ErbB Expression: The Mouse Inner Ear and Maturation of the Mitogenic Response to Heregulin

CLIFFORD R. HUME 0 METTE KIRKEGAARD 0 ELIZABETH C. OESTERLE 0 0 Department of Otolaryngology-Head and Neck Surgery, Virginia Merrill Bloedel Hearing Research Center, University of Washington , Seattle, WA 98195, USA In humans, hair cell loss often leads to hearing and balance impairments. Hair cell replacement is vigorous and spontaneous in avians and nonmammalian vertebrates. In mammals, in contrast, it occurs at a very low rate, or not at all, presumably because of a very low level of supporting cell proliferation following injury. Heregulin (HRG), a member of the epidermal growth factor (EGF) family of growth factors, is reported to be a potent mitogen for neonatal rat vestibular sensory epithelium, but its effects in adults are unknown. We report here that HRG-a stimulates cell proliferation in organotypic cultures of neonatal, but not adult, mouse utricular sensory epithelia. Our findings support the idea that the proliferative capabilities of the adult mammalian vestibular sensory epithelia differ significantly from that seen in neonatal animals. Immunohistochemistry reveals that HRG-binding receptors (erbBs 2-4) and erbB1 are widely expressed in vestibular and auditory sensory epithelia in neonatal and adult mouse inner ear. The distribution of erbBs in the neonatal and adult mouse ear is consistent with the EGF receptor/ligand family regulating diverse cellular processes in the inner ear, including cell proliferation and differentiation. - The vertebrate inner ear contains multiple types of sensory hair cells that are responsible for the detection of sound and motion. In birds, if the sensory epithelium is damaged, supporting cell precursors are stimulated to proliferate and differentiate into new hair cells (Corwin and Cotanche 1988; Ryals and Rubel 1988; Weisleder and Rubel 1993). As these new hair cells are reinnervated, this efficient and highly ordered process results in the restoration of auditory and vestibular function (reviewed in Cotanche et al. 1994; Carey et al. 1996; Stone et al. 1998; Cotanche 1999; Smolders 1999). In contrast to birds, mammals are unable to regenerate auditory hair cells and have a very limited ability to regenerate vestibular hair cells via a mitotic pathway if the sensory epithelium is damaged (Warchol et al. 1993; Lambert 1994; Rubel et al. 1995; Tanyeri et al. 1995; Li and Forge 1997; Zheng and Gao 1997; Kuntz and Oesterle 1998a; Forge et al. 1998). Little is known about the signals that regulate the regeneration of hair cells. Despite the testing of extensive numbers of known growth factors by several groups, factors capable of stimulating proliferation in the mammalian auditory epithelium, the organ of Corti, have not yet been identified. Only a few factors show some promise of increasing proliferation and promoting new hair cell formation in the mammalian vestibular sensory epithelium (reviewed in Staecker and Van de Water 1998; Oesterle and Hume 1999). The most effective of these, epidermal growth factor (EGF) (Yamashita and Oesterle 1995; Zheng et al. 1997, 1999), transforming growth factor alpha (TGFa) (Lambert 1994; Yamashita and Oesterle 1995; Zheng et al. 1997, 1999; Kuntz and Oesterle 1998a; Oesterle et al. 2003), glial growth factor 2 (GGF2) (Montcouquiol and Corwin 2001), heregulin (HRG) (Zheng et al. 1999), and neu-differentiation factor (NDF) (Gu et al. 1998, 1999), are structurally related members of the EGF-ligand family. GGF2, HRG, and NDF are also members of the neuregulin family, a subfamily of the larger EGF-ligand family that consists of over 15 splice isoforms arising from a single gene, NRG1. The most studied of these ligands in the inner ear to date, TGF-a, has been shown to stimulate supporting cell proliferation in cultured utricular sensory epithelium from neonatal rats and adult mice (Lambert 1994; Yamashita and Oesterle 1995; Zheng et al. 1997, 1999). TGF-as mitogenic effects are potentiated by insulin (Yamashita and Oesterle 1995; Kuntz and Oesterle 1998a), and TGF-a plus insulin infusion into the adult rat ear stimulates the production of new supporting cells, and possibly new hair cells, in the utricular sensory epithelium in vivo (Kuntz and Oesterle 1998a; Oesterle et al. 2003). EGF, when used in combination with insulin, also stimulates supporting cell proliferation in an in vitro organotypic culture system from adult mice (Yamashita and Oesterle 1995) and in isolated sheets of neonatal utricular sensory epithelium (Zheng et al. 1997, 1999). Several members of the neuregulin family, GGF2, NDF, and heregulin (HRG) b, are mitogenic for neonatal vestibular sensory epithelia (Gu et al. 1998, 1999; Zheng et al. 1999; Montcouquiol and Corwin 2001). In the adult, their effects are unknown (NDF, HRG) or incompletely characterized (GGF2, Gu et al. 1997). HRG-b appears to be a potent mitogen for neonatal vestibular sensory epithelia; a tenfold increase in the numbers of sensory epithelial (SE) cells synthesizing DNA was reported in cultures supplemented with HRG-b relative to that seen in control cultures (Zheng et al. 1999). Its effects need to be explored in adult tissues to determine its capabilities as a potential therapeutic agent for alleviating sensory-neural hearing loss or vestibular disorders in mammals, possibly in humans. The mitogenic effects of growth factors often vary developmentally in many tissues; hence, factors that are mitogenic for neonatal inner ear sensory epithelia may not necessarily have similar effects in adult tissues. As summarized in Figure 1, signaling by members of the EGF family of ligands is mediated by four interacting transmembrane tyrosine kinase receptors: erbB1 (EGFR, HER), erbB2 (HER2, neu), erbB3 (HER3), and erbB4 (HER4) (for review, see Carraway and Cantley 1994; Lemke 1996; Burden and Yarden 1997; Riese and Stern 1998; Adlkofer and Lai 2000). The erbB receptors differ in both their ligand affinity and signaling activity. ErbB receptor family members, with the exception of erbB2, bind multiple ligands. Ligand binding induces receptor dimerization and activation of the intrinsic tyrosine kinase followed by activation of downstream signaling pathways. Receptor dimerization can take place between identical receptors (homodimers) or with any of the other erbB family members (heterodimers), depending on which receptors are expressed in a given cell (Riese et al. 1996a, b; Sliwkowski et al. 1994). ErbB2 does not have an identified ligand, but it is frequently activated as a result of receptor heterodimerization (Alroy and Yarden 1997; Sliwkowski et al. 1994). ErbB3 is an unusual receptor in that it does not possess any kinase activity, although it can recruit other receptors to form active heterodimers (Guy et al. 1994). There are three functional groups of EGF-related ligands based on their ability to bind individual erbB receptors (Fig. 1) (reviewed in Riese and Stern 1998). One group of ligands (EGF, TGF-a, and amphiregulin) binds to receptors cont (...truncated)