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 live r. They stilt retained expression of receptors for vascular endothelia l growth factor (VEGF), Flt-1, and kinase domain-containing receptor ( KDR). NP31 and NP32 turned out to be incapable of tubulogenesis in bas ement membrane matrix (Matrigel), which belongs to endothelial propert ies, as shown by SECs in primary culture. Expression of temperature-se nsitive, virally activated Ras (ts-v-Ras) restored tubulogenic behavio rs back to NP31 only at permissive temperature. Matrigel induced longl asting tyrosine phosphorylation of Shc, with recruitment of Grb-2 and microtubule-associated protein kinase (MAPK) activation in both parent al NP31 and NP31 transformed by ts-v-Ras, which was blocked by anti-be ta 1 integrin antibody. Tubulogenesis was inhibited by adenovirus-medi ated expression of dominant-negative Ras in human umbilical vein endot helial 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 to xin, an inhibitor for Rho, caused fragmentation of branching cords in networks formed by NP31 that expressed ts-v-Ras at permissive temperat ure. These data suggest that the integrin-mediated Ras signals may be necessary but are not sufficient for tubulogenesis and that an artific ial expression of v-Ras might substitute for the second signal require d in this system. (C) 1998 Wiley-Liss, Inc.