还与 CaM 以 PDGF-BB 依赖的方式共同免疫沉淀,表明 Akt 和 CaM 之间的直接相互作用参与了 Akt 的持续磷酸化。此外,我们提供的证据表明,W7 可以抑制 PDGF-BB 对血清剥夺的 ST88-14 细胞的抗凋亡作用,这表明 PDGF-BB 诱导的钙/CaM 激活在促进细胞存活中起作用,可能通过持续激活 Akt。我们得出结论,过度表达的 PDGF 受体-β刺激引起的钙/CaM/Akt 途径的激活可能有助于 MPNST 细胞的存活和致瘤性。