Cutaneous basement membrane formation in organotypic culture

Woenne, E., Schmidt, C., Mirancea, N., Nischt, R., Smyth, N., Werner, U., Fusenig, N.E., Gerl, M. and Breitkreutz, D. (2004) Cutaneous basement membrane formation in organotypic culture. Journal of Investigative Dermatology, 123 (5), A87-A97. (doi:10.1111/j.1523-1747.2004.23519_53.x).

Abstract

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.

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