The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands form a unique cell-cell contact-mediated system for controlling cell localization and organization. Their high expression in a wide variety of human tumors indicates a role in tumor progression, and relatively low Eph and ephrin levels in normal tissues make these proteins potential targets for anticancer therapies. The monoclonal antibody IIIA4, previously used to isolate EphA3, binds with subnanomolar affinity to a conformation-specific epitope within the ephrin-binding domain that is closely adjacent to the ''low-affinity'' ephrin-A5 heterotetramerization site. We show that similar to ephrin-A5, preclustered IIIA4 effectively triggers EphA3 activation, contraction of the cytoskeleton, and cell rounding. BIAcore analysis, immunoblot, and confocal microscopy of wild-type and mutant EphA3 with compromised ephrin-A5 or IIIA4-binding capacities indicate that IIIA4 binding triggers an EphA3 conformation which is permissive for the assembly of EphA3/ephrin-A5-type signaling clusters. Furthermore, unclustered IIIA4 and ephrin-A5 Fc applied in combination initiate greatly enhanced EphA3 signaling. Radiometal conjugates of ephrin-A5 and IIIA4 retain their affinity, and in mouse xenografts localize to, and are internalized rapidly into EphA3-positive, human tumors. These findings show the biological importance of EphA3/ ephrin-A5 interactions and that ephrin-A5 and IIIA4 have great potential as tumor targeting reagents. (Cancer Res 2005; 65(15): 6745-54)

Studies have shown that high levels of glucose and angiotensin II (Ang II) stimulate hypertrophy and the expression of matrix protein genes in mouse proximal tubular cells in vitro. The present study tested the hypothesis that blockade of the renin-angiotensin system (RAS) inhibits the stimulatory effect of high levels of glucose on the expression of the renal angiotensinogen (ANG) gene and the formation of Ang II and subsequently attenuates the induction of hypertrophy in kidney proximal tubular cells. Immortalized rat proximal tubular cells (IRPTC) were cultured in monolayer. The levels of expression of rat ANG and ANG mRNA in the IRPTC were quantified by specific radioimmunoassays for rat ANG (RIA-rANG) and by a reverse-transcription polymerase chain reaction (RT-PCR) assay, respectively. Hypertrophy of IRPTC was analyzed by flow cytometry (FACScan) and cellular protein assay. Our studies showed that losartan (an Ang II (AT₁)-receptor blocker), perindopril and captopril (inhibitors of angiotensin-converting enzyme) blocked the stimulatory effect of a high level of glucose (i.e. 25 mM) on the expression of the rat ANG gene and hypertrophy in IRPTC but not by the Ang II (AT₂)-receptor blocker. Our studies indicate that the blockade of RAS is effective in inhibiting the stimulatory effect of hyperglycemia on the expression of the ANG gene and hypertrophy in IRPTC, supporting the notion that the local formation of intrarenal Ang II may play a role in the development of renal hypertrophy during early diabetes.

Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure of the homologous EphB2⅐ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E ␣-helix, the E-F loop, the underlying H ␤-strand, as well as the nearby B-C loop, which define a distinct third surface required for oligomerization and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct protein-interaction interfaces to assemble functional EphA3 signaling complexes.

Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF)-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs), but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29+/CD73+/CD90+/CD146+ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.