Limits...
Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo.

Ågren MS, Schnabel R, Christensen LH, Mirastschijski U - Eur. J. Cell Biol. (2014)

Bottom Line: Levels of the collagenases MMP-8 and MMP-13 were insignificant and neither MMP-2 nor MMP-14 were associated with increased collagen degradation.Type I collagen formation was down-regulated in cultured compared with native skin explants but was not reduced further by TNF-α.TNF-α had no significant effect on epidermal apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Copenhagen Wound Healing Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark; Digestive Disease Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark; Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address: saag0005@regionh.dk.

Show MeSH

Related in: MedlinePlus

MMP-1 and MMP-3 expression in control and TNF-α-treated skin analyzed by ELISA (A and D), Western blot (B and E) and β-casein zymography (F), and MMP-1 immunohistochemistry (C) and MMP-3 correlation to ICTP (G) after 8 days of incubation. (A and D) Pooled tissue extracts and media from five explants per donor and group were assayed. Mean ± SEM. **p < 0.01, ***p < 0.005 versus control. n = number of skin donors. (B and E) The PVDF membrane was first probed with the polyclonal MMP-1 antibody (B), then stripped and reprobed with the polyclonal MMP-3 antibody (E). Lanes 1 and 3, control; 2 and 4, TNF-α; 1 and 2, pooled tissue extract (18 μl/lane) from 30 individual 8-mm skin explants from the 6 donors (1–6) per group; lanes 3 and 4, pooled media (18 μl/lane) from 24 individual 8-mm skin explants from 5 donors (media from donor 1 was lost) per group. Std.: 42.7 kDa rhMMP-1 (14 ng). Glycosylated and nonglycosylated latent and active MMP-1/MMP-3 doublets are indicated. (C) Representative sections of native (a, d and g), or cultured skin explants from control (b, e and h) and TNF-α groups (c, f and i) treated with primary monoclonal MMP-1 antibody that detects latent and active forms (a–f) or with isotype (negative) control (g–i). (a–c, 40×; d–i, 900×). (F) Casein gels were incubated in the absence (Buffer) or presence of 10 μM GM6001. Lane 1, rhMMP-3 (5 ng); 2, pooled media from control-treated explants (15 μl); 3, pooled media from TNF-α-treated explants (15 μl); 4, rhMMP-1 (2 ng). Mark12™ (Life Technologies) molecular weight marker was run in parallel lane. Upper doublets represent latent forms of MMP-3 (upper bands) and MMP-1 (lower bands) and lower doublets active forms of MMP-3 (upper bands) and MMP-1 (lower bands). (G) Tissue MMP-3 contents and corresponding ICTP in media. Each symbol as indicated in Fig. 1 represents pooled tissue extracts and media from five explants per donor. The TNF-α-treated skin explants of donor 1 is missing due to lost pooled media sample. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4300401&req=5

fig0040: MMP-1 and MMP-3 expression in control and TNF-α-treated skin analyzed by ELISA (A and D), Western blot (B and E) and β-casein zymography (F), and MMP-1 immunohistochemistry (C) and MMP-3 correlation to ICTP (G) after 8 days of incubation. (A and D) Pooled tissue extracts and media from five explants per donor and group were assayed. Mean ± SEM. **p < 0.01, ***p < 0.005 versus control. n = number of skin donors. (B and E) The PVDF membrane was first probed with the polyclonal MMP-1 antibody (B), then stripped and reprobed with the polyclonal MMP-3 antibody (E). Lanes 1 and 3, control; 2 and 4, TNF-α; 1 and 2, pooled tissue extract (18 μl/lane) from 30 individual 8-mm skin explants from the 6 donors (1–6) per group; lanes 3 and 4, pooled media (18 μl/lane) from 24 individual 8-mm skin explants from 5 donors (media from donor 1 was lost) per group. Std.: 42.7 kDa rhMMP-1 (14 ng). Glycosylated and nonglycosylated latent and active MMP-1/MMP-3 doublets are indicated. (C) Representative sections of native (a, d and g), or cultured skin explants from control (b, e and h) and TNF-α groups (c, f and i) treated with primary monoclonal MMP-1 antibody that detects latent and active forms (a–f) or with isotype (negative) control (g–i). (a–c, 40×; d–i, 900×). (F) Casein gels were incubated in the absence (Buffer) or presence of 10 μM GM6001. Lane 1, rhMMP-3 (5 ng); 2, pooled media from control-treated explants (15 μl); 3, pooled media from TNF-α-treated explants (15 μl); 4, rhMMP-1 (2 ng). Mark12™ (Life Technologies) molecular weight marker was run in parallel lane. Upper doublets represent latent forms of MMP-3 (upper bands) and MMP-1 (lower bands) and lower doublets active forms of MMP-3 (upper bands) and MMP-1 (lower bands). (G) Tissue MMP-3 contents and corresponding ICTP in media. Each symbol as indicated in Fig. 1 represents pooled tissue extracts and media from five explants per donor. The TNF-α-treated skin explants of donor 1 is missing due to lost pooled media sample. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Mentions: There was no difference in total tissue MMP-1 contents between the TNF-α and control groups determined by the ELISA assay. On the other hand, MMP-1 content in media was increased with TNF-α present during incubation (Fig. 7A). Western blot analysis indicated that MMP-1 was in catalytically active glycosylated and nonglycosylated forms in the tissue while both latent and active MMP-1 glycosylated and nonglycosylated forms were found in about equal proportions in the media (Fig. 7B). Immunohistological staining of cultured skin explants clearly showed that MMP-1 was increased throughout the epidermis, being almost exclusively cytoplasmic (++/+++), and slightly reduced (+) extracellularly in the stroma compared with native skin. There were no apparent differences in localization or intensity of the MMP-1 immunoreactivity between control and TNF-α-treated skin explants. Native skin showed predominance of MMP-1 extracellularly in the stroma (++), most intensely in the papillary dermis, and in the nucleus (++) of scattered keratinocytes in epidermis (Fig. 7C). Nuclear MMP-1 was also observed in sections without pre-treatment for antigen retrieval although the signal was weaker than with pre-treatment. In contrast, MMP-1 was entirely cytoplasmic in human oral epithelium (Supplementary Fig. S1).


Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo.

Ågren MS, Schnabel R, Christensen LH, Mirastschijski U - Eur. J. Cell Biol. (2014)

MMP-1 and MMP-3 expression in control and TNF-α-treated skin analyzed by ELISA (A and D), Western blot (B and E) and β-casein zymography (F), and MMP-1 immunohistochemistry (C) and MMP-3 correlation to ICTP (G) after 8 days of incubation. (A and D) Pooled tissue extracts and media from five explants per donor and group were assayed. Mean ± SEM. **p < 0.01, ***p < 0.005 versus control. n = number of skin donors. (B and E) The PVDF membrane was first probed with the polyclonal MMP-1 antibody (B), then stripped and reprobed with the polyclonal MMP-3 antibody (E). Lanes 1 and 3, control; 2 and 4, TNF-α; 1 and 2, pooled tissue extract (18 μl/lane) from 30 individual 8-mm skin explants from the 6 donors (1–6) per group; lanes 3 and 4, pooled media (18 μl/lane) from 24 individual 8-mm skin explants from 5 donors (media from donor 1 was lost) per group. Std.: 42.7 kDa rhMMP-1 (14 ng). Glycosylated and nonglycosylated latent and active MMP-1/MMP-3 doublets are indicated. (C) Representative sections of native (a, d and g), or cultured skin explants from control (b, e and h) and TNF-α groups (c, f and i) treated with primary monoclonal MMP-1 antibody that detects latent and active forms (a–f) or with isotype (negative) control (g–i). (a–c, 40×; d–i, 900×). (F) Casein gels were incubated in the absence (Buffer) or presence of 10 μM GM6001. Lane 1, rhMMP-3 (5 ng); 2, pooled media from control-treated explants (15 μl); 3, pooled media from TNF-α-treated explants (15 μl); 4, rhMMP-1 (2 ng). Mark12™ (Life Technologies) molecular weight marker was run in parallel lane. Upper doublets represent latent forms of MMP-3 (upper bands) and MMP-1 (lower bands) and lower doublets active forms of MMP-3 (upper bands) and MMP-1 (lower bands). (G) Tissue MMP-3 contents and corresponding ICTP in media. Each symbol as indicated in Fig. 1 represents pooled tissue extracts and media from five explants per donor. The TNF-α-treated skin explants of donor 1 is missing due to lost pooled media sample. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300401&req=5

fig0040: MMP-1 and MMP-3 expression in control and TNF-α-treated skin analyzed by ELISA (A and D), Western blot (B and E) and β-casein zymography (F), and MMP-1 immunohistochemistry (C) and MMP-3 correlation to ICTP (G) after 8 days of incubation. (A and D) Pooled tissue extracts and media from five explants per donor and group were assayed. Mean ± SEM. **p < 0.01, ***p < 0.005 versus control. n = number of skin donors. (B and E) The PVDF membrane was first probed with the polyclonal MMP-1 antibody (B), then stripped and reprobed with the polyclonal MMP-3 antibody (E). Lanes 1 and 3, control; 2 and 4, TNF-α; 1 and 2, pooled tissue extract (18 μl/lane) from 30 individual 8-mm skin explants from the 6 donors (1–6) per group; lanes 3 and 4, pooled media (18 μl/lane) from 24 individual 8-mm skin explants from 5 donors (media from donor 1 was lost) per group. Std.: 42.7 kDa rhMMP-1 (14 ng). Glycosylated and nonglycosylated latent and active MMP-1/MMP-3 doublets are indicated. (C) Representative sections of native (a, d and g), or cultured skin explants from control (b, e and h) and TNF-α groups (c, f and i) treated with primary monoclonal MMP-1 antibody that detects latent and active forms (a–f) or with isotype (negative) control (g–i). (a–c, 40×; d–i, 900×). (F) Casein gels were incubated in the absence (Buffer) or presence of 10 μM GM6001. Lane 1, rhMMP-3 (5 ng); 2, pooled media from control-treated explants (15 μl); 3, pooled media from TNF-α-treated explants (15 μl); 4, rhMMP-1 (2 ng). Mark12™ (Life Technologies) molecular weight marker was run in parallel lane. Upper doublets represent latent forms of MMP-3 (upper bands) and MMP-1 (lower bands) and lower doublets active forms of MMP-3 (upper bands) and MMP-1 (lower bands). (G) Tissue MMP-3 contents and corresponding ICTP in media. Each symbol as indicated in Fig. 1 represents pooled tissue extracts and media from five explants per donor. The TNF-α-treated skin explants of donor 1 is missing due to lost pooled media sample. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Mentions: There was no difference in total tissue MMP-1 contents between the TNF-α and control groups determined by the ELISA assay. On the other hand, MMP-1 content in media was increased with TNF-α present during incubation (Fig. 7A). Western blot analysis indicated that MMP-1 was in catalytically active glycosylated and nonglycosylated forms in the tissue while both latent and active MMP-1 glycosylated and nonglycosylated forms were found in about equal proportions in the media (Fig. 7B). Immunohistological staining of cultured skin explants clearly showed that MMP-1 was increased throughout the epidermis, being almost exclusively cytoplasmic (++/+++), and slightly reduced (+) extracellularly in the stroma compared with native skin. There were no apparent differences in localization or intensity of the MMP-1 immunoreactivity between control and TNF-α-treated skin explants. Native skin showed predominance of MMP-1 extracellularly in the stroma (++), most intensely in the papillary dermis, and in the nucleus (++) of scattered keratinocytes in epidermis (Fig. 7C). Nuclear MMP-1 was also observed in sections without pre-treatment for antigen retrieval although the signal was weaker than with pre-treatment. In contrast, MMP-1 was entirely cytoplasmic in human oral epithelium (Supplementary Fig. S1).

Bottom Line: Levels of the collagenases MMP-8 and MMP-13 were insignificant and neither MMP-2 nor MMP-14 were associated with increased collagen degradation.Type I collagen formation was down-regulated in cultured compared with native skin explants but was not reduced further by TNF-α.TNF-α had no significant effect on epidermal apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Copenhagen Wound Healing Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark; Digestive Disease Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark; Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address: saag0005@regionh.dk.

Show MeSH
Related in: MedlinePlus