Cutaneous basement membrane formation in organotypic culture

The cutaneous basement membrane (BM) consists mainly of polymeric collagen-IV and laminin-10 and associated mono-/oligomeric laminin-5, nidogen, and perlecan. Since BM-defects in transgenic or knockout mice are mostly lethal at early developmental stages, we have studied the role of nidogen specifically in 3D-cocultures of human keratinocytes (HK) and fibroblasts (human/mouse, HF/MF) by either blocking interactions or implementing molecular deficiencies. HK or HaCaT cells were grown on collagen gels harboring HF or MF from normal or ko-mice. Nidogen-laminin interaction was blocked by the laminin-fragment (gamma-1-III3-5, L-gamma-f) binding nidogen. BM-formation was surveyed by immunofluorescence (IF), regular (EM), immuno-electron microscopy (IEM), and Western blots of protein extracts of separated epithelial and `dermal' tissue. In 3D-cocultures of HK and HF L-gamma-f blocked mainly deposition of nidogen, laminin-10, and perlecan. Whereas the hemidesmosomal/BM components laminin-5, BP180, and integrin alpha6beta4 were still detectable (IF), by EM and IEM any BM-structures or hemidesmosomes (insertion of keratins) were absent. The fibroblast-made nidogen was eliminated by employing MF from nidogen-1/-2 ko-mice. In 3D-cocultures with HaCaT cells nidogen1/2 (??/++)-MF abolished nidogen-1 staining, but (??/+?)-MF reduced additionally nidogen-2, collagen-IV, and laminin-10. Absence of nidogens (??/??) further abolished collagen-IV and laminin-5; integrins such as alpha6beta4 appear normal (IIF). BM-formation could be reinstalled with recombinant nidogen-1 or -2. BM-perlecan, for comparison, is apparently synthesized also by keratinocytes. Thus, deficiency in either cell type did not affect BM-formation, demonstrated by growing perlecan (?/?)-MF or HaCaT antisense-perlecan cells with normal keratinocytes or fibroblasts, respectively. Accordingly, BM-components are efficiently recruited for ultrastructural assembly in this skin model.