The Ectodermal Dysplasia Receptor Activates the Nuclear Factor-κB, JNK, and Cell Death Pathways and Binds to Ectodysplasin A

Abstract

The ectodermal dysplasia receptor (EDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to play a key role in the process of ectodermal differentiation. We present evidence that EDAR is capable of activating the nuclear factor-B, JNK, and caspase-independent cell death pathways and that these activities are impaired in mutants lacking its death domain or those associated with anhidrotic ectodermal dysplasia and the downless phenotype. Although EDAR possesses a death domain, it did not interact with the death domain-containing adaptor proteins TRADD and FADD. EDAR successfully interacted with various TRAF family members; however, a dominant-negative mutant of TRAF2 was incapable of blocking EDAR-induced nuclear factor-B or JNK activation. Collectively, the above results suggest that EDAR utilizes a novel signal transduction pathway. Finally, ectodysplasin A can physically interact with the extracellular domain of EDAR and thus represents its biological ligand.Anhidrotic (or hypohidrotic) ectodermal dysplasia is a disorder of ectodermal differentiation characterized by a triad of signs consisting of sparse hair, abnormal or missing teeth, and an inability to sweat (1). A similar phenotype is seen in mice with mutations involving the downless locus, suggesting the existence of a common underlying genetic defect. Recently, mutations in EDAR, 1 a novel receptor of the TNFR family, were found in several families with autosomal dominant and recessive forms of anhidrotic ectodermal dysplasia and in downless mice (2, 3). Although the above studies established the role of EDAR in ectodermal differentiation, the signaling pathways activated by this receptor remain to be elucidated.Death domain-containing receptors of the TNFR family are believed to activate two main signaling cascades: a kinase cascade leading to NF-B and JNK activation and a caspase cascade leading to cell death (4). As the cytoplasmic domain of EDAR was reported to contain a region with partial homology to the death domain, we decided to test its abilities to activate the above signaling cascades (2, 3). In this report, we present evidence that, like the classical death domain-containing receptors, EDAR is capable of activating the NF-B, JNK, and cell death pathways. However, EDAR does not interact with the death domain-containing adaptor proteins TRADD or FADD and does not activate the caspase cascade. Our results suggest the existence of a novel signaling pathway utilized by EDAR. MATERIALS AND METHODS Cell Lines and Reagents-293Tand MCF7 cells were obtained from Dr. David Han (University of Washington, Seattle, WA). 293 EBNA cells were obtained from Invitrogen. Rabbit polyclonal antibodies against FLAG, Myc, and hemagglutinin tags were obtained from Santa Cruz Biotechnology. FLAG beads and control beads were obtained from Sigma. The pull-down kinase assay kit for JNK was obtained from New England Biolabs Inc., and the constructs for the Pathdetect luciferase reporter assay were purchased from Stratagene....

Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia

“…The emerging model has the EDA ligand, a transmembrane protein, binding its receptor, EDAR (15,16,50), and signaling to the target cell through activation of the NF-B system of transcriptional regulation (50,51). During tooth development in mouse, signaling between EDA and its receptor, EDAR, is required to establish and maintain enamel knot morphology and function (16,52).…”

NF-κB-related genetic diseases

“…44,45 It encodes a death-domain containing member of the TNF-R family, EDAR, and is a specific receptor for ectodysplasin. 46,47 Its expression is restricted to placodes, thickenings of epithelia where epidermal appendages begin to form. 48,49 The third locus whose disruption leads to EDA has been identified only recently in crinkled mice and subsequently found mutated in a human family.…”

“…Additional biochemical studies have confirmed that ectodysplasin/EDAR interaction indeed results in NF-kB activation. 31,47,52 Therefore, mutations in three genes that encode members of a signaling cascade that leads to NF-kB activation result in anhidrotic ectodermal dysplasia. Although the details of the ectodysplasin/EDAR signaling pathway are not fully characterized, it is clear from analyzing the mouse models of EDA pathology that this pathway is involved very early during development of hair follicle morphogenesis, 44 assigning a previously unrecognized role for NF-kB in this process.…”

Traf6 is essential for murine tooth cusp morphogenesis

“…Eda signaling and Troy are believed to be mediated by NF-B (Kojima et al, 2000;Yan et al, 2000;Kumar et al, 2001). Indeed mutations in certain components of the NF-B pathway have molar cusp abnormalities (Hu et al, 1999b;Li et al, 1999;Takeda et al, 1999;Makris et al, 2000;Zonana et al, 2000;Dö ffinger et al, 2001; Ohazama et al, manuscript submitted for publication).…”