Dlg-1 Interacts With and Regulates the Activities of Fibroblast Growth Factor Receptors and EphA2 in the Mouse Lens.

The formation and maintenance of the architecture of specialized organs are dependent on growth factor receptor signaling pathways that regulate cell proliferation, differentiation, cell–cell adhesion, cytoskeletal structure, apical–basal polarity, and planar cell polarity (PCP).1–3 The ocular lens is an organ whose distinctive structure is known to be dependent on multiple growth factor receptor signaling pathways that regulate these diverse biologic processes. Identifying factors that coordinate these signaling pathways is crucial to our understanding of normal lens development and the lens pathology that forms when signaling pathways are disrupted. Dlg-1 (Discs large-1), the mouse homolog of Drosophila dlg, is required for developmental processes in multiple organs4–6 through its regulation of cell proliferation, cell–cell adhesion, cell shape, and apical–basal and PCP.7–9 Previously, we showed that Dlg-1 is required for lens fiber cell differentiation and maintenance of the architecture of the lens8 and also is a modulator of the fibroblast growth factor receptor (Fgfr) signaling pathway in the mouse lens.10 In this study, we address the possibility that Dlg-1 is a factor that regulates the interaction of the ephrin/Eph11–16 and Fgf/Fgfr17–19 signaling pathways, two signaling pathway that are crucial for fiber cell structure and differentiation. The lens is composed of a monolayer of epithelial cells that overlies a mass of derivative, differentiated, highly elongated cells, the fiber cells. After formation of the lens vesicle by day E10.5, cells in the anterior form the lens epithelial cells, whereas cells in the posterior undergo a terminal differentiation process to form the primary fiber cells. Subsequently, cells in the periphery of the epithelium undergo differentiation to form secondary fibers. Primary and secondary fiber cell differentiation is characterized by cell cycle withdrawal, cell elongation, and expression of differentiation-specific proteins. Notably, the secondary fibers take on a distinct hexagonal structure and are tightly packed into organized rows of cells that migrate along the capsule and epithelial cells until they meet their counterpart forming the lens sutures.20,21 Among the various growth factors that are expressed in the lens, Fgfs are the only known growth factors capable of inducing lens fiber cell differentiation.2,22,23 In mice, simultaneous deletion of Fgfrs 1, 2, and 3 led to complete arrest of lens fiber differentiation, demonstrating that Fgfr signaling is required for lens development.24 EphA2, a member of another family of receptor tyrosine kinases (RTKs), is expressed in the cortical lens fiber cells.12–14 EphA2 and ephrin A-5, the ligand for EphA2 in the lens, have been shown to be required for the hexagonal structure of the lens fiber cells11–16 and for linking the adherens junction protein, N-cadherin, to β-catenin, thereby promoting linkage to the cytoskeleton and cell–cell adhesion.12 Interplay between the Eph/ephrin and Fgfr signaling pathways has been shown to regulate cell fate determination in Ciona embryos.25 In mammalian cells, Fgfr and EphA4 have been shown to phosphorylate each other and costimulation of both receptors resulted in the potentiation of mitogen-activated protein kinase (MAPK) signaling.26 Recently, we discovered that Dlg-1, the mouse homolog of the Drosophila gene discs-large (dlg), is expressed in the lens and is required for cell adhesion, apical–basal polarity, and fiber cell structure and differentiation in the lens using lens-specific ablation of Dlg-1.9,27 We also showed that loss of Dlg-1 in the lens resulted in reduced levels of Fgfr2 and downstream signaling intermediates but increased levels of Fgfr1 and 3, indicating that Dlg-1 is a modulator of the Fgfr signaling pathway.10 Dlg-1 is a Psd95/Dlg/ZO-1 (PDZ) domain containing protein. These proteins are thought to act as scaffolding molecules that assemble large macromolecular complexes at the cell membrane.28 PDZ proteins interact directly with proteins that contain a PDZ binding motif, usually positioned at the C terminus of the protein.28 Because Fgfrs are not known to contain PDZ binding motifs, it is possible that the regulation of Fgfr signaling by Dlg-1 is mediated by other proteins. Interestingly, the lenses of mice deficient for Dlg-1,8 Epha2,13,15,16 and Efna512 show similar disruption in the hexagonal architecture of the fiber cell and cell adhesion defects, suggesting that these proteins may mediate similar processes in the lens. EphA2 does contain a PDZ binding motif,29 suggesting that Dlg-1 and EphA2 may interact to regulate fiber cell structure and cell–cell adhesion. In this study, we first addressed the finding that Fgfr1 and Fgfr2 levels are oppositely affected in the Dlg-1–deficient state and the impact of this imbalance on Fgfr signaling. We found that levels of Fgfr1 negatively correlated with levels of Fgfr2 and that the levels of activated Fgfr signaling intermediates positively correlated with Fgfr2 levels. Second, we addressed the hypothesis that Dlg-1, EphA2, and Fgfrs interact, and Dlg-1 affects the activities of these RTKs. We found that ablation of Dlg-1 led to reduced EphA2 levels and to disruption of the association of N-cadherin and β-catenin. We found that Dlg-1 complexes with EphA2, that EphA2 complexes with Fgfr1, Fgfr2, and Fgfr3 and that Dlg-1 complexes with Fgfr1, Fgfr2, and Fgfr3, albeit more weakly than does EphA2. Finally, we found that loss of Dlg-1 led to reduced complexing of EphA2 with N-cadherin, β-catenin, and Fgfr2 but increased complexing of EphA2 with Fgfr1 and Fgfr3, and these changes correlated with the altered levels of activation of EphA2, Fgfr1, and Fgfr2. Taken together, these data suggest that Dlg-1 regulates EphA2 function and the interaction between Dlg-1 and EphA2 mediates, at least in part, the effect of Dlg-1 on Fgfr signaling and adherens junction formation.