Pouliot N, Connolly L M, Moritz R L, Simpson R J, Burgess A W
Melbourne Branch, The Ludwig Institute for Cancer Research, Victoria, 3050, Australia.
Exp Cell Res. 2000 Dec 15;261(2):360-71. doi: 10.1006/excr.2000.5065.
Epidermal growth factor (EGF) receptor ligands such as EGF and transforming growth factor-alpha (TGF-alpha) play an important role in controlling the proliferation, survival, morphology, and motility of colonic epithelial cells. There is also increasing evidence that growth factors and extracellular matrix (ECM) proteins cooperate to regulate these cellular processes. We have reported previously that autocrine TGF-alpha and an unidentified ECM protein in the serum-free conditioned medium of the human colon carcinoma cell line LIM1215 synergize to induce spreading of these cells in low-density cultures. We have now purified the ECM protein secreted by LIM1215 cells and show that it synergizes with EGF to induce spreading of LIM1215 cells and other human cell lines from the colon and other tissues. The purified ECM migrated as a single protein band with an apparent molecular mass of approximately 800 kDa on SDS-PAGE under nonreducing conditions and, under reducing conditions, as three protein bands of approximately 360, 210, and 200 kDa. Immunoblotting experiments and mass spectrometry analysis of tryptic digests on the purified protein identified the 360-, 210-, and 200-kDa protein bands as laminin alpha5, beta1, and gamma1 chains, respectively, indicating that LIM1215 cells secrete laminin-10 (alpha5 beta1 gamma1). In serum-free medium, LIM1215 cells adhere to laminin-10 primarily via alpha2 beta1 and alpha3 beta1 integrin receptors. EGF-induced spreading of LIM1215 cells on laminin-10 is partially inhibited by pretreatment of the cells with blocking antibodies directed against integrin alpha3 or beta1 but not alpha2, alpha6, or beta4 subunits. Spreading is almost completely inhibited by blocking alpha3 + alpha2, alpha3 + alpha6, or beta1 + beta4 integrin chains and results in cell death. Increased spreading in the presence of EGF correlates with up-regulation of alpha6 beta4 integrins in these cells after exposure to EGF. These results indicate that colon cancer cells attach and spread on laminin-10 via multiple integrin receptors and suggest a critical role for alpha3 beta1 integrins in the spreading response. Together, our results support the concept that the adhesive properties of colon cancer cells are modulated by autocrine production of TGF-alpha and laminin-10 and autocrine induction of appropriate integrins.
表皮生长因子（EGF）受体配体，如表皮生长因子（EGF）和转化生长因子-α（TGF-α），在控制结肠上皮细胞的增殖、存活、形态和运动方面发挥着重要作用。越来越多的证据表明，生长因子和细胞外基质（ECM）蛋白协同调节这些细胞过程。我们之前报道过，人结肠癌细胞系LIM1215无血清条件培养基中的自分泌TGF-α和一种未鉴定的ECM蛋白协同作用，诱导这些细胞在低密度培养中铺展。我们现在已经纯化了LIM1215细胞分泌的ECM蛋白，并表明它与EGF协同作用，诱导LIM1215细胞以及来自结肠和其他组织的其他人类细胞系铺展。纯化的ECM在非还原条件下的SDS-PAGE上以单一蛋白条带迁移，表观分子量约为800 kDa，在还原条件下，以约360、210和200 kDa的三条蛋白条带迁移。对纯化蛋白进行胰蛋白酶消化后的免疫印迹实验和质谱分析分别将360 kDa、210 kDa和200 kDa的蛋白条带鉴定为层粘连蛋白α5、β1和γ1链，表明LIM1215细胞分泌层粘连蛋白-10（α5β1γ1）。在无血清培养基中，LIM1215细胞主要通过α2β1和α3β1整合素受体粘附于层粘连蛋白-10。用针对整合素α3或β1但不针对α2、α6或β4亚基的阻断抗体预处理细胞，可部分抑制EGF诱导的LIM1215细胞在层粘连蛋白-10上的铺展。阻断α3 + α2、α3 + α6或β1 + β4整合素链几乎完全抑制铺展，并导致细胞死亡。在EGF存在下铺展增加与这些细胞在暴露于EGF后α6β4整合素的上调相关。这些结果表明，结肠癌细胞通过多种整合素受体附着并在层粘连蛋白-10上铺展，并提示α3β1整合素在铺展反应中起关键作用。总之，我们的结果支持这样一种概念，即结肠癌细胞的粘附特性受到TGF-α和层粘连蛋白-10的自分泌产生以及适当整合素的自分泌诱导的调节。
