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研究開発
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1998年度

International Investigative Dermatology '98

SHISEIDO MGH/Harvard CBRC Symposium
EMPHASIS ON CELL-CELL COMUNICATION

開催年月 :
1998年5月
共催 :
MGH/Harvard CBRC・資生堂

概要

1998年5月、ドイツのケルンで開催された
IID'98(INTERNATIONAL INVESTIGATIVE DERMATOLOGY 1998)において、
当社はMGH/Harvard CBRC
(Massachusetts General Hospital / Harvard Cutaneous Biology Research Center)
と共同でサテライトシンポジウムを開催。
「細胞間情報伝達」をテーマに、MGH/Harvard CBRCでの基礎研究、
培ってきた研究ネットワークをもとに、世界の第一線の研究者を招聘して開催されました。

1)Chairperson

1Mr. Hideoki Ogawa
M.D., Ph.D., Juntendo Univ., School of Med., Tokyo

2Mr. Robert E. Burgeson
Ph.D., MGH/Harvard CBRC, Boston

2)Molecular Assembly and Functions of Tight Junctions

Mr. Shoichiro Tsukita
M.D., Ph.D., Kyoto Univ., School of Medicine, Kyoto

3)Role of cadherins and integrins in regulating keratinocyte growth and differentiation

Ms. Fiona M. Watt
Ph.D., Imperial Cancer Research Fund, London

4)Signal Transduction through Cell Adhesion Components

Mr. Jürgen Behrens
Ph.D., Max-Delbrck Center for Molecular Medicine, Berlin

5)Epidermal Laminins: In vivo Functions and Potential Clinical Applications

Mr. Robert E. Burgeson
Ph.D., MGH/Harvard CBRC, Boston


Chairperson

1 Mr. Hideoki Ogawa
M.D., Ph.D.,
Juntendo Univ., School of Med., Tokyo

Hideoki Ogawa PHOTO

■ Qualifications
M.D. March, 1966
Ph.D. March, 1972

■ Training and positions
1966 M.D. :
Juntendo University School of Medicine,Tokyo

1981- Professor and Chairman:
Department of Dermatology, Juntendo University School of Medicine

1996- Dean :
Juntendo University School of Medicine

■ Scientific activities

  1. (1) Board of Directors

    1994-1997 Vice president of ISHAM (International Society of Human and Animal Mycology)
    1994- Vice president of ISD (International Society of Dermatology)
    1995- Chairman of JSID (Japanese Society for Investigative Dermatology)

  2. (2) Guest Professor

    1979 Duke University Medical Center, U.S.A.
    1988- Beijin Medical University, China.
    1994- Japan-China Friendship Medical Center, China.

  3. (3) Honorary Member

    1985- Korean Dermatology Association.
    1987- Chinese Medical Association.
    1991- American Dermatology Association: ADA
    1993- British Association of Dermatologist

  4. (4) Awards

    1987 1st "Yasuda-Sakamoto Memorial Award" for Dermatological Researcher
    1988 The Award from the Governor of the Japan International Cooperation Agency (JICA)
    1993 The 2nd Class of Most Noble Order of the Crown & Title of "Commondore Knight.", from Royal Thai Government

  5. (5) Editor

    1990-1993 : Editor in Chief / Journal of Dermatological Science (Amsterdam)
    1992- : Editor of Journal of the European Academy of Dermatology and Venereology(Amsterdam)
    1992 : Editor of Journal of Medical and Veterinary Mycology (UK)

  6. (6) Chief Organizer of a Diploma Course in Dermatology

    The Diploma Course extends for a period of 10 months each year in Bangkok, Thailand.
    It is conducted jointly by the Royal Thai Government and The Japanese Government which contributes as part of its "Technical Cooperation Programme". The purpose of this course is for the further education of young dermatologists in developing Asian and Oceania countries. Past attendance has numbered approximately 400 during the past 14 years with representatives from over 25 Asian and Oceania countries.

  7. (7) Special Interest in Dermatology (key word)

    keratin, keratinization, dyskeratosis, hair growth, wound healing, medical mycology, autoimmune and/or hereditary blistering diseases etc.


Chairperson

2 Mr. Robert E. Burgeson

Ph.D., MGH/Harvard CBRC, Boston

Robert E. Burgeson PHOTO

■ Research and/or Professional Experience
1968 - 1973 UCLA - Institute of Molecular Biology: NIH Predoctoral Fellow of Dr. John Fessler.

1973 - 1974 UCLA School of Dentistry Postdoctoral training with Dr. Alfred Weinstock.

1974 - 1975 Harbor-UCLA Medical Center: Medical Genetics. NIH Postdoctoral Fellow with Dr. David Rimoin

1983 - 1991 Shriners Hospital for Crippled Children, Portland Research Unit, Senior Investigator, Oregon Health Sciences University, Departments of Biochemistry and Molecular Biology, Cell Biology and Anatomy and Dermatology, Research Professor

1991-Present Professor of Dermatology (Cellular Biology and Anatomy), Harvard Medical School, Associate Director for Research, Cutaneous Biology Research Center, Massachusetts General Hospital


Molecular Assembly and Functions of Tight Junctions
A Mystery of the Molecular Architecture of Tight Junctions

Mr. Shoichiro Tsukita

M.D., Ph.D.,
Kyoto Univ.,
School of Medicine, Kyoto

Shoichiro Tsukita PHOTO

Tight junction (TJ), an element of epithelial and endothelial junctional complexes, seals cells to create a primary barrier to the diffusion of solutes through the paracellular pathway. TJ is also thought to function as a boundary between the apical and basolateral plasma membrane domains, which differ in protein/lipid composition, to create and maintain epithelial and endothelial cell polarity. TJ is thus a fundamental structure for the establishment of compositionally distinct fluid compartments in various tissues. In ultrathin section electron micrographs, TJ appears as a series of discrete sites of apparent fusion, involving the outer leaflet of the plasma membranes of adjacent cells. By freeze fracture electron microscopy, these apparent fusion sites are observed as a set of continuous, anastomosing intramembranous particle strands (TJ strands). Recent technical progress has enabled the identification of several TJ-associated peripheral membrane proteins such as ZO-1 and ZO-2. Although detailed analyses of these proteins have led to better understanding of the structure and function of TJ, lack of information concerning the TJ-specific integral membrane proteins hampered more direct assessment of the function of TJ at the molecular level.

Five years ago, we identified occludin which was the only known integral membrane protein localizing at TJ. Accumulating evidence has shown that occludin is directly involved in the formation and functions of TJ. For example, we have shown the direct interaction of the cytoplasmic domain of occludin with ZO-1/ZO-2, the formation of TJ strands in occludin-overexpressed cells, and the importance of phosphorylation of occludin in TJ formation. To examine functions of occludin and to clarify whether occludin is the only integral membrane protein in TJ, occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene.To our surprise, the occludin-deficient ES cells differentiated into polarized epithelial cells, which bore morphologically and functionally well-developed TJ. These findings indicate that there are as yet unidentified integral membrane protein(s) in TJ, which can form strand structures, recruit ZO-1, and function as a barrier without occludin. Identification of integral membrane proteins of TJ has been regarded as a difficult issue, but now we can utilize occludin as a probe to find such putative TJ components. We will overview our recent data on the identification of novel TJ integral membrane proteins, and discuss the mystery of the molecular architecture of TJ.


Role of cadherins and integrins in regulating keratinocyte growth and differentiation

Ms. Fiona M. Watt

Ph.D.,
Imperial Cancer Research Fund,
London

Fiona M. Watt PHOTO

Adhesion of epidermal keratinocytes to the underlying basement membrane is mediated by extracellular matrix receptors of the integrin family. My laboratory is studying the functions of integrins that share a common β subunit,β1. In addition to mediating keratinocyte adhesion, spreading and migration on extracellular matrix, theβ1 integrins play a role in regulating the onset of terminal differentiation: loss of bound legend serves as a potent differentiation stimulus. We have analysed a series of mutations in the fA1 cytoplasmic domain in order to determine whether the signalling pathways that control adhesion and differentiation are distinct. We have also found that epidermal stem cells have higher levels ofβ1 integrins than keratinocytes of lower proliferative potential, the transit amplifying cells. Exit from the stem cell compartment can be triggered by c-Myc and this is associated with decreasedβ1 integrin expression. We are currently investigating whether highβ1 integrin levels are required for maintenance of the stem cell phenotype.

Another class of adhesive receptor that influences keratinocyte differentiation comprises the classical cadherins. Expression of a dominant negative cadherin mutant in keratinocytes results in decreased growth rate and stimulation of terminal differentiation. We are now studying the events downstream of E-cadherin-mediated adhesion that regulate keratinocyte differentiation.

References:
Gandarillas, A. and Watt, F.M. c-Myc promotes differentiation of human epidermal stem cells. Genes & Devel., 11:2869-2882, 1997 Jones, P.H., Harper, S. and Watt, F.M. Stem cell Patterning and Fate in Human Epidermis. Cell, 80:83-93, 1995 Zhu, A. J. and Watt, F.M. Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes. J. Cell Sci., 109:3013-3023, 1996


Signal Transduction through Cell Adhesion Components

Mr. Jürgen Behrens

Ph.D.,
Max-Delbrck Center
for Molecular Medicine, Berlin

Jrgen Behrens PHOTO

Cadherins constitute a family of cell adhesion molecules responsible for homophilic cell interactions in a variety of tissues. The major cadherin in epithelial cells, E-cadherin is frequently downregulated or mutated in carcinomas which is a prerequisite for invasiveness and metastasis formation. Cadherins interact at their cytoplasmic domain with intercellular proteins termed α-, β-, γ- catenins which provide a link to the cytoskeleton.β-Catenin and its Drosophila homologue armadillo are also involved in the wnt/wingless signal transduction pathway and interact with the tumor suppressor protein APC. We found previously thatβ-catenin binds to the HMG box transcription factor LEF-1. In mammalian cells, coexpressed LEF-1 andβ-catenin form a complex that is localized to the cell nucleus. Moreover, LEF-1 andβ-catenin form a ternary complex with DNA that displays an altered DNA bend. Microinjection of LEF-1 into Xenopus embryos induces axis duplication, which is augmented by interaction withβ-catenin indicating that downstream signaling in the wnt pathway is mediated by the LEF-1/-βcatenin complex. We have further evidence thatβ-catenin also interacts with factors involved in the remodeling of chromatin. Thus,β-catenin might activate gene expression by providing the link between specific transcription factors such as LEF-1 and regulators of chromatin function. We have also identified a novel protein, named conductin, which interacts with bothβ-catenin and APC. In colon carcinoma cells, conductin downregulates cytoplasmic and nuclear pools ofβ-catenin; in Xenopus embryos, conductin inhibits axis formation by interfering with the wnt pathway. Our data suggest that conductin control wnt signaling by directing -βcatenin to the APC degradation pathway. The potential role of conductin as a tumor suppressor protein will be discussed.


Epidermal Laminins: In vivo Functions and Potential Clinical Applications
Laminin 5 improves the take of keratinocyte sheet grafts

Mr. Robert E. Burgeson
Ph.D.,
MGH/Harvard CBRC, Boston

Robert E. Burgeson PHOTO

Laminin 5 is critical to the stability of epithelial attachment. Within the anchoring complex at the dermal-epidermal junction, laminin 5 bridges the transmembrane hemidesmosomal protein, integrin α6β4 with the anchoring fibril protein type VII collagen. Mutations in the genes enconding the chains of laminin 5 cause dermal-epidermal separations characteristic of junctional epidermolysis bullosa. Cultured keratinocytes preferentially adhere to laminin 5 through interactions with the integrin α3β1. Laminin 5 also induces the assembly of hemidesmosomes by cultured epithelial cells. These observations suggest that laminin 5 might be clinically useful in situations where increased adhesion of epithelial cells to their substrate would be desirable.

Autologous keratinocyte sheet grafts have been applied to human burn wounds with variable results. In many cases, the "take" of the epithelial sheet grafts is low. Therefore, autografting of keratinocyte sheets would appear to be an excellent test of the hypothesis that added laminin 5 might increase the take of these grafts.

Two animal models have been tested with promising results. Human keratinocyte sheets were either treated with a solution of laminin 5 (1μg/ml) or were untreated prior to grafting to the panniculus carnosus of nude mice. The take rate was assessed by photometric measurement of the are of surviving epithelium at 7 days post grafting by NIH image analysis. The area of epithelization of the treated group was significantly larger than that of the untreated group. Immunohistochemical and ultrastructural analyses indicated that at day 3 post grafting, the laminia densa was more continuous in the laminin 5 treated group than in the controls,

A second animal model was used to test the hypothesis that continuous treatment of grafted epithelial sheets with laminin 5 solution would further improve the graft take. Keratinocyte sheets were transferred to the fascia under a 1cm diameter full-thickness wound on the backs of 45 nude mice. The transplanted sheets were covered with a Fusenig chamber, and purified laminin 5 (final applied concentration 10μg/ml) or Ringer's solution was applied every 12 hours following transplant. The take of the grafts was evaluated by routine histology and by transmission electron microscopy at 2 days. 23 of 26 (88.5%) treated and 10 of 19 (53%) untreated grafts were judged to have stratified and appeared firmly affixed to the wound bed. The extent of epithelial necrosis was less in the treated grafts that in the untreated. Among the grafts judged to have taken, the treated grafts consistently were more fully stratified and both rudimentary hemidesmosomes and basement membranes were readily detected beneat h treated but not untreated graft sites.

Keratinocytes from a JEB patient genotyped as laminin β3-/- were similarly grafted to 50 mice and laminin 5 was applied as above. The grafts were evaluated by histology, immunofluorescence microscopy for basement membrane components, and transmission electron microscopy at 7 days. Establishment of the treated grafts occurred in 12 of 25 (48%) cases and in 4 of 25 (16%) untreated cases. In all cases, transmission electron microscopy analyses demonstrated detachment of the neoepithelium upon histological processing and the complete absence of recognizable basement membrane. The applied laminin 5 could not be detected by immunofluorescence in either case following 7 days.

From these studies we conclude that (1) laminin 5 significantly increases the adhesion of epithelial sheets to wound surfaces; (2) the viability of grafts in enhanced by exogenous laminin 5 due to an increase in the rate of basement membrane formation; and a decrease in the incidence of necrosis (apoptosis?); and (3) while laminin 5 increases the initial attachment of laminin 5-/- sheets to the graft bed, continuous laminin 5 production is required to support basement membrane assembly and stability.

Based upon these encouraging findings, laminin 5 solutions have been incorporated into the protocols for the treatment of human burn patients. Latticed full thickness grafts have been augmented with laminin 5 treated keratinocyte sheets in attempts to increase the area potentially covered by the latticed grafts. The outcomes of these studies will be discussed.

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