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

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

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

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 decreases Rac1 activity in migrating keratinocytes.

<p>A: The amount of active Rac1 was determined by affinity precipitation with purified GST-PAK[PBD]-fusion protein from keratinocyte lysates 3 h after induction of migration. SST treatment reduces active Rac1 compared to untreated cells. Precipitation with GST alone was used as negative control. (I) input, (P) precipitate. B: Relative quantification of Rac1 activity (control = 100%; n = 4). Results are shown as means+/−SEM, ** P<0.01.</p

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

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

Somatostatin receptor activation reduces keratinocyte cell counts but does not influence apoptosis and necrosis.

<p>A: Cells were stimulated with 1 µM SST for 72 h and the effect on cell counts was evaluated. Results are percentages compared to untreated cells (n = 13). B: Effect of selective SSTR agonists on cell counts (n = 4). Only the SSTR5/1-specific agonist inhibits keratinocyte proliferation. C: Effect of 1 µM SST on apoptosis. D: Effect of 1 µM SST on necrosis (positive controls for C and D as included in the assays; positive controls = 100%; n = 3). Results are shown as means+/−SEM, *** P<0.005.</p

SSTR subtypes are expressed in human skin and are functionally coupled to the cAMP pathway in primary keratinocytes.

<p>mRNA isolated from normal human skin (A) and primary human keratinocytes (B) was analyzed for the presence of mRNAs coding for the five somatostatin receptor subtypes SSTR1-5 by RT-PCR using subtype specific primer combinations. In (B) an example for SSTR-expression in epidermal skin equivalents (SSTR5) and in rat brain (SSTR2; positive control) is shown additionally. C: Inhibition of cAMP signalling by increasing concentrations of SST in forskolin-stimulated keratinocytes. Keratinocytes were stimulated with 10 µM forskolin for 10 min without SST (100% cAMP synthesis) or with 10–1000 nM SST. Results are shown as means+/−SD. Data were fitted by non-linear regression using GraphPad Prism software; the calculated IC₅₀ value for SST is 32+/−5 nM. D: Effect of different SST receptor agonists on cAMP levels after stimulation of cAMP synthesis by forskolin (FSK, means+/−SEM, * P<0.05, *** P<0.005.). 1 µM SST as well as selective receptor agonists (sst2-sst5/1 at a concentration of 1 µM) inhibited cAMP synthesis, showing that all receptor subtypes couple to cAMP signalling pathways.</p