Necl2 regulates epidermal adhesion and wound repair

Abstract

Differential expression of cell adhesion molecules regulates stem cell location, self-renewal and lineage selection under steady state conditions and during tissue repair. We show that the intercellular adhesion protein nectin-like molecule 2 (Necl2) is highly expressed in bulge stem cells of adult human and mouse hair follicles. Overexpression of Necl2 in cultured human keratinocytes led to upregulation of calcium/calmodulin-associated Ser/Thr kinase (CASK), increased calcium-independent intercellular adhesion, and inhibition of cell motility and in vitro wound healing. Although the rate of cell proliferation was reduced, terminal differentiation was unaffected. To assess the role of Necl2 in vivo, we examined the epidermis of Necl2-null mice and developed transgenic mice that expressed Necl2 in the basal layer of murine epidermis. Necl2 overexpression led to a reduction in S-phase cells and an increase in quiescent cells retaining DNA label in the bulge. Although epidermal homeostasis appeared normal in both transgenic and knockout mice, wound healing was markedly delayed. Necl2 overexpression resulted in reduced proliferation and increased levels of CASK and E-cadherin at the leading edge of healing wounds, consistent with its effects in culture. Our results demonstrate that Necl2 is involved in regulating epidermal stem cell quiescence and location.

Fig. 1.

Necl2 expression in developing and adult epidermis. (A-D) E17.5 (A,B) or E20.5 mouse skin (C,D) was stained for Necl2 (red) and E-cadherin (green, A,C) or P-cadherin (green, B,D) with DAPI nuclear counterstain (blue). (E,F) Adult mouse back skin section (E) and tail epidermal whole-mount (F) stained with antibodies against Necl2 (red, E; green, F) and keratin-15 (green, E) or CD34 (red, F) with DAPI nuclear counterstain (blue). Note staining of sebaceous glands (asterisk, F) is nonspecific. (G,H) Flow cytometric analysis of Necl2 expression (H) in adult integrin-α6 (Itga6)-positive basal cells (red box, G) and CD34- and Itga6-positive bulge cells (blue box, G). (I,J) Adult human hair follicles stained for Necl2 (green) and keratin-14 (red, I) or keratin-15 (red, J). (K) Necl2 (green) and Ki67 (red) immunostaining of adult mouse anagen follicles (tail epidermal whole mount) with DAPI nuclear counterstain (blue). Arrowhead indicates Necl2 expression in hair follicle bulb. (L,M) Adult tail whole mounts of K14ΔNβ-cateninER epidermis treated with 4-hydroxy-tamoxifen for 2 weeks and stained for Necl2 (green) and CD34 (red, L) or Ki67 (red, M) with DAPI nuclear counterstain (blue). Arrowheads indicate ectopic hair follicles arising from sebaceous glands and existing follicles. White lines (A-F, I-M) indicate position of basement membrane or delineate hair follicles and sebaceous glands. Scale bars: 100 μm in A-F,I-M.

Fig. 2.

Effects of Necl2 overexpression on growth and differentiation of cultured human keratinocytes. (A) Flow cytometry of keratinocytes transduced within empty vector (EV, red) or Necl2 (blue) and labelled with anti-Necl2. (B) Necl2 immunoblots of Necl2-transduced or control (EV) AM12 packaging cells (A) and keratinocytes (K). Molecular mass markers (kDa) are indicated. (C,D) Representative dishes of control(C) and Necl2- (D) transduced keratinocyte colonies 2 weeks after plating. (E) Quantification of relative size of individual control (black) and Necl2 transduced (red) clones. All clones (n>300) from one experiment are shown; data are representative of three separate experiments. (F) Keratinocyte growth was assessed by measuring mitochondrial activity (OD₄₉₀; ^*P<0.05). (G) Flow cytometric determination of proportion of involucrin-positive Necl2-transduced and control keratinocytes. (H,I) Quantification of basal cell density (H) and epidermal thickness (I) in epidermis reconstituted by control (EV) and Necl2-transduced cells cultured on de-epidermised dermis. (J,K) H&E-stained sections of control (J) and Necl2-transduced (K) keratinocytes grown on DED substrates for 2 weeks. (L) Western blot of control and Necl2-transduced keratinocytes cultured in the absence of HGF (0) or stimulated with HGF for the times indicated. Blot was probed with antibodies against CASK or, as a loading control, β-tubulin. (M,N) Representative images of colonies transduced with Necl2 (M) or empty vector (N) stained with antibodies against CASK (red) and Necl2 (green). Error bars in G-I represent s.e.m. Scale bars: 100 μm in J,K; 10 μm in M,N.

Fig. 3.

Necl2 overexpression influences keratinocyte adhesion and motility. (A) Identification of cell doublets based on Draq5 nuclear area/aspect ratio (left panel; blue gate) and characterisation of doublets by labelling with FITC- and RPE-conjugated anti-α6-integrin antibodies (right). Insert shows representative RPE-RPE (top), RPE-FITC (middle) and FITC-FITC (bottom) stained cell doublets. (B) Quantification of relative RPE-FITC labelled EV-EV, Necl2-EV and Necl2-Necl2 doublets. (C-G) Representative images (C-F) and quantification (G) of Necl2 expression in control (C,E) and Necl2-transduced (D,F) cells cultured under standard (high; C,D) and low-calcium (E,F) conditions. (H-L) Representative images (H-K) and quantification (L) of E-cadherin expression in control (H,J) and Necl2-transduced (I,K) cells cultured under standard (high; H,I) and low-calcium (J,K) conditions. (G,L) Quantification of pixel intensity at cell-cell borders. (M-O) Control (M) and Necl2-transduced (N) cell motility tracks and maximum migration distances (O) relative to a starting position (x,y=0,0) at time=0 minutes. Each line in M,N shows motility of an individual cell. (B,G,L) Error bars represent s.e.m.; ^*P<0.05. Scale bars: 10 μm in C-F,H-K.

Fig. 4.

Overexpression or deletion of Necl2 does not disturb epidermal homeostasis. (A-C) H&E-stained sections of dorsal skin of wild-type (A), K14Necl2 transgenic (B) and Necl2-knockout mice (C). (D-F) Tail epidermal whole mounts of wild-type (D), K14Necl2 transgenic (E) and Necl2-knockout (F) mice stained for keratin-14 (green) and Necl2 (red), with DAPI nuclear counterstain (blue). (G-I) Wild-type (G), K14Necl2 transgenic (H) and Necl2 null (I) dorsal skin sections stained with anti-CASK (red) and DAPI nuclear counterstain (blue). (J-L) Tail epidermal whole mounts of wild type (J), K14Necl2 transgenic (K) and Necl2-null (L) mice stained for keratin-14 (red) and CD34 (green). (M-P) Flow cytometric analysis of α6-inegrin and CD34 double-positive (orange boxes) and α6-integrin-positive, CD34-negative (green boxes) cells in wild-type (M), K14Necl2 transgenic (N) and Necl2-knockout (O) mouse epidermis. (P) Relative abundance of cells in M-O. IFE, interfollicular epidermis. Asterisk in J indicates nonspecific staining of sebaceous gland. Scale bars: 100 μm in A-L.

Fig. 5.

Necl2 overexpression reduces epidermal proliferation. (A-E) Determination of proportion of cells in G0-G1, S, G2-M phases of the cell cycle by flow cytometry of cells labelled with Hoechst 33342. (A) Total undifferentiated cells (α6-integrin positive; Itga6⁺) or bulge cells (CD34 and α6-integrin double positive; CD34⁺) are shown beneath the appropriate wild-type controls. Data shown are means±s.e.m. from four transgenic and four matched wild-type controls and from three knockout and three wild-type control mice. Values in red are significantly different from controls (P≤0.05). (B-E) Representative flow cytometry profiles of CD34⁺ bulge populations, showing position of cell cycle gates in wild-type versus K14-Necl2 transgenic mice (B,C) and wild-type versus Necl2-knockout mice (D,E). (F,G) Tail epidermal whole mounts from wild-type (F) and transgenic (G) mice labelled with anti-BrdU (to detect LRC; red) and K15 (green). (H,I) Representative flow cytometry plots of BrdU-labelled LRC (red boxes) in wild-type (H) and K14Necl2 transgenic (I) epidermis. Numbers refer to percentage BrdU-positive cells. Cells were double labelled with anti-Necl2. Scale bars: 100 μm in F,G.

Fig. 6.

Skin wound healing is modulated by Necl2. (A) Wild-type skin collected 7 days after wounding stained for Necl2 (red), keratin-6 (green) and DAPI (blue). (B) Wound size, expressed as a percentage of starting wound area, with time after wounding. Data are represented as mean values±s.e.m. (C-E) Representative H&E-stained sections of wild-type (C), K14Necl2 transgenic (D), and Necl2-knockout (E) mouse skin 7 days after wounding. Brackets show distance between wound margins. (F-H) Keratin-6 (red) and DAPI (green) labelling of the leading edge of re-epithelialisation 7 days after wounding wild-type (F), K14Necl2 transgenic (G) and Necl2-knockout (H) mice. (I) Quantification of average keratin-6-positive area per section prepared as in F-H, expressed as a percentage of wild-type controls. Data represent average of two sections per wound from a minimum of five animals. Error bars in B and I represent s.e.m.; ^*P<0.05. Scale bars: 100 μm in A,C-E; 50 μm in F-H.

Fig. 7.

Necl2 overexpression decreases proliferation and leads to upregulation of E-cadherin and CASK during wound healing. (A-C) BrdU-labelled S-phase cells (brown, A-C) at the leading edge of healing wild-type (A), transgenic (B) and Necl2-knockout (C) mouse epidermis. (D) Quantification of BrdU-positive cells as a function of distance from the wound edge in wild-type (black bars), transgenic (red bars), and knockout mice (blue bars). Data are presented relative to appropriate congenic wild-type controls (n=4 knockout mice/group; n=8 transgenic mice/group). Error bars show s.e.m.; ^*P<0.05. (E-H) Wounded wild-type (E,F) or K14Necl2 transgenic (G,H) epidermis stained with anti-CASK (red) and DAPI nuclear counterstain (blue; E,G). (I-N) E-cadherin (green, I-N) and Necl2 (red, I,K,M) expression at the leading edge of re-epithelialising wounds, 7 days after wounding. (E,G,I-N) DAPI nuclear counterstain (blue). Lines (A-C,E-N) denote dermal-epidermal boundary and boxes (E,G,I,K,M) denote regions shown at higher magnification in adjacent panels. Scale bars: 50 μm in A-C; 100 μm in E-N.