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-171- Keratinocyte(2) 9/5 Room-D
EXPRESSION OF ENTEROPEPTIDASE IN HUMAN EPIDERMAL KERATINOCYTES.
Jotaro Nakanishi, Junko Sato, Junichi Koyama, Yasuhisa Nakayama.
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 intercelluler cohesive structure. In this study, we examined the expression of enteropeptidase, which specifically cleaves trypsinogen to yield active trypsin, in human keratinocytes by RT-PCR and in situ hybridization. Enteropeptidase was not expressed in cultured human keratinocytes at growth phase and at confluency, even though in high calcium (1.5mM) condition, while the expression was observed in the cells exposed to the air. Furthermore, in situ hybridization study revealed that enteropeptidase was expressed only in the uppermost granular cells in human epidermis. These results suggest that trypsinogens expressed in suprabasal cells are activated to trypsin by enteropeptidase expressed in uppermost cells and that the active trypsin may play important roles in terminal differentiation and desquamation.
-172- Keratinocyte(2) 9/5 Room-D
TARGETED DISRUPTION OF THE TRANSGLUTAMINASE 1 GENE.
Masato Matsuki1, Keiko Yamada1, Eiichiro Ueda1, Yohchi Morishima1, Kohichi Tabata1, Kiyofumi Yamanishi1, Hirokazu Yasuno1, Masahito Ikawa2, Masaru Okabe2, Junji Takeda3, Taroh Kinoshita3.
1Department of Dermatology, Kyoto Prefectural University of Medicine, Kyoto, 2Department of Science for Laboratory Animal Experimentation, 3Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.
Transglutaminase 1 (TGase 1) is expressed during the process of keratinization and is believed to produce cell envelope by catalyzing protein cross-linking reaction. The gene for TGase 1 is a disease gene of autosomal recessive lamellar ichthyosis. In this study, to elucidate in vivo function of TGase 1 gene, we generated mice lacking TGase 1 gene by targeted disruption of the 2-kb mouse TGase 1 gene containing exons from 1 to 3. The heterozygous mutant mice were normal in phenotype, but the homozygous mutant mice showed neonatal lethality with severe morphological changes of the stratum corneum. Thus, the TGase 1 gene is essential to development of the stratum corneum and for survival of mice.
-173- Keratinocyte(2) 9/5 Room-D
BCL-XL ACTIVATES HUMAN TRANSGLUTAMINASE 1 PROMOTER IN KERATINOCYTES.
Kohichi Tabata1, Eiichiro Ueda1, Kiyofumi Yamanishi1, Hirokazu Yasuno1, Yutaka Eguchi2, Yoshihide Tsujimoto2.
1Department of Dermatology, Kyoto Prefectural University of Medicine, Kyoto, 2Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School, Suita, Japan.
Transglutaminase 1 (TGase1) is expressed at the late stage of terminal differentiation of keratinocytes to form cell envelope by protein cross-linking reaction. Bcl-xL, a member of the bcl-2 family regulating programmed cell death, is expressed in the upper spinous and granular layers of the epidermis. The localization of bcl-xL is closely related to that of TGase1 mRNA, suggesting its regulatory role in TGase1 gene expression. When the 5' flanking region of human TGase1 gene linked to a luciferase reporter gene was co-transfected with a chicken bcl-xL expression plasmid, luciferase activity was increased in keratinocytes, but not in non-keratinocytic HT1080 cells. Thus, bcl-xL may be involved in the activation of the human TGase 1 promoter in keratinocytes.
-174- Keratinocyte(2) 9/5 Room-D
CORNIFIED CELL ENVELOPE (CCE) PRECURSOR PROTEINS AND TRANSGLUTAMINASE (TGASE) 1 ARE COORDINATELY EXPRESSED IN THE SITES OF HAIR CANAL MORPHOGENESIS DURING HUMAN HAIR FOLLICLE DEVELOPMENT.
Masashi Akiyama1, Lynne T. Smith2, Kozo Yoneda3, Karen A. Holbrook4, Hiroshi Shimizu5.
1Division of Dermatology, Kitasato Institute Hospital, Tokyo, Japan. 2Departments of Biological Structure and Medicine/Dermatology, University of Washington School of Medicine, Seattle, WA, U.S.A. 3Department of Dermatology, Kyoto University Faculty of Me
CCE formation is a key step of the final stage of keratinization. CCE precursor proteins including loricrin, small proline-rich proteins (SPRPs) 1 and 2 and involucrin are cross-linked by keratinocyte TGase 1 to the inner surface of plasma membrane of cornified cells in CCE formation and its outer surface is coated with content of lamellar granules (LG). To clarify the time and the site of expression of these CCE associated molecules in hair follicle development, skin samples from human fetuses of a series of estimated gestational ages (EGAs) were examined. Loricrin was seen in cells of the neck of hair peg (85-105 d EGA) and TGase 1 and LG content were observed in the inner cells of hair peg. Immunoreactivities of loricrin, SPRPs 1 & 2, involucrin, TGase 1 and LG content were detected in inner root sheath cells of the bulbous hair peg (105-135 d EGA) and in primitive hair canals in intraepidermal portion of hair follicle (>135 d EGA). TGase 1 was also positive in outer root sheath and sebaceous gland. These data indicated that CCE proteins, TGase 1 and lamellar granule lipid content are coordinately expressed in the sites defined to form hair canal during human hair follicle development.
-175- Keratinocyte(2) 9/5 Room-D
INDUCTION OF KERATIN 17 EXPRESSION BY IFN-g IN RECONSTRUCTED SKIN.
Gen Nakanishi, Wataru Fujimoto, Jiro Arata.
Department of Dermatology, Okayama University Medical School, Okayama, Japan.
In normal skin, expression of keratin 17 is restricted to specific skin appendages such as outer root sheath and nail matrix cells, whereas it is highly expressed in hyperplastic epidermis such as in psoriatic plaques. It has been suggested that the induced expression of K17 in psoriatic epidermis is due to IFN-g activity. In this study, to characterize IFN-g action in skin, we examined induction of K17 expression by IFN-g in reconstructed skin by immunohistochemistry, and compared it with K17 expression in NHEKs of monolayer cultures. Immunohistochemical study demonstrated that K17 protein was not expressed in reconstructed skin without IFN-g treatment whereas it was induced in the supralayers after IFN-g treatment. Control experiment showed that keratin 16 protein was expressed in suprabasal area in reconstructed skin without IFN-g treatment. In cultured NHEKs K17 was highly expressed without IFN-g treatment at mRNA and protein level. Northern blot analysis demonstrated that K17 mRNA was not up-regulated in NHEKs by IFN-g , contact inhibition, or TPA treatment. These data suggest that reconstructed skin may be a useful in vitro model system to elucidate mechanisms of IFN-g inducible genes expression by IFN-g .
-176- Keratinocyte(2) 9/5 Room-D
EXPRESSION OF TETRA-SPANS TRANSMEMBRANE FAMILY AND INTEGRINS IN BASAL CELL CARCINOMA AND SEBORRHEIC KERATOSIS.
Hitoshi Okochi, Masutaka Furue.
Department of Dermatology, Tokyo University Branch Hospital, Tokyo, Japan.
Tetra-spans transmembrane family members (CD9, CD37, CD53, CD63, CD81, CD82) are known to be associated with integrins in various cells. We have already shown that CD9, CD81 and CD82 are expressed in the entire living layers of the normal epidermis and CD53 is expressed in upper spinous layers. a2, a3, a6 and b1 integrin are also expressed in the normal epidermis. In this study we compared the expression of those molecules in basal cell carcinoma (n=5) and seborrheic keratosis (n=9) immunohistochemically. The expression of CD9, CD81, CD82 and a6 integrin was markedly down-regulated in basal cell carcinoma, while a2, a3, a6 and b1 integrin were up-regulated in seborrheic keratosis. These results suggest that the expression of tetra-spans transmembrane family members is not closely related with that of integrins in these tumors.