There is temporal and spatial expression of alpha 1 (IV), alpha 2 (IV), alpha 5 (IV), alpha 6 (IV) collagen chains and beta 1 integrins during the development of the basal lamina in an ''in vitro'' skin model

Temporal and spatial expression of alpha 1 (IV), alpha 2 (IV), alpha 3 (IV), alpha 4 (IV), alpha 5 (IV), and alpha 6 (IV) collagen chains was studied during the formation of the basal lamina in an ''in vitro'' skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta 1, beta 4, alpha 1, alpha 2, alpha 3, alpha 5, alpha 6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha 2 (IV) and alpha 6 (IV) chains was estimated by northern blots. The earliest expression of alpha 1 (IV) and alpha 2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha 1 (IV) and alpha 2 (IV) chains were noted in basal keratinocytes and as a broad band (10 mu m) in the adjacent dermis. At this stage 80% of the alpha 2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha 1 (IV) and alpha 2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha 6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha 5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. alpha 6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta 1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta 1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha 1 beta 1, alpha 2 beta 1 integrins, altered the pattern of collagen IV deposition.