|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Overexpression of the gap junction protein Cx43 as found in diabetic foot ulcers can retard fibroblast migration
Ariadna Mendoza‑Naranjo*1, Peter Cormie*1, Antonio E. Serrano*, Chuihui M. Wang*, Christopher Thrasivoulou*, Jessica E.S. Sutcliffe*, Daniel J. Gilmartin*, Janice Tsui†, Thomas E. Serena‡, Anthony R.J. Phillips§ and David L. Becker*2
*Department of Cell and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, U.K., †Department of General Surgery, University College London, Gower Street, London, WC1E 6BT, U.K., ‡311 Pennsylvania Avenue West, Warren, PA 16365, U.S.A., and §CoDa Therapeutics Inc, 111 Jervois Road, Herne Bay, Auckland, New Zealand
Poor healing of DFUs (diabetic foot ulcers) is a major clinical problem that can be extremely debilitating and lead to lower limb amputation. In the normal acute wound, the Cx43 (connexin 43) gap junction protein is down-regulated at the wound edge as a precursor to cell migration and healing. In fibroblasts from the human chronic DFU wound edge there was a striking and significant 10-fold elevation of Cx43 protein, as well as a 6-fold increase in N-cadherin and a 2-fold increase in ZO-1 (zonular occludin-1), compared with unwounded skin. In streptozotocin diabetic rats, Cx43 was found to be up-regulated in intact dermal fibroblasts in direct proportion to blood glucose levels and increased 2-fold further in response to wounding of the skin. To mimic diabetes, NIH 3T3 fibroblasts were cultured under different concentrations of glucose or mannitol and Cx43 protein intercellular communication and migration rates were determined. Cultures of fibroblasts in very high (40 mM) glucose conditions showed significantly elevated Cx43 protein levels, as shown by immunostaining and Western blotting, and significantly increasing gap junctional communication, as shown by dye transfer. In scratch wound-healing assays, increased levels of Cx43 from high glucose resulted in repressed filopodial extensions and significantly slower migration rates than in either standard conditions (5.5 mM glucose) or the osmotic control of mannitol. Conversely, when glucose-induced Cx43 up-regulation was prevented with Cx43shRNA (Cx43 short-hairpin RNA) transduction, the fibroblasts extended long filopodia and migrated significantly faster. Cx43 protein was up-regulated in fibroblasts in DFUs as well as after high glucose exposure in culture which correlated with inhibition of fibroblast migration and is likely to contribute to impaired wound healing.
Key words: antisense, chronic wound, Connexin 43, diabetic foot ulcer, migration, wound healing
Abbreviations: Cx43, connexin 43, Cx43shRNA, Cx43 short-hairpin RNA, DFU, diabetic foot ulcer, FGF, fibroblast growth factor, GAP27, GTPase-activating protein 27, p.Sup, pSuppressor, STZ, streptozotocin, ZO-1, zonular occludin-1
1These authors equally contributed to this work.
2To whom correspondence should be addressed (email firstname.lastname@example.org).
Received 15 November 2011/2 March 2012; accepted 28 March 2012
Published as Cell Biology International Immediate Publication 28 March 2012, doi:10.1042/CBI20110628
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology