- Email: [email protected]
Hyaluronan increases the level of activation protein-1 (AP-1) in cultured epidermal keratinocytes
ESDR / JSID I SID Abstracts
0397 BRAIN-DERIVED NEUROTROPHIC FACTOR, NEUROTROPHIN-3, AND NEUROTROPHlN-4 STIMULATE MURINE EPIDERMAL KERATINOCYTE PROLIFERATION IN SITU N.V.Botchkareva’ .V.A.Botchkarev’d;d. M.Lommatzsch2&H.RenZ..Paus’. ‘Department of Dermatology, Charit&, ‘Institute of Clinical Chemistry, Charit&Virchow Hospital, Humboldt-Universitl LU Berlin, Berlin, Gellllrlny Nerve growth factor (NGF) is produced by keratinocyter (KCs) and modulates their proliferation and apoptosis. However, it is as yet unknown whether other members of the NGF family of neurotrophins, brain-derived neurotmphic factor (BDNF), neurotrophin-3 (NT-3) and newotrophin-4 (NT-4) also modulate KC proliferation in situ. We determined by ELISA that, in C57BU6 mouse skin NT-3 protein levels were-substantially higher than those of BDNF. By immunofluorescence, NT-3-immunoreactivity (IR) was strongly expressed by the subcutaneous panniculus carnosos muscle and by tbe arreetor pili muscle of CS7BL/6 skin, while BDNF-IR was foundonly in skin nerve bundles, NT-CIR was noted in single epidermal KCs. TrkB, tbe high affinity receptor for BDNF and NT-4, was detected in basal epidennal KCs, while the high affinity NT-3 receptor, TrkC, was expressed by skin nerve bundles. Compared to the corresponding age-matched wild type littennates, BDNF or NT-3 overexpressing transgenic mice showed a significantly enhanced number of Ki 67+ epidemtal KCs in viva, while the ounber of Ki 67+ cells was substantially reduced in BDNF knockout mice. In skin organ adtore of C57BV6 mice, BDNF, NT-3, or NT-4 all significantly increased BrdU-incorporation into epidermal KCs. Thus normal murine epidennal KCs in siti, some of which may even serve a source for NT-4, are target cells for BDNF, NT-3 and NT-4 stimulation. This strongly suggests that neurotrophins also act as “epdbehotrophins”, end are intimately involved in the control of epidermal homeostasis.
0400 s"PsRANTrGBN(SAG)~ACTIVETCELLSI,~D"~ESIGNIPICAN KERATINOCYTES (KC) WHEN SUPERANTlGEN IS CONTINUOUSLY PRESENT. Tm Sm!tb & Brian J NekolotT Departmentof Pathology,Loyola University, Maywood, 1L Bacterial SAGs are potent inducersofT cell aetivatlonand trigger autoimmune direeses,but bow SAGs contribute to autoimmonay is not clear. One mechanismpostulatesSAG-directedcellmediatedcytotoxicity in which SAG-activatedT cell kill MHC class II-expressingtarget cells. An mvitro assaywas developedto determine if T cells induce KC cytoparbiceffeca (CPE) in co-presenceof SAGs Human KCr were grown to 70% confluency. IO6blood-dewed lymphocyteswere overlaid on KCa with or without SAGr (SESISEC2at and observedfor KC CPE. No CPE was observedwhen freshly isolatedlymphocyteswere co-cultured wth KCs in presenceof SAGs during a 24-48 hour incubation However, if lymphocyteswere pre-activatedby SAG (72 hrs), and then co-culturedwxb KCs in continuouspresenceof SAGs, s~gniticamCPE ofKCs occurredin lessthan 18 boors. CPE metuded significant detachmentof KCs from the plate. Maximal KC CPE effect was dependentupon presenceof SAG%;reromulated SAG-reactivelymphocytescocotturedwith KCr without SAGs had decreasedet&t. Direct eonmetwith activatedlymphocyteswas necessaryfor CPE as separating lymphocytesand KCS using ~~SWCIISpreventedinduction of KC detachment However,vacuoleformation of KCs still occurredsuggestmgCPE was in part mediatedby soluble mediators Pre-treating KCs (IFN-7: ,013 UnmIml: 24 hrs) to !mduceHf..&DR produced increasedCPE when T cells and SAGEwere subsequently added Lymphocytesderived from normal controlr (n=3) or psoriaticpatients(n=3) producedsimilar effects. In addition, SAG-reactiveCD4+ T cell lines derived from psonaticpacnts also mediated signdicant KC CPE in presenceof SAGs. Neutralizing Abr to LFA-1, CD95, IFN-y, or TNFa drd not havedramatic cffeeu when usedalone; however,eombinatmn of theseAbs signifieantty inhlbad the detachmentof KCs. In concIuston,SAG-reactwe,but not resting blwd-dewed lymphocytesmediate suggestactivatedT cells cxposedte prominent CPE of KCs m continuoospresenceof SAGs These~~suI~E bacterial SAGEet mflammatory sitesin skin may not only enhanceKC ectivation,but also induce KC CPE. Such KC reactionsmay releasenormally sequesteredselfproteins that can stimulate autoreaCtiveT cells. In this way, pathogenicT cells initially activatedby SAGr may be secondarilyre-somutatedleading to expansionof autoreactiveT cells
I pghl)
0398
0401
INDUCTION OF SKALPiELAFlN GENE EXPRESSION IN CULTURED HUMAN KERATINOCYTES BY INFLAMMATORY CYTOKINES. B Patrick i Department of Dermatology, Un~verslty Hospital Nijmegen. N#negen, The Netherlands Keratinccytes of inflamed epidermis (psoriasis. wound healing) are hyperpmliferative and display an abnormal differentiation program. This regenerative differentiation pathway is characterlsed by the Induction of a set of genes that are not expressed by keratinocytes in normal skin, such as the cytokeratins CK6 and CKIS, and the pmtelnase InhIbItor SUALPlelafin Here we have studied the induction and regulation of SKALPlelafin gene expression in epldermal keratinacytes as a paradigm for the regeneratwe ddferentlailon program. We have constructed deletion mtiants of the SKALPlelafin promoter region that conferred keratinocyte-specific expression upon tramlent transfeCtion Deletion of a region that contaned two potential AP-1 binding sites and several potential NF-ILS bmdlng s+s.s resulted I” a 90% reduction of promoter activity. Usmg SKALPlelafin evprewon at the mRNA and protein level as a readout, various cytokines and growth factors. that are found ,n elevated levels psoriatic lesions and healing skin wounds, were tested in an in vitro model on thetr abikty to Induce SKALPlelafin gene expresslo” in cultured human kerahncqtes Tumour Necrosis Factor-alpha (TNF-a) was by far the most potent inducer of SKALPlelafin gene expression; Transfo”“mg Growth Factor-alpha (TGF-a) and Interferon-gamma (IFN-.,) gave a moderate !nductlon of SKALPlelafin gene expression. InductIon of SKALPlelafin expresslo” by TNF-a was preceded by actwation of the mltogen activated protem (MAP) klnase family member c-fun N-terminal kmase 1 (JNK-I) On the basis of our 8” vdro rindlngs, we speculate that Induction of SKALPlelafin expresston m wo IS part of an epldermal stress response pathway, that leads to the regenerative differentiation program. which IS characteristic for psoriatic ep!derm!s and healing skm wounds.
STRATUM CORNEUM GELATINASE; A NOVEL LATE DIFFERENTIATION, EPIDERMAL cell Biology & Physiolo~ Unit, Unilevcr Resexcb, CYSTEME PROTBASE. mq Celwmtb Bse, Bedfor& U.K. Biochemical pnrsrcs involved in Nahuo comeam foneatioo and maturaion utilize several proteotytic enzymes to mcdixj a wide range ofstrwmmt proteins. During studies to investigate rbe physlologicaUy impaiant Smtum conrum pmteases.a novel gelatbmlytic proteasewas idendlial, which was named Svahlm Comeem (SC) geJa”nase. The emyme was detected using getado zymograpby, the proteasebeing present ap a de@ band of seratin0lysi.swith apparent omteatar we~gbtaof 33 and 34 kDa. No a&by was d&e&d when wein or baa&oh were wed a~ subswatee SC gelatina% denmmtrated acid prete% anrvity with a pB optimum of 54 and mild Inhibitor tiea sbowd redwing coeditians (JmM cysteine or D?T) wre an absolote requiremeot. that SC gelatinaxe was a cystebx protea& being totally inhibited by lOoM E64 and lOoM Z-phearg-dia.zom&me; similar a&ity ofthe 33 and 34 kDa band was seen in all experiments indicating with antisera raised against tkat they were variants of the same enzyme. Pultber c-cm bornan catheosin B. B or I,. all wevioesly idatified witbin fhe erridermis. demoostrated the tmwl CallIre of & enzyme. Brat?SC-g&tin& bands WM gfycuprc&s, rev&d by coA aftimty chromatography. Fwtbemmre, removal of the otigmacobartde8with N-flycarrax am6rmed the ningk identity of the enzyme by prcdwiog an naive core pmteie cd apparent molecular weight of 32 kDa. Pretiminnrv imwtieations to identifv the ohwioltigl timction of this emvme demoostmtcd that it xv& untforn& dtstrtbuted withdeptb & && SC. sod p-t in a9 SC ;ites of the body. altbougb it wad viriuatly absent in patmo-ptantar SC. SC gelatinax was dewted in It is calcium-induced diEerentited cultwed keratinocytes and was secreted into the medium hypoulaizerl that SC gelat&% plays a mle in rixemodihcatiionof the epidermet exvaccllti mati during coticaton and/or strafllm comeon, maturation. In concluaioo: (I) eaacts of human stratum comeom mntaio a novel gelatieotyitc protease. (2) the enzyme, SC getatinase,is an acidic qsretne proteasepresentas tw gtywytated bands of 34 and 35 kDa and as a core protein of 32 kDa: (3) SC gelatinate is predd and secretedby dt&reotiated keradnocytesand is locatedin all examined SC 61tesexceptfor patm~plantru stratum eomeom.
0399
0402
AF’-2y, THE RELEVANT AP-2 ISOFORM FOR SKIN DIFFERENTIATION7 && Buehner I. Wilhelm St& ’ and Reinhard Buettner I. Department of Pathology (‘I and University of Regensburg, Germany Department ofDermatology Transcription factor AP-2 was shown to be an important factor m morphogenesis and differentiation ofthe skin Three dd%erent murine and human isofortns encoded bv
HYALURONAN INCREASES THE LEVELOF ACTIVATION PROTEIN-I ,AP-1, IN CULTURED ~mi’. EPIDERMAL KERATINOCYTES. !QdI&@wn. ,QI Kaamiranta.RailaTamo Lammi_ Juba-Pekb Pttnimeiki and Markku Tm Department of Anatomy, University of Kuopio, Kuopio. Finland. Hyaluronan (HA) has been considered a passive extracellular m&ix (ECM) component, but recent studies have shown its importance in mntmlling many cell functions including motlkty proliferation and adhesion. which imply signaling from ECM to cytosot. Keratmocytes express at least two cell surface receptors for HA: CL& and the receptor for hyalumnic acid mediated motility (RHAMM). Acbvatton protein-l (AP-1) is a common transcrtptmn mfactor that we found actwated by exogenous HA in keratinocytes. In the present study we oharacterlzed the role of call surface HA-receptors in AP-1 activation. Epidermal keratincqies (HaCat) war@treated with intact longcham HA, HA-oligosaccharides and other glyvasaminoglycans. Cells were harvested, nuclear proteins isolated and AP-1 level determined with gel mobility shit assay. CD44. and RHAMM-antibodies and HA+ligomers were used to block the HA-binding site in the receptors Hyaluronan imreased the amount of AP-I rapidly. with maximum effect !n two hours. Intact HA concentrations down tot n@ml gave a detectable increase. Hyaluronan oligomers HA4 and HAS Induced AP-1, like intaot HA, white oligomers HAS and HA10 reduced AP-t below control level. A CD44 antlbodv sliahtlv increased AP-1 level and did not influence the resoonse to HA. RHAMM andibodies reduced AP-1 activity and pwented the increase by H’A. Other glycasaminoglycans did not affect AP-1 level. AP-1 was not activated bv HA in chondrowtes or f,broblasts. In this study w showed that exogenous HA increases AP-1 level in keratinocytes. This Induction seems spsciflc to keratinocytes and involves RHAMM-receptor in sIgnal tmnsductmn and is blocked by HAS-10 ollgomers. Interactions between CD44 and RHAMM in cell surface HA management may explain the AP-I 1ncmas6 by CD44 antibodies and HA4-6 oligomen.
“’,
three different genes have been isolated:
AI’-2~.
AP-2R and AP-2~. However,
neither
the AF-2a nor the AP-28 knockout mice showed any detectable skin phenotype. In this study we could reveal by immunohistochemical stainings and RT-PCR that AF’-2 is highly expressed in basal layers of normal skin as well as in basal cell carcinomas and that all three known AF-2 isoforms can be detected in both tissues. We further could demonstrate by lunferase reporter analysis that the three Ap-2 isoforms showed significant differences m their activation potentials for the basal keratin promoter K14 when transfected into the keratinocyte cell line HaCaT Cotransfection of K14 promoter together wth plasmid pCMX-AP-2a lead to a weak additional increase in luciferase expression whereas cotransfected plasmid p&f?AP-2l! did not iniluence the activity of K14 promoter In contrast K14 promoter was highly acttvated after cantransfection of pCMX-AP-2y. Mutation ofthe putative AP-2 bmding rite of the K14 promoter resulted in a significant decrease in luciferax expression In summary, we here show that all three AP-2 isofomu are present in nomad skin and BCCs and that AP-2y is the most potential stimulator of K14 promoter activity These results may suggest that this isoform may be the most relevant activator of keratinocvte specific gene expression.
. _.
0397 BRAIN-DERIVED NEUROTROPHIC FACTOR, NEUROTROPHIN-3, AND NEUROTROPHlN-4 STIMULATE MURINE EPIDERMAL KERATINOCYTE PROLIFERATION IN SITU N.V.Botchkareva’ .V.A.Botchkarev’d;d. M.Lommatzsch2&H.RenZ..Paus’. ‘Department of Dermatology, Charit&, ‘Institute of Clinical Chemistry, Charit&Virchow Hospital, Humboldt-Universitl LU Berlin, Berlin, Gellllrlny Nerve growth factor (NGF) is produced by keratinocyter (KCs) and modulates their proliferation and apoptosis. However, it is as yet unknown whether other members of the NGF family of neurotrophins, brain-derived neurotmphic factor (BDNF), neurotrophin-3 (NT-3) and newotrophin-4 (NT-4) also modulate KC proliferation in situ. We determined by ELISA that, in C57BU6 mouse skin NT-3 protein levels were-substantially higher than those of BDNF. By immunofluorescence, NT-3-immunoreactivity (IR) was strongly expressed by the subcutaneous panniculus carnosos muscle and by tbe arreetor pili muscle of CS7BL/6 skin, while BDNF-IR was foundonly in skin nerve bundles, NT-CIR was noted in single epidermal KCs. TrkB, tbe high affinity receptor for BDNF and NT-4, was detected in basal epidennal KCs, while the high affinity NT-3 receptor, TrkC, was expressed by skin nerve bundles. Compared to the corresponding age-matched wild type littennates, BDNF or NT-3 overexpressing transgenic mice showed a significantly enhanced number of Ki 67+ epidemtal KCs in viva, while the ounber of Ki 67+ cells was substantially reduced in BDNF knockout mice. In skin organ adtore of C57BV6 mice, BDNF, NT-3, or NT-4 all significantly increased BrdU-incorporation into epidermal KCs. Thus normal murine epidennal KCs in siti, some of which may even serve a source for NT-4, are target cells for BDNF, NT-3 and NT-4 stimulation. This strongly suggests that neurotrophins also act as “epdbehotrophins”, end are intimately involved in the control of epidermal homeostasis.
0400 s"PsRANTrGBN(SAG)~ACTIVETCELLSI,~D"~ESIGNIPICAN KERATINOCYTES (KC) WHEN SUPERANTlGEN IS CONTINUOUSLY PRESENT. Tm Sm!tb & Brian J NekolotT Departmentof Pathology,Loyola University, Maywood, 1L Bacterial SAGs are potent inducersofT cell aetivatlonand trigger autoimmune direeses,but bow SAGs contribute to autoimmonay is not clear. One mechanismpostulatesSAG-directedcellmediatedcytotoxicity in which SAG-activatedT cell kill MHC class II-expressingtarget cells. An mvitro assaywas developedto determine if T cells induce KC cytoparbiceffeca (CPE) in co-presenceof SAGs Human KCr were grown to 70% confluency. IO6blood-dewed lymphocyteswere overlaid on KCa with or without SAGr (SESISEC2at and observedfor KC CPE. No CPE was observedwhen freshly isolatedlymphocyteswere co-cultured wth KCs in presenceof SAGs during a 24-48 hour incubation However, if lymphocyteswere pre-activatedby SAG (72 hrs), and then co-culturedwxb KCs in continuouspresenceof SAGs, s~gniticamCPE ofKCs occurredin lessthan 18 boors. CPE metuded significant detachmentof KCs from the plate. Maximal KC CPE effect was dependentupon presenceof SAG%;reromulated SAG-reactivelymphocytescocotturedwith KCr without SAGs had decreasedet&t. Direct eonmetwith activatedlymphocyteswas necessaryfor CPE as separating lymphocytesand KCS using ~~SWCIISpreventedinduction of KC detachment However,vacuoleformation of KCs still occurredsuggestmgCPE was in part mediatedby soluble mediators Pre-treating KCs (IFN-7: ,013 UnmIml: 24 hrs) to !mduceHf..&DR produced increasedCPE when T cells and SAGEwere subsequently added Lymphocytesderived from normal controlr (n=3) or psoriaticpatients(n=3) producedsimilar effects. In addition, SAG-reactiveCD4+ T cell lines derived from psonaticpacnts also mediated signdicant KC CPE in presenceof SAGs. Neutralizing Abr to LFA-1, CD95, IFN-y, or TNFa drd not havedramatic cffeeu when usedalone; however,eombinatmn of theseAbs signifieantty inhlbad the detachmentof KCs. In concIuston,SAG-reactwe,but not resting blwd-dewed lymphocytesmediate suggestactivatedT cells cxposedte prominent CPE of KCs m continuoospresenceof SAGs These~~suI~E bacterial SAGEet mflammatory sitesin skin may not only enhanceKC ectivation,but also induce KC CPE. Such KC reactionsmay releasenormally sequesteredselfproteins that can stimulate autoreaCtiveT cells. In this way, pathogenicT cells initially activatedby SAGr may be secondarilyre-somutatedleading to expansionof autoreactiveT cells
I pghl)
0398
0401
INDUCTION OF SKALPiELAFlN GENE EXPRESSION IN CULTURED HUMAN KERATINOCYTES BY INFLAMMATORY CYTOKINES. B Patrick i Department of Dermatology, Un~verslty Hospital Nijmegen. N#negen, The Netherlands Keratinccytes of inflamed epidermis (psoriasis. wound healing) are hyperpmliferative and display an abnormal differentiation program. This regenerative differentiation pathway is characterlsed by the Induction of a set of genes that are not expressed by keratinocytes in normal skin, such as the cytokeratins CK6 and CKIS, and the pmtelnase InhIbItor SUALPlelafin Here we have studied the induction and regulation of SKALPlelafin gene expression in epldermal keratinacytes as a paradigm for the regeneratwe ddferentlailon program. We have constructed deletion mtiants of the SKALPlelafin promoter region that conferred keratinocyte-specific expression upon tramlent transfeCtion Deletion of a region that contaned two potential AP-1 binding sites and several potential NF-ILS bmdlng s+s.s resulted I” a 90% reduction of promoter activity. Usmg SKALPlelafin evprewon at the mRNA and protein level as a readout, various cytokines and growth factors. that are found ,n elevated levels psoriatic lesions and healing skin wounds, were tested in an in vitro model on thetr abikty to Induce SKALPlelafin gene expresslo” in cultured human kerahncqtes Tumour Necrosis Factor-alpha (TNF-a) was by far the most potent inducer of SKALPlelafin gene expression; Transfo”“mg Growth Factor-alpha (TGF-a) and Interferon-gamma (IFN-.,) gave a moderate !nductlon of SKALPlelafin gene expression. InductIon of SKALPlelafin expresslo” by TNF-a was preceded by actwation of the mltogen activated protem (MAP) klnase family member c-fun N-terminal kmase 1 (JNK-I) On the basis of our 8” vdro rindlngs, we speculate that Induction of SKALPlelafin expresston m wo IS part of an epldermal stress response pathway, that leads to the regenerative differentiation program. which IS characteristic for psoriatic ep!derm!s and healing skm wounds.
STRATUM CORNEUM GELATINASE; A NOVEL LATE DIFFERENTIATION, EPIDERMAL cell Biology & Physiolo~ Unit, Unilevcr Resexcb, CYSTEME PROTBASE. mq Celwmtb Bse, Bedfor& U.K. Biochemical pnrsrcs involved in Nahuo comeam foneatioo and maturaion utilize several proteotytic enzymes to mcdixj a wide range ofstrwmmt proteins. During studies to investigate rbe physlologicaUy impaiant Smtum conrum pmteases.a novel gelatbmlytic proteasewas idendlial, which was named Svahlm Comeem (SC) geJa”nase. The emyme was detected using getado zymograpby, the proteasebeing present ap a de@ band of seratin0lysi.swith apparent omteatar we~gbtaof 33 and 34 kDa. No a&by was d&e&d when wein or baa&oh were wed a~ subswatee SC gelatina% denmmtrated acid prete% anrvity with a pB optimum of 54 and mild Inhibitor tiea sbowd redwing coeditians (JmM cysteine or D?T) wre an absolote requiremeot. that SC gelatinaxe was a cystebx protea& being totally inhibited by lOoM E64 and lOoM Z-phearg-dia.zom&me; similar a&ity ofthe 33 and 34 kDa band was seen in all experiments indicating with antisera raised against tkat they were variants of the same enzyme. Pultber c-cm bornan catheosin B. B or I,. all wevioesly idatified witbin fhe erridermis. demoostrated the tmwl CallIre of & enzyme. Brat?SC-g&tin& bands WM gfycuprc&s, rev&d by coA aftimty chromatography. Fwtbemmre, removal of the otigmacobartde8with N-flycarrax am6rmed the ningk identity of the enzyme by prcdwiog an naive core pmteie cd apparent molecular weight of 32 kDa. Pretiminnrv imwtieations to identifv the ohwioltigl timction of this emvme demoostmtcd that it xv& untforn& dtstrtbuted withdeptb & && SC. sod p-t in a9 SC ;ites of the body. altbougb it wad viriuatly absent in patmo-ptantar SC. SC gelatinax was dewted in It is calcium-induced diEerentited cultwed keratinocytes and was secreted into the medium hypoulaizerl that SC gelat&% plays a mle in rixemodihcatiionof the epidermet exvaccllti mati during coticaton and/or strafllm comeon, maturation. In concluaioo: (I) eaacts of human stratum comeom mntaio a novel gelatieotyitc protease. (2) the enzyme, SC getatinase,is an acidic qsretne proteasepresentas tw gtywytated bands of 34 and 35 kDa and as a core protein of 32 kDa: (3) SC gelatinate is predd and secretedby dt&reotiated keradnocytesand is locatedin all examined SC 61tesexceptfor patm~plantru stratum eomeom.
0399
0402
AF’-2y, THE RELEVANT AP-2 ISOFORM FOR SKIN DIFFERENTIATION7 && Buehner I. Wilhelm St& ’ and Reinhard Buettner I. Department of Pathology (‘I and University of Regensburg, Germany Department ofDermatology Transcription factor AP-2 was shown to be an important factor m morphogenesis and differentiation ofthe skin Three dd%erent murine and human isofortns encoded bv
HYALURONAN INCREASES THE LEVELOF ACTIVATION PROTEIN-I ,AP-1, IN CULTURED ~mi’. EPIDERMAL KERATINOCYTES. !QdI&@wn. ,QI Kaamiranta.RailaTamo Lammi_ Juba-Pekb Pttnimeiki and Markku Tm Department of Anatomy, University of Kuopio, Kuopio. Finland. Hyaluronan (HA) has been considered a passive extracellular m&ix (ECM) component, but recent studies have shown its importance in mntmlling many cell functions including motlkty proliferation and adhesion. which imply signaling from ECM to cytosot. Keratmocytes express at least two cell surface receptors for HA: CL& and the receptor for hyalumnic acid mediated motility (RHAMM). Acbvatton protein-l (AP-1) is a common transcrtptmn mfactor that we found actwated by exogenous HA in keratinocytes. In the present study we oharacterlzed the role of call surface HA-receptors in AP-1 activation. Epidermal keratincqies (HaCat) war@treated with intact longcham HA, HA-oligosaccharides and other glyvasaminoglycans. Cells were harvested, nuclear proteins isolated and AP-1 level determined with gel mobility shit assay. CD44. and RHAMM-antibodies and HA+ligomers were used to block the HA-binding site in the receptors Hyaluronan imreased the amount of AP-I rapidly. with maximum effect !n two hours. Intact HA concentrations down tot n@ml gave a detectable increase. Hyaluronan oligomers HA4 and HAS Induced AP-1, like intaot HA, white oligomers HAS and HA10 reduced AP-t below control level. A CD44 antlbodv sliahtlv increased AP-1 level and did not influence the resoonse to HA. RHAMM andibodies reduced AP-1 activity and pwented the increase by H’A. Other glycasaminoglycans did not affect AP-1 level. AP-1 was not activated bv HA in chondrowtes or f,broblasts. In this study w showed that exogenous HA increases AP-1 level in keratinocytes. This Induction seems spsciflc to keratinocytes and involves RHAMM-receptor in sIgnal tmnsductmn and is blocked by HAS-10 ollgomers. Interactions between CD44 and RHAMM in cell surface HA management may explain the AP-I 1ncmas6 by CD44 antibodies and HA4-6 oligomen.
“’,
three different genes have been isolated:
AI’-2~.
AP-2R and AP-2~. However,
neither
the AF-2a nor the AP-28 knockout mice showed any detectable skin phenotype. In this study we could reveal by immunohistochemical stainings and RT-PCR that AF’-2 is highly expressed in basal layers of normal skin as well as in basal cell carcinomas and that all three known AF-2 isoforms can be detected in both tissues. We further could demonstrate by lunferase reporter analysis that the three Ap-2 isoforms showed significant differences m their activation potentials for the basal keratin promoter K14 when transfected into the keratinocyte cell line HaCaT Cotransfection of K14 promoter together wth plasmid pCMX-AP-2a lead to a weak additional increase in luciferase expression whereas cotransfected plasmid p&f?AP-2l! did not iniluence the activity of K14 promoter In contrast K14 promoter was highly acttvated after cantransfection of pCMX-AP-2y. Mutation ofthe putative AP-2 bmding rite of the K14 promoter resulted in a significant decrease in luciferax expression In summary, we here show that all three AP-2 isofomu are present in nomad skin and BCCs and that AP-2y is the most potential stimulator of K14 promoter activity These results may suggest that this isoform may be the most relevant activator of keratinocvte specific gene expression.
. _.