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Message of protease nexin-1 is expressed in human dermal papilla cells and is affected by androgen
ESDR / JSID I SID Abstracts
0835
0838
EXPRESSION OF ENTEROPEPTIDASE IS RESTRICTED IN UPPERMOST GRANULAR CELLS IN HUMAN EPIDERMIS. Jotaro Nakanishi, Junko Sate. Junicbi Kovama and Yasuhisa Nakavama. Shiseido Research Center, Yokohama, Japan. We have previously shown that two types of trypsinogens are expressed in human epidermal keratinocytes and may play an important role in desquamation by catalyzing the degradation of desmosomal proteins. In this stody, we examined the expression of enteropeptidase, which specifically cleaves trypsinogen to yield active trypsin, in human epidennal keratinocytes. We performed in situ hybridization using normal human skin. The cells which express enteropeptidase were studded only in the uppermost granular cell layer just under the stratum comeurn, and this result was similar to the localization of the apoptotic cells by TUNEL staining. In cultured human keratinocytes, enteropeptidase was not expressed at proliferating phase, while the weak expression was observed after confluency by RT-PCR. The expression level was not affected by high calcium (1.5&f) condition. In situ hybridization study revealed that enteropeptidase was expressed only in the stratifiedcells, in which comification step was going on, while the expression was not observed in monolayer area. This result seems to correspond with the observation in the skin. Thus, OUTfindings indicate that trypsinogen is activated by enteropeptidase at the final stage of terminal differentiation including cell death process in uppermost glanular layer. And it is also suggested that the active trypsin may play important roles not only in desquamation, but also in comification.
MESSAGE OF PROTEASE NEXIN-1 IS EXPRESSED IN H"MAN DERKAL PAPILLA CELLS AND IS AFFECTED BY ANDROGEN. Tadashiae Sonoda, Sotaro Kurata. Yuli Asada. Susumu Takavasu, Department of Dermatology, Oita MedIcal Unlverslty, Oita,
0836
0839
IDENTIFICATION OF NOVEL GENES DIFFERENTIALLY REGULATED IN PSORIATIC SKIN BY DDRT-PCR. Giinter Michel, Alireza Mirmohammadsadegh, Harry F. Abts, Thomas Ruzicka, Department of Dermatology, Heinrich-
DEtECTION OF CRYPTDIN 5 IN MICE SKIN Yoshinori Shirafuii. Takashi Oono, Hiroko Kantii. and Jir6 Amta Depabnent of Dermatology, Okayama University Medical School, Sbikata-cho
Heine-University of Dtlsseldotf, Germany. The characteristics of psoriasis are epidennal inflammatory infiltrate. However, the exact ettology and comprehensive understanding of the disease
hyperproliferation
and
Japan.
Protease nexin-1 (PN-11, a serine protease inhibitor, plays important colas in requlatuw cell crrowth. blfierentiation and death b; modulatlnq p&teolytic activity. Recently it has been shown that PN-1 message 1s expressed in rat dermal papilla cells 1x1a hair cycledependent fashion and its levels correlate with the ablllties to support hair growth. In this study we investigated the expression of PN-1 and 1t.scontrol by androgen in cultured human dermal papilla cells (DPC) by reverse transcription polymerase chain reaction CRT-PCR). DPC were derived from beard(n=2), balding(n=3), and nonbalding(n=4) scalp hair follicles. PN-1 messages were identified in any type of cells. Then, DPC were incubated with 1O-8 M dihydrotestosterone (DHT) in the presence or absence of cyproterone acetate(lO-' M) for 24 hours. The PN-1 expression was negatively regulated by DHT in a dose-dependent manner II?two DPC from balding scalp. These data suggest that PN-1 plays a passable role 1x1 controlling hair growth in human and participates in the development of male pattern baldness.
an
of psoriasis is still unclear requires knowledge of all
genes improperly regulated (overexpressed or down-modulated) in lesional skin including the identification of so far unknown genes. For this purpose we performed a differential display analysis, comparing RT-PCR products from RNA prepared from lesional psoriatic skin of muittple patients to those from nonlesional skin. Products with different signal intensities in neighboring lanes were eluted from the sequencing gel, reamplifled, cloned and sequenced Genetic data bank searches were performed to identify known sequences. So far 6 of 149 products were already described in a different context: the “bullous pemphigoid autoantigen” (BPI80), the DNA-binding RNA-binding protein “nucleolysin”, the ubiquitln-conjugating
protein “NF-IV’, the enzyme E2, the
macropain subunit iota and the oxitocin receptor. However, these clones did not show differential expression in the slot blot analysis. Differential expression could be verified by northern blot analyses using clones 2, 46 and 110 as probes with clone 110 showing the most pronounced difference. These clones represent candtdate
0837
unknown sequences and are now further genes potentially involved in the pathogenesis
examined as of psoriasis.
2-5-1, Okayama 700, Japan. Defensins are widely-distributed and broad-spectrumantimicrobial peptides against bacteria fungi, and enveloped viruses. Defensins have been isolated from granules of neuuophils of humans, rabbits, rats, and guinea pigs. They have also been found in lung macrophages as weU as in human rabbit, and mouse small-intestioal Paneth cells. Defensins derived from mice are called cryptdins. The human- B -defensin-2 was recently isolated from human skin, however. defensins have not yei been detected in skins of experimental animals. In this study, we detected expression of ua: cryptdin messenger ribonucleic acid m the skin ot BALB/c mice using reverse uanscriptase po1ymera.w chain reaction. Sequence analysis demonstrated a near-perfect identity with nyptdin 5. It will conhibute to further analysis of defensin function in the skin.
possible
0840
MEAsuREmNT OF h4ATRlx MET4LLGPRGTEINASES BY QUANTITATlVE RT-PCR ANTTMONYLPOTASSIUM TARmTE (APT’)C$JSEs CELL DEATH OF Cl-CL ASSAY:AWLK!ATIONTOTHEST"DYOF GENEREGULATIONINFIBROBLASTS BY (CUT+NEOUS T CELL LYMPHOMA) CELLS Om. m Dermatology Depariment, Universiy Hospital Zurich Glotiastras~e 31, CH-8091 Zitrich, Swiaerland. Cutaneoos T cell lymphoma (CrCL) is Blymphoproliferative disorder of the skin. Fe most frequent forms are the skin restricted Mycosis fumgoides0 and its leskemlc varisnt the Waty syndrome (SS). Until now there is no cure for these diseaes. AFT is wed against several pam&s (Schizostoma,Leiihmaoia) and can cause cell death of “adous cell types. To test whether APT CMinduce cell death of CTCL cells under “ontoxic conditions we incubated tht CTCL ceUlines in the presence of varioos concenuadons of APT and investigated whether d% expression of cell sorvival genes bcl-2, bclXL,and mcl-1 is influenced by this agent Siace it has been found that CrcL ceUs $0~ consti~tiveNFlcB~sc~ptioofanora~~~andthatNFIcB~anenhan~ecell~UrnV~. wealsoanalysedUleeffectofAPTonNFkB. Theexpression ofW-2, bcl-xl_ bcl-x.s,and mcl-1 was tested by Wesam blotting of cytoplasmtic extras from three CTCL cell lines (HUT%. Mylar 2059, SeAx) and NFkB DNA-bindiig activity was measured by elecaophoredc mobility shift assays (EMSAs). A ndioacdvely labeled DNA-oligonucleotide representig dx. NFkB binding site was incubated with nuclear exerts of CTCL cells and the DNA-W complexes were identified by electmphoresis and sulwquent autoradiography. The addition of 5-10 ti APT induced apoptosis after 3-10 days of all three cell Lines and let to a marked decrease of NFkB DNA binding activity. The expression of bcl-2, ~&XL, and, mcl-1 was strongly decreasedwhen compared totheconstitutively expressedbetaactinpne.?heseresultsindicatethrt APTcan induceceUdeath ofCTCLcells in viva at non-toxic concentrations and that the expression ofcell survival genes is markedly decreased by this drug.
0835
0838
EXPRESSION OF ENTEROPEPTIDASE IS RESTRICTED IN UPPERMOST GRANULAR CELLS IN HUMAN EPIDERMIS. Jotaro Nakanishi, Junko Sate. Junicbi Kovama and Yasuhisa Nakavama. Shiseido Research Center, Yokohama, Japan. We have previously shown that two types of trypsinogens are expressed in human epidermal keratinocytes and may play an important role in desquamation by catalyzing the degradation of desmosomal proteins. In this stody, we examined the expression of enteropeptidase, which specifically cleaves trypsinogen to yield active trypsin, in human epidennal keratinocytes. We performed in situ hybridization using normal human skin. The cells which express enteropeptidase were studded only in the uppermost granular cell layer just under the stratum comeurn, and this result was similar to the localization of the apoptotic cells by TUNEL staining. In cultured human keratinocytes, enteropeptidase was not expressed at proliferating phase, while the weak expression was observed after confluency by RT-PCR. The expression level was not affected by high calcium (1.5&f) condition. In situ hybridization study revealed that enteropeptidase was expressed only in the stratifiedcells, in which comification step was going on, while the expression was not observed in monolayer area. This result seems to correspond with the observation in the skin. Thus, OUTfindings indicate that trypsinogen is activated by enteropeptidase at the final stage of terminal differentiation including cell death process in uppermost glanular layer. And it is also suggested that the active trypsin may play important roles not only in desquamation, but also in comification.
MESSAGE OF PROTEASE NEXIN-1 IS EXPRESSED IN H"MAN DERKAL PAPILLA CELLS AND IS AFFECTED BY ANDROGEN. Tadashiae Sonoda, Sotaro Kurata. Yuli Asada. Susumu Takavasu, Department of Dermatology, Oita MedIcal Unlverslty, Oita,
0836
0839
IDENTIFICATION OF NOVEL GENES DIFFERENTIALLY REGULATED IN PSORIATIC SKIN BY DDRT-PCR. Giinter Michel, Alireza Mirmohammadsadegh, Harry F. Abts, Thomas Ruzicka, Department of Dermatology, Heinrich-
DEtECTION OF CRYPTDIN 5 IN MICE SKIN Yoshinori Shirafuii. Takashi Oono, Hiroko Kantii. and Jir6 Amta Depabnent of Dermatology, Okayama University Medical School, Sbikata-cho
Heine-University of Dtlsseldotf, Germany. The characteristics of psoriasis are epidennal inflammatory infiltrate. However, the exact ettology and comprehensive understanding of the disease
hyperproliferation
and
Japan.
Protease nexin-1 (PN-11, a serine protease inhibitor, plays important colas in requlatuw cell crrowth. blfierentiation and death b; modulatlnq p&teolytic activity. Recently it has been shown that PN-1 message 1s expressed in rat dermal papilla cells 1x1a hair cycledependent fashion and its levels correlate with the ablllties to support hair growth. In this study we investigated the expression of PN-1 and 1t.scontrol by androgen in cultured human dermal papilla cells (DPC) by reverse transcription polymerase chain reaction CRT-PCR). DPC were derived from beard(n=2), balding(n=3), and nonbalding(n=4) scalp hair follicles. PN-1 messages were identified in any type of cells. Then, DPC were incubated with 1O-8 M dihydrotestosterone (DHT) in the presence or absence of cyproterone acetate(lO-' M) for 24 hours. The PN-1 expression was negatively regulated by DHT in a dose-dependent manner II?two DPC from balding scalp. These data suggest that PN-1 plays a passable role 1x1 controlling hair growth in human and participates in the development of male pattern baldness.
an
of psoriasis is still unclear requires knowledge of all
genes improperly regulated (overexpressed or down-modulated) in lesional skin including the identification of so far unknown genes. For this purpose we performed a differential display analysis, comparing RT-PCR products from RNA prepared from lesional psoriatic skin of muittple patients to those from nonlesional skin. Products with different signal intensities in neighboring lanes were eluted from the sequencing gel, reamplifled, cloned and sequenced Genetic data bank searches were performed to identify known sequences. So far 6 of 149 products were already described in a different context: the “bullous pemphigoid autoantigen” (BPI80), the DNA-binding RNA-binding protein “nucleolysin”, the ubiquitln-conjugating
protein “NF-IV’, the enzyme E2, the
macropain subunit iota and the oxitocin receptor. However, these clones did not show differential expression in the slot blot analysis. Differential expression could be verified by northern blot analyses using clones 2, 46 and 110 as probes with clone 110 showing the most pronounced difference. These clones represent candtdate
0837
unknown sequences and are now further genes potentially involved in the pathogenesis
examined as of psoriasis.
2-5-1, Okayama 700, Japan. Defensins are widely-distributed and broad-spectrumantimicrobial peptides against bacteria fungi, and enveloped viruses. Defensins have been isolated from granules of neuuophils of humans, rabbits, rats, and guinea pigs. They have also been found in lung macrophages as weU as in human rabbit, and mouse small-intestioal Paneth cells. Defensins derived from mice are called cryptdins. The human- B -defensin-2 was recently isolated from human skin, however. defensins have not yei been detected in skins of experimental animals. In this study, we detected expression of ua: cryptdin messenger ribonucleic acid m the skin ot BALB/c mice using reverse uanscriptase po1ymera.w chain reaction. Sequence analysis demonstrated a near-perfect identity with nyptdin 5. It will conhibute to further analysis of defensin function in the skin.
possible
0840
MEAsuREmNT OF h4ATRlx MET4LLGPRGTEINASES BY QUANTITATlVE RT-PCR ANTTMONYLPOTASSIUM TARmTE (APT’)C$JSEs CELL DEATH OF Cl-CL ASSAY:AWLK!ATIONTOTHEST"DYOF GENEREGULATIONINFIBROBLASTS BY (CUT+NEOUS T CELL LYMPHOMA) CELLS Om. m Dermatology Depariment, Universiy Hospital Zurich Glotiastras~e 31, CH-8091 Zitrich, Swiaerland. Cutaneoos T cell lymphoma (CrCL) is Blymphoproliferative disorder of the skin. Fe most frequent forms are the skin restricted Mycosis fumgoides0 and its leskemlc varisnt the Waty syndrome (SS). Until now there is no cure for these diseaes. AFT is wed against several pam&s (Schizostoma,Leiihmaoia) and can cause cell death of “adous cell types. To test whether APT CMinduce cell death of CTCL cells under “ontoxic conditions we incubated tht CTCL ceUlines in the presence of varioos concenuadons of APT and investigated whether d% expression of cell sorvival genes bcl-2, bclXL,and mcl-1 is influenced by this agent Siace it has been found that CrcL ceUs $0~ consti~tiveNFlcB~sc~ptioofanora~~~andthatNFIcB~anenhan~ecell~UrnV~. wealsoanalysedUleeffectofAPTonNFkB. Theexpression ofW-2, bcl-xl_ bcl-x.s,and mcl-1 was tested by Wesam blotting of cytoplasmtic extras from three CTCL cell lines (HUT%. Mylar 2059, SeAx) and NFkB DNA-bindiig activity was measured by elecaophoredc mobility shift assays (EMSAs). A ndioacdvely labeled DNA-oligonucleotide representig dx. NFkB binding site was incubated with nuclear exerts of CTCL cells and the DNA-W complexes were identified by electmphoresis and sulwquent autoradiography. The addition of 5-10 ti APT induced apoptosis after 3-10 days of all three cell Lines and let to a marked decrease of NFkB DNA binding activity. The expression of bcl-2, ~&XL, and, mcl-1 was strongly decreasedwhen compared totheconstitutively expressedbetaactinpne.?heseresultsindicatethrt APTcan induceceUdeath ofCTCLcells in viva at non-toxic concentrations and that the expression ofcell survival genes is markedly decreased by this drug.