HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 27, 20742-20747, July 7, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/27/20742    most recent M002691200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kojima, T. Articles by Kitamura, T. Search for Related Content PubMed PubMed Citation Articles by Kojima, T. Articles by Kitamura, T. Social Bookmarking                      What's this?

TROY, a Newly Identified Member of the Tumor Necrosis Factor Receptor Superfamily, Exhibits a Homology with Edar and Is Expressed in Embryonic Skin and Hair Follicles^*

Tetsuo Kojima^§, Yoshihiro Morikawa^¶, Neal G. Copeland, Debra J. Gilbert, Nancy A. Jenkins, Emiko Senba^¶, and Toshio Kitamura^**

From the  Department of Hematopoietic Factors, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan, ^§ Cytokine Research Program, Chugai Research Institute for Molecular Medicine, Inc., Niihari, Ibaraki 300-4101, Japan, ^¶ Department of Anatomy and Neurobiology, Wakayama Medical School, Kimiidera, Wakayama 641-8509, Japan, and  Mouse Cancer Genetics Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702

Received for publication, March 30, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In a signal sequence trap screening of the murine brain, we identified a new member of the tumor necrosis factor receptor superfamily designated TROY. TROY is a type I membrane protein of 416 amino acids with characteristic cysteine-rich motifs in the extracellular domain and a tumor necrosis factor receptor-associated factor (TRAF) 2 binding sequence in the cytoplasmic domain of 223 amino acids. In fact, activation of nuclear factor B was induced by the overexpression of TROY and inhibited by dominant negative forms of TRAF2, TRAF5, and TRAF6, indicating that TRAFs and nuclear factor B are involved in the signal transduction of TROY. We also cloned a cDNA for a human counterpart, which showed a 75% homology with mouse TROY at the amino acid level. The extracellular domain of TROY exhibits an extensive homology with that of Edar, a receptor that specifies hair follicle fate. TROY mRNA is strongly expressed in brain and embryo and moderately expressed in the heart, lung, and liver but not the spleen. In the embryo, the expression level is particularly strong in the skin. Interestingly, in situ hybridization analysis of the embryo showed that TROY mRNA was exclusively expressed in the epithelium of many tissues. On the other hand, in neonatal mice, TROY is expressed in hair follicles like Edar as well as in the cerebrum, suggesting pleiotropic functions of TROY in development as well as in the adult mice. The Troy gene is located near the waved coat (Wc) locus, a mutant related to abnormalities in skin and hair.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Tumor necrosis factor-related cytokines form a large family of pleiotropic mediators of host defense and immune systems that act either locally as membrane proteins or on distant target cells as secreted proteins. Members of the tumor necrosis factor receptor superfamily (TNFRSF)¹ mediate the action of tumor necrosis factor-related cytokines leading to cell death or to cell proliferation and differentiation (1-3). Most of the genes for TNFRSF encode type I transmembrane glycoproteins with an extracellular ligand-binding domain, a single membrane-spanning region, and a cytoplasmic region that activates cell functions. The characteristic pattern is found within the extracellular domain formed by cysteine-rich 40-residue repeats with a 25-30% homology. The majority of conserved positions are cysteine residues. The cysteine-rich repeats reflect the characteristic structural domain of the TNFRSF (4-6). Overall, this family of glycoproteins shows a relatively low level of sequence conservation, despite sharing a common fundamental structure. Cytoplasmic regions of the receptors show considerably more diversity than do extracellular regions in sequence and size. Although there is no common motif found in all members of the TNFRSF, some elements are shared between subsets of family members. For instance, tumor necrosis factor (4, 7, 8), CD95/FAS (9), and tumor necrosis factor-related apoptosis-inducing ligand receptor (10-12) share a domain of approximately 80 amino acids near the C-terminal called the "death domain" (13-15) that is required for induction of apoptosis by these receptors. On the other hand, the majority of TNFRSF members recruit the TNFR-associated factor (TRAF) family of intracellular adaptor molecules through cytoplasmic tails to promote cell survival by activation of downstream protein kinase cascades and, ultimately, transcription factors of the NF-B and activator protein 1 family (16, 17). A major TRAF2-binding consensus sequence, (P/S/A/T)X(Q/E)E, and a minor consensus motif, PXQXXD, were defined, based on structural analyses (18).

The members of the TNFRSF are not only involved in the immune system but are also involved in many other biological systems. For example, osteoprotegerin and receptor activator of NFB has been shown to play critical roles in osteoclast differentiation and function (19, 20). The downless (dl) gene isolated by positional cloning encodes Edar, a new member of the TNFRSF (21, 22). Mice with mutations in this gene have defects in hair follicle induction, lack sweat glands, and have malformed teeth. Based on the mutant phenotype and Edar expression pattern, it was proposed that Edar specifies the fate of hair follicles.

We have now identified a new member of the TNFRSF expressed on the mouse embryo (TROY), using a newly established signal sequence trap method SST-REX (23). TROY shows a unique tissue distribution in the embryo as well as after birth; in the embryo, TROY is exclusively expressed in the epithelium including the neuroepithelium, skin, bronchiolar epithelium, conjunctiva, and so forth, whereas after birth, TROY is strongly expressed in hair follicles like Edar as well as in neurons.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents and Cell Lines-- Recombinant murine interleukin 3 was produced in silk worms (24). Ba/F3 cells (25) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and 1 ng/ml murine interleukin 3. Human embryonic kidney 293T cells (26) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. An ecotropic retrovirus packaging cell line BOSC23 (26) was maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and a guanine phosphoribosyltransferase selection reagent (Specialty Media, Lavallette, NJ). Two days before transfection, cells were transferred to Dulbecco's modified Eagle's medium/10% fetal calf serum that did not contain guanine phosphoribosyltransferase selection reagents. Nullipotent embryonal carcinoma NF-1 was obtained from ATCC, and human glioma U251 and gliosarcoma GI-1 were from RIKEN (Tsukuba, Japan).

Cloning of a cDNA for TROY-- Total RNA was prepared from mouse brain, and poly(A)⁺ RNA was further purified using oligo(dT)-cellulose chromatography. cDNA was synthesized by random hexamers primed with SuperScript Choice System (Life Technologies, Inc.), separated through a SizeSep 400 spun column (Pharmacia), and inserted into the BstXI site of a cloning vector for SST, pMX-SST, using BstXI adapters (Invitrogen). The ligated DNA was introduced into DH10B cells (Electromax; Life Technologies, Inc.) by electroporation. Plasmid DNA was prepared using standard methods. Infection of Ba/F3 cells with retroviruses carrying a cDNA library for SST and isolation of cDNA fragments by polymerase chain reaction from factor-independent clones were performed as described previously (23). Full-length cDNA clones for TROY were obtained from a mouse brain and an embryonic day 17.5 skin cDNA library synthesized by oligo(dT) and random priming, respectively. A biotinylated probe of 460 bp was obtained by polymerase chain reaction from TROY cDNA using primers 5'-CAAGGTCCTACCTCTACACA and 5'-AAGGTTCACCTTGCTGGTAC, using 50 µM biotin-21-dUTP (CLONTECH), 200 µM dATP, dGTP, and dCTP, and 10 µM dTTP. Mouse brain and embryonic skin cDNA libraries were screened using biotinylated linear DNA probes coated with RecA proteins, as described previously (27-29). To isolate a cDNA clone encoding a human counterpart of TROY, a human gliosarcoma cell line GI-1 cDNA library synthesized by random priming was screened using the mouse TROY cDNA coding region as a probe.

RNA Blot Hybridization-- Total RNAs of cell lines were extracted with Trizol (Life Technologies, Inc.), and mouse embryo poly(A)⁺ RNA was purified with FastTrack2.0 (Invitrogen). Total RNA (30 µg) and 3 µg of poly(A)⁺ RNA were fractionated in a 0.7% agarose gel containing formaldehyde and transferred to a Hybond N+ nylon filter (Amersham). Mouse Multiple Tissue Northern blot was obtained from CLONTECH. The Northern blot was hybridized with radiolabeled TROY cDNA.

In Situ Hybridization-- A 456-bp BamHI-HindIII cDNA fragment (coding region 151-607) of mouse TROY was inserted into a pBluescriptII KS vector. Digoxigenin-labeled riboprobes for TROY were transcribed in vitro from linearized, gel-purified plasmids using T7 polymerase (antisense cRNA probe) and T3 polymerase (sense cRNA probe). Embryos were embedded in OCT compound (Miles, Elkhart, IN), frozen in n-hexane, and then cut and analyzed by in situ hybridization using a modification of previously reported methods (30, 31). When control sections were hybridized with identical quantities of sense cRNA, signals were nil (data not shown).

NF-B-dependent Reporter Assays-- Human embryonic kidney 293T cells were plated in 6-well plates at a concentration of 10⁶ cells/well. On the following day, using FuGENE6 (Roche Molecular Biochemicals), the cells were transfected with 100 ng of pTK-lacZ, a thymidine kinase promoter-driven -galactosidase expression plasmid, to normalize for transfection efficiency, together with 100 ng of a reporter plasmid and various amounts of each expression vector. Total DNA (3 µg) was kept constant by supplementation with DNA derived from the pCOSI vector. A reporter plasmid, B-luc, has five repeats of the NF-B site upstream of a promoter derived from human T-cell lymphotrophic virus 1 and a luciferase reporter gene (32). Twenty-four h after the transfection, the cells were harvested in phosphate-buffered saline and lysed in the luciferase lysis buffer. The lysates were assayed for luciferase and -galactosidase activities (33).

Interspecific Mouse Backcross Mapping-- Interspecific backcross progenies were generated by mating (C57BL/6J × Mus spretus)F₁ females and C57BL/6J males, as described previously (34, 35). A total of 205 N2 mice were used to map the Troy locus (see text for details). The probe, a 1690-bp fragment of mouse cDNA, was labeled with [-³²P]dCTP using nick translation labeling kits (Roche Molecular Biochemicals). A fragment of 9.6 kb was detected in PstI-digested C57BL/6J DNA, and a fragment of 15.5 kb was detected in PstI-digested M. spretus DNA. The presence or absence of the 15.5-kb PstI M. spretus-specific fragment was monitored in the backcross mice.

A description of the probes and restriction fragment-length polymorphisms for the loci linked to Troy including Ctsg, Gjb2, and Fzd3 has been reported previously (36, 37). One locus for our interspecific backcross has not been reported. The Blk probe, an 854-bp EcoRI fragment of mouse cDNA, detected co-segregated KpnI fragments. Recombination distances were calculated using Map Manager, version 2.6.5. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation of the Mouse TROY cDNA Clone Encoding a Newly Identified Member of the TNFR Superfamily-- In the SST-REX method (23), Ba/F3 clones transduced with cDNAs containing signal sequences showed factor-independent growth through surface expression of a constitutively active receptor for thrombopoietin as a fusion protein. Among the cDNA clones isolated by SST-REX from a mouse brain cDNA library, the LTI9 clone showed homology with members of the TNFR superfamily at the amino acid sequence level. Positions of cysteine residues in the cysteine-rich repeat were perfectly conserved. Full-length cDNAs obtained from oligo(dT)-primed cDNA libraries derived from mouse brain using the RecA screening method (27-29) were confirmed to contain a sequence that was identical to LTI9.

These cDNAs contain an open reading frame of 1251 nucleotides. The putative initiation codon is preceded by a sequence (AGAGCC) in good agreement with the Kozak's consensus sequence for initiation of translation in eukaryotes (38). The termination codon is followed by a 3' untranslated region of 2590 bp. A canonical polyadenylation signal is present 36 bp upstream of the poly(A) tail (39). The protein putatively encoded by the TROY mRNA is a cysteine-rich protein of 416 amino acids with a calculated molecular mass of 45 kDa (Fig. 1). Two hydrophobic regions are present in the protein, representing the signal sequence and the transmembrane region. A hydrophobic stretch of 25 amino acids toward the C terminus (amino acids 169-193) was assigned as a transmembrane domain because it had a potentially single helical span. A cleavage site for the signal peptide was found between Cys (at position 29) and Glu (at position 30).

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Comparison between the protein sequence of mouse and human TROY. The signal sequence and transmembrane domain are underlined, and common amino acid residues are shaded. Two perfect TNFR cysteine-rich motifs are dot underlined, conserved cysteine residues and conserved amino acid residues are indicated by the asterisks and dots, respectively.

Like other members of the TNFRSF, TROY contains the characteristic cysteine-rich motifs (C - x(4,6) - [FYH] - x(5,10) - C - x(0,2) - C - x(2,3) - C - x(7,11) - C - x(4,6) - [DNEQSKP] - x(2)- C) that have been shown by x-ray crystallography to represent a repetitive structural unit (40, 41) (Fig. 1). TROY contains two perfect TNFR motifs and one imperfect motif in which C2 and C6 are not present. TROY exhibited a significant and extensive homology (33%) with all three motifs of Edar, the dl gene product (Fig. 2) (21, 22).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Alignment of the extracellular domain of mouse TROY with that of mouse Edar. Identical residues are indicated with an asterisk, and similar residues are indicated by a dot.

The cytoplasmic tail of TROY spans amino acids 194-416 of the precursor protein and does not harbor the death domain discovered in the intracellular domains of CD95 (9), TNFR (4, 7, 8), tumor necrosis factor-related apoptosis-inducing ligand receptor (10-12), and Edar; however, it does contain a major TRAF2-binding consensus sequence, TLQE (amino acids 276-279).

A shorter clone, dTROY, was also identified from a cDNA library of mouse embryonic day 17.5 skin. The extracellular and transmembrane domains of dTROY were identical to those of TROY. However, the cytoplasmic tail of dTROY has only 21 amino acids and does not contain a TRAF2-binding consensus sequence (Fig. 3). A consensus sequence of the 5' boundary of the intron was present on the transition point from the common sequence to the unique sequence of dTROY, suggesting that dTROY contains the same sequence from the intron at the 3' end. To confirm that dTROY was not an artifact, a dTROY-specific reverse transcription-polymerase chain reaction was performed using a sense primer of the common sequence and an antisense primer of the unique sequence of dTROY. A band of dTROY was amplified from the total RNA of embryonic day 17.5 skin but not from mouse brain or liver (data not shown).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Structure of dTROY, a putative decoy receptor. The region of dTROY that is structurally different from TROY is boxed. The consensus sequence of the 5' boundary of the intron is shaded. The dTROY-specific antisense primer region is underlined.

Isolation of a cDNA Clone Encoding a Human Counterpart of TROY-- A human counterpart of TROY expression in the human gliosarcoma cell line GI-1 was detected in the Northern blot hybridization (Fig. 4). A human counterpart was obtained from a cDNA library derived from the GI-1 cell line using RecA screening. This cDNA contains an open reading frame of 1269 nucleotides and encodes a cysteine-rich protein of 423 amino acids with a calculated molecular mass of 46 kDa. The overall identity between human TROY and murine TROY at the amino acid level is 75%, with a 92% identity in the extracellular and transmembrane domain. The cytoplasmic tail of human TROY was 234 amino acids long and had a 57% homology with murine TROY (Fig. 1).

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Northern blot analysis for detection of TROY transcripts. Blotted membranes were probed with a [32P]dCTP-labeled TROY cDNA fragment in PerfectHyb solution (Toyobo) and then washed with 0.2× saline/sodium phosphate/EDTA at 65 °C. Hybridized signals were identified using BAS2000 or x-ray films (Fuji Film).

Expression of TROY RNA-- Expression of TROY mRNA was examined by Northern blot analysis using a cDNA for the open reading frame of TROY as a probe (Fig. 4). TROY mRNA was strongly expressed in the brain, with an approximate molecular size of 4.5 kb, which was close to the length of the TROY cDNA clone (3970 nucleotides) identified. TROY mRNA was detected in most tissues, but not in the spleen. In the embryo, the expression level was periodically increased and was particularly strong in the skin. TROY mRNA was also detected in human glioma U251 cells, GI-1 cells, A3-1 embryonic stem cells, and nullipotent embryonal carcinoma NF-1 cells (data not shown). Cellular localization of TROY mRNA was investigated by in situ hybridization in mouse embryo day 13.5. Interestingly, TROY mRNA was detected exclusively in the epithelium (i.e. neuroepithelium in the frontal and lateral lobes, the epidermis of the skin, bronchiolar epithelium, epithelium of the tongue, gastric epithelium, conjunctiva, and cochlea), whereas in neonatal mice, TROY mRNA was mainly detected in hair follicles like Edar and in neuron-like cells in the cerebrum but not in the epidermis of the skin (Fig. 5).

View larger version (176K): [in this window] [in a new window]   Fig. 5.   In situ hybridization of TROY. Sections of an embryo at embryonic day 13.5 (A, lung; B, oculus; C, skin; D, frontal lobe) and neonatal (E, skin; F, cerebrum) were incubated with 0.1 ml of hybridization buffer containing 40 ng of the cRNA and then washed for 1 h in 0.1× SSC at 65 °C. The sections were then stained with an alkaline phosphatase-conjugated anti-digoxigenin Fab fragment (Roche Molecular Biochemicals), nitro blue tetrazolium (Roche Molecular Biochemicals), 5-bromo-4-chloro-3-indolyl phosphate (Roche Molecular Biochemicals), and levamisole. TROY mRNA is seen in the epithelium (i.e. bronchiolar epithelium (arrowhead), conjunctiva (arrowhead), epidermis of the skin (arrowhead), and neuroepithelium (arrowhead)), whereas in neonatal mice, TROY mRNA is detected in hair follicles (arrowhead) and in neuron-like cells (arrowhead) but not in the epidermis of the skin. Bar, 100 µm.

TROY Induces NF-B Activation-- Because the TRAF2-binding consensus sequence TLQE was found in the cytoplasmic domain of TROY, we asked whether TROY activates NF-B through TRAF2. Human embryonic kidney 293T cells were transiently transfected with an interleukin 2 promoter-derived NF-B-luciferase reporter gene (32) and a TROY expression vector (Fig. 6). Co-transfection of TROY induced a >3.5-fold higher luciferase activity, as compared with that seen with the vector control. To determine whether TRAF2, TRAF5, and TRAF6 are involved in NF-B activation by TROY, we tested whether TROY-induced NF-B activation was inhibited by dominant negative mutants of TRAF2, TRAF5, and TRAF6 lacking the N-terminal zinc-binding domain required for NF-B activation (42-48). Overexpression of dominant negative TRAF2, TRAF5, or TRAF6 blocked NF-B activation in the reporter assay. These results demonstrated that TRAFs were involved in TROY-mediated NF-B activation. The dTROY with a small cytoplasmic domain did not activate NF-B.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   TROY mediates NF-B activation. 293T cells were transiently transfected with 100 ng of luc reporter plasmid, 100 ng of -galactosidase plasmid, and the indicated expression plasmids. Twenty-four h later, the cells were collected, and the luciferase activity in each sample was determined. The values were normalized to the expression of -galactosidase. The level of induction in luciferase activity is indicated as compared with cells transfected with the control vector. Data are shown as the average ± S.D. of triplicate samples and represent one of three independent experiments, all of which had similar results.

Troy Is Located Near Wc-- The mouse chromosomal location of Troy was determined by interspecific backcross analysis, using progeny derived from matings of [(C57BL/6J × M. spretus)F₁ × C57BL/6J] mice. This interspecific backcross mapping panel has been typed for over 2900 loci that are well distributed among all of the autosomes as well as the X chromosome (34). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms using a mouse cDNA TROY probe. The 15.5-kb PstI M. spretus restriction fragment length polymorphisms (see "Materials and Methods") was used to follow the segregation of the Troy locus in backcross mice. Mapping showed that Troy is located in the central region of mouse chromosome 14 linked to Ctsg, Gjb2, Blk, and Fzd3. Although 161 mice were analyzed for every marker (shown in the segregation analysis; Fig. 7), up to 191 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies, using additional data. Ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are as follows: centromere - Ctsg (1:191) - Gjb2 (2:180) - Troy - (2:171) - Blk - (4:175) - Fzd3. The recombination frequencies [expressed as genetic distances in cM ± the S.E.] are: centromere - Ctsg (0.5 ± 0.5) - Gjb2 (1.1 ± 0.8) - Troy (1.2 ± 0.8)- Blk (2.3 ± 1.1) - Fzd3.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Troy maps to the central region of mouse chromosome 14. Troy was placed on mouse chromosome 14 by interspecific backcross analysis. The segregation patterns of Troy and flanking genes in 161 backcross animals typed for all loci are shown at the top of the figure. For individual pairs of loci, more than 161 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny inherited from the (C57BL/6J × M. spretus)F1 parent. , the presence of a C57BL/6J allele; , the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 14 linkage map showing the location of Troy in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in cM are shown to the left of the chromosome, and the positions of loci in human chromosomes (where known) are shown to the right. References for the human map positions of loci cited in this study can be obtained from the Genome Data Base, a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

We compared our interspecific map of chromosome 14 with a composite mouse linkage map that shows the location of many uncloned mouse mutations (provided by the Mouse Genome Data Base, a computerized database maintained at The Jackson Laboratory, Bar Harbor, Maine) and found that Troy localized in the vicinity of the waved coat (Wc) locus, a mutant that presents abnormality in the skin and hair (49-51).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We isolated and characterized a newly identified member of TNFRSF, TROY, from a cDNA library of the murine brain using SST-REX. In structural analysis of the cDNA, the translation product of TROY was shown to be a type I transmembrane protein of 416 amino acid residues. Whereas the extracellular domain of TROY retains the characteristic structure of TNFRSF, the intracellular domain does not contain the death domain.

During our characterization of TROY, Hu et al. (52) reported a novel member of the TNFRSF (TNFRSF19) that was identical to TROY, except that the cytoplasmic tail was shorter than that of TROY by 68 amino acids. In a search of the expressed sequence tag database, three clones (GenBank^TM accession numbers AI551729, AA445805, and AI48211) encoding the C-terminal region of TROY were identified, all of which were identical to the C-terminal region of TROY, but not to that of TNFRSF19. Moreover, the cytoplasmic tail of human TROY identified in this study was similar to murine TROY but not to TNFRSF19 in size and sequence, indicating that TROY is a major and complete cDNA clone.

When compared with other members of the TNFRSF, the closest known relative of TROY is a receptor called Edar (21, 22) that specifies hair follicle fate and is 33% identical to TROY in the extracellular domain. Mice with mutations in the Edar gene not only have defects in hair follicle induction but also lack sweat glands and have malformed teeth (21, 22). The expression of TROY is strong in embryonic skin and hair follicles, as is the case for Edar. This high homology in the extracellular domain and the similarity of distribution between TROY and Edar strongly suggest that TROY also plays some role in the development of embryonic skin and induction of hair follicles. Whereas Edar harbors a death domain, TROY does not contain one and activates NF-B through TRAF proteins. Therefore, it would be interesting to know how TROY and Edar functionally interact with each other.

In this context, it is of interest that the gene for Troy is located near the waved coat (Wc) locus, a mutant of which presents abnormality in skin and hair. Heterozygotes of the Wc locus have a wavy coat and whiskers and dry and scaly skin, and homozygotes die in utero (49-51). The central region of mouse chromosome 14 shares regions of homology with human chromosomes 7q, 8p, 13q, and 14q. The draft sequence of human chromosome13q12.11-3 from the Sanger center has been registered in the European Molecular Biology Laboratory database and includes the sequence of human TROY (nucleotides 624-1586). It would be interesting to examine whether there is a similar genetic disease in the human.

TROY exhibits a major TRAF2-binding consensus sequence in the cytoplasmic tail, and overexpression of TROY induced NF-B activation, as noted in the reporter assay using luciferase. This activation was blocked by overexpression of dominant negative TRAF2, TRAF5, and TRAF6. Like most members of the TNFRSF, TROY is perhaps homotrimerized by stimulation of its ligands and recruits TRAF2, TRAF5, and TRAF6 to the cytoplasmic tail, which promotes cell survival and proliferation by activating downstream protein kinase cascades and eventually activating transcription factors in the NF-B and activator protein 1 family. Hu et al. (52) reported that NF-B activation is not induced by TNFRSF19, a truncated version of TROY lacking the C-terminal 77 amino acid residues of the cytoplasmic tail, indicating that the C-terminal region of TROY is essential for activation of NF-B and for signal transduction.

We also found a truncated form of TROY, dTROY, that harbors a smaller cytoplasmic tail than TROY. The consensus sequence of the 5' boundary of the intron was found on the transition point from the TROY/dTROY common nucleotide sequence to the dTROY unique sequence, indicating that dTROY is an alternative splicing form containing an unspliced intron. Unlike TROY, dTROY can not induce NF-B activation and may play a role as a decoy receptor such as tumor necrosis factor-related apoptosis-inducing ligand receptor 3 (53) and decoy receptor 3 (54) for the CD90 ligand to negate a signal from a ligand for TROY. Expression of dTROY mRNA, detected using dTROY-specific reverse transcription-polymerase chain reaction, was found in the embryonic skin but not in the brain or liver. In the embryo, the expression level of TROY appears to be significantly increased during the late stages of embryogenesis and fluctuated from embryonic day 11.5 to embryonic day 15.5. Moreover, the TROY sequence was found as an expressed sequence tag in the mouse four-cell embryo (GenBank^TM accession number AU041881). These results suggest that TROY may play pleiotropic roles in embryogenesis. Of particular interest is the fact that homozygotes for the Wc locus result in embryonic lethality.

In summary, TROY is a novel member of the TNFRSF that exhibits a similarity with Edar in structure and expression patterns, and the gene for TROY is located in the vicinity of Wc. The present data also suggest that TROY is involved in embryonic development and the development of skin and hair follicles. For further study, it is important to identify the cognate ligand, which is now under investigation.

	ACKNOWLEDGEMENTS

We thank Dedorah B. Householder for excellent technical assistance, Dr. Hiroyoshi Nakano (Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan) for the TRAF expression vector, and Mariko Ohara for editorial assistance.

	FOOTNOTES

^* This research was supported by a grant-in-aid from the Ministry of Health and Welfare and a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan and in part by the United States National Cancer Institute, Department of Health and Human Services. The Department of Hematopoietic Factors of the Institute of Medical Science, University of Tokyo (Tokyo, Japan) is supported in part by Chugai Pharmaceutical Ltd.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequences for the mouse TROY, dTROY, and human TROY reported in this paper have been submitted to the DDBJ/GenBank^TM/EMBL Data Bank with accession numbers AB040432, AB040433, and AB040434, respectively.

^** To whom correspondence should be addressed. Tel.: 81-35449-5758; Fax: 81-35449-5453; E-mail: kitamura@ims.u-tokyo.ac.jp.

Published, JBC Papers in Press, April 6, 2000, DOI 10.1074/jbc.M002691200

	ABBREVIATIONS

The abbreviations used are: TNFR, tumor necrosis factor receptor; TNFRSF, tumor necrosis factor receptor superfamily; TRAF, tumor necrosis factor receptor-associated factor; NF, nuclear factor; bp, base pair(s); kb, kilobase(s); SST, signal sequence trap.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. Pispa, M. Pummila, P. A. Barker, I. Thesleff, and M. L. Mikkola Edar and Troy signalling pathways act redundantly to regulate initiation of hair follicle development Hum. Mol. Genet., November 1, 2008; 17(21): 3380 - 3391. [Abstract] [Full Text] [PDF]

				S. Pantalacci, A. Chaumot, G. Benoit, A. Sadier, F. Delsuc, E. J. P. Douzery, and V. Laudet Conserved Features and Evolutionary Shifts of the EDA Signaling Pathway Involved in Vertebrate Skin Appendage Development Mol. Biol. Evol., May 1, 2008; 25(5): 912 - 928. [Abstract] [Full Text] [PDF]

				R. Schmidt-Ullrich, D. J. Tobin, D. Lenhard, P. Schneider, R. Paus, and C. Scheidereit NF-{kappa}B transmits Eda A1/EdaR signalling to activate Shh and cyclin D1 expression, and controls post-initiation hair placode down growth Development, March 15, 2006; 133(6): 1045 - 1057. [Abstract] [Full Text] [PDF]

				M. Yuhki, M. Yamada, M. Kawano, T. Iwasato, S. Itohara, H. Yoshida, M. Ogawa, and Y. Mishina BMPR1A signaling is necessary for hair follicle cycling and hair shaft differentiation in mice Development, April 15, 2004; 131(8): 1825 - 1833. [Abstract] [Full Text] [PDF]

				Y. Wang, X. Li, L. Wang, P. Ding, Y. Zhang, W. Han, and D. Ma An alternative form of paraptosis-like cell death, triggered by TAJ/TROY and enhanced by PDCD5 overexpression J. Cell Sci., March 15, 2004; 117(8): 1525 - 1532. [Abstract] [Full Text] [PDF]

				A. C. Bharti, Y. Takada, S. Shishodia, and B. B. Aggarwal Evidence That Receptor Activator of Nuclear Factor (NF)-{kappa}B Ligand Can Suppress Cell Proliferation and Induce Apoptosis through Activation of a NF-{kappa}B-independent and TRAF6-dependent Mechanism J. Biol. Chem., February 13, 2004; 279(7): 6065 - 6076. [Abstract] [Full Text] [PDF]

				S. Ruiz, C. Segrelles, A. Bravo, M. Santos, P. Perez, H. Leis, J. L. Jorcano, and J. M. Paramio Abnormal epidermal differentiation and impaired epithelial-mesenchymal tissue interactions in mice lacking the retinoblastoma relatives p107 and p130 Development, June 1, 2003; 130(11): 2341 - 2353. [Abstract] [Full Text] [PDF]

				J. Laurikkala, J. Pispa, H.-S. Jung, P. Nieminen, M. Mikkola, X. Wang, U. Saarialho-Kere, J. Galceran, R. Grosschedl, and I. Thesleff Regulation of hair follicle development by the TNF signal ectodysplasin and its receptor Edar Development, March 7, 2003; 129(10): 2541 - 2553. [Abstract] [Full Text] [PDF]

				S. K. Sinha, S. Zachariah, H. I. Quinones, M. Shindo, and P. M. Chaudhary Role of TRAF3 and -6 in the Activation of the NF-kappa B and JNK Pathways by X-linked Ectodermal Dysplasia Receptor J. Biol. Chem., November 15, 2002; 277(47): 44953 - 44961. [Abstract] [Full Text] [PDF]

				A. Naito, H. Yoshida, E. Nishioka, M. Satoh, S. Azuma, T. Yamamoto, S.-i. Nishikawa, and J.-i. Inoue TRAF6-deficient mice display hypohidrotic ectodermal dysplasia PNAS, June 25, 2002; 99(13): 8766 - 8771. [Abstract] [Full Text] [PDF]

				I. Thesleff and M. L. Mikkola Death Receptor Signaling Giving Life to Ectodermal Organs Sci. Signal., May 7, 2002; 2002(131): pe22 - pe22. [Abstract] [Full Text] [PDF]

				M. Priolo and C. Lagana Ectodermal dysplasias: a new clinical-genetic classification J. Med. Genet., September 1, 2001; 38(9): 579 - 585. [Abstract] [Full Text] [PDF]

				G. L. Sica, G. Zhu, K. Tamada, D. Liu, J. Ni, and L. Chen RELT, a new member of the tumor necrosis factor receptor superfamily, is selectively expressed in hematopoietic tissues and activates transcription factor NF-{kappa}B Blood, May 1, 2001; 97(9): 2702 - 2707. [Abstract] [Full Text] [PDF]

				M. Yan, L.-C. Wang, S. G. Hymowitz, S. Schilbach, J. Lee, A. Goddard, A. M. de Vos, W.-Q. Gao, and V. M. Dixit Two-Amino Acid Molecular Switch in an Epithelial Morphogen That Regulates Binding to Two Distinct Receptors Science, October 20, 2000; 290(5491): 523 - 527. [Abstract] [Full Text]

				A. Kumar, M. T. Eby, S. Sinha, A. Jasmin, and P. M. Chaudhary The Ectodermal Dysplasia Receptor Activates the Nuclear Factor-kappa B, JNK, and Cell Death Pathways and Binds to Ectodysplasin A J. Biol. Chem., January 19, 2001; 276(4): 2668 - 2677. [Abstract] [Full Text] [PDF]

P. Schneider, S. L. Street, O. Gaide, S. Hertig, A. Tardivel, J. Tschopp, L. Runkel, K. Alevizopoulos, B. M. Ferguson, and J. Zonana Mutations Leading to X-linked Hypohidrotic Ectodermal Dysplasia Affect Three Major Functional Domains in the Tumor Necrosis Factor Family Member Ectodysplasin-A J. Biol. Chem., May 25, 2001; 276(22): 18819 - 18827. [Abstract] [Full Text]