Iida J, Meijne A M, Spiro R C, Roos E, Furcht L T, McCarthy J B
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455, USA.
Cancer Res. 1995 May 15;55(10):2177-85.
In this study, we evaluated the potential role for a specific melanoma-associated chondroitin sulfate proteoglycan core protein, termed NG2, to collaborate with alpha 4 beta 1 integrin in focal contact formation in human melanoma cells. Although melanoma cells adhered to substrata coated with either the alpha 4 beta 1 integrin binding fibronectin synthetic peptide CS1-OVA or anti-NG2 mAbs, no spreading or focal contact formation was observed on either substratum. However, melanoma cells spread and formed focal contacts on "chimeric substrata" coated with CS1-OVA and the anti-NG2 mAb, 9.2.27, indicating that engaging both adhesion receptors changes the adhesion phenotype of melanoma cells by reorganizing the cytoskeleton. The collaboration between the two receptors is specific to fibronectin, since cells adherent on substrata coated with low concentrations of either laminin and 9.2.27 or type IV collagen and 9.2.27 failed to spread, while cells adherent on low concentrations of fibronectin and 9.2.27 exhibited a fully spread morphology. Two selective tyrosine kinase inhibitors, genistein and herbimycin A, totally inhibited cell spreading on the substrata coated with CS1-OVA and 9.2.27, indicating that tyrosine kinase(s) is important for cell spreading and focal contact formation. When cells were cultured on substrata coated with CS1-OVA and 9.2.27, two proteins (M(r) 130,000 and 120,000) were tyrosine phosphorylated in a genistein- and herbimycin A-sensitive fashion. These proteins were not immunologically related to pp125FAK or alpha 4 beta 1 integrin. Importantly, when melanoma cells were cultured on substrata coated with CS1 and then stimulated with 9.2.27-conjugated microsphere beads, formation of focal contacts and stress fibers was also observed, indicating that NG2 can collaborate with alpha 4 beta 1 integrin when each receptor is engaged on distinct and separate substrata. These results demonstrate that NG2 acts as a coreceptor for spreading and focal contact formation in association with alpha 4 beta 1 integrin in melanoma cells and suggest a model in which the NG2 core protein communicates to alpha 4 beta 1 integrin by an inside-out signaling mechanism.
在本研究中，我们评估了一种特定的黑色素瘤相关硫酸软骨素蛋白聚糖核心蛋白（称为NG2）在人黑色素瘤细胞粘着斑形成过程中与α4β1整合素协同作用的潜在作用。尽管黑色素瘤细胞可粘附于包被有α4β1整合素结合纤连蛋白合成肽CS1-OVA或抗NG2单克隆抗体的基质上，但在这两种基质上均未观察到细胞铺展或粘着斑形成。然而，黑色素瘤细胞在包被有CS1-OVA和抗NG2单克隆抗体9.2.27的“嵌合基质”上能够铺展并形成粘着斑，这表明激活两种粘附受体可通过重组细胞骨架改变黑色素瘤细胞的粘附表型。两种受体之间的协同作用对纤连蛋白具有特异性，因为粘附于包被有低浓度层粘连蛋白和9.2.27或IV型胶原和9.2.27的基质上的细胞未能铺展，而粘附于低浓度纤连蛋白和9.2.27的细胞则呈现出完全铺展的形态。两种选择性酪氨酸激酶抑制剂染料木黄酮和赫曲霉素A完全抑制了细胞在包被有CS1-OVA和9.2.27的基质上的铺展，这表明酪氨酸激酶对细胞铺展和粘着斑形成很重要。当细胞在包被有CS1-OVA和9.2.27的基质上培养时，两种蛋白（分子量130,000和120,000）以染料木黄酮和赫曲霉素A敏感的方式发生酪氨酸磷酸化。这些蛋白与pp125FAK或α4β1整合素无免疫相关性。重要的是，当黑色素瘤细胞在包被有CS1的基质上培养，然后用9.2.27偶联的微球珠刺激时，也观察到了粘着斑和应力纤维的形成，这表明当每个受体分别与不同的基质结合时，NG2可与α4β1整合素协同作用。这些结果表明，在黑色素瘤细胞中，NG2作为与α4β1整合素相关的铺展和粘着斑形成的共受体，并提示了一种模型，即NG2核心蛋白通过由内向外的信号传导机制与α4β1整合素进行通讯。
