Control of basement membrane formation in skin-organotypic 3d-coculture

Abstract

Basement membrane (BM) formation was functionally dissected in 3d-cocultures of human keratinocytes (HK) and fibroblasts (human/mouse, HF/MFf) by either blocking interactions or implementing molecular deficiencies. This was supposed to complement knockout mouse studies, where loss or functional defects of collagen-IV, laminins, nidogen, or perlecan are causing embryonic or neonatal death. HK or HaCaT cells were grown on collagen gels harboring hf or mf from normal or ko-mice. To block nidogen-binding to laminin-10 the corresponding laminin-fragment (gamma1-iii3-5, L-gamma-f) was applied. BM-formation was surveyed by immunofluorescence (IF), regular (EM) and immuno-electron microscopy (IEM). In 3d-cocultures of HK and HF L-gamma-f blocked deposition of nidogen, laminin-10, and perlecan, while collagen-IV appeared normal. Although the hemidesmosome components laminin-5, BP180, and integrin alpha6beta4 were only mildly affected, EM and IEM revealed complete absence of BM, hemidesmosomes, and basal insertion of keratin filaments. To eliminate nidogen, made by fibroblasts, MF from nidogen1/nidogen2 ko-mice or crossbreds were employed. In 3d-cocultures with HaCaT cells nidogen1/2 (??/++)-MF abolished nidogen1-staining, but (??/+?)-mf reduced also largely nidogen2, collagen-IV, and drastically laminin-10. Total absence of nidogen (??/??) also deleted collagen-IV & laminin-5, integrins e.g. alpha6beta4 appearing still normal (IF). BM-formation could be entirely rescued by applying recombinant nidogens. In skin, perlecan can be apparently synthesized by both keratinocytes & fibroblasts. Accordingly, deficiency in either cell type did not affect BM-formation, demonstrated by combining either perlecan (?/?)-mf or HaCaT anti-sense-perlecan cells with respective normal partner cells. Thus, in this skin model BM-components are efficiently transported to their actual assembly site.