The splice variant of the ECM1 gene is expressed during the late phases of terminal differentiation of the epidermis

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ESDR I JSID I MD Abstracts

0424 TELOMERASE REGULATION IN NORMAL HUMAN SKIN KERATlNOCYTES V. Vormwald-Dogan*, R. Figueroa*, H. Lindenmnier, and P. Boukamp German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg Telomerase is a ribonucleoprotein which is active in germ lie. immortal and tumor cells while absent in many normal somatic cells. However, it is expressed in continuously renewing tissues such as the epidermis and here telomerase activity is high in cells of the basal layer. This constitutive expression, although often ignored, suggests that telomerase has an important function in the epidermis. In tumor cells, telomerase is believed to counteract t&mere erosion, which occurs in e.g. normal telomerase-deficient iibroblasts due to the ,,endreplication problem“. We now show that despite telomerase activity, also the telomeres of normal human skin keratinocytes shorten with increasing passage numbers suggesting that telomerase either may indeed be insufficient or may fulfill a different yet unknown function. Surprisingly, in keratinocytes,

telomerase

is only active for a short time period - during the proliferative

phase in the basal layer. Tbereafier it is switched off and by treating the cells with calcium ionophore we are able to demonstrate that this downregulation is closely correlated with Ca++-induced differentiation. We further show that also in a differentiation-independent system telomemse is downregulated by Ca++. This regulation dose-dependent and reversible. Furthermore, regulation does not seem to be PKCdependent.

Thus, our data demonstrate

that Ca++ is a key regulator

activity and suggest that this preceeds the process of Ca++-induced * both authors have contributed equally to the work.

is

of telomerase differentiation.

0425 TYROSINE CELL-CELL

PHOSPHORYLATION OF AODUCIN PLAYS A ROLE IN REGULATED CONTACT FORMATION OF E6/E7- TRANSFECTED KERATINOCYTES

CJohnen’. B.Hinz’. E O’Keefe’. H.W.Kaiser’ ‘Dept. of Dermatology. Theoretical Biology.Universlty Bonn, Germany; ‘Department of Dermatology, of North Carolina. USA

‘Dept. of University

We examined the role of adducin for tyrosine phospholylation-dependent regulation of cell-cell contact fonation. Adducin, a 200 kDa heterodimeric protein, is one of the first proteins present in adhesion junctions after induction of cell-cellcontacts. In vitro adducin bundles F-actin and enhances the binding affinity of spectnn to actin. In viva it IS associated with the cytoskeleton and actively involved in stabilizing cell-cell contacts. Its locelisation in adherence junctions suggests a role in calcium dependent cell-cell contact formation. We have raised e specific antibody from human red blood cells to demonstrate the presence of edducin in normal keratinocytes (nHEK) and in keratinowtes transfected with aenes EBiE7 of human oaoilloma virus (trHEK) Under hidh [Ca”] conditions (l.imM) adducin is localiied’ai the plasma membrane of nHEK. whereas cuitivabon in low I&‘1 medium 10.03mMI leads to a diffuse intracellular distribution. In contrast to &EK. ‘sdducin was not detectable at sites of cell cell-contacts I” trHEK even under high [Ca’+l condibons. Incubation with Genh?in, a tyrosme kinase InhIbitor. induced the redistribution of adducin to cell membranes of nelghbouring cells. This redistnbutioo was associated with the form&on of desmosomes. previously absent in trHEK. Changes in single cell morphology and overall cell formation were observed by scanning electron microscopy. lmmunoprecipitation with phosphotyrosine and subseauent western-blot studies demonstrated adducin es being tyiosine phosphorylatad. Generally, the tyrosine phospholylation level of cell contact proteins es adducin and vinculin is decreased after Genistefn treatment.

0423 THE SPLICE VARIANT OF THE ECMI GENE IS EXPRESSED DURING THE LATE PHASES OF TERMINAL DIFFERENTIATION OF THE EPIDERMIS. P. Smits’. Y &&!!I&. P. Tvlzanowski. V. Sarafian. J. Wouters. E. Hauben’. D. Huvlebroeck. E: Van Marck” and J. Merreaaert. l Departments of Biochemistry and “Pathology, U.I A., Antwero and ‘Deoaltment of Histoloav. Universitv of Namur. Namur. Belaium. The human chromosome &sins at’lq21 a cluster of genes that are involved in terminal differentiation of the epidermis, the Epidermal DifferentNation Complex (EDC). The Extracellular Matw protein 1 gene (Ecml) was localized either centromeric or telomeric of the EDC. Transcriots of Ecml exist in two forms. a 1.8 kb and an alternatively spliced 1.4 kb mRNA for human and a 1.9 kb and 1.5 kb for mouse The alternative splicing event has only been found I” the epidermis and in tonsils. We have analyzed the expression of Ecml RNA/protein during the differentiation of keratmocytes by IR sifu hybridization. Northern analysis and immunohistochemistry. We found that the splice variant is expressed in the upper layers of the epidermis of embryonic mice from day 16.5 d.p.c. onwards. This is the time period in the development of the embryonic epidermis around which the spinous layers are being formed. Cultures of differentiating human keratinocytes express the splice variant after the spinous layer markers type II keratin Kl and type I keratin KIO. The protein corresponding to the Ecml splice variant was localizedin the upper sp~nous and lower granular layers of the human epidermis Furthermore, the expression of the Ecml gene was found to be upregulated by sodium butyrate, an inducer of termlnal differentiation of keratnocytes. Refinement of the genomic localization placed the Ecmt gene centromeric of the marker DiS442. but at an estimated distance from the EDC of et least 2 Mbp. We conclude that the Ecml splice variant plays an es yet unknown role in the terminal differentiation of keratinocyfes and maps outside the EDC as it has been defined up to now.

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0426 RlDD SYNDROME:

RETINA,. DEGENERATION, ICHTHYOSIS, DEAFNESS AND DEVELOPMENTAL DELAY ASSOCIATGD WITH AN ABNORMA,JTY OF CHROMOSOME a,. shani Depamnem of Dermatology,