CONTROL OF CYCLIN D1, P27(KIP1), AND CELL-CYCLE PROGRESSION IN HUMAN CAPILLARY ENDOTHELIAL-CELLS BY CELL-SHAPE AND CYTOSKELETAL TENSION

The extracellular matrix (ECM) plays an essential role in the regulati on of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both act ivate intracellular signaling cascades and mediate tension-dependent c hanges in cell shape and cytoskeletal structure. Although the growth c ontrol field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally cr itical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was co ntrolled by culturing cells on microfabricated substrates containing E CM-coated adhesive islands with defined shape and size on the micromet er stale or on plastic dishes coated with defined ECM molecular coatin g densities. Cells that were prevented from spreading in medium contai ning soluble growth factors exhibited normal activation of the mitogen -activated kinase (erk1/erk2) growth signaling pathway. However, in co ntrast to spread cells, these cells failed to progress through GI and enter S phase. This shape-dependent block in cell cycle progression co rrelated with a failure to increase cyclin D1 protein levels, down-reg ulate the cell cycle inhibitor p27(kip1) and phosphorylate the retinob lastoma protein in late GI. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disr upting the actin network using cytochalasin or by inhibiting cytoskele tal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structu re, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alo ne are not sufficient. Subsequent cell cycle progression and, hence, c ell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.