Stathmin Regulates Keratinocyte Proliferation and Migration during Cutaneous Regeneration

<div><p>Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes <i>in vitro</i>; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. <i>In vitro</i>, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration.</p> </div

Increased Stathmin levels correlate with keratinocyte proliferating in an OTC wound healing model.

<p>(A) Schematic display of sample segmentation for automated analysis of OTC wound specimens. Segmentation in 10 areas was defined using HE-staining, while quantitative evaluation was performed using consecutive sections after Ki67/Stathmin double staining. Opposite areas were pooled (A/A’, B/B’, C/C’, D/D’, E/E’) to increase the cell number suitable for statistical analysis. (B) Ki67 and Stathmin double staining of OTC wound healing sections. Exemplary wound edges were shown for 0 (immediately after wounding), 1, 4, and 6 days after punching. (C) Percentage of Stathmin (left) and Ki67 (right) positive cells in different regions of the OTC specimens at indicated time-points. Please note that the regions E/E' (for day 1) and D/D’; E/E’ (for day 0) are not detectable due to incomplete wound closure at early time points. (D) Exemplarily, Ki67/Stathmin double staining is shown. Results of automated quantitative analysis of Ki67 and Stathmin positivity in keratinocytes of the basal epidermal layer are depicted. Black arrows: Ki67-negative/Stathmin-positive cells; White arrows: Ki67/Stathmin-positive cells. Dashed line in bar graph indicates the mathematical regression for the whole experimental time-course. Two independent time-courses were analyzed showing similar results.</p

Differential Stathmin expression in skin in wound healing.

<p>(A) High-power magnification of hair follicle in unwounded mouse skin after Stathmin staining. Black bordered arrows: Stathmin-positive interfollicular keratinocytes; black arrows: keratinocytes surrounding the papilla; arrowheads: cells encircling the sebaceous gland. (B) Exemplary pictures for Stathmin- and H&E overview staining in unwounded and wounded mouse skin at different time-points after wounding (day 1, 3, and 15 post-wounding). Black bordered arrows: migration tongue; black arrows: keratinocytes after reepithelialization; arrowheads: epidermal keratinocytes with high Stathmin positivity. (C) Stathmin staining of OTC samples consisting of primary human keratinocytes and fibroblasts after including 8 µm punches (2 independent sets of samples were analyzed). Black bordered arrows: migration tongue; black arrows: keratinocytes after reepithelialization; arrowheads: epidermal keratinocytes with high Stathmin positivity.</p

Bimodal activation of Stathmin via HGF/c-Met signaling.

<p>(A) Real-time PCR kinetics of Stathmin mRNA expression for 8 hours after administration of HGF (20 ng/ml). For each time-point the ratio of stimulated to untreated primary human keratinocytes was calculated. Data are shown as mean +/- SEM (n=3) and were normalized to transcript levels of untreated cells. (B) Scheme depicting the two major signaling pathways activated by HGF. Chemical inhibitors Akti-1/2 (5 µM) and GW5074 (2 µM) were utilized for selective inhibition of PI3K/AKT- and Ras/Raf/MAPK-pathways. Stathmin transcript levels of keratinocytes were analyzed 1h after stimulation with HGF (20 ng/ml) and administration of both substances for 15 and 30 min, respectively. Data are shown as mean +/- SEM (n=3). (C) Quantitative western immunoblotting analysis of Stathmin and phospho-Stathmin in keratinocytes after stimulation with HGF and administration of Akti-1/2 or GW5074. Total AKT and ERK1/2 as well as phospho-AKT (pAKT) and phospho-ERK1/2 (pERK1/2) served as controls for successful HGF stimulation and pathway inhibition. Numbers indicate quantitative densitometric values of phospho-Stathmin and total Stathmin levels compared to untreated cells. (D) Influence of c-Met inhibition on total Stathmin levels and its phosphorylation status using a receptor-specific inhibitor. Primary keratinocytes were treated with the c-Met inhibitor (PHA-665752, 0.5µM). Numbers indicate quantitative densitometric values of phospho-Stathmin and total Stathmin. (E) Stathmin and c-Met protein expression in keratinocytes after siRNA-mediated inhibition of Stathmin and c-Met after 48 hours (final concentration: 20 nM). Two independent nonsense siRNAs served as negative controls. Different parts of one gel are shown (dividing lines). For all western immunoblots, actin served as loading control.</p

Increased Stathmin levels support keratinocyte proliferation.

<p>(A) DNA incorporation assay (SYBR-green assay) after siRNA-mediated inhibition of Stathmin in keratinocytes (final concentration: 20 nM). Nonsense siRNA served as negative controls and was used for statistical comparison (#). Data are shown as mean + SEM (n=3). (B) Keratinocyte viability assay (MTT assay) after siRNA-mediated inhibition of Stathmin (final concentration: 20 nM). Nonsense siRNA served as negative controls and was used for statistical comparison (#). Data are shown as mean + SEM (n=3). (C) Mitotic events of individual keratinocytes were automatically detected after Stathmin or c-Met inhibition based on morphological features. (D) FACS cell cycle analysis. The number of S-phase keratinocytes is indicated. Statistical test: Mann-Whitney U, p*<0.05, p**<0.01, p***<0.001.</p