Delayed cutaneous wound healing and aberrant expression of hair follicle stem cell markers in mice selectively lacking Ctip2 in epidermis

Abstract

Background: COUP-TF interacting protein 2 [(Ctip2), also known as Bcl11b] is an important regulator of skin homeostasis, and is overexpressed in head and neck cancer. Ctip2(ep-/-) mice, selectively ablated for Ctip2 in epidermal keratinocytes, exhibited impaired terminal differentiation and delayed epidermal permeability barrier (EPB) establishment during development, similar to what was observed in Ctip2 null (Ctip2(-/-)) mice. Considering that as an important role of Ctip2, and the fact that molecular networks which underlie cancer progression partially overlap with those responsible for tissue remodeling, we sought to determine the role of Ctip2 during cutaneous wound healing.

Methodology/principal findings: Full thickness excisional wound healing experiments were performed on Ctip2(L2/L2) and Ctip2(ep-/-) animals per time point and used for harvesting samples for histology, immunohistochemistry (IHC) and immunoblotting. Results demonstrated inherent defects in proliferation and migration of Ctip2 lacking keratinocytes during re-epithelialization. Mutant mice exhibited reduced epidermal proliferation, delayed keratinocyte activation, altered cell-cell adhesion and impaired ECM development. Post wounding, Ctip2(ep-/-) mice wounds displayed lack of E-Cadherin suppression in the migratory tongue, insufficient expression of alpha smooth muscle actin (alpha SMA) in the dermis, and robust induction of K8. Importantly, dysregulated expression of several hair follicle (HF) stem cell markers such as K15, NFATc1, CD133, CD34 and Lrig1 was observed in mutant skin during wound repair.

Conclusions/significance: Results confirm a cell autonomous role of keratinocytic Ctip2 to modulate cell migration, proliferation and/or differentiation, and to maintain HF stem cells during cutaneous wounding. Furthermore, Ctip2 in a non-cell autonomous manner regulated granulation tissue formation and tissue contraction during wound closure.

Figure 1. Expression of Ctip2 in skin of adult mice following tape stripping (TS), during full thickness wound healing and in hair cycling.

Immunoblot analyses of Ctip2 protein expression in wild type mice skin 24 and 48 hrs post tape stripping (A), and in wound biopsies obtained at Days 7, 9 and 11 after wounding (B). β-actin is used as control. (C) Bar chart showing an increase in the percentage of Ctip2 positive cells 48 hours post TS and on days 5 & 7 post wounding (*p<0.05). Bar chart showing, percentage of (D) intrafollicular and (E) extrafollicular Ctip2 expressing cells in depilation induced hair cycling in adult mouse skin. Significant (* P<0.05; **P<0.005) increase in Ctip2 expression was observed in induced anagen compared to telogen and catagen stages (D and E). (F) An overview of Ctip2 localization by immunofluorescence (IF) in the adult unwounded mice skin, 48 hr after mechanical injury and at day 7 post wounding using anti-Ctip2 antibody. (G) Overview of Ctip2 localization by IF in depilation induced hair cycle in mice skin. HE-hyperproliferative epithelium; HF-hair follicle; E-epidermis; D-dermis; Bu-bulge; DP-dermal papilla; bu-bulge. Yellow dotted lines separates the epidermis from dermis (F) and white dotted lines outlines the HF (G). Scale bars: 50 µm (F) and 25 µm (G).

Figure 2. Impaired wound healing in skin of adult Ctip2^ep−/− mice.

(A) Macroscopic images of time course of healing of 5 mm full-thickness excisional wounds. Note, that in Ctip2^L2/L2 mice at day 7, 9 and 11 post-wounding, the wound area was significantly reduced compared to the Ctip2^ep−/− mouse. (B) At indicated time points, the diameter of the wound was measured using photography and digital analyses. Data from three independent experiments (9 Ctip2^L2/L2 mice and 9 Ctip2^ep−/− mice were used for this study) were pooled. The difference in the rate of wound closure was statistically significant between two groups (P<0.001). (C) Graph showing increased migratory tongue distances between the two migratory epithelial tongue on the wound bed from Ctip2^L2/L2 and Ctip2^ep−/− mice. (*p<0.05). (D) Hematoxylin and Eosin (H & E) stained images of the day 5 and 9 post wounding samples. The black broken brackets in day 5 and day 9 show the outline of the wound area and the migratory tongue distance (day 5). Hyperproliferative epithelium; E- Epidermis; D-Dermis. Scale bar: 200 µm (D).

Figure 3. Altered keratinocyte activation (K6 & K16), proliferation (PCNA), differentiation (K10 & K8), and impaired expression of epithelial stem cell marker (K15) during wound healing in mutant mice.

(A) IHC analysis of K6 (red) expression on Day 5 post wounding samples from Ctip2^L2/L2 and Ctip2^ep−/− mice. The white dotted lines indicate the migratory tongue on the wound bed (Day5 samples) and separation of the epidermis from dermis in (A). All sections were counterstained with DAPI (blue). HE- hyperproliferative epithelium; E- epidermis; D- dermis; HF- hair follicle. Scale bar: 50 µm. (B) Immunoblot analyses of PCNA, K10, K6 and K16 in adult skin of Ctip2^L2/L2 and Ctip2^ep−/− mice after wounding (Day 7, Day 9 and Day 11). (C) Immunoblot analyses of keratin markers, K8 and K15 induction in the skin of Ctip2^L2/L2 and Ctip2^ep−/− adult mice on days 3, 5, 7, 9 & 11 post wounding. * indicates undetectable K8 expression in the unwounded wild type skin (day 0). β-actin is used as an internal control.

Figure 4. Reduced epidermal proliferation and differentiation in skin wounds from Ctip2^ep−/− mice.

(A–C) Expression of PCNA in skin sections from Ctip2^L2/L2 and Ctip2^ep−/− mice on day 5 (A and B) and day 7(C) post wounding, using antibodies against PCNA (in red). (B &C) Co-localization of K10 and PCNA on days 5 and 7 wound biopsies from dorsal skin of Ctip2^L2/L2 and Ctip2^ep−/− mice using anti-K10 antibody (green) and PCNA (red). All slides were counterstained with DAPI. The white arrow points to the directions of the wound (A). Yellow arrow with dotted lines indicates the direction of wound (B) and separates the epidermis from the dermis (C). MT-migratory tongue; HE- hyperproliferative epithelium; HF- hair follicle; E-epidermis and D- dermis. (D) Bar graph showing significant decrease (*P<0.05) in percentage of PCNA positive cells during wound healing on day 5 and 7 in the skin of Ctip2^ep−/− mice. Green and red bars represent Ctip2^L2/L2 and Ctip2^ep−/− mice, respectively. Scale bars: 50 µm (A), 100 µm (B and C).

Figure 5. Altered expression of E-cadherin, Phalloidin and alpha SMA in Ctip2^ep−/− mutant day 5 skin wounds.

(A) Expression of E-Cadherin is down regulated in the Ctip2^L2/L2 control basal layer migratory tongue but not in Ctip2^ep−/− mutants. The insets are magnified. (B) Expression of Phalloidin (red) is down regulated in the Ctip2^L2/L2 control but not in Ctip2^ep−/− mutant migratory tongue. (C) Reduced accumulation of myofibroblast (alpha SMA: red) in Ctip2^ep−/− mice wounds compared to the wild type mice. (D) Quantification of alpha SMA. Bar graph shows reduced pixel intensities in Ctip2^ep−/− compared to the Ctip2^L2/L2 control. Data are represented as means ± SD. Significant differences between Ctip2^L2/L2 and Ctip2^ep−/− (P<0.05). (E) Increased numbers of blood vessels in Day 5 post wounding samples inCtip2^L2/L2 compared to Ctip2^ep−/− mice but not significant. Values are the mean and SEM (n = 4 mice). MT-migratory tongue; E-epidermis and D- dermis. Arrows point to the directions of the wound. Dotted lines demarcate the epidermis from the dermis. Scale bars: 50 µm (A and B), 100 µm (C).

Figure 6. Aberrant expression of markers of HF stem cells in Ctip2^ep−/− mice skin during wound repair.

IHC analysis of (A) K15, (B) NFATc1 (red), (C) CD133 and (D) CD34 and (E) Lirg1 expression in HF on day 5 post wound healing samples from Ctip2^L2/L2 and Ctip2^ep−/− mice, using specific antibodies (* indicates loss of CD133 positive cells). The yellow dotted lines indicate the separation of epidermis from dermis near the wound bed (Day5 samples) in (A) and white dotted lines outline the HF. All sections were counterstained with DAPI (blue). Arrow heads indicate K15 positive cells (A). HE-hyperproliferative epithelium; E- epidermis; D- dermis; HF- Hair follicle; Bu-Bulge; JZ-junctional zone. Scale bars: 50 µm.