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Activation of p75 nerve growth factor receptor is blocked by cyclic peptides containing a lysine-glycine-alanine (KGA) motif
ESDR I JSID I SID Abstracts
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TYROSINE PHOSPHORYLATION AND SRC FAMILY KINASES CONTROL KERATINOCYTE CELL-CELL ADHESION Enzo Calautti. Sara Cabcdi. Nancy Kedersha Cutaneous Biology research Center, Massachusetts General Hosmta, and Harwd’Medeal School. 13th Street. Charlesfawn. MA 02129 In their progression from the basal tc upper differentiateh layers of tic epidermis. keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is hew these early structural events are related tc the biochemical pathways which trigger differentiation. We show here that tymsine phosphcrylation, and Src-related kinases in particular, play a direct and important role m the changes in keratinocyte cell adhesion which are connected wtb diffenntiatmn, and that this regulatory function is important both in virm and in viva. I” particular. B-catenin, y-cate”i”/plakoglcbi” and plZO-Cas are all significantly tyrosine phosphorylated in cultured mcuse keratinocytes within 9 hours cf calctum-induced differentiation. In parallel with these changes, there is an increased association of a-catenin and plZO-Cas with EInhibitton of tyrcsine cadherin. whxh is prevented by tyrcsine kininhibition phosphorylation suppresses adherens junctions formation and causes a sigmficant reduction in adhesive strength of diffcrenttating keratmocytes. Consistent wtth the involvement of the Fyn tyrcsine kinase in keratinccyte differentiation, fyn-dehcient keratinocytes have strongly decreased tyrosine phcsphorylation levels of B- and ycatcnins and pl2OCas. Moreover, the structural and functional abnormabties I” cell adhesion caused by tyrcsine kinase inhibition are also observed in the cultured jjw deficient keratinocytes. While skin of jyn -/- truce appears normal. skm of mace with a disruption in both the fyn and src genes shows mtrinsically reduced tyrosme phosphorylaticn of B-catenm, strongly decreased pl20-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, tymsme phosphcrylation contrcls keratinccyte cell cell adhesion both in wro and in viva, and Fyn and related kinases are key elements m thts process.
ACTlVATlON OF p75 NERVE GROWTH FACTOR RECEPTOR IS BLOCKED BY CYCLIC PEPTlDES CONTATNTNG A LYSINE-GLYCME-ALANME (KGA) MOW. 3 Zhai. K. Traber,M. Year and B.A. Gilchrest. Boston University School of Medicine, B&on, MA Cutaneusmelanocyter(M) sharea common embryologic origin and signaling molecules with neurons (N). including receptarrfor nerve~w,, factor (NGF). Recendy, d,e 75 kD NGF receptor(~75’~) has been implicated in apoptosir of W-irradiated M, and N exposedto p amyloid (Ap), presumablyvia ctustermg andaggregation of% receptor.TOconfirmthat+inc”ced apoptosis is mediatedby binding an* actlvafl””(If pnp75 sl#almg ~thways wen mves”@edm APstimulatedNM 3T3 cells 3T3) or the samecells transfectedwith plasmid vectw cngineendto expresshuman~75 (~75 (pCM” 3T3) as a c”“trol. Witldi 8 hours, even low dose AB (250 nM) prominently ,,,creasedthe 2.7 and 3.2 kb apoptosis assc&ted c-jun tmwxiptS in p7SNn 3T3 cells but not in conholr. Po,y(ADpribose)polymerase,the substratefor caspase-3, known to be cleavedduring apoptasia,was then studied by westernblot analysis. Only the full length protein was seen in pCMV 3’111cells, but in p75”R 3T3 cells a&x AB there WBSa gradual increaseof the 89 kD cleavageproduct, demonsnati,,g actwatlon of easpese-3 by Ai3 binding to ~75~. Computer analysis of Af3 srmcmre revealed3 amino acids. similar to the NGF binding site for ~75”“. likely to Lx in a &t~p turn, creatinga receptm lncreaslng ~“ce”trati”“s (Obinding site: KGA. To determineif KGA mediatesAD bindingto ~75 100“M) of a” HPLC purifiedcychcpeptide CI(GBIC was added to p75* ;n cells in the presenced “’ I-A!3 CariaIC. containing the AOA sequenceknown t&m NGF mutants not to bind p7sNTR,sewed as a negativecontrol. CaIC competitively inhibited I” t-A!3 binding to cells (50% inhibition at -7 nM), while C,&C&IC bad no wrct on I” I-A$ bindmg.We the” askedif cycbcpeptides expectedto interferewith p7SNRclustering canpreventp75-mediated M and N apaptosa. M were “V irradiated with 25 ml/cm’ UVB and then supplemented with one of three HPLC purified cyclic peptider containingthe KGA motif, cr a cycbcpeptide containing AGA (negativecontrol). In W-irradiated M supplemented wtb KCA-contaming pcptides there was no significant di&zmce in cell yields behveen shamand UV-trmdiated M. I” contrast,cell yletds of UV-irmdiafed M supplemented with AGA contliningpeptidewere significantlylower(p
116 kD
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CHARACTERIZATION OF NOVEL PROTEIN INTERACTIONS IN THE PZlc’P’ PATHWAY OF CELL CYCLE CONTROL. dens Oliver Funk. Andrew McShea 8 Denise A. Galloway. Program in Cancer Biology, Fred Hutchinson Cancer Research Center, Seattle, Washinaton. USA. p21c’p1 mediates cell cycle arrest-by various p53-dependent and -independent stimuli and therebv reoulates the GqlS checkooint. differentiation, and senescence. ~21 ‘inhibzs cyclin-dependent kinase {CDKj activity and proliferating cell nuclear antigen (PCNA)-dependent DNA replication by binding to CDWcyclin complexes and to PCNA through distinct domains. Our previous analyses showed that the carboxyl (C)-terminus of p.21 simultaneously modulates both CDK activity and PCNA-dependent DNA replication and that a single protein, the HPV type 16 E7 oncoprotein (16E7), can override this modulation to disrupt normal cell cycle control. To identify cellular proteins which share this ability of 16E7 a yeast two-hybrid assay was employed using p21 as bait. A novel human cDNA whose product bound to p21C was cloned (21BP). This interaction was confirmed in vitro with purified and in vitro translated proteins and in viva with 293 cells cotransfected with p21- and 21BP-encoding plasmids. Notihern blotting showed abundant 21BP RNA levels in many tissues. Subsequent cloning of the mouse and rat homologs revealed a very conserved amino acid sequence indicating a potentially important function of 21BP in the p21 pathway. The functions of 21BP and its mutual relationship with p21 are studied in isogenic p21-positive and p21-null cell lines. Our results illuminate a new model for the regulation of the various p21 activities by complex protein interactions as well as for uncoupling of cellular differentiation and proliferation by p21 antagonism.
MATERNAL UNIPARENTAL MEROISODISOYY IN THE LAMB3 REGlON 0 F CHROMOSOME 1 IS A NOVEL MECHANISM RESULTING IN LETHAL JUNCTIONAL EPIDERYOLYSIS BULLOSA. Jouni YasukcTaldzawa’2. P Ikkinen’ Himshi__$timizd. Takeii Nishikwa2. ‘Department of Dermatolcg~ and Cutaneous Biology, Jefferson Medii College, Philadelphia. PA 19107; ‘Depad~ent of Dermatology, Keio University School of Medicine, To$c. Japan. Hedii junctkmal epidermolysis bullosa (HJEB; OMIMlt2267W) $ a lethal. autcscmal recesswe blisterino disorder caused bv mutat~cns in one of the three oenes. LAMAI. LAMB3 OTLAMCi encoding the cc&titutive pclypeptide subunits cf lamink 5. The majority of cases result from ronsense or frameshi mutations in both alleles cf any one of these genes. In this study, we describe a patient homozygous for a novel &sense mutation P93SX m excn 19 of LAMBB. which has been mapped to chromosome fq32, resulting frcm a complex chmmoscmal aberration. The patient w born with extensive blrstermg and demonstrated negative immuncfluOreScence staining for larmnin 5, and transmission electrcn miCrOsCOWrevealed tiSSUe SepamtiCn within twina lucida of the denal-epidermal junction, diagncstic of HJEB. The mother of the proband was found to be heterczygcus carder for this mutation, while the father demonstrated the wild-type LAMB3 allele only. Nonpaternity was excluded by 13 micmsatellite madws in six different chromosomes. Genotype ar&ysis using 28 miCrOSatWllii markers spanning the entire chromosome 1 revealed that the patient had maternal primary hetercdlsomy. as well as mercisodisomy wnhln two reglcns of chromosome 1, one on Ip and the other one on Iq, the latter region spanmng -12 CM and containing the maternal LAMB3 mutation. Thus. HJEB in this patlent developed as a result from reduction to homozygosity of the maternal LAMB3 mutation on chromosome lq32. Mechanistiially, these chromcsomal aberratrcns could be due to nondisjunction at the first meiotic division preceded by recombinationa, events. and followed by edher gamete ccmplementation at fertilization cr. more likely. friscmy rescue at postzygotic miioss.
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NEW ROLES FOR NEUROTROPHINJ, NEUROTROPHIN-4 AND BRAIN-DERIVED NEUROTROPHIC FACTOR: INVOLVEMENT IN HAIR GROWTH CONTROL
RESTORATION OF OPEN READING FRAME DUE TO SKIPPING OF AN MON Vi,-“, AN ,NTERNAL DELETION IN THE COL7Af GENE. J. Frank. P &8pmer@. H.C. C&etz* and A.&!&i&w, Dept. d Dermatology, Columbia University, New Ych NY; “Dept. of DenatoloW. NorthWestem University. Chicago. IL.; ‘Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore. MD. Exon skipping as a result of intra-exonk deletion has been infrequently describec as a mechanism of action of a deletion mutation, and when reported, leads to a frameshii and premature termination codon. In miS study, two pedigrees with dominant dystrcphic epldermclySis bullosa (DDEB) in three generations were investigated. Screening of aHexcns of COL‘IAI revealed a 16 bp deletion within excn 87. This result was discordant with the growing body ewdence supporting the paradigm that frameshirt mutations in COL7Al only cause disease when inherited in frans wiih a secccd COL7Al mutation In a recessive manner. Because of the dominant mheritance of thi mutation, we investigated the effect of this nut&on at the transcript level, using RNA extracted fmm cuitured keatinccytes of an affected family member. Southern blot snalys~sof an RT-PCR pmdwt spanning excns 85-92 showed two different size pmducts, cf 363 and 294 bp. Dimd sequencing of the 294 bp product revealed that excn 87 was skipped in Is entirety, while the flanking excn sequences remained intact and in-fame, creating a transctfpt encoding a protein with dommant negative potential, and thus reconciling the dcminant inlwdtance observed in this famtb. Hcwever, in both families, affected children born to parents wilb DDEB appeared dinicalfy to have a much mere severe type of dystmphic EB. Mutation analysts revealed that the severely affected children in both families were compound heterzygctes for one DDEB and cne RDEB mtiaticn in COL7Al. The second mutations (a premature termination codon in excn 15 and a splice site mutabcn in excn 3) were mhedted in fransfmm unaffected oarants. Thi Infomwtan used tc perform prenatal diagnosis of the fetal genotype in Family’A. at 25% risk fcr a child with either DDEB or RDEB. Restoration of open reading frame by excn skipping is a rarely repcrted phenomenon which may be more prevalent than previously appreciated, and hightens an awareness of novel mechanisms of acbcn for mutatnns.
~.L.2P.Welkerl.M.Metzl. van der V&_& E&us’. ‘Dept of Dermatology, Charit&, Humboldt-Universit6< D-10117, Berlin, &pt of Pathology&Laboratory Medicine, Univ. of Kentucky, Lexington, KY, USA Increasing evidencesuggeststhat neurotrcphinsnot only control the development of skin innervation, but are also involved in the regulation of tissue mcrphcgenesis and remcdelling. Here we have studied the expressionand functionsof neurctrcphin-3 (NT-3), neurctrcphin-4 (NT-4) and brain-derived neumtmphic factor (BDNF) during the murine hair cycle. In the hack skin of C57BLi6 mice with all follicles in telogen, NT-3 protein levels reached 0.77 “gimg. Anagen developmentwas accompaniedby a decline of both NT-3 mRNA and protein levels, while maximal levels ofNT-3 and BDNF mRNA, and cfNT-3 protein (1 n&g) were fc”“d during synchronizedhair follicle regression(c&age”). I” early catage”kemtinocytes(KC) of the regressinghair matrix expressedmaximal BDNF mRNA, BSwell as maximal BDNF-, TrkB-, NT-3., and TrkC-immunoreactivi@ (IR), whereas dermal papilla fibrcblasts were TrkB+, NT-3+, and TrkC+. During late catagen, all of these antigens disappeared fmm the demnl papilla, while KCs of the regressing epithelial strand became TrkB+, NT-3+, NT-4+, and TrkC+, and thoseof the secondaryhair germ were BDNF+, NT-3+, and TrkC+. Compared to corresponding wild type mice, precacious catagen development was found in neonatal NT-3. or BDNF- overexpressing transgenic mice, while catagen retardation was seen in heterozygcus NT-3 (+I-) and homczygcus BDNF or NT-4 knockout(-/-) mice. Finally, NT-3, NT-4, and BDNF promoted catagen development in murine skin organ culture. Thus, NT-3, NT-4, and BDNF may be important elements in the ccntml of hair follicle regression, and TrkC and TrkB agonists/antagonists may be therapeutically exploitable as novel hair growth-modulatory drugs.
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TYROSINE PHOSPHORYLATION AND SRC FAMILY KINASES CONTROL KERATINOCYTE CELL-CELL ADHESION Enzo Calautti. Sara Cabcdi. Nancy Kedersha Cutaneous Biology research Center, Massachusetts General Hosmta, and Harwd’Medeal School. 13th Street. Charlesfawn. MA 02129 In their progression from the basal tc upper differentiateh layers of tic epidermis. keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is hew these early structural events are related tc the biochemical pathways which trigger differentiation. We show here that tymsine phosphcrylation, and Src-related kinases in particular, play a direct and important role m the changes in keratinocyte cell adhesion which are connected wtb diffenntiatmn, and that this regulatory function is important both in virm and in viva. I” particular. B-catenin, y-cate”i”/plakoglcbi” and plZO-Cas are all significantly tyrosine phosphorylated in cultured mcuse keratinocytes within 9 hours cf calctum-induced differentiation. In parallel with these changes, there is an increased association of a-catenin and plZO-Cas with EInhibitton of tyrcsine cadherin. whxh is prevented by tyrcsine kininhibition phosphorylation suppresses adherens junctions formation and causes a sigmficant reduction in adhesive strength of diffcrenttating keratmocytes. Consistent wtth the involvement of the Fyn tyrcsine kinase in keratinccyte differentiation, fyn-dehcient keratinocytes have strongly decreased tyrosine phcsphorylation levels of B- and ycatcnins and pl2OCas. Moreover, the structural and functional abnormabties I” cell adhesion caused by tyrcsine kinase inhibition are also observed in the cultured jjw deficient keratinocytes. While skin of jyn -/- truce appears normal. skm of mace with a disruption in both the fyn and src genes shows mtrinsically reduced tyrosme phosphorylaticn of B-catenm, strongly decreased pl20-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, tymsme phosphcrylation contrcls keratinccyte cell cell adhesion both in wro and in viva, and Fyn and related kinases are key elements m thts process.
ACTlVATlON OF p75 NERVE GROWTH FACTOR RECEPTOR IS BLOCKED BY CYCLIC PEPTlDES CONTATNTNG A LYSINE-GLYCME-ALANME (KGA) MOW. 3 Zhai. K. Traber,M. Year and B.A. Gilchrest. Boston University School of Medicine, B&on, MA Cutaneusmelanocyter(M) sharea common embryologic origin and signaling molecules with neurons (N). including receptarrfor nerve~w,, factor (NGF). Recendy, d,e 75 kD NGF receptor(~75’~) has been implicated in apoptosir of W-irradiated M, and N exposedto p amyloid (Ap), presumablyvia ctustermg andaggregation of% receptor.TOconfirmthat+inc”ced apoptosis is mediatedby binding an* actlvafl””(If pnp75 sl#almg ~thways wen mves”@edm APstimulatedNM 3T3 cells 3T3) or the samecells transfectedwith plasmid vectw cngineendto expresshuman~75 (~75 (pCM” 3T3) as a c”“trol. Witldi 8 hours, even low dose AB (250 nM) prominently ,,,creasedthe 2.7 and 3.2 kb apoptosis assc&ted c-jun tmwxiptS in p7SNn 3T3 cells but not in conholr. Po,y(ADpribose)polymerase,the substratefor caspase-3, known to be cleavedduring apoptasia,was then studied by westernblot analysis. Only the full length protein was seen in pCMV 3’111cells, but in p75”R 3T3 cells a&x AB there WBSa gradual increaseof the 89 kD cleavageproduct, demonsnati,,g actwatlon of easpese-3 by Ai3 binding to ~75~. Computer analysis of Af3 srmcmre revealed3 amino acids. similar to the NGF binding site for ~75”“. likely to Lx in a &t~p turn, creatinga receptm lncreaslng ~“ce”trati”“s (Obinding site: KGA. To determineif KGA mediatesAD bindingto ~75 100“M) of a” HPLC purifiedcychcpeptide CI(GBIC was added to p75* ;n cells in the presenced “’ I-A!3 CariaIC. containing the AOA sequenceknown t&m NGF mutants not to bind p7sNTR,sewed as a negativecontrol. CaIC competitively inhibited I” t-A!3 binding to cells (50% inhibition at -7 nM), while C,&C&IC bad no wrct on I” I-A$ bindmg.We the” askedif cycbcpeptides expectedto interferewith p7SNRclustering canpreventp75-mediated M and N apaptosa. M were “V irradiated with 25 ml/cm’ UVB and then supplemented with one of three HPLC purified cyclic peptider containingthe KGA motif, cr a cycbcpeptide containing AGA (negativecontrol). In W-irradiated M supplemented wtb KCA-contaming pcptides there was no significant di&zmce in cell yields behveen shamand UV-trmdiated M. I” contrast,cell yletds of UV-irmdiafed M supplemented with AGA contliningpeptidewere significantlylower(p
116 kD
0050
0053
CHARACTERIZATION OF NOVEL PROTEIN INTERACTIONS IN THE PZlc’P’ PATHWAY OF CELL CYCLE CONTROL. dens Oliver Funk. Andrew McShea 8 Denise A. Galloway. Program in Cancer Biology, Fred Hutchinson Cancer Research Center, Seattle, Washinaton. USA. p21c’p1 mediates cell cycle arrest-by various p53-dependent and -independent stimuli and therebv reoulates the GqlS checkooint. differentiation, and senescence. ~21 ‘inhibzs cyclin-dependent kinase {CDKj activity and proliferating cell nuclear antigen (PCNA)-dependent DNA replication by binding to CDWcyclin complexes and to PCNA through distinct domains. Our previous analyses showed that the carboxyl (C)-terminus of p.21 simultaneously modulates both CDK activity and PCNA-dependent DNA replication and that a single protein, the HPV type 16 E7 oncoprotein (16E7), can override this modulation to disrupt normal cell cycle control. To identify cellular proteins which share this ability of 16E7 a yeast two-hybrid assay was employed using p21 as bait. A novel human cDNA whose product bound to p21C was cloned (21BP). This interaction was confirmed in vitro with purified and in vitro translated proteins and in viva with 293 cells cotransfected with p21- and 21BP-encoding plasmids. Notihern blotting showed abundant 21BP RNA levels in many tissues. Subsequent cloning of the mouse and rat homologs revealed a very conserved amino acid sequence indicating a potentially important function of 21BP in the p21 pathway. The functions of 21BP and its mutual relationship with p21 are studied in isogenic p21-positive and p21-null cell lines. Our results illuminate a new model for the regulation of the various p21 activities by complex protein interactions as well as for uncoupling of cellular differentiation and proliferation by p21 antagonism.
MATERNAL UNIPARENTAL MEROISODISOYY IN THE LAMB3 REGlON 0 F CHROMOSOME 1 IS A NOVEL MECHANISM RESULTING IN LETHAL JUNCTIONAL EPIDERYOLYSIS BULLOSA. Jouni YasukcTaldzawa’2. P Ikkinen’ Himshi__$timizd. Takeii Nishikwa2. ‘Department of Dermatolcg~ and Cutaneous Biology, Jefferson Medii College, Philadelphia. PA 19107; ‘Depad~ent of Dermatology, Keio University School of Medicine, To$c. Japan. Hedii junctkmal epidermolysis bullosa (HJEB; OMIMlt2267W) $ a lethal. autcscmal recesswe blisterino disorder caused bv mutat~cns in one of the three oenes. LAMAI. LAMB3 OTLAMCi encoding the cc&titutive pclypeptide subunits cf lamink 5. The majority of cases result from ronsense or frameshi mutations in both alleles cf any one of these genes. In this study, we describe a patient homozygous for a novel &sense mutation P93SX m excn 19 of LAMBB. which has been mapped to chromosome fq32, resulting frcm a complex chmmoscmal aberration. The patient w born with extensive blrstermg and demonstrated negative immuncfluOreScence staining for larmnin 5, and transmission electrcn miCrOsCOWrevealed tiSSUe SepamtiCn within twina lucida of the denal-epidermal junction, diagncstic of HJEB. The mother of the proband was found to be heterczygcus carder for this mutation, while the father demonstrated the wild-type LAMB3 allele only. Nonpaternity was excluded by 13 micmsatellite madws in six different chromosomes. Genotype ar&ysis using 28 miCrOSatWllii markers spanning the entire chromosome 1 revealed that the patient had maternal primary hetercdlsomy. as well as mercisodisomy wnhln two reglcns of chromosome 1, one on Ip and the other one on Iq, the latter region spanmng -12 CM and containing the maternal LAMB3 mutation. Thus. HJEB in this patlent developed as a result from reduction to homozygosity of the maternal LAMB3 mutation on chromosome lq32. Mechanistiially, these chromcsomal aberratrcns could be due to nondisjunction at the first meiotic division preceded by recombinationa, events. and followed by edher gamete ccmplementation at fertilization cr. more likely. friscmy rescue at postzygotic miioss.
0051
0054
NEW ROLES FOR NEUROTROPHINJ, NEUROTROPHIN-4 AND BRAIN-DERIVED NEUROTROPHIC FACTOR: INVOLVEMENT IN HAIR GROWTH CONTROL
RESTORATION OF OPEN READING FRAME DUE TO SKIPPING OF AN MON Vi,-“, AN ,NTERNAL DELETION IN THE COL7Af GENE. J. Frank. P &8pmer@. H.C. C&etz* and A.&!&i&w, Dept. d Dermatology, Columbia University, New Ych NY; “Dept. of DenatoloW. NorthWestem University. Chicago. IL.; ‘Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore. MD. Exon skipping as a result of intra-exonk deletion has been infrequently describec as a mechanism of action of a deletion mutation, and when reported, leads to a frameshii and premature termination codon. In miS study, two pedigrees with dominant dystrcphic epldermclySis bullosa (DDEB) in three generations were investigated. Screening of aHexcns of COL‘IAI revealed a 16 bp deletion within excn 87. This result was discordant with the growing body ewdence supporting the paradigm that frameshirt mutations in COL7Al only cause disease when inherited in frans wiih a secccd COL7Al mutation In a recessive manner. Because of the dominant mheritance of thi mutation, we investigated the effect of this nut&on at the transcript level, using RNA extracted fmm cuitured keatinccytes of an affected family member. Southern blot snalys~sof an RT-PCR pmdwt spanning excns 85-92 showed two different size pmducts, cf 363 and 294 bp. Dimd sequencing of the 294 bp product revealed that excn 87 was skipped in Is entirety, while the flanking excn sequences remained intact and in-fame, creating a transctfpt encoding a protein with dommant negative potential, and thus reconciling the dcminant inlwdtance observed in this famtb. Hcwever, in both families, affected children born to parents wilb DDEB appeared dinicalfy to have a much mere severe type of dystmphic EB. Mutation analysts revealed that the severely affected children in both families were compound heterzygctes for one DDEB and cne RDEB mtiaticn in COL7Al. The second mutations (a premature termination codon in excn 15 and a splice site mutabcn in excn 3) were mhedted in fransfmm unaffected oarants. Thi Infomwtan used tc perform prenatal diagnosis of the fetal genotype in Family’A. at 25% risk fcr a child with either DDEB or RDEB. Restoration of open reading frame by excn skipping is a rarely repcrted phenomenon which may be more prevalent than previously appreciated, and hightens an awareness of novel mechanisms of acbcn for mutatnns.
~.L.2P.Welkerl.M.Metzl. van der V&_& E&us’. ‘Dept of Dermatology, Charit&, Humboldt-Universit6< D-10117, Berlin, &pt of Pathology&Laboratory Medicine, Univ. of Kentucky, Lexington, KY, USA Increasing evidencesuggeststhat neurotrcphinsnot only control the development of skin innervation, but are also involved in the regulation of tissue mcrphcgenesis and remcdelling. Here we have studied the expressionand functionsof neurctrcphin-3 (NT-3), neurctrcphin-4 (NT-4) and brain-derived neumtmphic factor (BDNF) during the murine hair cycle. In the hack skin of C57BLi6 mice with all follicles in telogen, NT-3 protein levels reached 0.77 “gimg. Anagen developmentwas accompaniedby a decline of both NT-3 mRNA and protein levels, while maximal levels ofNT-3 and BDNF mRNA, and cfNT-3 protein (1 n&g) were fc”“d during synchronizedhair follicle regression(c&age”). I” early catage”kemtinocytes(KC) of the regressinghair matrix expressedmaximal BDNF mRNA, BSwell as maximal BDNF-, TrkB-, NT-3., and TrkC-immunoreactivi@ (IR), whereas dermal papilla fibrcblasts were TrkB+, NT-3+, and TrkC+. During late catagen, all of these antigens disappeared fmm the demnl papilla, while KCs of the regressing epithelial strand became TrkB+, NT-3+, NT-4+, and TrkC+, and thoseof the secondaryhair germ were BDNF+, NT-3+, and TrkC+. Compared to corresponding wild type mice, precacious catagen development was found in neonatal NT-3. or BDNF- overexpressing transgenic mice, while catagen retardation was seen in heterozygcus NT-3 (+I-) and homczygcus BDNF or NT-4 knockout(-/-) mice. Finally, NT-3, NT-4, and BDNF promoted catagen development in murine skin organ culture. Thus, NT-3, NT-4, and BDNF may be important elements in the ccntml of hair follicle regression, and TrkC and TrkB agonists/antagonists may be therapeutically exploitable as novel hair growth-modulatory drugs.