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patched signalling pathway in basal cell carcinomas is associated with expression of the transcription factor GLI-1
ESDR I JSID I SID Abstracts
0133
0136
REPRESSION OF CELLULAR GENES BY UVB RADIATION: A NEW MECHANISM IN THE UVB RESPONSE OF HUMAN KERATINOCYTES? w F. Abts T&na.8 Weiss. GClnler Michel. Depl. of Dermatology and Biologisch Meclizinisches Forschungszentrum (BMFZ) He&h-Heine-University, Di%seldorf, Genany The midrange ultraviolet radiation (UVB) turned out to be the most relevant physical injury to human skin. In order to elucidate the biological consequence of solar irradiation it is therefore important to uncover the molecular events in epidermal keratinocytes after UVEirradiation (UVR). To tdentify and done mediators and targets of the LIVE response in systematic wey. we trace the changes in steady-state mRNA levels in keratinocytes after irradiation by mRNA differential display PCR (DD-PCR). Due to the potential of the DD-PCR to detect also UVB-repressed genes. we were able to analyze this scarcely investigated effect of the UVB radiation on gene expression. Appmximately one third of all diirenbally expressed genes turned out to be down-regulated during 24h after UVR. The identification and analysis of these new UVB-repressed genes offers the opportunity to idenbfy unrecognized molecular mechanisms in the UV response of human cells. For one of these UV-repressed genes full-length cDNA clones have been isolated. They encode a potential protein with a predicted molecular maes of -44 kDa. This novel protein displayed all of the structural features unique to the ovalbumin family of the serine pmteinase inhibitors (serpins). Expression characteristics of this new serpin indicates functions during proliferation/differentiation and possibly apoptosis in human keratinocytee. Further analysis of thts new serpin at the protein level will now help to elucidate the physiological function and its mode of action during the UVB response of keratinocytes.
ALPHA-MELANOCYTE-STIMULATING HORMONE REDUCES ENDOTOXININDUCED ACTIVATION OF NUCLEAR FACTOR-KBIN ENDOTHELIAL CELLS. D.-H. Kalden. M Fastrich T. &Q&& Scholzen M. Iiarhnever. T. SC war and T A. Luger. Ludwig Boltzmenn Cellbiology end Innnu”obiolor$ of”he Ski”, Department ofDermatology, Univemity of Muenster, Muenster, Germany The neuropeptide a-Melanocyte-stimulating hormone (a-MSH) is a potent antiin&nmnatoty and antipyretic agent. To identify a potential mechanism of the antiinflammatory action of a-M%% we tested its e&&s on regulation of adhesion molecules Since many adhesion molecules, including vascular cell adhesion molecule 1 (VCAM-1) and E-s&c&, contain regulatory NF-XB sites in their promoter regions, we also investigated the role of a-MSH in the a&ado” of the transc~ption factor NF-KB Monolayer cell cultures of transfomwd human microvascular endotheiial cells (HMEC-I) as well as dennel ndcrovascular endothelial cells (HDMEC) were treated with LPS (lWng/“d) alone and in combination with a-MSH (10‘8-10”6I@. The ~IRNA WBS isolated a&r 24h and expression of the adhesion molecules VCAM-I and E-se&tin was investigated by Northern blot enaJysis and semiquantitative RT-PCR. Nuclear extracts were prepared at early time points for EMSA using ‘zP-labeled specific oligonucleotides to detect NF-KB. The expression of VCAM and Eselectin was stronalv uoremdated a&r 24h of LPS treatment A markedly reduction ww shown in comb~&+ti~”;;ith aMSH (lo’- Iv’* M). whereas VCAhi and E-a&ctin nRNA was also induced by a-MSH ( lCi’O-lO~‘z hi) alone 30 tin after LPS stimulation the activrdio” (nuclear tmnslocation) of NF-WBwas strongly induced Silas to the adhesion molecules mRNA data, thi$ effect was reduced by catimuletion with a-MSH in a dose-dependent manna, at which lo-’ M a-MSH showed the grongest suppression. tis, a-MSH, which is released by stin”daed in&mletoly cells, may act via the moduletio” of tre”acripti0” factor NF-al suggesting that a-MSH has an et&t very early in the intlmnmatory cascade.
Institut~~f
0137
0134 lND”CTlON OFPROINFLAMMATORY
MOLECULES BY ANTHRALIN IS MEDIATED VIA REACTWE OXYGEN SPEClES AND ACTlVATlON OF NF-KB IN HUMAN KERATINOCYTES M. Podda. C Schulz V. SchmCKetih’. W.H. Boehncke. R. Milbradt, R Kauhnann. TM. Zollner Dept. of Denatology, ‘Dept of Physiology, J W Goethe-University, Frankfurt Germany Anthralin IS a widely used and highly effinent drug m the treatment of psonasts, however, ifs mode of &on 1sstill not completely understood Prevuxs studies demonstrated that anthral~n acbvates me transcrlptlon factor NF-rB in the murme epidermal cell line JB6 via the production of reactive oxygen species (ROS). This findlog 1s intnguing. since NF-rB regulates the transcription of many proinflammatory cytokines and rather an inhibition than an acbvabon would be expected to be bendcial m the treatment of psoriasis. We therefore asked fl anthrelin achvates NF-rB in the human keratmocyte cell line HaCaT and inCreaseS the expression of NF-rB regulated proinflammatory cytoklnes (IL-8 IL-8. TNFa). the adhesion molecule ICAM-I and the indwble mtrlc oxide synthase (INOS), all known to be induced !n acbve psonabc skm As assessed by electromoblllry-shict-assay anthralin (1, 2, 5. 10, 20 pM) actwates the transcription factor NF-rB in human kerat~nocytes in a dose dependent manner A concurrent dose dependent ~mcreaseof nit& production, an lndlrect measure of INOS activity. staltlng at 1 pM (,4,4t,,8 vs 5,2+0.6 pM ,n untreated ceils) with a maxmum at 10 +t (29.5&4,0 PM) 1s present in the supematent analyzed by the Griesoreection Stating et 2 pM the release of IL-8 and ICAM-I expression is also markedly mcrased, determined respectively by ELISA and Row cytometry. Interestingly. no significant change could be found for TNFa and IL-8 N-Acetyl-Lcystane (NAC) and pyrrclidmedithiocarbamate (PDTC). two stwcturelly unrelated antioxidant compaunds, were used to determine the mvotvement of reactwe oxygen spates m NF-kB activation and the downstream increased expression of INOS, ICAM-I and IL-8 Non-toxic concentrations of NAC (25 mM) or PDTC (100 PM) inhIbIted NF-rB activabon and all prewously determined pranflammatory effects of anthralin We conclude that in human keratlnocytes anthrabn not only actjvates the transcription factor NFlrB, but also induces pranRammatory NF-xB regulated proteins via the production of ROS. In the clinical practice It is well know that Our m vitro findmgs substantiate me clinical psorlasas ,,burns-away in anthralln’s bre’ observation that anthralln acts by the mductlon and not through inhlbltlon of lnflammatlon
TRANSFORMATION AND IMMORTALISATION OF SKIN KERATINOCYTES
0135
0138
a-MELANOCYTE STIMULATING HORMONE DOWNREGULATES IL-I MEDIATED TARGET GENE EXPRESSION BY AfBTlENUATlON W&JFxB waz ACTIVATION. T.Brzoska. D.-H. Kelden T.Scholzen S etz K.Bbert. and T.A.Luaer, Ludwig Boltzmann Institute of Cellbiology and Inmnmobiology of the Skin, Department of Dermatology, University of Muenster, Muenster, Gemany The Proopiomelanocortin derived hormone a-Melanocyte stimulating hormone (aMSH) not only has an important role in pigmentation but also functions aa potent inummomodulator. Although the proinflammatory cytokine IL-I is a strong inducer of a-MSH expression by epidemml cells, a-MSH downregulates systemic prointlammatory activities of IL-1 such as fever and leukocytosis. Keratinocytes upon stimulation with IL-1 readily express and secrete large amounts of chemokineq which are responsible for activation and chemotaxis of infbonmatory cells. This effect of IL1 is mediated via activation of the transcription factors NFKB and AP-1. Using the keratinocyte cell lines HaCaT and KB we investigated the effect of a-MSH on the IL10 induced activation of NFKB br EMSA 10 min r&r simultaneous stimulation of the cells with a-MSH (lOd-IO~’ M) and IL-ID (5 “g/ml) NFKB activation was markedly reduced compared to cells treated with IIz~~ only. 30 ti a&r stimulation this effect still was observed in cells costinwlated with 10” M a-MSH and IL-lp. To test the effect of the a-MSH mediated attenuation of NF& activation on the outcome of IL-Lp stimulation cells were analysed for chemokine mRNA expression (semiquantitative RT-PCR) and release (ELISA). The production of IL-S, Groa and RANTES was significantly inhibited after 48 h when elfi stimulated HaCaT cells were treated with a-MSH (106-10~‘*M). These data indicate that a-MSH, mainly via modulating the activities of transcription factors such as NFKB, functions as a potent antagonist of IL-1 mediated proinflammatory e&xts.
IS ASSOCIATED WITH CHANGES IN GENOblIC DNA METHYLATION OF HtCl BUT NOT P16 Fiona Cockerill, Patricia Purkis, Anthony G Ouinn, Centre for Cutaneous Research St Bartholomew’s & The Royal London Hospital School of Medicine & Dentisny, 2 Newark St, London Alterabons m the level and pattern of genomic DNA methylation are a common findina in many human cancers. DNA hypamethylation in specdic turnour types has been ihown to involve the same chromaome .regions th% show frequeni?oss of hetwxygosity (LOH) and recent work has shown a relationshIp between increased DNA methylation of the promoter region of a number of tomoor suppressor genes and transcnptional inactivanon of these genes. ‘Ibe role of DNA methylation in skin carcinogenesis is poorly understood. In previous work we have shown a high frequency of LOH of markers from chromosome 17~ and % in cutaneous SC& In this study ue have mvestigated the metiylation status of ‘candid& mmour suppressor genes from ihex regions in normal and transformed keratinocytes. DNA methylation was mvestigated by Southern blot analysis using methylation sensitlvc rsstriction enzymes. In normal skm, fibroblasts and primary keratinocytes we found no evidence of methylanon of the promoter region of the pl6 gene which maps to chromosome 9p. Homozygous delettons of p16 were ldentitied in 3 of 3 bead & neck (H&N) XC lines and 2 of 5 cutaneous SCC lines. Hypermethylation of pl6 occurred in I of the 3 cutaneous SCC lines heterozygous for ~16. None of the 5 immortalised skin keratmocyte lines studied showed pl6 hypermethylation. The HICl gene which maps to chromosome 17p was found to be unmethylued in normal skin, fibrohlasrs and primary keratinocytes In contract to our findings for ~16, hypermethylation of HICl was a common finding in immortahsed and transformed keratinocytes wth alterations in 3 of 4 XC lines and 3 of 3 immortalised keratinocyte hnes These fmdmgs prowde evidence of a role for DNA methylaeon m kerammcyte transformation. The frequent occurrence of HICl hypcrmethylation m immortahsed keratmocytes suggests that this gene may be impownt in human skm carcinogenesis.
OF THE HSDGEHOGIPATCHED SIGNAILING PATHWAY IN BASAL CELL CARCINOMASIS ASSOCIATED WITH FXPRESSION OF THE ACTIVATION
IM
TRANSCRIPTION FACTOR GLI-I Judith Green. L&h. AG Ouinn, Cutaneous Research, St Bartholomew’s &The Royal London School of Dentistry, 2 Newark St, London.
Centre for
Medicine &
0133
0136
REPRESSION OF CELLULAR GENES BY UVB RADIATION: A NEW MECHANISM IN THE UVB RESPONSE OF HUMAN KERATINOCYTES? w F. Abts T&na.8 Weiss. GClnler Michel. Depl. of Dermatology and Biologisch Meclizinisches Forschungszentrum (BMFZ) He&h-Heine-University, Di%seldorf, Genany The midrange ultraviolet radiation (UVB) turned out to be the most relevant physical injury to human skin. In order to elucidate the biological consequence of solar irradiation it is therefore important to uncover the molecular events in epidermal keratinocytes after UVEirradiation (UVR). To tdentify and done mediators and targets of the LIVE response in systematic wey. we trace the changes in steady-state mRNA levels in keratinocytes after irradiation by mRNA differential display PCR (DD-PCR). Due to the potential of the DD-PCR to detect also UVB-repressed genes. we were able to analyze this scarcely investigated effect of the UVB radiation on gene expression. Appmximately one third of all diirenbally expressed genes turned out to be down-regulated during 24h after UVR. The identification and analysis of these new UVB-repressed genes offers the opportunity to idenbfy unrecognized molecular mechanisms in the UV response of human cells. For one of these UV-repressed genes full-length cDNA clones have been isolated. They encode a potential protein with a predicted molecular maes of -44 kDa. This novel protein displayed all of the structural features unique to the ovalbumin family of the serine pmteinase inhibitors (serpins). Expression characteristics of this new serpin indicates functions during proliferation/differentiation and possibly apoptosis in human keratinocytee. Further analysis of thts new serpin at the protein level will now help to elucidate the physiological function and its mode of action during the UVB response of keratinocytes.
ALPHA-MELANOCYTE-STIMULATING HORMONE REDUCES ENDOTOXININDUCED ACTIVATION OF NUCLEAR FACTOR-KBIN ENDOTHELIAL CELLS. D.-H. Kalden. M Fastrich T. &Q&& Scholzen M. Iiarhnever. T. SC war and T A. Luger. Ludwig Boltzmenn Cellbiology end Innnu”obiolor$ of”he Ski”, Department ofDermatology, Univemity of Muenster, Muenster, Germany The neuropeptide a-Melanocyte-stimulating hormone (a-MSH) is a potent antiin&nmnatoty and antipyretic agent. To identify a potential mechanism of the antiinflammatory action of a-M%% we tested its e&&s on regulation of adhesion molecules Since many adhesion molecules, including vascular cell adhesion molecule 1 (VCAM-1) and E-s&c&, contain regulatory NF-XB sites in their promoter regions, we also investigated the role of a-MSH in the a&ado” of the transc~ption factor NF-KB Monolayer cell cultures of transfomwd human microvascular endotheiial cells (HMEC-I) as well as dennel ndcrovascular endothelial cells (HDMEC) were treated with LPS (lWng/“d) alone and in combination with a-MSH (10‘8-10”6I@. The ~IRNA WBS isolated a&r 24h and expression of the adhesion molecules VCAM-I and E-se&tin was investigated by Northern blot enaJysis and semiquantitative RT-PCR. Nuclear extracts were prepared at early time points for EMSA using ‘zP-labeled specific oligonucleotides to detect NF-KB. The expression of VCAM and Eselectin was stronalv uoremdated a&r 24h of LPS treatment A markedly reduction ww shown in comb~&+ti~”;;ith aMSH (lo’- Iv’* M). whereas VCAhi and E-a&ctin nRNA was also induced by a-MSH ( lCi’O-lO~‘z hi) alone 30 tin after LPS stimulation the activrdio” (nuclear tmnslocation) of NF-WBwas strongly induced Silas to the adhesion molecules mRNA data, thi$ effect was reduced by catimuletion with a-MSH in a dose-dependent manna, at which lo-’ M a-MSH showed the grongest suppression. tis, a-MSH, which is released by stin”daed in&mletoly cells, may act via the moduletio” of tre”acripti0” factor NF-al suggesting that a-MSH has an et&t very early in the intlmnmatory cascade.
Institut~~f
0137
0134 lND”CTlON OFPROINFLAMMATORY
MOLECULES BY ANTHRALIN IS MEDIATED VIA REACTWE OXYGEN SPEClES AND ACTlVATlON OF NF-KB IN HUMAN KERATINOCYTES M. Podda. C Schulz V. SchmCKetih’. W.H. Boehncke. R. Milbradt, R Kauhnann. TM. Zollner Dept. of Denatology, ‘Dept of Physiology, J W Goethe-University, Frankfurt Germany Anthralin IS a widely used and highly effinent drug m the treatment of psonasts, however, ifs mode of &on 1sstill not completely understood Prevuxs studies demonstrated that anthral~n acbvates me transcrlptlon factor NF-rB in the murme epidermal cell line JB6 via the production of reactive oxygen species (ROS). This findlog 1s intnguing. since NF-rB regulates the transcription of many proinflammatory cytokines and rather an inhibition than an acbvabon would be expected to be bendcial m the treatment of psoriasis. We therefore asked fl anthrelin achvates NF-rB in the human keratmocyte cell line HaCaT and inCreaseS the expression of NF-rB regulated proinflammatory cytoklnes (IL-8 IL-8. TNFa). the adhesion molecule ICAM-I and the indwble mtrlc oxide synthase (INOS), all known to be induced !n acbve psonabc skm As assessed by electromoblllry-shict-assay anthralin (1, 2, 5. 10, 20 pM) actwates the transcription factor NF-rB in human kerat~nocytes in a dose dependent manner A concurrent dose dependent ~mcreaseof nit& production, an lndlrect measure of INOS activity. staltlng at 1 pM (,4,4t,,8 vs 5,2+0.6 pM ,n untreated ceils) with a maxmum at 10 +t (29.5&4,0 PM) 1s present in the supematent analyzed by the Griesoreection Stating et 2 pM the release of IL-8 and ICAM-I expression is also markedly mcrased, determined respectively by ELISA and Row cytometry. Interestingly. no significant change could be found for TNFa and IL-8 N-Acetyl-Lcystane (NAC) and pyrrclidmedithiocarbamate (PDTC). two stwcturelly unrelated antioxidant compaunds, were used to determine the mvotvement of reactwe oxygen spates m NF-kB activation and the downstream increased expression of INOS, ICAM-I and IL-8 Non-toxic concentrations of NAC (25 mM) or PDTC (100 PM) inhIbIted NF-rB activabon and all prewously determined pranflammatory effects of anthralin We conclude that in human keratlnocytes anthrabn not only actjvates the transcription factor NFlrB, but also induces pranRammatory NF-xB regulated proteins via the production of ROS. In the clinical practice It is well know that Our m vitro findmgs substantiate me clinical psorlasas ,,burns-away in anthralln’s bre’ observation that anthralln acts by the mductlon and not through inhlbltlon of lnflammatlon
TRANSFORMATION AND IMMORTALISATION OF SKIN KERATINOCYTES
0135
0138
a-MELANOCYTE STIMULATING HORMONE DOWNREGULATES IL-I MEDIATED TARGET GENE EXPRESSION BY AfBTlENUATlON W&JFxB waz ACTIVATION. T.Brzoska. D.-H. Kelden T.Scholzen S etz K.Bbert. and T.A.Luaer, Ludwig Boltzmann Institute of Cellbiology and Inmnmobiology of the Skin, Department of Dermatology, University of Muenster, Muenster, Gemany The Proopiomelanocortin derived hormone a-Melanocyte stimulating hormone (aMSH) not only has an important role in pigmentation but also functions aa potent inummomodulator. Although the proinflammatory cytokine IL-I is a strong inducer of a-MSH expression by epidemml cells, a-MSH downregulates systemic prointlammatory activities of IL-1 such as fever and leukocytosis. Keratinocytes upon stimulation with IL-1 readily express and secrete large amounts of chemokineq which are responsible for activation and chemotaxis of infbonmatory cells. This effect of IL1 is mediated via activation of the transcription factors NFKB and AP-1. Using the keratinocyte cell lines HaCaT and KB we investigated the effect of a-MSH on the IL10 induced activation of NFKB br EMSA 10 min r&r simultaneous stimulation of the cells with a-MSH (lOd-IO~’ M) and IL-ID (5 “g/ml) NFKB activation was markedly reduced compared to cells treated with IIz~~ only. 30 ti a&r stimulation this effect still was observed in cells costinwlated with 10” M a-MSH and IL-lp. To test the effect of the a-MSH mediated attenuation of NF& activation on the outcome of IL-Lp stimulation cells were analysed for chemokine mRNA expression (semiquantitative RT-PCR) and release (ELISA). The production of IL-S, Groa and RANTES was significantly inhibited after 48 h when elfi stimulated HaCaT cells were treated with a-MSH (106-10~‘*M). These data indicate that a-MSH, mainly via modulating the activities of transcription factors such as NFKB, functions as a potent antagonist of IL-1 mediated proinflammatory e&xts.
IS ASSOCIATED WITH CHANGES IN GENOblIC DNA METHYLATION OF HtCl BUT NOT P16 Fiona Cockerill, Patricia Purkis, Anthony G Ouinn, Centre for Cutaneous Research St Bartholomew’s & The Royal London Hospital School of Medicine & Dentisny, 2 Newark St, London Alterabons m the level and pattern of genomic DNA methylation are a common findina in many human cancers. DNA hypamethylation in specdic turnour types has been ihown to involve the same chromaome .regions th% show frequeni?oss of hetwxygosity (LOH) and recent work has shown a relationshIp between increased DNA methylation of the promoter region of a number of tomoor suppressor genes and transcnptional inactivanon of these genes. ‘Ibe role of DNA methylation in skin carcinogenesis is poorly understood. In previous work we have shown a high frequency of LOH of markers from chromosome 17~ and % in cutaneous SC& In this study ue have mvestigated the metiylation status of ‘candid& mmour suppressor genes from ihex regions in normal and transformed keratinocytes. DNA methylation was mvestigated by Southern blot analysis using methylation sensitlvc rsstriction enzymes. In normal skm, fibroblasts and primary keratinocytes we found no evidence of methylanon of the promoter region of the pl6 gene which maps to chromosome 9p. Homozygous delettons of p16 were ldentitied in 3 of 3 bead & neck (H&N) XC lines and 2 of 5 cutaneous SCC lines. Hypermethylation of pl6 occurred in I of the 3 cutaneous SCC lines heterozygous for ~16. None of the 5 immortalised skin keratmocyte lines studied showed pl6 hypermethylation. The HICl gene which maps to chromosome 17p was found to be unmethylued in normal skin, fibrohlasrs and primary keratinocytes In contract to our findings for ~16, hypermethylation of HICl was a common finding in immortahsed and transformed keratinocytes wth alterations in 3 of 4 XC lines and 3 of 3 immortalised keratinocyte hnes These fmdmgs prowde evidence of a role for DNA methylaeon m kerammcyte transformation. The frequent occurrence of HICl hypcrmethylation m immortahsed keratmocytes suggests that this gene may be impownt in human skm carcinogenesis.
OF THE HSDGEHOGIPATCHED SIGNAILING PATHWAY IN BASAL CELL CARCINOMASIS ASSOCIATED WITH FXPRESSION OF THE ACTIVATION
IM
TRANSCRIPTION FACTOR GLI-I Judith Green. L&h. AG Ouinn, Cutaneous Research, St Bartholomew’s &The Royal London School of Dentistry, 2 Newark St, London.
Centre for
Medicine &