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Interaction with the extracellular matrix (ECM) component hyaluronan (HA) induces maturation of human dendritic cells (DC)
ESDR I JSID I SID Abstracts
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CLEAVAGE OF BP1 80 (lYPE XVII COLLAGEN) YIELDS A 120 KDA COLLAGENOUSEXTRACELLULAR POLYPEPTIDE. Y. Hirako*, J. Usukura#, J. Uematsu*, T. Hashimotot. Y. KitaiimaP and K. Owaribe*. ‘Biosyst., Grad. Sch. of Human Informat. and #Dept. of Anat., Sch. of Med., Nagoya Univ., Nagoya. tDept. of Dermatol, Kurume Univ. Sch. of Med., Fukuoka. gDept. of Dermatol, Glfu Univ. Sch. of Med.. Gifu, Japan. One of the putative cell-matrix adhesion molecules present In the hemidesmosome (HO) is the 180 kDa bullous pemphigoid antigen (BP180). A monoclonal antibody (mAb) 1337 stained the basement membrane zone of bovine skin and recognized a 120 kDa collagenase-sensitive polypeptide in the HD fraction isolated from bovine cornea. The 120 kDa Dolypepnde was not detected in HDS of cultured cells but rather in iheculiure medium. These results Indicate a localization of the polypeptide distinct from EP180. However, the 120 kDa polypeptide was also recognized by monoclonal antibodies to the extracellular but not the cytoplasmic part of BP180, and found to react with a polyclonal antibody against the non-collagenous 16A domain of BP180. Together with other data, the 120 kDa polypeptide was ldentifled as an eXtraCellUlar fragment of i3P180. The mAb-1337 apparently recognizes a unique epitope that is exposed or formed by the cleavage. Rotary shadow electron microscopy of 120 kDafragments demonstrated that they have the unique molecular shape consisting of a central rod and a flexible tail, without the globular head that is present in the BP180 molecule. From these results, we conclude that mAb-1337 shows unique epitope specificity, recognizing only the 120 kDa extracellular fragment of BP180 which is constitutively cleaved on the cell surface as a 120 kDa fragment both in in ViVo and in vitro.
EXPRESSIONS OF MATRIX METALLOPROTEINASE (MMP)-9 AND INVOLUCRIN FROM KERATINOCYTES ARE SIMULTANEOUSLY INDUCED BY KERATINCCYTE DIFFERENTIATING FACTOR (KDF)-1 AFTER THE STIMULATION OF HIGH CALCIUM CONCENTRATION. Takashi KobavashL Jim Kishimoto. Ritsuko l&ma. Da Id Hudson. Robert F Buraeson, Cutaneous Bioloqv Research Cent&, Massachusetts General Hospital I Harvard, Boston,%A Since matrix metalloproteinase (MMP)-9 has been suggested to play many important roles in the metabolism of e&cellular mat&we studied tile expression of MMP-9 from cultured human keratinocytes (KCs). Using zymography of conditioned cutture media, the secretion of MMP-9 from KCs was shown to be induced by high calcium concentration [Ca++]and by the addition of TGF-beta. both of which are known to cause apoptosis of KCs. In immunofluorescenl studies, we observed that MMP-9 and involucrin are localized in the same KCs after increasing the [Ca*]. Within the promotor region of human MMP-9 gene, we found a sequence of 10 base pairs highly homologous to KRE-4, which is present within the promotor region of the human involucrin gene and is reported to bind the keratinocyte differentiation factor (KDF)-1. Gel shift assays showed that this unique sequence in MMP-9 gene reacted with the nuclear extract of differentiated cultured KCs in [Ca*‘] In the same manner as a KRE-4 oligo did. We confirmed that the promotor constructs including this sequence transfected in KCs showed higher transcriptional activities in high [Ca+*] than in low [Ca*+] by luciferase assays. In conclusion, MMP-9 from KCs might play an important role in KC differentiation including apoptosis.
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FUNCTIONAL ANALYSIS OF LAMININ-5 DEMONSTRATES THE PIVOTAL ROLE OF THE 440kDa EXTRACELLULAR FORM OF THE PROTEIN IN CELL ADHESlON.Laurent Gaenoux, Marvline Alleera, Flavia SoidtQ, Jo& Vailly, lean_ P-c, and Guerrino Meneeu& INSERM U385, University of Nice. France. Laminin-S is the main epithelial adhesion ligand which is involved in cell mxgration, wound healing. tumoral invaston and morphogeucsis. Iu the extracellular matnx (ECM) deposited by keratinocytes, this lamiuin a3g3$ heterotrimer is detected as a 440 kDa protein and a more abundant 400 kDa form resulting from the proteolytical cleavage of the 72 chain (155 kDa) into a polypeptide (105 kDa) with a truncated N-terminal domain. To analyze the role of the two extracellu,ar forms of larmnin-5, we have transfected human LSVS keratinocytes bearing a genetic mutation that abolishes synthesis of the lamiuin Q chain with eukqotic vectors expressing mutated lamiuin 72 cDNAs. Keratinocytes LSVS-NC and LSV5.C were obtained that restore the assembly of recombinant laminin-5 molecules with either the mtact 155 kDa or the truncated 105 kDaq2 chain, respechvely. Analysis of monolayer and organotypic cultures of LSV5NC and LSVS-C cells showed that: -i) Integrity of the putative cleavage srte of the laminin $?. chain by protease BMPI 1s required for the extra~ellular processing of laminin-5; -ii) In the early phase of cell adhesion, the two extracellular forms of lamiuin5 interact with lutegrin a3pl; -ii]) Only the 440 kDa fonu of lamiuin-5 incorporates the ECM. where it acts as au adhesion ligand: -iv) Iu contrast to what previously suggested by others, the stabilization of laminin-5 in the ECM is not mediated by fibulin-2. Iu addition, trausfection of uomxxl and laminin p- keratinocytes using a eukaryotic vector expressing the short arm of lamiuiu p chain demonstrated that the N-terminal domain of the polypeptlde undergoes polarized secretion and incorporation into the ECM. Our results show that Integrity of the short arm of the p chain is essential to the polarized secretion and integration of laminin-5 m the ECM, where the protein is proteolitically cleaved. They also reveal that the unprocessed 440 kDa form of laminin-5 plays au active role in cell attachment.
A RAPID ACTIVATION OF SRC TYROSINE KINASE AND ITS BINDING TO MICROTUBULES AND NUCLEAR ENVELOPE IN CELLS AT CUT EDGES AS A CULTURED WOUND MODEL. Takah~roYam&. M. Koii Owada*,Yasuo tira!ima . H&?&i Kawakauu** Dept of Dermatol. Chfu UNV. School of Med., Glfu, *:Inst. of Mol. and Cell BIO,.for Pharmaceul. Scl., Kyoto Phammceut. Uruv., Kyoto Japan **:UCSF Lung Biology Center San Frauax”, US In human c&c, the phosphorylauon of Try419 increases m kmase actlvlty, whereas that of Try-530 decreases the kinase activity. We previously produced a mouoclonal antibody, clone 28, which IS speclhc for the active form (Try-530 nonphosphorylated). In order to study the fuucuon of c-Src m keratlnocytes, we studied the ,ntracellular d,st”buhon of ,ts acUve and mac”ve form ,n cultured human carcmoma cell hne (DJM-1) as a mode, system by immunofluorescence micr,oscopy and Immuuoblothng. usmg clone 28 and mAb327, wluch recagnzes both actn’e and uxuxctlveforms. Smce c-&c has been suggested to be mvolved in the control of celladhesum m other cells. we produced a dynamic conditions of cell-mlgrstron by woundmg the cell culture by cutting mto squares hke a mesh-pattern with a blade (culture wound model). Before cuttmg the culture, the active form of c-Src was expressed m cells located only at the penphery of colomes or tsolated uugratmg cells 2nd it was assocmted with microtubules. centnole and nucleus (especially nuclear envelopes) as revealed by unmunofluorescencc The actwe fonu of c-Src bound to mlcrotubules was readily extracted. while that bound fo nucleus and nuclear envelopes was not, by Tnton X-100 treatment. Wounding the colony generated a dramatic and rapid acLLvat,onof c-Src m a lew lows of cells along the cutting edges even a the middle of colony and tie active c-Src was assocmted wxth microtubules and nucleus 3nd this mcrease of the active form was also detectedby Immunoblottmg.These results suggest that achvatlon of C-SICmay play a role in the function of microtubules !n the cell uugrauon, especmlly generated at an early stage oi wound heahng
0147
0150 1ntmactk.n with (HA) CC.
induces
the eatracdtdar mstrIx @CM) corn maturation of human dcndritic cells (&?)
nenthysluronsn
Termeer, J. Hennies. J.M. Weiss, U. Voith, B. Mai, E. Sch(ipf and J.C. Simon
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CLEAVAGE OF BP1 80 (lYPE XVII COLLAGEN) YIELDS A 120 KDA COLLAGENOUSEXTRACELLULAR POLYPEPTIDE. Y. Hirako*, J. Usukura#, J. Uematsu*, T. Hashimotot. Y. KitaiimaP and K. Owaribe*. ‘Biosyst., Grad. Sch. of Human Informat. and #Dept. of Anat., Sch. of Med., Nagoya Univ., Nagoya. tDept. of Dermatol, Kurume Univ. Sch. of Med., Fukuoka. gDept. of Dermatol, Glfu Univ. Sch. of Med.. Gifu, Japan. One of the putative cell-matrix adhesion molecules present In the hemidesmosome (HO) is the 180 kDa bullous pemphigoid antigen (BP180). A monoclonal antibody (mAb) 1337 stained the basement membrane zone of bovine skin and recognized a 120 kDa collagenase-sensitive polypeptide in the HD fraction isolated from bovine cornea. The 120 kDa Dolypepnde was not detected in HDS of cultured cells but rather in iheculiure medium. These results Indicate a localization of the polypeptide distinct from EP180. However, the 120 kDa polypeptide was also recognized by monoclonal antibodies to the extracellular but not the cytoplasmic part of BP180, and found to react with a polyclonal antibody against the non-collagenous 16A domain of BP180. Together with other data, the 120 kDa polypeptide was ldentifled as an eXtraCellUlar fragment of i3P180. The mAb-1337 apparently recognizes a unique epitope that is exposed or formed by the cleavage. Rotary shadow electron microscopy of 120 kDafragments demonstrated that they have the unique molecular shape consisting of a central rod and a flexible tail, without the globular head that is present in the BP180 molecule. From these results, we conclude that mAb-1337 shows unique epitope specificity, recognizing only the 120 kDa extracellular fragment of BP180 which is constitutively cleaved on the cell surface as a 120 kDa fragment both in in ViVo and in vitro.
EXPRESSIONS OF MATRIX METALLOPROTEINASE (MMP)-9 AND INVOLUCRIN FROM KERATINOCYTES ARE SIMULTANEOUSLY INDUCED BY KERATINCCYTE DIFFERENTIATING FACTOR (KDF)-1 AFTER THE STIMULATION OF HIGH CALCIUM CONCENTRATION. Takashi KobavashL Jim Kishimoto. Ritsuko l&ma. Da Id Hudson. Robert F Buraeson, Cutaneous Bioloqv Research Cent&, Massachusetts General Hospital I Harvard, Boston,%A Since matrix metalloproteinase (MMP)-9 has been suggested to play many important roles in the metabolism of e&cellular mat&we studied tile expression of MMP-9 from cultured human keratinocytes (KCs). Using zymography of conditioned cutture media, the secretion of MMP-9 from KCs was shown to be induced by high calcium concentration [Ca++]and by the addition of TGF-beta. both of which are known to cause apoptosis of KCs. In immunofluorescenl studies, we observed that MMP-9 and involucrin are localized in the same KCs after increasing the [Ca*]. Within the promotor region of human MMP-9 gene, we found a sequence of 10 base pairs highly homologous to KRE-4, which is present within the promotor region of the human involucrin gene and is reported to bind the keratinocyte differentiation factor (KDF)-1. Gel shift assays showed that this unique sequence in MMP-9 gene reacted with the nuclear extract of differentiated cultured KCs in [Ca*‘] In the same manner as a KRE-4 oligo did. We confirmed that the promotor constructs including this sequence transfected in KCs showed higher transcriptional activities in high [Ca+*] than in low [Ca*+] by luciferase assays. In conclusion, MMP-9 from KCs might play an important role in KC differentiation including apoptosis.
0146
0149
FUNCTIONAL ANALYSIS OF LAMININ-5 DEMONSTRATES THE PIVOTAL ROLE OF THE 440kDa EXTRACELLULAR FORM OF THE PROTEIN IN CELL ADHESlON.Laurent Gaenoux, Marvline Alleera, Flavia SoidtQ, Jo& Vailly, lean_ P-c, and Guerrino Meneeu& INSERM U385, University of Nice. France. Laminin-S is the main epithelial adhesion ligand which is involved in cell mxgration, wound healing. tumoral invaston and morphogeucsis. Iu the extracellular matnx (ECM) deposited by keratinocytes, this lamiuin a3g3$ heterotrimer is detected as a 440 kDa protein and a more abundant 400 kDa form resulting from the proteolytical cleavage of the 72 chain (155 kDa) into a polypeptide (105 kDa) with a truncated N-terminal domain. To analyze the role of the two extracellu,ar forms of larmnin-5, we have transfected human LSVS keratinocytes bearing a genetic mutation that abolishes synthesis of the lamiuin Q chain with eukqotic vectors expressing mutated lamiuin 72 cDNAs. Keratinocytes LSVS-NC and LSV5.C were obtained that restore the assembly of recombinant laminin-5 molecules with either the mtact 155 kDa or the truncated 105 kDaq2 chain, respechvely. Analysis of monolayer and organotypic cultures of LSV5NC and LSVS-C cells showed that: -i) Integrity of the putative cleavage srte of the laminin $?. chain by protease BMPI 1s required for the extra~ellular processing of laminin-5; -ii) In the early phase of cell adhesion, the two extracellular forms of lamiuin5 interact with lutegrin a3pl; -ii]) Only the 440 kDa fonu of lamiuin-5 incorporates the ECM. where it acts as au adhesion ligand: -iv) Iu contrast to what previously suggested by others, the stabilization of laminin-5 in the ECM is not mediated by fibulin-2. Iu addition, trausfection of uomxxl and laminin p- keratinocytes using a eukaryotic vector expressing the short arm of lamiuiu p chain demonstrated that the N-terminal domain of the polypeptlde undergoes polarized secretion and incorporation into the ECM. Our results show that Integrity of the short arm of the p chain is essential to the polarized secretion and integration of laminin-5 m the ECM, where the protein is proteolitically cleaved. They also reveal that the unprocessed 440 kDa form of laminin-5 plays au active role in cell attachment.
A RAPID ACTIVATION OF SRC TYROSINE KINASE AND ITS BINDING TO MICROTUBULES AND NUCLEAR ENVELOPE IN CELLS AT CUT EDGES AS A CULTURED WOUND MODEL. Takah~roYam&. M. Koii Owada*,Yasuo tira!ima . H&?&i Kawakauu** Dept of Dermatol. Chfu UNV. School of Med., Glfu, *:Inst. of Mol. and Cell BIO,.for Pharmaceul. Scl., Kyoto Phammceut. Uruv., Kyoto Japan **:UCSF Lung Biology Center San Frauax”, US In human c&c, the phosphorylauon of Try419 increases m kmase actlvlty, whereas that of Try-530 decreases the kinase activity. We previously produced a mouoclonal antibody, clone 28, which IS speclhc for the active form (Try-530 nonphosphorylated). In order to study the fuucuon of c-Src m keratlnocytes, we studied the ,ntracellular d,st”buhon of ,ts acUve and mac”ve form ,n cultured human carcmoma cell hne (DJM-1) as a mode, system by immunofluorescence micr,oscopy and Immuuoblothng. usmg clone 28 and mAb327, wluch recagnzes both actn’e and uxuxctlveforms. Smce c-&c has been suggested to be mvolved in the control of celladhesum m other cells. we produced a dynamic conditions of cell-mlgrstron by woundmg the cell culture by cutting mto squares hke a mesh-pattern with a blade (culture wound model). Before cuttmg the culture, the active form of c-Src was expressed m cells located only at the penphery of colomes or tsolated uugratmg cells 2nd it was assocmted with microtubules. centnole and nucleus (especially nuclear envelopes) as revealed by unmunofluorescencc The actwe fonu of c-Src bound to mlcrotubules was readily extracted. while that bound fo nucleus and nuclear envelopes was not, by Tnton X-100 treatment. Wounding the colony generated a dramatic and rapid acLLvat,onof c-Src m a lew lows of cells along the cutting edges even a the middle of colony and tie active c-Src was assocmted wxth microtubules and nucleus 3nd this mcrease of the active form was also detectedby Immunoblottmg.These results suggest that achvatlon of C-SICmay play a role in the function of microtubules !n the cell uugrauon, especmlly generated at an early stage oi wound heahng
0147
0150 1ntmactk.n with (HA) CC.
induces
the eatracdtdar mstrIx @CM) corn maturation of human dcndritic cells (&?)
nenthysluronsn
Termeer, J. Hennies. J.M. Weiss, U. Voith, B. Mai, E. Sch(ipf and J.C. Simon