Basement Membrane Reconstruction in Human Skin Equivalents Is Regulated by Fibroblasts and/or Exogenously Activated Keratinocytes

Abstract

This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence of serum and exogenous growth factors. The expression of various BM components was analyzed on the protein and mRNA level. Irrespective of the presence or absence of fibroblasts, keratin 14, hemidesmosomal proteins plectin, BP230 and BP180, and integrins alpha1beta1, alpha2beta1, alpha3beta1, and alpha6beta4 were expressed but laminin 1 was absent. Only in the presence of fibroblasts or of various growth factors, laminin 5 and laminin 10/11, nidogen, uncein, type IV and type VII collagen were decorating the dermal/epidermal junction. These findings indicate that the attachment of basal keratinocytes to the dermal matrix is most likely mediated by integrins alpha1beta1 and alpha2beta1, and not by laminins that bind to integrin alpha6beta4 and that the epithelial-mesenchymal cross-talk plays an important role in synthesis and deposition of various BM components.

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

“…Factors released by fibroblasts resulted in the expression of basement membrane components (e.g. collagen type IV and VII, laminin 332, nidogen, BP-180, and LAD-1) in organotypic keratinocyte monocultures (32). In this study, similar results have been obtained for the ECM1 molecule as well as for PLSCR1, demonstrating that ECM1 and PLSCR1 were deposited and localize at the DEJ zone in reconstructed skin.…”

“…In this study, we used human skin equivalents to identify whether growth factors are able to stimulate the expression of ECM1 and/or PLSCR1. We tested EGF as a growth factor because this protein is known to induce basement membrane formation in organotypic keratinocyte monocultures (32). EGF is also able to activate PLSCR1 by inducing PLSCR1 to bind to the EGF receptor (EGFR) (40,41).…”

“…EGF is also able to activate PLSCR1 by inducing PLSCR1 to bind to the EGF receptor (EGFR) (40,41). Furthermore, human skin equivalents have been used because of the crucial role fibroblasts play in basement membrane formation (27,32). Factors released by fibroblasts resulted in the expression of basement membrane components (e.g.…”

“…The effects of EGF on ECM1 and PLSCR1 expression in organotypic keratinocyte monocultures were investigated as well. This cytokine is an excellent control to follow basement membrane formation because it activates the expression of various basement membrane proteins (32). For this purpose, 1 ng/ml EGF was supplemented to the culture medium of skin cultures, cultured for 2 weeks at the air/liquid interface.…”

Desenvolvimento de epiderme equivalente sobre membrana do tipo transwell e membrana biopolimérica

“…A citoqueratina 14 (CK14), por exemplo, é expressa majoritariamente na camada basal do epitélio (seta vermelha CK14), em células indiferenciadas altamente proliferativas já a citoqueratina 10 (CK10) e a involucrina são expressas na camada suprabasal e extratos mais exernos da epiderme (estrato espinhoso e granuloso), representando marcadores de diferenciação tardia do epitélio. Estes dois últimos marcadores são os primeiros a serem expressos quando a célula inicia seu processo de diferenciação (Fuchs, 1990;El Ghalbzouri et al, 2005;Lamb e Ambler, 2013). Na figura 23 B é possível verificar a expressão de citoqueratina 10 na camada suprabasal (seta vermelha CK10), conforme observado na maioria dos modelos de epiderme equivalente e na pele humana (Boelsma et al, 2000).…”