[en] Interactions between the Eph receptor tyrosine kinase and ephrin ligands transduce short-range signals regulating axon pathfinding, development of the cardiovascular system, as well as migration and spreading of neuronal and non-neuronal cells. Some of these effects are believed to be mediated by alterations in actin dynamics. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. EphrinA1 is proposed to contribute to angiogenesis as it is strongly expressed at sites of neovascularization. Moreover, angiogenic factors induce the expression of ephrinA1 in endothelial cells. In this study, using rat vascular smooth muscle cells (VSMCs), we investigated the contribution of the small Rho GTPases in ephrinA1-induced integrin inactivation. EphrinA1 did not significantly affect early adhesion of VSMCs on purified laminin or fibronectin, but strongly impaired cell spreading. The Rho kinase inhibitor Y-27632 partly reversed the ephrinA1 effect, suggesting involvement of Rho in this model. However, inhibition of RhoA synthesis with short interfering (si)RNA had a modest effect, suggesting that RhoA plays a limited role in ephrinA1-mediated inhibition of spreading in VSMCs. The ephrinA1-mediated morphological alterations correlated with inhibition of Rac1 and p21-activated kinase 1 (PAK1) activity, and were antagonized by the expression of a constitutively active Rac mutant. Moreover, repression of Rac1 synthesis with siRNA amplifies the ephrinA1-induced inhibition of spreading. Finally, sphingosine-1-phosphate (S1P), a lipid mediator known to inhibit Rac activation in VSMCs amplifies the ephrinA1 effect. In conclusion, our results emphasize the role of the Rac/PAK pathway in ephrinA1-mediated inhibition of spreading. In this way, ephrinA1, alone or in synergy with S1P, can participate in blood vessel destabilization, a prerequisite for angiogenesis.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Deroanne, Christophe ; Institute of Signaling, Developmental Biology and Cancer Research > CNRS-UMR 6543,Centre Antoine Lacassagne, Nice
Vouret-Craviari, Valerie; Institute of Signaling, Developmental Biology and Cancer Research > CNRS-UMR 6543,Centre Antoine Lacassagne, Nice
Wang, Bingcheng; Case Western Reserve University, School of Medicine, Cleveland-USa > Rammelkamp Center for Research, > R421 and DPt of Pharmacol and Ireland Cancer Center
Pouyssegur, Jacques; Institute of Signaling, Developmental Biology and Cancer Research > CNRS-UMR 6543,Centre Antoine Lacassagne, Nice
Language :
English
Title :
EphrinA1 inactivates integrin-mediated vascular smooth muscle cell spreading via the Rac/PAK pathway.
Boyd, A. W. and Lackmann, M. (2001). Signals from Eph and ephrin proteins: a developmental tool kit. Sci STKE 2001, RE20.
Bruckner, K. and Klein, R. (1998). Signaling by Eph receptors and their ephrin ligands. Curr. Opin. Neurobiol. 8, 375-382.
Carter, N., Nakamoto, T., Hirai, H. and Hunter, T. (2002). EphrinA1-induced cytoskeletal re-organization requires FAK and p130(cas). Nat. Cell Biol. 4, 565-573.
Cheng, N., Brantley, D. M. and Chen, J. (2002). The ephrins and Eph receptors in angiogenesis. Cytokine Growth Factor Rev. 13, 75-85.
Coleman, M. L. and Marshall, C. J. (2001). A family outing: small GTPases cyclin' through G1. Nat. Cell Biol. 3, E250-251.
Daniel, T. O., Stein, E., Cerretti, D. P., St John, P. L., Robert, B. and Abrahamson, D. R. (1996). ELK and LERK-2 in developing kidney and microvascular endothelial assembly. Kidney Int. 57, S73-81.
Davies, S. P., Reddy, H., Caivano, M. and Cohen, P, (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95-105.
Davy, A. and Robbins, S. M. (2000). Ephrin-A5 modulates cell adhesion and morphology in an integrin-dependent manner. EMBO J. 19, 5396-5405.
Davy, A., Gale, N. W., Murray, E. W., Klinghoffer, R. A., Soriano, P., Feuerstein, C. and Robbins, S. M. (1999). Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion. Genes Dev. 13, 3125-3135.
Deroanne, C. F., Colige, A. C., Nusgens, B. V. and Lapiere, C. M. (1996). Modulation of expression and assembly of vinculin during in vitro fibrillar collagen-induced angiogenesis and its reversal. Exp. Cell Res. 224, 215-223.
Dodelet, V. C. and Pasquale, E. B. (2000). Eph receptors and ephrin ligands: embryogenesis to tumorigenesis. Oncogene 19, 5614-5619.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498.
Flenniken, A. M., Gale, N. W., Yancopoulos, G. D. and Wilkinson, D. G. (1996). Distinct and overlapping expression patterns of ligands for Eph-related receptor tyrosine kinases during mouse embryogenesis. Dev. Biol. 179, 382-401.
Gale, N. W., Holland, S. J., Valenzuela, D. M., Flenniken, A., Pan, L., Ryan, T. E., Henkemeyer, M., Strebhardt, K., Hirai, H., Wilkinson, D. G. et al. (1996). Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. Neuron 17, 9-19.
Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science 279, 509-514.
Hall, C., Brown, M., Jacobs, T., Ferrari, G., Cann, N., Teo, M., Monfries, C. and Lim, L. (2001). Collapsin response mediator protein switches RhoA and Rac1 morphology in N1E-115 neuroblastoma cells and is regulated by Rho kinase. J. Biol. Chem. 276, 43482-43486.
Holder, N. and Klein, R. (1999). Eph receptors and ephrins: effectors of morphogenesis. Development 126, 2033-2044.
Huynh-Do, U., Stein, E., Lane, A. A., Liu, H., Cerretti, D. P. and Daniel, T. O. (1999). Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through αvβ3 and α5β1 integrins. EMBO J. 18, 2165-2173.
Katsumi, A., Milanini, J., Kiosses, W. B., del Pozo, M. A., Kaunas, R., Chien, S., Hahn, K. M. and Schwartz, M. A. (2002). Effects of cell tension on the small GTPase Rac. J. Cell Biol. 158, 153-164.
Kiosses, W. B., Shattil, S. J., Pampori, N. and Schwartz, M. A. (2001). Rac recruits high-affinity integrin αvβ3 to lamellipodia in endothelial cell migration. Nat. Cell Biol. 3, 316-320.
Knoll, B. and Drescher, U. (2002). Ephrin-As as receptors in topographic projections. Trends Neurosci. 25, 145-149.
Lambert, C. A., Soudant, E. P., Nusgens, B. V. and Lapiere, C. M. (1992). Pretranslational regulation of extracellular matrix macromolecules and collagenase expression in fibroblasts by mechanical forces. Lab. Invest. 66, 444-451.
Lawrenson, I. D., Wimmer-Kleikamp, S. H., Lock, P., Schoenwaelder, S. M., Down, M., Boyd, A. W., Alewood, P. F. and Lackmann, M. (2002). Ephrin-A5 induces rounding, blebbing and de-adhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling. J. Cell Sci. 115, 1059-1072.
Le Gall, M., Grall, D., Chambard, J. C., Pouyssegur, J. and van Obberghen-Schilling, E. (1998). An anchorage-dependent signal distinct from p42/44 MAP kinase activation is required for cell cycle progression. Oncogene 17, 1271-1277.
Mellitzer, G., Xu, Q. and Wilkinson, D. G. (1999). Eph receptors and ephrins restrict cell intermingling and communication. Nature 400, 77-81.
Mellitzer, G., Xu, Q. and Wilkinson, D. G. (2000). Control of cell behaviour by signalling through Eph receptors and ephrins. Curr. Opin. Neurobiol. 10, 400-408.
Miao, H., Burnett, E., Kinch, M., Simon, E. and Wang, B. (2000). Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation. Nat. Cell Biol. 2, 62-69.
Mooney, D. J., Hansen, L. K., Langer, R., Vacanti, J. P. and Ingber, D. E. (1994). Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover. Mol. Biol. Cell 5, 1281-1288.
Myers, C., Charboneau, A. and Boudreau, N. (2000). Homeobox B3 promotes capillary morphogenesis and angiogenesis. J. Cell Biol. 148, 343-351.
Ogawa, K., Pasqualini, R., Lindberg, R. A., Kain, R., Freeman, A. L. and Pasquale, E. B. (2000). The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization. Oncogene 19, 6043-6052.
Pandey, A., Shao, H., Marks, R. M., Polverini, P. J. and Dixit, V. M. (1995). Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF-α-induced angiogenesis. Science 268, 567-569.
Ren, X. D., Kiosses, W. B. and Schwartz, M. A. (1999). Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18, 578-585.
Richard, D. E., Berra, E. and Pouyssegur, J. (2000). Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1α in vascular smooth muscle cells. J. Biol. Chem. 275, 26765-26771.
Ryu, Y., Takuwa, N., Sugimoto, N., Sakurada, S., Usui, S., Okamoto, H., Matsui, O. and Takuwa, Y. (2002). Sphingosine-1-phosphate, a platelet-derived lysophospholipid mediator, negatively regulates cellular Rac activity and cell migration in vascular smooth muscle cells. Circ. Res. 90, 325-332.
Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R. A. and Collard, J. G. (1999). Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J. Cell Biol. 147, 1009-1022.
Schoenwaelder, S. M., Petch, L. A., Williamson, D., Shen, R., Feng, G. S. and Burridge, K. (2000). The protein tyrosine phosphatase Shp-2 regulates RhoA activity. Curr. Biol. 10, 1523-1526.
Sells, M. A., Boyd, J. T. and Chernoff, J. (1999). p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts. J. Cell Biol. 145, 837-849.
Sells, M. A., Pfaff, A. and Chernoff, J. (2000). Temporal and spatial distribution of activated Pak1 in fibroblasts. J. Cell Biol. 151, 1449-1458.
Shamah, S. M., Lin, M. Z., Goldberg, J. L., Estrach, S., Sahin, M., Hu, L., Bazalakova, M., Neve, R. L., Corfas, G., Debant, A. et al. (2001). EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin. Cell 105, 233-244.
Stein, E., Lane, A. A., Cerretti, D. P., Schoecklmann, H. O., Schroff, A. D., van Etten, R. L. and Daniel, T. O. (1998). Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses. Genes Dev. 12, 667-678.
Vinals, F. and Pouyssegur, J. (1999). Confluence of vascular endothelial cells induces cell cycle exit by inhibiting p42/p44 mitogen-activated protein kinase activity. Mol. Cell. Biol. 19, 2763-2772.
Wahl, S., Barth, H., Ciossek, T., Aktories, K. and Mueller, B. K. (2000). Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase. J. Cell Biol. 149, 263-270.
Wang, H. U., Chen, Z. F. and Anderson, D. J. (1998). Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93, 741-753.
Xu, Q., Mellitzer, G. and Wilkinson, D. G. (2000). Roles of Eph receptors and ephrins in segmental patterning. Philos. Trans. R. Soc. London B Biol. Sci. 355, 993-1002.
Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J. and Holash, J. (2000). Vascular-specific growth factors and blood vessel formation. Nature 407, 242-248.
