Somatostatin Inhibits Cell Migration and Reduces Cell Counts of Human Keratinocytes and Delays Epidermal Wound Healing in an Ex Vivo Wound Model

The peptide hormone somatostatin (SST) and its five G protein-coupled receptors (SSTR1-5) were described to be present in the skin, but their cutaneous function(s) and skin-specific signalling mechanisms are widely unknown. By using receptor specific agonists we show here that the SSTRs expressed in keratinocytes are functionally coupled to the inhibition of adenylate cyclase. In addition, treatment with SSTR4 and SSTR5/1 specific agonists significantly influences the MAP kinase signalling pathway. As epidermal hormone receptors in general are known to regulate re-epithelialization following skin injury, we investigated the effect of SST on cell counts and migration of human keratinocytes. Our results demonstrate a significant inhibition of cell migration and reduction of cell counts by SST. We do not observe an effect on apoptosis and necrosis. Analysis of signalling pathways showed that somatostatin inhibits cell migration independent of its effect on cAMP. Migrating keratinocytes treated with SST show altered cytoskeleton dynamics with delayed lamellipodia formation. Furthermore, the activity of the small GTPase Rac1 is diminished, providing evidence for the control of the actin cytoskeleton by somatostatin receptors in keratinocytes. While activation of all receptors leads to redundant effects on cell migration, only treatment with a SSTR5/1 specific agonist resulted in decreased cell counts. In accordance with reduced cell counts and impaired migration we observe delayed re-epithelialization in an ex vivo wound healing model. Consequently, our experiments suggest SST as a negative regulator of epidermal wound healing

Interfering with VE-PTP stabilizes endothelial junctions in vivo via Tie-2 in the absence of VE-cadherin

Vascular endothelial (VE)-protein tyrosine phosphatase (PTP) associates with VE-cadherin, thereby supporting its adhesive activity and endothelial junction integrity. VE-PTP also associates with Tie-2, dampening the tyrosine kinase activity of this receptor that can support stabilization of endothelial junctions. Here, we have analyzed how interference with VE-PTP affects the stability of endothelial junctions in vivo. Blocking VE-PTP by antibodies, a specific pharmacological inhibitor (AKB-9778), and gene ablation counteracted vascular leak induction by inflammatory mediators. In addition, leukocyte transmigration through the endothelial barrier was attenuated. Interference with Tie-2 expression in vivo reversed junction-stabilizing effects of AKB-9778 into junction-destabilizing effects. Furthermore, lack of Tie-2 was sufficient to weaken the vessel barrier. Mechanistically, inhibition of VE-PTP stabilized endothelial junctions via Tie-2, which triggered activation of Rap1, which then caused the dissolution of radial stress fibers via Rac1 and suppression of nonmuscle myosin II. Remarkably, VE-cadherin gene ablation did not abolish the junction-stabilizing effect of the VE-PTP inhibitor. Collectively, we conclude that inhibition of VE-PTP stabilizes challenged endothelial junctions in vivo via Tie-2 by a VE-cadherin-independent mechanism. In the absence of Tie-2, however, VE-PTP inhibition destabilizes endothelial barrier integrity in agreement with the VE-cadherin-supportive effect of VE-PTP. © 2015 Frye et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http ://www .rupress .org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http ://creativecommons .org /licenses /by -nc -sa /3 .0 /).133351sciescopu

SST delays epidermal wound healing in a porcine <i>ex vivo</i> model.

<p>A: <i>Ex vivo</i> wound healing models from porcine ear skin were treated with SST for 48 h and compared to control models. Examples for hematoxylin/eosin stainings of control (upper picture) and SST treated (lower picture) models. The wound margin is indicated by an arrowhead and the regenerated epidermis is depicted by a dashed line above the model. While the control model shows complete re-epithelialization, application of SST inhibits wound closure. B Quantification of epidermal wound healing. Re-epithelialization was measured at both wound margins by an investigator blind to the experimental conditions. Data are depicted as mean +/− SEM; *, p<0.05. n = 6.</p

Inhibition of migration by SST is cAMP-independent.

<p>Quantification of cell migration in scratch assays after treatment with SST (1 µM), FSK (10 µM) or LPA (5 µM) as well as combinations of these substances. Data are presented as percentages of the recovered scratch area relative to untreated control cells (n = 5) Results are shown as means+/−SEM, * P<0.05, compared to controls; * P<0.05 between different treatment groups.</p

SSTR activation in keratinocytes modulates the MAP kinase pathway.

<p>A: MAP kinase activity assay. Cells were treated with 1 µM SST or 5% FCS (or both) for 5 or 10 min and lysates were analyzed with antibodies against ERK1/2 or phospho-ERK1/2 by Western blotting. B: The relative intensities (control value = 1) of 6 experiments after 5 min treatment were quantified and are shown as means +/− SEM (* P<0.05, *** P<0.005.). C: Effect of different SST receptor agonists on ERK phosphorylation (n = 5; * P<0.05, ** P<0.01), a representative blot is shown below. SST as well as the SSTR4-specific agonist significantly induces ERK phosphorylation, while treatment with agonist sst5/1 results in significantly decreased phospho-ERK levels.</p

Activation of SST receptors results in the inhibition of keratinocyte migration.

<p>A: Keratinocyte monolayers were scratch wounded and the scratched area was examined directly after scratching (0 h) and every 6 hours during an incubation period of 24 h. To prevent proliferative effects, cells were pre-exposed to X-ray irradiation to induce cell cycle arrest. B: Quantification of cell migration in scratch assays after SST stimulation (1 µM SST for 24 h). Data are presented as percentages of the recovered scratch area relative to untreated control cells (n = 7). C: Effect of selective SSTR agonists (1 µM for 24 h) on cell migration compared to untreated cells (n = 5). All subtype-specific agonists tested inhibit keratinocyte migration. Results are shown as means+/−SEM, * P<0.05, ** P<0.01, *** P<0.005.</p

SST delays lamellipodium formation in the early phase of keratinocyte migration.

<p>A: Lamellipodium formation in migrating keratinocytes. Cells were scratch wounded, treated as indicated, fixed after 3 h of migration and the actin cytoskeleton was visualized with fluorophor-labelled phalloidin. Lamellipodia are marked by an overlay of red pseudocolor. B: Areas of extending lamellipodia were measured after 3 h for each treatment and compared to control cells (n = 3, total number of analyzed cells is indicated inside bars, means+/−SEM, * P<0.05).</p