Figure 1.
Skin wound healing is accelerated in 8-week-old Med1epi−/− mice.
A: Representative macroscopic views of skin wounds on days 1, 3, 5 and 7 after wounding in 8 week old wild-type and Med1epi−/− mice. Full-thickness wounds (4 mm in diameter) were made on the middle of the backs of mice to synchronize tension and wound healing was monitored by taking digital photographs. Note the acceleration of wound healing in Med1epi−/− mice. B: Evaluation of wound closure by morphometrical analysis of the wound areas. The % of the wound area to the initial area was calculated from the photographs. N = number of mice; n = number of measurements. Bars = means ± SE. *P<0.05. C: Representative histological view of skin wound healing on day 3. Arrowheads and arrows indicate original wound edges and re-epithelialized leading edges, respectively. Scale bar = 500 µm. D: Time-course of changes of the re-epithelialization ratio after wounding in wild-type and Med1epi−/− mice. The % re-epithelialization was calculated by measuring the distance between the leading edges and the width between original wound edges as described in the Materials and Methods. N = number of mice; n = number of sections. Bars = means ± SE. *P<0.05, **P<0.01.
Figure 2.
Migrating epithelial tongues are elongated and the proliferation of keratinocytes is enhanced in 8-week-old Med1epi−/− mice.
A: The lengths of migrating epithelial tongues were measured on days 1 and 3 after injury in 8-week-old wild-type and Med1epi−/− mice. N = number of mice; n = number of measurements. Bars = means ± SE. **P<0.01. B: Analysis of keratinocyte proliferation at the re-epithelialized leading edges in 8-week-old wild-type and Med1epi−/− mice. Images show representative high-power fields of immunohistochemistry for Ki67 in epidermal cells in the transitional epidermis and in the migrating epithelial tongues on days 1, 3 and 5 after injury. Scale bar = 50 µm. C: Quantification of proliferating cells on days 1, 3 and 5 after injury. Ki67-positive cells were counted in the transitional epidermis and the epithelial tongues [2] of wound sites in 8-week-old wild-type and Med1epi−/− mice and were related to the area of the same part of the epidermis. N = number of mice; n = number of measurements. Bars = means ± SE. *P<0.05, **P<0.01. D: Evaluation of distance between the original wound edges in 8-week-old wild-type and Med1epi−/− mice on days 1, 3 and 5 after injury. N = number of mice; n = number of sections. Bars = means ± SE. N.S., not significant. E: Immunohistochemistry of α-SMA for the detection of myofibroblasts in the granulation tissue on days 5 and 7 after wounding. F: The area stained with α-SMA was determined by planimetric image analysis using ImageJ software. N = number of mice; n = number of sections. Bars = means ± SE. N.S., not significant.
Figure 3.
Follistatin expression is decreased and the MAPK signaling pathway is activated in Med1epi−/− mice keratinocytes in vitro.
A: Western blot analysis of follistatin, JNK, phospho-JNK, ERK and phospho-ERK in wild-type and Med1epi−/− keratinocytes (left). Quantification of the expression of each protein (right) (n = 3). Bars = means ± SE. *P<0.05, **P<0.01. B: Representative microscopic views in migration assays. Keratinocytes were cultured in KBM to form confluent monolayers and then were serum deprived for 24 h. The cells were subsequently incubated with mitomycin C (0.5 mg/ml) for 2 h and were then scratched with a p200 pipette tip, followed by incubation in KBM for 72 h. The cells were analyzed by phase contrast microscopy and were photographed at the indicated time points. C: Quantification of the number of migrating keratinocytes. The number of cells which had migrated into the wounded space at the indicated time points was counted microscopically and related to the wounded area. Bars = means ± SE. *P<0.05, **P<0.01. D: Western blot analysis of JNK phosphorylation after treatment with activin A (5 ng/mL). The level of JNK phosphorylation reached the highest value at 10 min after activin A treatment in wild-type and in Med1epi−/− keratinocytes cultured in KBM. Note that both the peak and the basal level of JNK phosphorylation were enhanced in MED1epi−/− keratinocytes compared with wild-type keratinocytes. E: Quantification of JNK phosphorylation after administration of activin A in Med1epi−/− and wild-type keratinocytes (n = 3). Bars = means ± SE. *P<0.05, **P<0.01. F: Cell cycle analysis of Med1epi−/− and wild-type keratinocytes. Cultured in KBM with acitivin A (5 ng/mL), Med1epi−/− keratinocytes showed an increased ratio of S phase cells compared with wild-type keratinocytes (left) while no difference was observed when those cells were cultured in KGM (right). Bars = means ± SE. **P<0.01, N.S., not significant.
Figure 4.
Skin wound healing is delayed in 6 month old Med1epi−/− mice.
A: Representative macroscopic views of skin wounds on days 1, 3, 5 and 7 after wounding in 6-month-old wild-type and Med1epi−/− mice. Full-thickness wounds (4 mm in diameter) were made on the middle of the back skins of mice and wound healing was monitored by taking digital photographs. Note that wound healing in 6-month-old Med1epi−/− mice was significantly delayed compared with the age-matched wild-type mice. B: Evaluation of wound closure by morphometrical analysis of the wound areas in 6-month-old wild-type and Med1epi−/− mice. The % of the wound area to the initial area was calculated from the photographs. N = number of mice; n = number of measurements. Bars = means ± SE. **P<0.01. C: Re-epithelialization ratio on days 1, 3 and 5 after wounding in 6-month-old wild-type and Med1epi−/− mice. The % re-epithelialization was calculated as mentioned above. N = number of mice; n = number of sections. Bars = means ± SE. *P<0.05, **P<0.01. D: The lengths of migrating epithelial tongues were measured on days 1, 3 and 5 after injury in 6-month-old wild-type and Med1epi−/− mice. N = number of mice; n = number of measurements. Bars = means ± SE. *P<0.05, **P<0.01. E: Evaluation of distance between original wound edges in 6-month-old wild-type and Med1epi−/− mice on days 1, 3 and 5 after injury. N = number of mice; n = number of sections. Bars = means ± SE. N.S., not significant.
Figure 5.
Proliferative keratinocytes and BrdU-positive label retaining cells are decreased in Med1epi−/− mice.
A: Images show representative high-power fields of immunohistochemistry for Ki67 in 6-month-old wild-type and Med1epi−/− mice keratinocytes in the transitional epidermis and the migrating epithelial tongues on days 1, 3 and 5 after injury. Scale bars = 50 µm. B: Quantification of proliferating cells on days 1, 3 and 5 after injury. Ki67-positive cells were counted in the transitional epidermis and the epithelial tongues of wound sites in 6-month-old wild-type and Med1epi−/− mice and were related to the area of the same part of the epidermis. N = number of mice; n = number of measurements. Bars = means ± SE. **P<0.01. C: BrdU-positive slow-cycling label retaining cells in hair follicles in 6-month-old wild-type and Med1epi−/− mice were detected on day 3 after injury (left). Arrowheads, BrdU-positive label retaining cells; Arrows, BrdU-positive label retaining cells migrating into epidermis adjacent to the wounds. The number of BrdU-positive cells in hair follicles was significantly decreased in 6-month-old Med1epi−/− mice compared with age-matched wild-type mice (right). Scale bars = 25 µm. N = number of mice; n = number of hair follicles. Bars = means ± SE. **P<0.01. D: BrdU label retaining cells in hair follicles in 8-week-old wild-type and Med1epi−/− mice (left). Arrowheads, BrdU-positive label retaining cells. The number of BrdU-positive cells in hair follicles in 8-week-old Med1epi−/− mice was comparable with age-matched wild-type mice (right). Scale bars = 25 µm. N = number of mice; n = number of hair follicles. Bars = means ± SE. N.S., not significant.
Figure 6.
Proposed model of altered cutaneous wound healing in Med1epi−/− mice.
In Med1epi−/− keratinocytes, the expression of follistatin is decreased. Consequently, activin, which is not sequestered by follistatin, is increased and activates the MAPK signaling pathway in keratinocytes in endocrine and/or paracrine manners. As a result, the proliferation and migration of Med1epi−/− keratinocytes are enhanced, contributing to rapid cutaneous wound healing process. However, as the number of hair follicle bulge stem cells decreases in old Med1epi−/− mice, the promoting effect of follistatin down-regulation on epidermal regeneration is overcome, resulting in impaired cutaneous wound healing.