There is temporal and spatial expression of α1 (IV), α2 (IV), α5 (IV), α6 (IV) collagen chains and β1 integrins during the development of the basal lamina in an 'in vitro' skin model

Temporal and spatial expression of α1 (IV), α2 (IV), α3 (IV), α4 (IV), α5 (IV), and α6 (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 β1, β4, α1, α2, α3, α5, α6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of α2 (IV) and α6 (IV) chains was estimated by northern blots. The earliest expression of α1 (IV) and α2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, α1 (IV) and α2 (IV) chains were noted in basal keratinocytes and as a broad band (10 μm) in the adjacent dermis. At this stage 80% of the α2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the α1 (IV) and α2 (IV) chains were present in a linear distribution at the epidermodermal junction and in the upper dermis. The α6 (IV) collagen chains were expressed much later at 36-d cultures and the α5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. α6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and β1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-β1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for α1β1, α2β1 integrins, altered the pattern of collagen IV deposition.