In a two-dimensional (2D) culture dish, the major activity of endothelial cells is proliferation with limited morphological change. When cultured in a three-dimensional (3D) collagen gel matrix, endothelial cells undergo a series of morphological changes starting with development of intracellular vacuoles and followed by cell elongation. Adjacent cells then coalesce to form tube-like structures. This process mimics the steps of capillary formation during angiogenesis. Using this model, we investigated the roles of extracellular signal-regulated kinase (ERK) and p38 MAP kinase (p38) in the tube formation from human umbilical vein endothelial cells (HUVEC). Proliferating HUVEC gradually lost their ability to divide after being transferred to 3D collagen matrices, where differentiation became the dominant cellular activity. The transition from proliferation to the differentiation state was accompanied by a drastic reduction of cyclin-dependent kinases CDC2, CDK4, and retinoblastoma (Rb) protein, but the expression of cyclin-dependent kinase inhibitor, p27kip1, was increased. Inhibition of p38 by SB203580 partially prevented these changes and increased the proliferation rate of HUVEC. However, cells under this condition exhibited unusually elongated cell bodies, and they were unable to coalesce to form tube structures. Inhibition of ERK neither affected the cell proliferation rate nor the expression levels of cell cycle regulators, but it completely blocked tube formation by inducing apoptosis, a finding different from the best-known role of ERK in cell proliferation in the 2D cell culture systems. We conclude that the major function of ERK is to maintain cell viability while p38 plays multiple roles in controlling cell proliferation, viability, and morphogenesis during tube formation.
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