Tight Junction Proteins Claudin-1 and Occludin Are Important for Cutaneous Wound Healing

Tight junction (TJ) proteins are known to be involved in proliferation and differentiation. These processes are essential for normal skin wound healing. Here, we investigated the TJ proteins claudin-1 and occludin in ex vivo skin wound healing models and tissue samples of acute and chronic human wounds and observed major differences in localization/expression of these proteins, with chronic wounds often showing a loss of the proteins at the wound margins and/or in the regenerating epidermis. Knockdown experiments in primary human keratinocytes showed that decreased claudin-1 expression resulted in significantly impaired scratch wound healing, with delayed migration and reduced proliferation. Activation of AKT pathway was significantly attenuated after claudin-1 knockdown, and protein levels of extracellular signal-related kinase 1/2 were reduced. For occludin, down-regulation had no impact on wound healing in normal scratch assays, but after subjecting the cells to mechanical stress, which is normally present in wounds, wound healing was impaired. For both proteins we show that most of these actions are independent from the formation of barrier-forming TJ structures, thus demonstrating nonbarrier-related functions of TJ proteins in the skin. However, for claudin-1 effects on scratch wound healing were more pronounced when TJs could form. Together, our findings provide evidence for a role of claudin-1 and occludin in epidermal regeneration with potential clinical importance

Comparison of scratch wound healing of human primary keratinocytes of various origins and under various culture conditions.

<p>Closed wound area per visual field (mm²) of (A) diabetic versus non-diabetic keratinocytes at 4, 8, 12, 24 and 36 h after wounding using K-SFM medium. n = 4 in duplicates (non-diabetic keratinocytes) and n = 3 in duplicates (diabetic keratinocytes) (B) Keratinocytes from juvenile donors under euglycaemic (6 mM glucose), hyperglycaemic (25 mM glucose) and hyperosmolar (6 mM glucose + 19 mM mannitol) conditions at 12 and 24 h after wounding in K-SFM, Dermalife and Epilife medium. n = 4 in duplicates. (C) Keratinocytes from adult, non-diabetic donors under euglycaemic (6 mM) and hyperglycaemic (25 mM) conditions 4, 8, 12, 24 and 36 h after wounding in K-SFM medium. n = 4 in duplicates. (D) Keratinocytes from juvenile donors under euglycaemic, hyperglycaemic and hyperosmolar conditions at 12 and 24 h after wounding in K-SFM without supplements. n = 4 in duplicates; mean ± SEM; *: statistically significant with p < 0.05.</p

Influence of TE on reepithelialization of hyperglycaemic and hyperosmolaric ex-vivo models.

<p>Length of the regenerated epidermis of porcine <i>ex-vivo</i> wound healing models that were treated with TE oleogel, W/O emulsion (60%) containing 10% TE oleogel, PDGF (120 ng/ml) or PBS directly after wounding for 48 h under hyperglycaemic or hyperosmolaric conditions (including preincubation); n = 16 in duplicates; mean ± SEM; *: statistically significant with p < 0.05.</p

Chemical composition and physical characteristics of the TE used.

<p>Chemical composition and physical characteristics of the TE used.</p

Influence of TE on cytokine expression under various glucose concentrations (n = 4).

<p>Influence of TE on cytokine expression under various glucose concentrations (n = 4).</p

<i>Ex-vivo</i> porcine wound healing models cultured under euglycaemic, hyperglycaemic and hyperosmolar conditions.

<p>(A) Length of the regenerated epidermis for the experimental settings with and without preincubation for 48 h prior to wounding under euglycaemic (6 mM glucose), hyperglycaemic (50 mM glucose) and hyperosmolar (6 mM glucose + 44 mM mannitol) conditions 48 h after wounding. n = 8 in triplicates; mean ± SEM; *: statistically significant with p < 0.05; (B) Yellow coloring of the skin biopsies cultured under hyperglycaemic conditions for a total of 96 h (48 h preincubation and 48 h after wounding).</p