EphB2 and ephrin-B2 regulate the ionic homeostasis of vestibular endolymph

Abstract

The ability to transport cations and anions across epithelia is critical for the regulation of pH, ionic homeostasis, and volume of extracellular fluids. Although the transporters and channels that facilitate ion and water movement across cell membranes are well known, the molecular mechanisms and signal transduction events that regulate these activities remain poorly understood. The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are well known to transduce bidirectional signals that control axon guidance and other cell migration/adhesion events during development. However, these molecules are also expressed in non-motile epithelial cells, including EphB2 in K⁺-secreting vestibular dark cells and ephrin-B2 in the adjacent transitional cells of the inner ear. Consistent with these expression patterns, mice with cytoplasmic domain mutations that interfere with EphB2 forward signaling or ephrin-B2 reverse signaling exhibit a hyperactive circling (waltzing) locomotion associated with a decreased amount of endolymph fluid that normally fills the vestibular labyrinth. Endolymph is unusual as an extracellular fluid in that it is normally high in K⁺ and low in Na⁺. Direct measurement of this fluid in live animals revealed significant decreases in K⁺ concentration and endolymphatic potential in both EphB2 and ephrin-B2 mutant mice. Our findings provide evidence that bidirectional signaling mediated by B-subclass Ephs and ephrins controls the production and ionic homeostasis of endolymph fluid and thereby provide the first evidence that these molecules can control the activities of mature epithelial cells.

Introduction

The vestibular apparatus is a bony chambered structure consisting of connecting tubes (the semicircular canals) and prominences (the utricle and saccule) that is essential for survival by functioning to detect gravity, linear acceleration, and angular motions of the head. The bony labyrinth is lined by a continuous sheet of epithelial cells that forms a closed, fluid-filled system, the membranous labyrinth. Ampullae and maculae mark the specialized regions of the membranous labyrinth involved in sensory transduction. Between the membranous labyrinth and bony labyrinth is a space containing extracellular fluid called perilymph, which is similar to cerebrospinal fluid with a high concentration of Na⁺ and a low concentration of K⁺. Within the lumen of the membranous labyrinth, a unique extracellular fluid called endolymph bathes the specialized sensory receptors located in the ampullae and maculae. Unlike perilymph, endolymph has a high concentration of K⁺ and a low concentration of Na⁺. The potassium-rich ionic composition of endolymph is maintained by vestibular dark cells; specialized secretory epithelial cells in the membranous labyrinth which lay adjacent to the sensory epithelium (Wangemann, 1995, Wangemann, 2002a). Endolymph is required for normal functioning of the vestibular system as disturbances in its production or ionic homeostasis lead to vestibular pathology such as the severe vertigo and positional nystagmus associated with Ménière’s disease (Strupp and Arbusow, 2001), which is characterized by an excessive amount of fluid within the inner ear (endolymphatic hydrops).

Our previous genetic studies indicated the receptor tyrosine kinase known as EphB2 is involved in the production of vestibular endolymph (Cowan et al., 2000). We showed that EphB2 knockout mice exhibit severe vestibular dysfunction that is associated with a drastic reduction in the volume of endolymph fluid within the vestibular apparatus. Consistent with a role for EphB2 in endolymph, we showed that this receptor is specifically expressed in the vestibular dark cells.

EphB2 is one member of the largest group of transmembrane receptor tyrosine kinases termed the Eph family (Eph Nomenclature Committee, 1997). Eph receptors are divided into two subclasses, EphA and EphB, based on sequence comparisons and ligand interactions. The extracellular portion of Eph receptors contains an N-terminal ligand-binding domain with 20 conserved cysteine residues (Himanen et al., 1998, Himanen et al., 2001) and two fibronectin type III repeats. Following the transmembrane segment, the cytoplasmic portion of Eph receptors contains a juxtamembrane region with two tyrosine residues involved in Src homology 2 (SH2) domain protein–protein interactions (Ellis et al., 1996, Hock et al., 1998a, Holland et al., 1997, Stein et al., 1998, Zisch et al., 1998) and kinase autoinhibition (Wybenga-Groot et al., 2001), a tyrosine kinase catalytic domain (Hanks et al., 1988), a sterile alpha motif (SAM) domain (Stapleton et al., 1999, Thanos et al., 1999), and a PSD-95/Dlg/Zo-1 (PDZ) domain binding site at the extreme C-terminal tail (Buchert et al., 1999, Cowan et al., 2000, Hock et al., 1998b, Torres et al., 1998).

The ephrins are cell surface-bound ligands that bind with high affinity to Eph molecules to relieve the receptor’s autoinhibition and activating its intrinsic tyrosine kinase catalytic domain (Himanen et al., 2001, Wybenga-Groot et al., 2001). The five A-subclass ephrins are tethered to the cell surface by a glycosylphosphatidylinositol (GPI) linkage, while the three B-subclass ephrins are anchored by a single transmembrane segment, which is followed by a short, highly conserved, cytoplasmic C-terminal tail. The cytoplasmic segment of B-subclass ephrins contains five tyrosine residues that can become phosphorylated (Bruckner et al., 1997, Cowan and Henkemeyer, 2001, Holland et al., 1996), leading to protein–protein interactions with the SH2/SH3 domain adaptor protein Grb4/Nckβ (Cowan and Henkemeyer, 2001). Furthermore, like Eph receptors, the extreme C-terminal tail of B-subclass ephrins contains sequences that are recognized and bound by a number of PDZ domain-containing proteins (Cowan and Henkemeyer, 2002). In general, each EphA receptor can promiscuously bind to any A-subclass ephrin but shows weak if any binding to B-subclass ephrins. Likewise, each EphB receptor promiscuously binds to any B-subclass ephrin but weakly or not at all to the A-subclass ephrins (Brambilla et al., 1995, Davis et al., 1994, Gale et al., 1996a, Gale et al., 1996b). However, some examples of cross-subclass interactions do exist (Bergemann et al., 1998, Gale et al., 1996a, Himanen et al., 2004). Strong activation of the Eph catalytic domain can only be achieved by co-culturing ephrin-expressing cells with Eph-expressing cells, or by artificially oligomerizing soluble forms of the ephrins (Davis et al., 1994). Importantly, the membrane association of ephrins and their need to be clustered indicates that signaling mediated by Eph:ephrin interactions occurs only at sites of cell–cell contact.

In addition to activating the Eph tyrosine kinase domain and transduction of signals into the Eph-expressing cell, the Eph:ephrin interaction also leads to transduction of signals into the ephrin-expressing cell (Bruckner et al., 1997, Cowan and Henkemeyer, 2001, Henkemeyer et al., 1996, Holland et al., 1996, Lu et al., 2001). This results in bidirectional signal transduction events that are propagated into both the Eph-expressing cell (forward signaling) and ephrin-expressing cell (reverse signaling). In this fashion, the Eph and ephrin molecules have both traditional receptor-like roles as well as ligand-like roles. This general idea of forward and reverse (bidirectional) signaling following cell–cell interactions of Eph and ephrin proteins has been an important feature to explain how these molecules function (Cowan and Henkemeyer, 2002, Davy and Soriano, 2005, Kullander and Klein, 2002).

The bidirectional signals stemming from cell–cell interactions of Eph and ephrin molecules have generally been thought to regulate cellular movement and/or adhesion, being most noted in axon guidance for their ability to induce the repulsion/retraction of pathfinding growth cones as the developing brain and spinal cord becomes wired (Flanagan and Vanderhaeghen, 1998, Frisen et al., 1999, Pasquale, 2005). Ephs and ephrins are also expressed in non-motile epithelial cells, such as those which form the membranous labyrinth of the inner ear. As mentioned above, the EphB2 receptor is expressed in the K⁺-secreting vestibular dark cells (Cowan et al., 2000). Consistent with this restricted expression, genetic analysis showed the EphB2 receptor is important for normal vestibular function in the CD1 strain of mouse as approximately 60% of protein-null mutant mice lacking EphB2 expression (EphB2^Δ/Δ) exhibit rapid head bobbing and a hyperactive circling locomotion; behavioral phenotypes typical of rodents with dysfunctional balance control (Cowan et al., 2000). We further reported that while EphB3^Δ/Δ null mice show no vestibular abnormalities, 100% of the compound mutant mice lacking both EphB2 and EphB3 expression (EphB2^Δ/Δ;EphB3^Δ/Δ) also run in circles and that the vestibular dysfunction correlates with a severe decrease in the amount of endolymph fluid that normally fills the vestibular labyrinth (Cowan et al., 2000). The shift in penetrance of vestibular dysfunction from 60% to 100% in the double mutants indicated both EphB2 and EphB3 have roles in endolymph production, with EphB2 being the central player. Furthermore, the EphB2^Ki mutation that results in the synthesis of a truncated, membrane-anchored EphB2-βgal fusion protein lacking the majority of its cytoplasmic region, including the kinase domain, also leads to vestibular dysfunction and reduced levels of endolymph (Cowan et al., 2000). This data indicated forward signals transduced by EphB2 into the dark cells have an important role in the regulation of endolymph production.

Here we identify the ephrin ligand that interacts with EphB2 to regulate endolymph production. Our data indicates ephrin-B2 may be a major player as it is expressed to high levels in the vestibular transitional cells which lay immediately adjacent to and make cell–cell contacts with the dark cells. Consistent with this expression pattern, we find that mutation of the ephrin-B2 gene in mice leads to severe vestibular dysfunction that is associated with a decreased amount of endolymph fluid in the inner ear. Remarkably, in addition to functioning as a ligand to bind EphB2 and activate receptor clustering and forward signaling into the dark cells, our genetic analysis indicates the EphB2:ephrin-B2 interaction also leads to transduction of reverse signals into the ephrin-B2-expressing transitional cells that are important for endolymph homeostasis. In vivo measurements from the inner ears of live mice show both EphB2 forward and ephrin-B2 reverse signaling are required to produce/maintain the high concentration of K⁺ ions in the endolymph fluid. Together, our data demonstrate important roles for both forward and reverse components of EphB2:ephrin-B2 bidirectional signaling in the ionic homeostasis of vestibular endolymph.