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

Digestion of native type I collagen by tissue-derived proteinases of native human skin (A), control (A and B) and TNF-α-treated human skin (A–C), active rhMMP-1 (D–F) or trypsin (F). (A–C) Tissue extract pools from 30 individual 8-mm skin explants (5 explants from each of the 6 donors) per group were concentrated 3× (Amicon® Ultra; Millipore). S, substrate. (B–D) Enzymes were incubated for 2 h with inhibitors before the substrate was added. (D) rhMMP-1 was incubated with substrate in the absence or presence of UK370106. (A–D, F) Collagenolytic activity in percentage of type I collagen degradation is shown below each lane. (E) Effect of rhMMP-1 on collagenolysis as a function of concentration and time of incubation (inset, 1 ng/ml). (F) Trypsin (Worthington, Lakewood, NJ, USA) treatment of native or denatured (56 °C, 30 min) substrate was carried out at identical assay conditions. rhMMP-1, 2.5 ng/ml. *, position of trypsin. D, denatured.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig0030: Digestion of native type I collagen by tissue-derived proteinases of native human skin (A), control (A and B) and TNF-α-treated human skin (A–C), active rhMMP-1 (D–F) or trypsin (F). (A–C) Tissue extract pools from 30 individual 8-mm skin explants (5 explants from each of the 6 donors) per group were concentrated 3× (Amicon® Ultra; Millipore). S, substrate. (B–D) Enzymes were incubated for 2 h with inhibitors before the substrate was added. (D) rhMMP-1 was incubated with substrate in the absence or presence of UK370106. (A–D, F) Collagenolytic activity in percentage of type I collagen degradation is shown below each lane. (E) Effect of rhMMP-1 on collagenolysis as a function of concentration and time of incubation (inset, 1 ng/ml). (F) Trypsin (Worthington, Lakewood, NJ, USA) treatment of native or denatured (56 °C, 30 min) substrate was carried out at identical assay conditions. rhMMP-1, 2.5 ng/ml. *, position of trypsin. D, denatured.

Mentions: We next assessed the collagenolytic activity using exogenous native type I skin collagen. Pooled tissue extracts of TNF-α-treated skin explants possessed increased collagenolytic activity (34%) compared with control-treated (19%) skin explants. The difference in collagenolytic activity between TNF-α and control-treated skin was more pronounced with the MMP-activator APMA during the assay. The collagenolytic activity of tissue extracts did not decline appreciably with length of incubation. No collagenolytic activity was detected in native skin (Fig. 5A). The MMP inhibitors GM6001 and TIMP-1 blocked collagenolysis. Moreover, a monoclonal neutralizing antibody against hMMP-1 abolished the collagenolytic activity of tissue extracts of incubated skin explants from the two groups (Fig. 5B). The MMP-1 antibody (10 μg/ml) also completely abrogated the collagenolytic activity (85%) of control day-8 pooled media from donor 1. Selective MMP-3 inhibition with UK370106 reduced type I collagenolysis by about 60% in the presence of APMA but less in the absence of APMA. UK370106 at 1 μM did not decrease the collagenolytic activity of rhMMP-1 (Fig. 5B–D). The assay was linear to about 30% collagenolysis (Fig. 5E). The type I collagen substrate was resistant to trypsin while denatured substrate was completely digested. rhMMP-1 generated the anticipated 3/4 and 1/4 fragments of the α1(I) and α2(I) chains (Fig. 5F).


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)

Digestion of native type I collagen by tissue-derived proteinases of native human skin (A), control (A and B) and TNF-α-treated human skin (A–C), active rhMMP-1 (D–F) or trypsin (F). (A–C) Tissue extract pools from 30 individual 8-mm skin explants (5 explants from each of the 6 donors) per group were concentrated 3× (Amicon® Ultra; Millipore). S, substrate. (B–D) Enzymes were incubated for 2 h with inhibitors before the substrate was added. (D) rhMMP-1 was incubated with substrate in the absence or presence of UK370106. (A–D, F) Collagenolytic activity in percentage of type I collagen degradation is shown below each lane. (E) Effect of rhMMP-1 on collagenolysis as a function of concentration and time of incubation (inset, 1 ng/ml). (F) Trypsin (Worthington, Lakewood, NJ, USA) treatment of native or denatured (56 °C, 30 min) substrate was carried out at identical assay conditions. rhMMP-1, 2.5 ng/ml. *, position of trypsin. D, denatured.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig0030: Digestion of native type I collagen by tissue-derived proteinases of native human skin (A), control (A and B) and TNF-α-treated human skin (A–C), active rhMMP-1 (D–F) or trypsin (F). (A–C) Tissue extract pools from 30 individual 8-mm skin explants (5 explants from each of the 6 donors) per group were concentrated 3× (Amicon® Ultra; Millipore). S, substrate. (B–D) Enzymes were incubated for 2 h with inhibitors before the substrate was added. (D) rhMMP-1 was incubated with substrate in the absence or presence of UK370106. (A–D, F) Collagenolytic activity in percentage of type I collagen degradation is shown below each lane. (E) Effect of rhMMP-1 on collagenolysis as a function of concentration and time of incubation (inset, 1 ng/ml). (F) Trypsin (Worthington, Lakewood, NJ, USA) treatment of native or denatured (56 °C, 30 min) substrate was carried out at identical assay conditions. rhMMP-1, 2.5 ng/ml. *, position of trypsin. D, denatured.
Mentions: We next assessed the collagenolytic activity using exogenous native type I skin collagen. Pooled tissue extracts of TNF-α-treated skin explants possessed increased collagenolytic activity (34%) compared with control-treated (19%) skin explants. The difference in collagenolytic activity between TNF-α and control-treated skin was more pronounced with the MMP-activator APMA during the assay. The collagenolytic activity of tissue extracts did not decline appreciably with length of incubation. No collagenolytic activity was detected in native skin (Fig. 5A). The MMP inhibitors GM6001 and TIMP-1 blocked collagenolysis. Moreover, a monoclonal neutralizing antibody against hMMP-1 abolished the collagenolytic activity of tissue extracts of incubated skin explants from the two groups (Fig. 5B). The MMP-1 antibody (10 μg/ml) also completely abrogated the collagenolytic activity (85%) of control day-8 pooled media from donor 1. Selective MMP-3 inhibition with UK370106 reduced type I collagenolysis by about 60% in the presence of APMA but less in the absence of APMA. UK370106 at 1 μM did not decrease the collagenolytic activity of rhMMP-1 (Fig. 5B–D). The assay was linear to about 30% collagenolysis (Fig. 5E). The type I collagen substrate was resistant to trypsin while denatured substrate was completely digested. rhMMP-1 generated the anticipated 3/4 and 1/4 fragments of the α1(I) and α2(I) chains (Fig. 5F).

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