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Virally activated Ras cooperates with integrin to induce tubulogenesis in sinusoidal endothelial cell lines
Y Maru1, S Yamaguchi, T Takahashi
1Department of Genetics, Institute of Medical Science, University of Tokyo, Japan. ymaru@hgc.ims.u-tokyo.ac.jp
Abstract:
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), Fit-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-beta1 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.
Insights
Researchers developed rat liver sinusoidal endothelial cell lines to study tubulogenesis. Activating Ras signaling restored this endothelial cell function, highlighting its role in blood vessel formation.
Area of Science:
- Cell Biology
- Vascular Biology
- Biochemistry
Background:
- Endothelial cells form blood vessels through tubulogenesis.
- Sinusoidal endothelial cells (SECs) from rat liver were used to establish cell lines.
- Vascular endothelial growth factor (VEGF) receptors (Fit-1, KDR) were expressed in these cell lines.
Purpose of the Study:
- To investigate the molecular mechanisms regulating endothelial cell tubulogenesis.
- To determine the role of Ras signaling and integrins in SEC tubulogenesis.
- To identify signaling pathways involved in the formation of vascular networks.
Main Methods:
- Establishment of four rat liver sinusoidal endothelial cell lines (NP11, NP26, NP31, NP32).
- Assessment of tubulogenesis in Matrigel.
- Expression of temperature-sensitive, virally activated Ras (ts-v-Ras).
- Analysis of tyrosine phosphorylation, Grb-2 recruitment, and MAPK activation.
- Inhibition studies using anti-beta1 integrin antibody, dominant-negative Ras, MEK inhibitor (PD 098059), and botulinum C3 toxin.
Main Results:
- NP31 and NP32 cells were initially incapable of tubulogenesis.
- Expression of ts-v-Ras restored tubulogenic behavior in NP31 cells.
- Matrigel-induced signaling involved Shc phosphorylation, Grb-2 recruitment, and MAPK activation, dependent on beta1 integrin.
- Dominant-negative Ras inhibited tubulogenesis in human umbilical vein endothelial cells (HUVECs).
- MEK inhibition blocked ts-v-Ras-induced tubulogenesis, and Rho inhibition fragmented vascular networks.
Conclusions:
- Integrin-mediated Ras signaling is necessary but not sufficient for tubulogenesis.
- Artificial expression of v-Ras can compensate for a missing second signal required for tubulogenesis.
- Ras and Rho signaling pathways, along with integrin engagement, are crucial for endothelial cell tubulogenesis.