Virally activated ras cooperates with integrin to induce tubulogenesis in sinusoidal endothelial cell lines

Four cell lines, named nonparenchymal 11 (NP11), NP26, NP31, and NP32, were established from sinusoidal endothelial cells (SECs) of rat liver. They still retained expression of receptors for vascular endothelial growth factor (VEGF), Flt‐1, and kinase domain‐containing receptor (KDR). NP31 and NP32 turned out to be incapable of tubulogenesis in basement membrane matrix (Matrigel), which belongs to endothelial properties, as shown by SECs in primary culture. Expression of temperature‐sensitive, virally activated Ras (ts‐v‐Ras) restored tubulogenic behaviors back to NP31 only at permissive temperature. Matrigel induced long‐lasting tyrosine phosphorylation of Shc, with recruitment of Grb‐2 and microtubule‐associated protein kinase (MAPK) activation in both parental NP31 and NP31 transformed by ts‐v‐Ras, which was blocked by anti‐β1 integrin antibody. Tubulogenesis was inhibited by adenovirus‐mediated expression of dominant‐negative Ras in human umbilical vein endothelial cells (HUVECs). PD 098059, a selective inhibitor of MAPK kinase (MEK), nearly perfectly blocked tubulogenesis by ts‐v‐Ras‐expressing NP31 cells at permissive temperature. Furthermore, the botulinum C3 toxin, an inhibitor for Rho, caused fragmentation of branching cords in networks formed by NP31 that expressed ts‐v‐Ras at permissive temperature. These data suggest that the integrin‐mediated Ras signals may be necessary but are not sufficient for tubulogenesis and that an artificial expression of v‐Ras might substitute for the second signal required in this system. J. Cell. Physiol. 176:223–234, 1998. © 1998 Wiley‐Liss, Inc.

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