Active synthesis of type I collagen homotrimer in Dupuytren’s fibrosis is unaffected by anti–TNF-α treatment
Kate Williamson, Katie J. Lee, Emma L. Beamish, Alan Carter, Jade A. Gumbs, Gabriella Cooper, Niamh S. O’Heneghan-Yates, Lisa A. Menezes, Graham Cheung, Daniel Brown, Rob Pettitt, Brendan Geraghty, Lucy A. Bosworth, Eithne J. Comerford, Peter D. Clegg, Elizabeth G. Canty-Laird
Kate Williamson, Katie J. Lee, Emma L. Beamish, Alan Carter, Jade A. Gumbs, Gabriella Cooper, Niamh S. O’Heneghan-Yates, Lisa A. Menezes, Graham Cheung, Daniel Brown, Rob Pettitt, Brendan Geraghty, Lucy A. Bosworth, Eithne J. Comerford, Peter D. Clegg, Elizabeth G. Canty-Laird
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Research Article Cell biology Therapeutics

Active synthesis of type I collagen homotrimer in Dupuytren’s fibrosis is unaffected by anti–TNF-α treatment

Abstract

Dupuytren’s disease is a common fibroproliferative disease of the palmar fascia of the hand, with advanced cases treated surgically. Anti-TNF injection has undergone phase 2 trials and may be effective in slowing early-stage disease progression. Here we sought to determine how new synthesis of type I collagen in Dupuytren’s differs from normal palmar fascia samples and to analyze the role of TNF in aberrant collagen synthesis. Model nonfibrotic but fibrous connective tissues were used to analyze active type I collagen protein synthesis in development, aging, and degenerative disease, where it was restricted to early development and ruptured tissue. Dupuytren’s tissue was shown to actively synthesize type I collagen, including abnormal type I collagen homotrimer. TNF-α reduced COL1A2 gene expression only in the presence of serum in 2D cell culture and had opposing effects on collagen protein production in the presence or absence of serum. TNF-α had only limited effects in 3D tendon–like constructs. Anti-TNF did not reduce type I collagen synthesis in 3D tendon–like constructs or prevent type I collagen homotrimer synthesis in Dupuytren’s tissue. Hence, modulation of the TNF-α pathway in Dupuytren’s disease is unlikely to prevent the pathological collagen accumulation that is characteristic of fibrosis.

Authors

Kate Williamson, Katie J. Lee, Emma L. Beamish, Alan Carter, Jade A. Gumbs, Gabriella Cooper, Niamh S. O’Heneghan-Yates, Lisa A. Menezes, Graham Cheung, Daniel Brown, Rob Pettitt, Brendan Geraghty, Lucy A. Bosworth, Eithne J. Comerford, Peter D. Clegg, Elizabeth G. Canty-Laird

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Figure 5

Normal palmar fascia and Dupuytren’s cells fully process procollagen, assemble de novo collagen fibrils, and create tendon-like structures in 3D culture.

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Normal palmar fascia and Dupuytren’s cells fully process procollagen, as...
(A–C) Representative gel images (1 of 4 replicates) of delayed reduction electrophoresis of the media (A and C) or salt extractions (B) of normal palmar fascia and Dupuytren’s cells grown in 2D (A) or 3D (B and C) culture and labeled with [14C]proline. Vertical lines delineate noncontiguous spliced lanes from the same gel (B) or different gels (C). Fully processed collagen was found in extracellular extracts of the tendon-like constructs (B), whereas proforms, and potentially other collagens, were found in the media of both 2D (A) and 3D cultures (C). The type I procollagen processing intermediates were identified by comparison to labeled standards analyzed under reducing, nonreducing, and delayed reduction conditions and are indicated: “d” indicates disulphide-linked dimers of the respective (/) chains, “pro” indicates procollagen, “pC” indicates pC collagen (lacking the N-propeptide), “pN” indicates pN collagen (lacking the C-propeptide), and “α1” or “α2” indicates the fully processed α chain. For A–C, lanes 1, 8, and 14 are the control for lanes 2, 9, and 15, which were treated with TNF-α in the absence of serum, and lanes 3, 10, and 16 are the control for lanes 4, 11, and 17, which were treated with TNF-α in the presence of serum. Samples in lanes 6 and 12 were treated with IgG control and in lanes 7 and 13 with anti–TNF-α. Lane 5 is a standard of labeled type I procollagen and processed forms. (D–F) Semithin toluidine blue–stained sections of 3D tendon–like constructs. Scale bars: 200 μm. (G–I) Transmission electron microscopy images of 3D tendon–like constructs. Scale bars: 2 μm. Constructs were derived from normal palmar fascia cells (D and G), Dupuytren’s nodule (E and H),or Dupuytren’s cord (F and I) (1 of 4 replicates). Sample numbers are given in Supplemental Table 3.