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

Control of basement membrane formation in skin-organotypic 3d-coculture
Control of basement membrane formation in skin-organotypic 3d-coculture
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.
0022-202X
A87-A90
Schmidt, C.
0797aee8-b87e-4d53-a425-1bda4deea665
Mirancea, N.
758b66cb-45bb-473d-9914-b340b8e25e8b
Nischt, R.
d9c0e203-a320-4d45-a6ef-d0d0da847acd
Smyth, N.
0eba2a40-3b43-4d40-bb64-621bd7e9d505
Werner, U.
6c6f826b-0535-4ee1-8fd6-6fa89668d577
Stark, H.J.
902f801c-a8be-4c54-bc48-c3553e96b8f4
Fusenig, N.E.
3bef1aa6-48c3-4857-a324-7f7962afb59a
Gerl, M.
07950999-1018-4e7f-9152-8e8a556a3c1a
Breitkreutz, D.
e59f7a35-f5de-41c9-bc5f-34436d738c1e
Schmidt, C.
0797aee8-b87e-4d53-a425-1bda4deea665
Mirancea, N.
758b66cb-45bb-473d-9914-b340b8e25e8b
Nischt, R.
d9c0e203-a320-4d45-a6ef-d0d0da847acd
Smyth, N.
0eba2a40-3b43-4d40-bb64-621bd7e9d505
Werner, U.
6c6f826b-0535-4ee1-8fd6-6fa89668d577
Stark, H.J.
902f801c-a8be-4c54-bc48-c3553e96b8f4
Fusenig, N.E.
3bef1aa6-48c3-4857-a324-7f7962afb59a
Gerl, M.
07950999-1018-4e7f-9152-8e8a556a3c1a
Breitkreutz, D.
e59f7a35-f5de-41c9-bc5f-34436d738c1e

Schmidt, C., Mirancea, N., Nischt, R., Smyth, N., Werner, U., Stark, H.J., Fusenig, N.E., Gerl, M. and Breitkreutz, D. (2004) Control of basement membrane formation in skin-organotypic 3d-coculture. Journal of Investigative Dermatology, 123 (5), A87-A90. (doi:10.1111/j.1523-1747.2004.23519_19.x).

Record type: Article

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.

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Published date: 1 November 2004

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Local EPrints ID: 56152
URI: http://eprints.soton.ac.uk/id/eprint/56152
ISSN: 0022-202X
PURE UUID: 48bebf2c-5df2-47d0-a7e4-5f8ce6b4925d

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Date deposited: 11 Aug 2008
Last modified: 08 Jan 2022 13:03

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Contributors

Author: C. Schmidt
Author: N. Mirancea
Author: R. Nischt
Author: N. Smyth
Author: U. Werner
Author: H.J. Stark
Author: N.E. Fusenig
Author: M. Gerl
Author: D. Breitkreutz

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