Elucidating the role of integrin-linked kinase in thymosin β4 induced migration of SW480 colon cancer cells

• Main Authors: Yu-Shan Tien, 田玉珊
• Other Authors: Yeu Su
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/36630717141051924201

碩士 === 國立陽明大學 === 生物藥學研究所 === 99 === Thymosin β4 (Tβ4) is a 5 kDa intracellular G-actin sequestering peptide which regulates F-actin formation. Aberrant expression of Tβ4 has been found not only in many malignant tumors but also to contribute to certain malignant phenotypes including mobility increase. Previous study done by others has reported that Tβ4 facilitates the migration of endothelial cells via its interaction with a lamellipodia-localized integrin-linked kinase (ILK) which in turn induces MMP-2 synthesis through activating Akt2. On the other hand, recent work from our laboratory provided evidence that Tβ4 can stimulate the migration of SW480 cells by activating Rac1 via upregulating ILK-IQGAP1 complex formation. However, contribution of the enzyme activity of ILK in this process in unclear. To address this question, genes encoding a hyperactive and a kinase-dead ILK were introduced respectively into SW480 cells via adenovirus infection. Results from transwell migration and wound healing assays showed that hyperactive ILK enhanced whereas kinase-dead ILK reduced Tβ4-induced cell migration. Accordingly, the levels of active Rac1 was increased and decreased respectively by hyperactive and kinase-dead ILK. Interestingly, shRNA-mediated knockdown of IQGAP1 expression in Tβ4-overexpression stable clone (Tb3) reduced not only their migration, but also Rac1 activation and the complex of IQGAP1/active Rac1. Meanwhile, upregulation of hyperactive ILK in IQGAP1-knockdowned Tb3 cells could partially restore their migration ability, suggesting that the residual IQGAP1 might be modified by ILK to sustain the activity of Rac1, leading to elevated migration. More intriguingly, higher RNA and protein levels of αPIX, a guanine nucleotide exchanger factor (GEFs) for Rac1 activation, were found in Tb3 cells whose knockdown resulted in a drastic reduction in the motility of these cells. Finally, we also found an involvement of MMPs in Tβ4-induced migration of SW480 cells since not only were the RNA levels and activities of MMP-2 and MMP-9 increased in Tb3 cells and in cells transiently overexpressing Tβ4 but also the migration ability of the former was partially abolished by GM6001, a broad-spectrum MMP inhibitor. Furthermore, the activities of MMP-2 and MMP-9 were decreased in Tb3 cells by ILK knockdown or by expressing a kinase-dead ILK. Besides, Tβ4-induced cell migration could be diminished by AKTi-1/2, an inhibitor for both AKT1 and AKT2, via reducing MMP-2 and MMP-9 activities as well as Rac1 activation. Taken together, our data demonstrate that in addition to ILK kinase activity, αPIX, AKT and MMPs are also crucial for Tβ4-facilitated migration of SW480 cells.