Figure 1.
The Akt-mTOR pathway is upregulated during wound healing.
(Upper panel). Representative histological sections of skin after incisional cutaneous wounds in mice. H&E stained section displaying the histological characteristics of the unwounded epithelium (normal adjacent) followed by an increase in the thickness of the spinous layer (acanthosis) (transitional), and the presence of migrating epithelial cells that form an epithelial tongue (epithelial tongue) at the wound edge. (second panel from top). Immunofluorescence for Cytokeratins 10 and 14 (K10-14, red) reveals the epidermal layer, which is delineated from the dermis by a white dotted line. Note the expansion of pAkt473 and pS6 (red) during wound healing from a single cell layer in the granular layer to multiple cell layers in the transitional epithelium, followed by expression in all cell layers including the basal layer in the migrating epithelial tongue (lower two panels). Cell nuclei were stained with DAPI (blue) and fibrin(ogen) with a FITC-conjugated antibody (green) in all panels. Scale bar, 50 µm and 100 µm, as indicated.
Figure 2.
Accelerated wound closure upon epidermal Pten deletion: Enhanced mTor activation and re-epithelization.
(A) Representative example of accelerated wound closure in Pten-deficient (K14Cre PtenF/F) mice compared to control mice 4 days after skin wounding. (B) Quantification of the wound area is represented by the box-and-whiskers graphic (horizontal line, median value; n = 13 for each group; **p = 0.0071). (C) Percentage of mice exhibiting open wounds after surgical incision in control mice (triangles, n = 8) and K14Cre PtenF/F mice (squares, n = 8) at the indicated days is depicted. There was a significant increase in the rate of wound closure (***p<0.0001). (D) Representative examples of K10 and K14 (red-upper panel), pAkt473 (red-middle panel), and pS6 (red–lower panel) expression in K14Cre PtenF/F wounds as determined by immunofluorescence. DAPI (blue) and fibrin(ogen) (green). Note the accumulation of pAkt473 and pS6 in the transitional zone and epithelial tongue at the wound edge (transitional and epithelial tongue), when compared to the adjacent epithelium (Normal). (E) Quantification of BrDU positive cells in histological slides at the transitional and wound edge of control and K14Cre PtenF/F mice (n = 10 for each group; *p = 0.0331). (F) Quantification of epithelial migration and re-epithelization. The length of the epithelial tongue was measured in histological slides of wounded control (n = 10) and K14Cre PtenF/F mice (n = 12) (mean; *p = 0.0268).
Figure 3.
Keratinocyte migration in vitro is enhanced upon Pten excision.
(A) Scratch wound assays in primary cultures of keratinocytes from control mice and K14Cre PtenF/F mice. Wounds were generated after cell confluence. In vitro cell migration and wound closure were assessed every 16 h. Areas of migration were measured in duplicates wells using keratinocytes from three distinct mice for each group (dotted line) and represented in (B) as mean ± s.e.m. (p = 0.0002) (C) Graphics shows cell proliferation represented as the percentage of BrDU incorporation by the primary keratinocytes. EGF was added where indicated (***p≤0.001).
Figure 4.
Rapamycin regulates the Akt-mTOR network in Pten-deficient keratinocytes and delays wound healing.
Primary cultures of keratinocytes isolated from control and K14Cre PtenF/F mice were treated with vehicle or rapamycin (50 nM), as indicated. Data are representative of three independent experiments performed in triplicate. (A) Western blot analysis showing very low Pten levels in K14Cre PtenF/F keratinocytes, which presented increased levels of both pAktThr308 and pAktSer473. Rapamycin effectively ablates S6 phosphorylation, decreases pAktS473, and increases pAktThr308 in K14Cre PtenF/F keratinocytes when compared to control keratinocytes. GAPDH provided loading controls. (B) Bar chart represents the percentage of BrDU positive cells in the primary culture of keratinocytes (***p≤0.001). (C and D) The graphic represents the percentage of animals with open wounds in the control mice (C) and K14Cre PtenF/F mice group (D) at each indicated day after cutaneous wounding. Rapamycin and vehicle administration was initiated 48 h before the wounding (see methods for details) and wound closure was used as endpoint; n = 8 for each genotype and time point; p values are indicated.
Figure 5.
Accelerated wound closure in upon epithelial Tsc1 excision.
Representative photographs of control and K14Cre Tsc1F/F mice at the indicated days after wounding. (B) Quantification of the wound area. Results represent the mean ± s.e.m; n = 8 for each time point and genotype. (***p<0.0001) (C) Graphic representation of the percentage of animals displaying open wounds in control mice (A) and K14Cre Tsc1F/F mice (B) at each depicted time (days) (*p = 0.0150).
Figure 6.
Acceleration of wound healing upon ablation of Tsc1 in the skin.
(A) Representative examples of immunofluorescence in histological sections of incisional skin wounds in mice; pS6 (red), fibrin(ogen) (green), DAPI (blue), as above. The white dotted line delineates the epithelial layer from the underlining dermis. Note the expansion of pS6 (red) expression in the normal, transitional and elongated migrating tongue of the epithelial compartment of the K14Cre Tsc1F/F mice when compared to control mice. Scale bar, 50 µm. (B) Representative H&E stained sections showing enhanced re-epithelization of the wound area as reflected by the length of the migrating epithelial tongue in the K14Cre Tsc1F/F mice when compared to the control mice. The black lines delineate the epithelial migrating tongue. Quantification of the epithelial tongue from each genotype is represented by the dot plot (n = 10 for each genotype; scale bar, 200 µm; *p = 0.0381). (C) Representative histological sections of wounded skin from control and K14Cre Tsc1F/F mice. The black line indicates the wound width, and the scatter plot represents the quantification of the wound width from control and K14Cre Tsc1F/F mice (n = 6 mice per genotype; scale bar, 400 µm; **p = 0.0048).