Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.

EphB1-mediated activation of extracellular-signalregulated kinase (ERK), was abrogated by overexpression of a caveolin-1 mutant lacking a functional scaffolding domain. Interaction between Ephs and caveolin-1 is not restricted to the B-subclass of receptors, since we show that EphA2 also interacts with caveolin-1. Furthermore, we demonstrate that the caveolin-binding motif within the kinase domain of EphB1 is primordial for its correct membrane targeting. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of EphB1. Journal of Cell Science 2300 of signaling (Engelman et al., 1998; Garcia-Cardena et al., 1996;Li et al., 1995). Caveolin-1 has a scaffolding domain, corresponding to amino acids 82-101, that can bind to a consensus sequence present in several signaling proteins, including epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors, the kinases Src and Fyn, and heterotrimeric G-proteins (Couet et al., 1997b;Li et al., 1995;Yamamoto et al., 1999). Two related caveolin-binding motifs (xxxxx and xxxxxx, where x is any amino acid and is one of the aromatic amino acids Trp, Phe or Tyr) have been identified, and these motifs exist within most caveolaeassociated proteins (Couet et al., 1997a).Incubation of endothelial cells with VEGF leads to a marked downregulation of both caveolae and caveolin-1 levels, whereas over-expression of caveolin-1 blocks VEGFdependent activation of Elk-1 promoter activity (Liu et al., 1999). In addition, VEGF receptor-2 (VEGFR-2) is shown to localize in endothelial caveolae and associates with caveolin-1, and this complex is rapidly dissociated upon stimulation with VEGF, suggesting that caveolin-1 acts as a negative regulator of VEGFR-2 activity (Labrecque et al., 2003). By contrast, caveolin has also been shown to function as an activator of insulin receptor (IR) signaling (Yamamoto et al., 1998). In this work, we show that the EphB1 receptor signaling is initiated in low-density caveolar membrane domains and that caveolin-1 has an important role in this pathway. To define a more global significance of the caveolin-1 interaction with Eph receptors, we also show that an A-subclass Eph receptor, EphA2, interacts with caveolin-1. In addition, we demonstrate that the caveolin-binding motif in the kinase domain of EphB1 receptor is important for its membrane targeting. Our findings identify caveolae and caveolin-1 as important players in the regulation of EphB1 expression and signaling. Results A caveolin-binding motif is located within the kinase domain of EphB1 and EphA2 receptorsA putative caveolin-binding motif has been identified within the conserved kinase domain of many RTKs, including EGF receptor, insulin receptor, PDGF receptor, erbB2 and fibroblast growth factor receptor (FGF-R) (Couet et al., 1997a). We searched the cytoplasmic tail of the EphB1 and EphA2 receptors for this caveolin-binding motif, and identified a single caveolin-binding motif (xxxxx) within the kinase domain of...