Project Details
Projekt
Regulation der Basalmembranbildung in organotypischen Kokulturen von Haut-Keratinozyten (inklusive Zell-Linien) und Fibroblasten
Applicant
Privatdozent Dr. Dirk Breitkreutz
Subject Area
Dermatology
Term
from 2000 to 2007
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 5250400
The course of basement membrane (BM)-formation is studied in organotypic cocultures (OTC) of normal (NHK) and transformed human keratinocytes on collagen gels with fibroblasts. Based on regular and serum-free media the influence of external factors is investigated such as transforming growth factor ß (TGFß) and retinoic acid (RA) but also of matrix components secreted by the keratinocytes or fibroblasts. In major focus are (i) laminin-5 (LN-5, made by keratinocytes only) and nidogen (ND, an exclusive fibroblast product) as early BM-constituents, (ii) their influence on expression and localization of the BM-laminin (LN-10), type IV collagen (CIV), perlecan, and further (iii) of the main components of hemidesmosomes (HD) integrin alpha6beta4, BP180, BP230, and plectin / HD1. The latter (HD) provide stable epidermal attachment. Besides NHK, HaCaT cells (lower LN-5 synthesis) and tumorigenic HaCaT-ras cells, represented by the benign I-5 and the malignant II-4 clone (lower BM synthesis in general), are employed. By pre-coating with matrix molecules (e.g. fibronectin, soluble LN 5, CIV) adhesion and HD-formation shall be promoted in OTC, particularly of II-4. This may provoke phenotypic changes (in II-4 towards "normalization"?) by influencing epithelial polarity and also growth. Besides IIF, regular EM and immuno-EM, RNA and protein analysis shall elucidate the dynamics (i) of synthesis and assembly, (ii) secondary modifications especially of ND and LN-5 (mainly the alpha3 and gamma2 chain), and (iii) if in the normal situation established BM-structures provide a negative feedback on BM-synthesis, down-regulating the expression of specific "early" genes (such as LN-5). (IV) With time, control by matrix molecules may become leaky in transformed epithelial cells dependent on the degree of malignancy.
DFG Programme
Priority Programmes
