Expression of laminin 5 by parental and c-Ha-ras-transformed HaCaT keratinocytes in organotypic cultures.
Tumor cells traverse the basement membrane zone and gain access to the underlying mesenchyme to eventually form metastases. Laminin 5 is a major component of the basement membrane and connects keratinocytes at the level of hemidesmosomes to the mesenchyme. Underneath invading tumor cells anti-laminin 5 staining is diminished, and laminin 5 degradation products can stimulate cell migration and epidermal growth factor (EGF) receptor signaling. To investigate laminin 5 expression in parental HaCaT and tumorigenic c-Ha-ras-transformed HaCaT II-4rt keratinocytes, the cells were cultivated under monolayer and organotypic culture conditions. In monolayer cultures, HaCaT and c-Ha-ras-transformed HaCaT II-4rt keratinocytes secreted comparable amounts of laminin 5. After 7 days of organotypic cultures, collagen IV, beta4-integrin, nidogen and laminin 5 were detected along the epithelial-mesenchymal interface of parental HaCaT keratinocytes, while staining for these proteins was patchy or absent in the organotypic cultures with c-Ha-ras-transformed HaCaT II-4rt cells. Immunoblotting analysis confirmed absence of laminin 5 deposition in organotypic cultures of c-Ha-ras-transformed HaCaT II-4rt while the protein was detected in organotypic cultures of HaCaT keratinocytes. Surprisingly, however, the alpha3 and gamma2 laminin chain transcripts were strongly induced in c-Ha-ras-transformed HaCaT II-4rt cells by organotypic culture conditions, indicating that invasive epidermal tumor cells retain high mRNA levels for laminin 5 chains and suggesting an autocrine/paracrine induction of the laminin chain mRNAs. Moreover, as laminin 5 was absent in organotypic cultures of c-Ha-ras-transformed HaCaT II-4rt cells, it suggests immediate degradation of the protein. Degradation products may further contribute to the malignant phenotype by enhancing cellular migration and EGF-receptor activation.
Zinn
Michaela
M
Department of Dermatology, University of Cologne, Cologne, Germany.
Aumailley
Monique
M
Krieg
Thomas
T
Smola
Hans
H
D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D011905:Genes, ras; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D046509:Tissue Culture Techniques; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D016207:Cytokines; D016326:Extracellular Matrix Proteins; D036341:Intercellular Signaling Peptides and Proteins; D000097794:Kalinin