De novo expression of the α5β1-fibronectin receptor in HT29 colon- cancer cells reduces activity of c-src. Increase of c-src activity by attachment on fibronectin

Abstract

Changes in integrin expression during malignant transformation have been observed in many tumors. Colon-carcinoma cells show reduced expression or even loss of the α5β1 integrin compared to normal or adenoma cells. To determine the significance of absent α5β1 integrin signaling, we translated the cDNA coding for the α5 integrin sub-unit into the human colon-carcinoma cell line HT29, which constitutively lacks this subunit but does express the β1 subunit. We show here that the newly expressed fibronectin receptor α5β1 generates multiple signals, causing marked changes in cytoskeletal arrangements within a few minutes of adhesion to fibronectin. Cells expressing the α5β1 integrin exhibit the formation of actin stress fibers and focal adhesions, as well as the induction of tyrosine phosphorylation of several proteins, within 10 min. We identified the focal adhesion kinase pp 125(FAK) and the cytoskeletal protein paxillin as major phosphorylation substrates in these cells. These proteins remained hypophosphorylated when α5-negative control cells were plated on fibronectin. The tyrosine kinase pp60(c-src), regarded as central in the regulation of cellular proliferation and constitutively over-expressed in HT29 and in colon-carcinoma cells, showed reduced intrinsic kinase activity in unstimulated MT29α5 cells. In contrast, fibronectin-induced signaling through α5β1 increased pp60(c-src) activity. Moreover, immunoprecipitation of pp60(c-src) from extracts of MT29α5 cells cultivated on fibronectin for 20 min revealed complex formation of pp60(c-src) and tyrosine-phosphorylated pp125(FAK). Our data suggest that de novo expression of the α5β1 integrin in HT29 colon-cancer cells restores signaling via pp125(FAK) and pp60(c-src). Thus, loss of this receptor during malignant transformation may contribute to tumor-cell autonomy, while reduced activity of pp60(c-src) in HT29α5-cells may participate directly in growth control.