16 research outputs found
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
The X chromosome and male infertility
The X chromosome is a key player in germ cell development, as has been highlighted for males in previous studies revealing that the mammalian X chromosome is enriched in genes expressed in early spermatogenesis. In this review, we focus on the X chromosome's unique biology as associated with human male infertility. Male infertility is most commonly caused by spermatogenic defects to which X chromosome dosage is closely linked; for example, any supernumerary X chromosome as in Klinefelter syndrome will lead to male infertility. Furthermore, because males normally only have a single X chromosome and because X-linked genetic anomalies are generally only present in a single copy in males, any loss-of-function mutations in single-copy X-chromosomal genes cannot be compensated by a normal allele. These features make X-linked genes particularly attractive for studying male spermatogenic failure. However, to date, only very few genetic causes have been identified as being definitively responsible for male infertility in humans. Although genetic studies of germ cell-enriched X-chromosomal genes in mice suggest a role of certain human orthologs in infertile men, these genes in mice and humans have striking evolutionary differences. Furthermore, the complexity and highly repetitive structure of the X chromosome hinder the mutational analysis of X-linked genes in humans. Therefore, we conclude that additional methodological approaches are urgently warranted to advance our understanding of the genetics of X-linked male infertility
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
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
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