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Human keratinocytes transduced with the β3 integrin subunit cDNA can interact with fibrin(ogen)
ESDR / JSID I SID Abstracts
S69
0409
0412
AND SIGNALTRANSDU~~ION Is KERATINOCYTE DIFFERENT~4~10~ DEFINEDBY ANfIFODALEXPRBSSION OFTWO HOMOLOGOUS MEMBYNE G’;“OPROT,ZIN? FOP-1 t+NIJ TROP-2. v ;; Ebe@rdKi I”*. ,‘+amo&wRtlnDanlre .Th ““~Ruzwb .a”dhi&wl scbpe Departments of Demmtoiogy, Unive:ties of DUsseldoff and WUnburg*, Depertnrent of Biochemistry and Molecular Biology.The Free University ofBerlin', Germany, and InstiNt di Ricer&e Fsrmacologiche “MarioNegri” ‘>S. Maria Irnbem, Italy We have studied two homologous members of a new family of ceil surface giycnproteins, Tropl and TmpZ, which arx expressed antipodaiiy on human keratinocytes. WhileTropl is expressed exclusively on immature cells of the germ ceil phenotype, such as primary and secondary epitheiiai gernrs, Trop2 is expressed by suprabasni keratinocyti committed to terminal differentiation. In addition, Tropi is strongly induced in basal ail carcinomas, while T-2 is markedly downregulated in these hnnom Funftional aspects of the two giycopmteins were studied in vitro using HaCaT cells and mouse L-ceil tmnsfect~ts expressing high levels of either Tropl or Trap-2. It was found that Trop2 mAbs induced transmission of intraccUular signals as
HUMAN KERATINOCYTES TRANSDUCED WITH THE 83 INTEGRIN SUB;NIT cDNA C$y IYTERACT WITH FIBRIN(OGEN). M. Kubo. &B. atz. M S mo”. Ta chman. and R.A.F. Clark. Departnxnt of Dermatology.
pH. When cell adbesim was stuclicdin a “Cr releaseasay, it was found that T-2 mAbsincreased honmphilic adhesion of keratinocytes. I” addition, possible roles in &I &ratio”were assessed using mcxliiiedBoyden chambers. I” contrast, Tmpl appeared to be involved in ceil proiiieratio”, a.9it was selectively upregulated on pmlifemting cells within colonies. Both expression petter” in human ski” end in-vim results suggest tbl the alternate expression of Tmpl and Tropl determines distinctive functional slates associated with de-differentiation or diffennliatio” of human kemtincqtes. Thus this novel family of kemtinocytemembrene glycoprosins may provide new insights into the reguistio” of crucial cellular functions such as organogenesis, tunror progression and terminai diffenntiaton.
School of Medicine, and Department of Oral Biology and Pathology, School of Dental Medicine, SUNY at Stony Brook. Stony Brook. NY. During the re-epilheiialization of cutaneous wounds, migrating epldermel cells dxsect a pathway between the fibrin clot and the collagen-rich demus or the tibronectin (FN). rich granulation tissue. These migrating epidermal cells up-regulate integrins asp,, a@ end a-46, but not ~43. uvp3 is a multi-ligand integrin recepior which interact with fibrintogen), FN and virronecrin (VN) in other cell types such as endorbehal cells and fibroblasts. a$3 has an important role in wound angiogenesis and granulation tissue formation. Previously we showed that human keratinocytes do not express a43 nor interact with tibrin(ogen). Based on these data, we hypatheszed that if the keratinofytes express 43 integrin, they will interact with ftbnn(ogen). To prove this hypothesis we transduced p3 integrin subunit cDNA into normal human keratinccytes m y&~ using a retrovims-mediated transduction method. Expression of avg3 on the cell surface was confirmed by immunofluorescence technique and FACS analysis using a monoclonai antibody to avp3 (23C6). These p3 cDNA-transduced keratinocytes attached to and spread on fibrin(ogen) sigruticantly compared with &Gal cDNAtransduced keratinocytes (control). The adhesion to fibrin was inhibited by LM6Cr9 (monoclonai anubody to ~43) in a dose-dependent fashion, but not by normal mouse IgG (control). p3 cDNA-transduced cells also attached to and spread on VN significantly more than the control cells. Thus failure of normal human kemtinocytes to adhere to ftbrm(ogen) is due to their lack of a$3 integrin receptor. 03 cDNAtransducedkeratinocyres will be useful iools to examine functlonnl role of aup3 invitro. Furthermore, we recently have produced Kl@3 cDNA transgenic mice to investigate mechanisms of wound re-epitheliaizatio” in
0410
0413
lNDUcTION OF KFBA’lIN 9 EXPRESSION IN NON-PALMOPIANTAR KERATINOCYITS BY PALMOPLANTAR FIBROBLASTS AT STFADY-STATE mRNA LEVELS. ” a and$ University, Osaka, Japan. Keratin 9 is au acidic type I keratin specific to the suprabasai keratinocytes of mainly palms and soles. Point mutations in kaatin 9 have recently brm show” to be involved in the etiology of painroplantar keratodenna. We report the sleady-state mRNA expression of keratkr 9 in human epidernrai &is cultured with or without pahnoplantar fibrobiasts. Cultured pahnoplantar keratinocytw not only from early passage but also from late passage expressed the same degree of kemiin 9 nrRNA, as measured by reverse transuiptase-polymaase chain reaction. Recombinant
IDENTIFICATION OF DEIMINATED ARGININE RESIDUES IN NEWBORN MOUSE EPIDERMAL KERATIN Kl. Tatsuo Senshu. Kvoichi Akiyama and Kohii Nomurat. Department of Ceil Chemistry and Department 01 Enzyme BiochemistryZ, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan Proteins containing citrulline residues are known to be present in the cornified layers of mammalian epidermis. Such citrulline residues are formed by enzymatic deimination of arginine residues by peptidylarginine deiminase. We have reported recently that major deiminated proteins in the cornified layer are partially degraded/disu~ide-cross-linked keratin Kl. To study the functional significance of this preferential deimination, we attempted to identify preferred acting sites of peptidylarginine deiminase Deiminated keratin Kl was obtained from newborn mouse epidermis by differential extraction using ureal2-mercaptoethanol followed by anion exchange chromatography. It was subjected to limited proteolytic cieavages, and the resulting deiminated peptides were fractionated by SDS-PAGE or HPLC for N-terminal sequencing and/or amino acid analysis. At least two sites were identified, one in the Vl and the other in the V2 regions. A tetrapeptide sequence (GRGG) covering the latter appears once in the mouse Kl sequence. A tridecapeptide sequence covering this residue shows 77% homohgy with a tridecapeptide sequence in the VZ region of human Kl, a presumptive site of deimination. It is probable that these regions are deiminated to modulate their interactions with other epidermal proteins during the terminal stage of epldermal differentiabon
evidenced by rapid iocrcases of intmcellularcalciurn levels and decmase of inhacellular
organotypic cultures, made of mltured non-pahnoplrmtar kemtinocytes and papillary pahnoplantar fibroblasts with the use of a mernbrax, also expressed the kerntin 9 spo5fic mRNA Reticular pabnoplantnr fibroblasts also paily induced keratin 9 mRNA expression. Non-p&noplantar kemtincxytea grown alone or with nonpahnopiantar iibroblasb showed no expression of keratm 9. Cultured pain~opiantar
tibrohiasb did not show keratin 9 expressk~n, either. Pahnopiantar kcmrinocytes grown with non-pahnoplantar fibrobiasts continued to express kerati” 9. These results suggest that 1) there are topological interaaions telween mesenchynrai and epithelial cells at steady-state mRNA levels, 2) the induction of kc&in 9 mRNA expression in heterotypic keralinocytcs is medkitcd by stromal permeable factors, 3) heterogeneity exists between papillary and reticular palmoplantar dermal fibmblasts in terms of keratin 9 inductiov, and 4) keratin 9 expression in painmplantar keratinoqtes is also controlled by intrhwc programs.
0411
0414
A NOVEL APOPTOSIS INDUCER. APOPTOSIS SIGNAL REGULATING KINASE (AS
KERA’IINOCYTE (KC) TRANSCRlPflON FACTORS AR!3 DIFFERENTIALLY INDUCED BY PATHOGENIC PSOKIATIC LYMPHOCYTES. X-M. Sun.T. Wrone-Smith. B.J. Nickeloff, Departmentof Pathology, Loyola University, Maywood, Illinois. Regulation of KC geneexpressionincludestranscriptionfactors(TFs)which bind to specific DNA sequenceelements. Since gene activation by TFs is mediated up and down rapidly, it hasbeendifficult to idenrify intranucleerlocalization ofTFs in establishedchrome psoriaticplaques. To define TF activation Pathwaysof relevanceto psoriasis,2 experimental approacheswere employed. First cultured KCs were exposed (3-6 hours)to various stimuli (IF?-y,TNF-a, W-B light, phorbal ester- TPA),end thecytoplasmictonucleartranslocation afTFs(STAT-l,NF-xB,ATF-Z,C-jun, Rel B)determined by immunostainingandconfirmed by gel-shit?assays. Well characterizedAbs were usedto localize specific TFs in cytoplasm end nuclearcompartmentsof KCs grown in S-well Lab Tek chambers.Gel shit?assayswere Performedusing standardprobesand isolated KC nuclei. While there was high coestitutive KC expressionof ATF-2 and c-jun, the other TFs could be differentially induced by the various stimuli. No differences in the responseof KCs obtained from either normal (n=3) or psoriatic patients (“4) was obsewed. Having establishedthis date-basefor which stimuh inducedvariousTFs, the secondapproachdeterminedthe inductionofKC TFs atier exposure to conditioned medium (CM) obtained from bacterial superantigen-activated T cells. T lymphocyteswere known toposserapathogenicphenotypebytheirabilityte inducepsoriasis using P SCID mouse:human skin model. 48 hour CM from theseaerivatedT cells induced KCs (“4) to significantly mcreesethe TFs NF-rB, STAT-I, and ATF-2 Taken together theseresultsindicatethat KCs rerpand rapidly to variousstimuli by translocatmgspecificTFs fmm the cy?aplasmto the nucleus Moreover, PathogenicT cells Producecytokines which differentially induce TFs in KCs. Using this approachit is possibleto exploreearly transcriptionaleventnthatoccurwhenpatbogenicTcellsenterskinandcausepsoriaticlesions.
Cancer Institute, Tokyo, Japan. A mitogen-activated protein (MAP) kinase kinase kinase, termed ASK1 , is a novel apoptosis inducer. ASK1 activates stress-activated protein kinase and p33 subgroups of MAP kinase via two different subgroups of MAP kinase kinase. In this study we examined the involvement of ASK1 in UVB-induced apoptosis of cultured human keratinocytes. in 100 mJ/cm* UVB irradiated-keratinocytes, characteristic DNA ladder was detected 24 h after the exposure. Apoptotic cells were determined by counting the nuclei labeled with TUNEL method at different times after irradiation. Positive cells were 3.3, 14. 65 and 98 % at 3. 6. 12 and 24 h after the irradiation, respectively. LIVB irradiation also increased the positiie cells for anti-ASK1 antibody determined by immunostaining. Doublestaining with anti-ASK1 antibody and TUNEL method revealed ASK1 positive keratinocytes and TUNEL positive cells were identical, which indicated ASK1 was induced during the process of apoptosis. Western blot analysis of UVB-irradiated keratinocytes showed an antiASK1 antibody-reactive protein which was not detected in control keratinocvtes. ASK1 is a kev element in the mechanism of UVB induced apoptosis of human keratindcytes.
S69
0409
0412
AND SIGNALTRANSDU~~ION Is KERATINOCYTE DIFFERENT~4~10~ DEFINEDBY ANfIFODALEXPRBSSION OFTWO HOMOLOGOUS MEMBYNE G’;“OPROT,ZIN? FOP-1 t+NIJ TROP-2. v ;; Ebe@rdKi I”*. ,‘+amo&wRtlnDanlre .Th ““~Ruzwb .a”dhi&wl scbpe Departments of Demmtoiogy, Unive:ties of DUsseldoff and WUnburg*, Depertnrent of Biochemistry and Molecular Biology.The Free University ofBerlin', Germany, and InstiNt di Ricer&e Fsrmacologiche “MarioNegri” ‘>S. Maria Irnbem, Italy We have studied two homologous members of a new family of ceil surface giycnproteins, Tropl and TmpZ, which arx expressed antipodaiiy on human keratinocytes. WhileTropl is expressed exclusively on immature cells of the germ ceil phenotype, such as primary and secondary epitheiiai gernrs, Trop2 is expressed by suprabasni keratinocyti committed to terminal differentiation. In addition, Tropi is strongly induced in basal ail carcinomas, while T-2 is markedly downregulated in these hnnom Funftional aspects of the two giycopmteins were studied in vitro using HaCaT cells and mouse L-ceil tmnsfect~ts expressing high levels of either Tropl or Trap-2. It was found that Trop2 mAbs induced transmission of intraccUular signals as
HUMAN KERATINOCYTES TRANSDUCED WITH THE 83 INTEGRIN SUB;NIT cDNA C$y IYTERACT WITH FIBRIN(OGEN). M. Kubo. &B. atz. M S mo”. Ta chman. and R.A.F. Clark. Departnxnt of Dermatology.
pH. When cell adbesim was stuclicdin a “Cr releaseasay, it was found that T-2 mAbsincreased honmphilic adhesion of keratinocytes. I” addition, possible roles in &I &ratio”were assessed using mcxliiiedBoyden chambers. I” contrast, Tmpl appeared to be involved in ceil proiiieratio”, a.9it was selectively upregulated on pmlifemting cells within colonies. Both expression petter” in human ski” end in-vim results suggest tbl the alternate expression of Tmpl and Tropl determines distinctive functional slates associated with de-differentiation or diffennliatio” of human kemtincqtes. Thus this novel family of kemtinocytemembrene glycoprosins may provide new insights into the reguistio” of crucial cellular functions such as organogenesis, tunror progression and terminai diffenntiaton.
School of Medicine, and Department of Oral Biology and Pathology, School of Dental Medicine, SUNY at Stony Brook. Stony Brook. NY. During the re-epilheiialization of cutaneous wounds, migrating epldermel cells dxsect a pathway between the fibrin clot and the collagen-rich demus or the tibronectin (FN). rich granulation tissue. These migrating epidermal cells up-regulate integrins asp,, a@ end a-46, but not ~43. uvp3 is a multi-ligand integrin recepior which interact with fibrintogen), FN and virronecrin (VN) in other cell types such as endorbehal cells and fibroblasts. a$3 has an important role in wound angiogenesis and granulation tissue formation. Previously we showed that human keratinocytes do not express a43 nor interact with tibrin(ogen). Based on these data, we hypatheszed that if the keratinofytes express 43 integrin, they will interact with ftbnn(ogen). To prove this hypothesis we transduced p3 integrin subunit cDNA into normal human keratinccytes m y&~ using a retrovims-mediated transduction method. Expression of avg3 on the cell surface was confirmed by immunofluorescence technique and FACS analysis using a monoclonai antibody to avp3 (23C6). These p3 cDNA-transduced keratinocytes attached to and spread on fibrin(ogen) sigruticantly compared with &Gal cDNAtransduced keratinocytes (control). The adhesion to fibrin was inhibited by LM6Cr9 (monoclonai anubody to ~43) in a dose-dependent fashion, but not by normal mouse IgG (control). p3 cDNA-transduced cells also attached to and spread on VN significantly more than the control cells. Thus failure of normal human kemtinocytes to adhere to ftbrm(ogen) is due to their lack of a$3 integrin receptor. 03 cDNAtransducedkeratinocyres will be useful iools to examine functlonnl role of aup3 invitro. Furthermore, we recently have produced Kl@3 cDNA transgenic mice to investigate mechanisms of wound re-epitheliaizatio” in
0410
0413
lNDUcTION OF KFBA’lIN 9 EXPRESSION IN NON-PALMOPIANTAR KERATINOCYITS BY PALMOPLANTAR FIBROBLASTS AT STFADY-STATE mRNA LEVELS. ” a and$ University, Osaka, Japan. Keratin 9 is au acidic type I keratin specific to the suprabasai keratinocytes of mainly palms and soles. Point mutations in kaatin 9 have recently brm show” to be involved in the etiology of painroplantar keratodenna. We report the sleady-state mRNA expression of keratkr 9 in human epidernrai &is cultured with or without pahnoplantar fibrobiasts. Cultured pahnoplantar keratinocytw not only from early passage but also from late passage expressed the same degree of kemiin 9 nrRNA, as measured by reverse transuiptase-polymaase chain reaction. Recombinant
IDENTIFICATION OF DEIMINATED ARGININE RESIDUES IN NEWBORN MOUSE EPIDERMAL KERATIN Kl. Tatsuo Senshu. Kvoichi Akiyama and Kohii Nomurat. Department of Ceil Chemistry and Department 01 Enzyme BiochemistryZ, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan Proteins containing citrulline residues are known to be present in the cornified layers of mammalian epidermis. Such citrulline residues are formed by enzymatic deimination of arginine residues by peptidylarginine deiminase. We have reported recently that major deiminated proteins in the cornified layer are partially degraded/disu~ide-cross-linked keratin Kl. To study the functional significance of this preferential deimination, we attempted to identify preferred acting sites of peptidylarginine deiminase Deiminated keratin Kl was obtained from newborn mouse epidermis by differential extraction using ureal2-mercaptoethanol followed by anion exchange chromatography. It was subjected to limited proteolytic cieavages, and the resulting deiminated peptides were fractionated by SDS-PAGE or HPLC for N-terminal sequencing and/or amino acid analysis. At least two sites were identified, one in the Vl and the other in the V2 regions. A tetrapeptide sequence (GRGG) covering the latter appears once in the mouse Kl sequence. A tridecapeptide sequence covering this residue shows 77% homohgy with a tridecapeptide sequence in the VZ region of human Kl, a presumptive site of deimination. It is probable that these regions are deiminated to modulate their interactions with other epidermal proteins during the terminal stage of epldermal differentiabon
evidenced by rapid iocrcases of intmcellularcalciurn levels and decmase of inhacellular
organotypic cultures, made of mltured non-pahnoplrmtar kemtinocytes and papillary pahnoplantar fibroblasts with the use of a mernbrax, also expressed the kerntin 9 spo5fic mRNA Reticular pabnoplantnr fibroblasts also paily induced keratin 9 mRNA expression. Non-p&noplantar kemtincxytea grown alone or with nonpahnopiantar iibroblasb showed no expression of keratm 9. Cultured pain~opiantar
tibrohiasb did not show keratin 9 expressk~n, either. Pahnopiantar kcmrinocytes grown with non-pahnoplantar fibrobiasts continued to express kerati” 9. These results suggest that 1) there are topological interaaions telween mesenchynrai and epithelial cells at steady-state mRNA levels, 2) the induction of kc&in 9 mRNA expression in heterotypic keralinocytcs is medkitcd by stromal permeable factors, 3) heterogeneity exists between papillary and reticular palmoplantar dermal fibmblasts in terms of keratin 9 inductiov, and 4) keratin 9 expression in painmplantar keratinoqtes is also controlled by intrhwc programs.
0411
0414
A NOVEL APOPTOSIS INDUCER. APOPTOSIS SIGNAL REGULATING KINASE (AS
KERA’IINOCYTE (KC) TRANSCRlPflON FACTORS AR!3 DIFFERENTIALLY INDUCED BY PATHOGENIC PSOKIATIC LYMPHOCYTES. X-M. Sun.T. Wrone-Smith. B.J. Nickeloff, Departmentof Pathology, Loyola University, Maywood, Illinois. Regulation of KC geneexpressionincludestranscriptionfactors(TFs)which bind to specific DNA sequenceelements. Since gene activation by TFs is mediated up and down rapidly, it hasbeendifficult to idenrify intranucleerlocalization ofTFs in establishedchrome psoriaticplaques. To define TF activation Pathwaysof relevanceto psoriasis,2 experimental approacheswere employed. First cultured KCs were exposed (3-6 hours)to various stimuli (IF?-y,TNF-a, W-B light, phorbal ester- TPA),end thecytoplasmictonucleartranslocation afTFs(STAT-l,NF-xB,ATF-Z,C-jun, Rel B)determined by immunostainingandconfirmed by gel-shit?assays. Well characterizedAbs were usedto localize specific TFs in cytoplasm end nuclearcompartmentsof KCs grown in S-well Lab Tek chambers.Gel shit?assayswere Performedusing standardprobesand isolated KC nuclei. While there was high coestitutive KC expressionof ATF-2 and c-jun, the other TFs could be differentially induced by the various stimuli. No differences in the responseof KCs obtained from either normal (n=3) or psoriatic patients (“4) was obsewed. Having establishedthis date-basefor which stimuh inducedvariousTFs, the secondapproachdeterminedthe inductionofKC TFs atier exposure to conditioned medium (CM) obtained from bacterial superantigen-activated T cells. T lymphocyteswere known toposserapathogenicphenotypebytheirabilityte inducepsoriasis using P SCID mouse:human skin model. 48 hour CM from theseaerivatedT cells induced KCs (“4) to significantly mcreesethe TFs NF-rB, STAT-I, and ATF-2 Taken together theseresultsindicatethat KCs rerpand rapidly to variousstimuli by translocatmgspecificTFs fmm the cy?aplasmto the nucleus Moreover, PathogenicT cells Producecytokines which differentially induce TFs in KCs. Using this approachit is possibleto exploreearly transcriptionaleventnthatoccurwhenpatbogenicTcellsenterskinandcausepsoriaticlesions.
Cancer Institute, Tokyo, Japan. A mitogen-activated protein (MAP) kinase kinase kinase, termed ASK1 , is a novel apoptosis inducer. ASK1 activates stress-activated protein kinase and p33 subgroups of MAP kinase via two different subgroups of MAP kinase kinase. In this study we examined the involvement of ASK1 in UVB-induced apoptosis of cultured human keratinocytes. in 100 mJ/cm* UVB irradiated-keratinocytes, characteristic DNA ladder was detected 24 h after the exposure. Apoptotic cells were determined by counting the nuclei labeled with TUNEL method at different times after irradiation. Positive cells were 3.3, 14. 65 and 98 % at 3. 6. 12 and 24 h after the irradiation, respectively. LIVB irradiation also increased the positiie cells for anti-ASK1 antibody determined by immunostaining. Doublestaining with anti-ASK1 antibody and TUNEL method revealed ASK1 positive keratinocytes and TUNEL positive cells were identical, which indicated ASK1 was induced during the process of apoptosis. Western blot analysis of UVB-irradiated keratinocytes showed an antiASK1 antibody-reactive protein which was not detected in control keratinocvtes. ASK1 is a kev element in the mechanism of UVB induced apoptosis of human keratindcytes.