Abstract
The alpha 5 alpha 1 integrin, a fibronectin receptor, has been implicated in the control of cell growth and the regulation of gene expression. We report that disruption of ligation between alpha 5 alpha 1 and fibronectin by integrin alpha 5 subunit or fibronectin monoclonal antibodies stimulated DNA synthesis in growth-arrested FET human colon carcinoma cells. This stimulation only occurred when monoclonal antibody was added in the early G1 phase of the cell cycle after release from quiescence by fresh medium. Stimulation of DNA synthesis by alpha 5 or fibronectin antibody was concentration- and time-dependent. FET cells expressed alpha 4 beta 1 integrin (another fibronectin receptor); however, addition of anti-human integrin alpha 4 monoclonal antibody had no effect on DNA synthesis. Treatment with alpha 5 monoclonal antibody led to a marked increase in the expression of CDK4 in G1 phase of the cell cycle and consequently increased the phosphorylation of retinoblastoma protein. alpha 5 monoclonal antibody treatment increased both cyclin A- and cyclin E-associated kinase activity which was accompanied by increased protein levels of CDK2 and cyclin A. Western blotting of immunoprecipitates demonstrated increased CDK2-cyclin E and CDK2-cyclin A complexes in cells treated with alpha 5 monoclonal antibody. Furthermore, disruption of alpha 5 alpha 1/fibronectin ligation activated mitogen-activated protein kinase p44 and p42 (extracellular signal-regulated kinase 1 and 2). Pretreatment of the cells with a specific inhibitor of MEK-1, PD98059, blocked the alpha 5 monoclonal antibody-induced mitogen-activated protein kinase activity. In addition PD98059 prevented alpha 5 monoclonal antibody-induced DNA synthesis. Since alpha 5 alpha 1 ligation to fibronectin is associated with decreased growth parameters, our results indicate that ligation of alpha 5 alpha 1 integrin to fibronectin results in suppressed mitogen-activated protein kinase activity which in turn inhibits cyclin-dependent kinase activity in growth-arrested cells.
Highlights
Integrins are a large family of cell-surface glycoproteins that mediate cell-cell and cell-extracellular matrix adhesion [1]
Stimulation of DNA Synthesis by ␣5 mAb in FET Cells— Previously we demonstrated that human colon carcinoma FET cells express cell surface ␣5␣1 integrin as well as fibronectin [41]
To determine whether FN/␣5␣1 integrin ligation contributed to the control of DNA synthesis, FET cells were rendered quiescent by growth factor and nutrient deprivation and released with fresh s 5A medium (SM) medium in the presence of anti-human integrin ␣5 subunit mAb for 8, 12, 18, 22, 24, and 26 h
Summary
Integrins are a large family of cell-surface glycoproteins that mediate cell-cell and cell-extracellular matrix adhesion [1]. Integrins can act as signaling receptors and transmit growth regulatory signals from the extracellular matrix to the interior of the cell [5]. Integrin ␣5␣1, a fibronectin (FN) receptor, has been implicated in the regulation of gene expression, cell growth, and tumorigenicity. A variant of K562 erythroleukemia cells selected for increased ability to attach to fibronectin showed a 5-fold up-regulation of ␣5␣1 expression and displayed significantly reduced growth in vitro as well as reduced turmorigencity [13]. Treatment of non-transformed FA-K562 cells overexpressing ␣5␣1 integrin with a synthetic peptide ligand results in an increase in CDC-2-dependent kinase activity [20, 21]. Cyclin-dependent kinases (CDK) complexed to regulatory cyclin subunits are key regulators of the cell cycle. CDK activity can be regulated by changes in expression of
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