Expansion of epidermal progenitors with high p63 phosphorylation during wound healing of mouse epidermis

Abstract

The transcription factor p63 plays an essential role in maintaining the proliferative potential of epidermal stem cells. We have shown recently that under homoeostatic conditions, phosphorylation of p63 increases during the early transition of stem cells to transit-amplifying cells in human epidermis. However, how p63 phosphorylation relates to the regenerative processes during wound healing remains unknown. In this study, we characterize epidermal cells that contribute to wound repair in mouse models using phosphorylated p63 as a marker for stem cell differentiation. Our studies reveal that epidermal progenitors with high p63 phosphorylation preferentially expand in response to wounding in both full-thickness wound and surface injury models. As phosphorylated p63 levels inversely correlate with the proliferative potential of epidermal progenitors, p63 phosphorylation may serve as a therapeutic target to modulate the function of these regenerative cells during wound healing.

Background

The mammalian epidermis is maintained by self-renewal of epidermal stem cells (SCs) that divide stochastically to generate transit-amplifying (TA) cells, which then undergo terminal differentiation after a few rounds of cell division (1,2). In response to injury, the epidermis initiates the process of tissue regeneration in concert with other cell types (3). Although epidermal SCs and TA cells are thought to play distinct roles in replenishing lost or damaged cells (1,2,4), their relative contributions to wound repair are not well understood due to the lack of specific markers that delineate early changes in SC differentiation.

We have shown previously that p63 plays an essential role in maintaining the proliferative potential of epidermal SCs (5). Recently, we have shown that phosphorylation of ΔNp63α, the major isoform of p63 in the epidermis, increases at the serine residues 66/68 as SCs differentiate into TA cells under homoeostatic conditions in a human epidermal model (6). As phosphorylated p63 (pp63) levels inversely correlate with the proliferative potential of epidermal progenitors (6), characterization of p63 phosphorylation during wound healing may provide insight for future therapeutic strategies geared at modulating function(s) of epidermal progenitors.

Questions addressed

To characterize epidermal progenitors during wound healing in mouse models using p63 phosphorylation as a marker for SC differentiation to TA cells.

Experimental design

Detailed methods are described in Supporting Information.

Results

We first asked whether our findings in human epidermis (6) are recapitulated in mouse models under homoeostatic conditions. For this, we stained sections of adult mouse skin with anti-p63 and anti-phosphorylated p63 (anti-pp63) antibodies that detect phosphorylated serine residues at positions 66/68 of ΔNp63α and quantified p63 and pp63 signals in individual epidermal cells. Our data show that p63 expression is highly variable within the basal layer (Fig. 1a, 1b: left) and that cells with high levels of p63 are found at a similar frequency in the basal and suprabasal layers (Fig. 1b: left, 1c). Because SCs in the interfollicular epidermis localize within the basal layer (1,2), these data suggest that, as in human epidermis (6), high expression of p63 is not restricted to SCs in mouse epidermis.

Figure 1.

Total and phosphorylated p63 levels in adult mouse epidermis.

(a) Representative immunofluorescence images of the basal cells in adult mouse epidermis stained with anti-p63 (red) and anti-pp63 (green) antibodies and counterstained with Hoechst 33342 (blue). The numbers on the top indicate relative p63 expression normalized by nuclear counterstaining (p63/DNA) and the pp63/p63 ratio (pp63/p63) with the highest being set to 1.0. Bar = 10 μm.

(b) Left: Histogram showing relative p63 levels in the basal (solid) and suprabasal (open) cells. Expression of p63 was normalized by nuclear counterstaining, and the highest p63/DNA value among all epidermal cells was set to 1.0. Right: Histogram showing the pp63/p63 ratio among all p63^hi epidermal cells with the highest ratio being set to 1.0. *, not detected. (c) Representative immunofluorescence images of adult mouse epidermis stained with anti-p63 (red) and anti-pp63 (green) antibodies and counterstained with Hoechst 33342 (blue). Arrowheads indicate representative p63^hi cells with relatively high pp63/p63 ratio in the suprabasal layers. Dotted lines indicate the epidermal–dermal border. Bars = 25 μm.

A comparison of the pp63/p63 ratio (pp63 levels in relation to total p63 expression) between the basal and suprabasal cells revealed that the overall pp63/p63 ratio was significantly higher in the suprabasal cells than basal cells (Fig. 1b: right). Our data also show that the basal layer contains a unique population of p63^hi cells with an extremely low pp63/p63 ratio that is completely absent from the suprabasal layers (Fig. 1b: right). Consistent with our findings in human epidermis (6), these data suggest that epidermal SCs in mice are included in the p63^hi basal layer population with relatively low p63 phosphorylation, while p63^hi cells with increased p63 phosphorylation represent more differentiated epidermal progenitors. Together, these data suggest that phosphorylation of p63 during SC differentiation is conserved between human and mouse.

Next, we created full-thickness wounds in the dorsal skin of adult mice and examined individual epidermal cells within the wound edge, a region that presumably serves as a reservoir for proliferating epidermal cells that ultimately cover the wounds (Fig. S1a).

A short pulse with 5-bromo-2′-deoxyuridine (BrdU) revealed that epidermal cells responded quickly to wound stimuli at day 1 postinjury (Fig. S1b-S1d). Moreover, quantification of p63 and pp63 signals in individual epidermal cells revealed that p63^hipp63^hi cells proliferated most robustly at day 1 and day 2 postinjury (Fig. 2a). Subsequently, the relative increase in p63^hipp63^hi cell number among all p63-positive epidermal cells was the highest at day 6 postinjury (Fig. 2b). As increased p63 phosphorylation leads to the proteasome-mediated degradation of p63 (7), we predict that the increase in the frequency of p63^lo cells at day 6 postinjury reflects differentiation of p63^hi cells to p63^lo cells. Together, our data indicate that epidermal cells with the highest proliferative expansion and accumulation in response to wounding exhibit a p63^hipp63^hi phenotype, the same cell type characterized as TA cells in homoeostasis (6) (Fig. 1).

Figure 2.

Expansion of epidermal progenitors with high p63 phosphorylation during wound healing in mice. (a) Frequency of BrdU-labelled p63^hipp63^lo, p63^hipp63^hi and p63^lo cells in each cell population at day 0, 1, 2 and 6 postinjury. n = 3; *P < 0.01, t-test. (b) Changes in relative cell number of p63^hipp63^lo, p63^hipp63^hi and p63^lo cells at day 0, 1, 2 and 6 postinjury. n = 3; *P < 0.01, t-test. In (a) and (b), high and low p63 levels and pp63/p63 ratio were set in adjacent uninjured epidermis, and the same values were applied to the wounds (see Supplemental Information for detail). (c) Representative images of surface-injured (right panels) and adjacent uninjured (left panels) mouse epidermis. Six days after the injury, cross sections were processed for haematoxylin and eosin (H&E) staining (top). Shown in the middle and bottom panels are corresponding images stained with anti-p63 (red) and anti-pp63 (green) antibodies and counterstained with Hoechst 33342 (blue) as indicated. Dotted lines indicate the epidermal–dermal border. Bars = 25 μm. (d) Western blot analysis of p63 and pp63 levels in uninjured epidermis and in the wounds at day 2 postsurface injury. The same membranes were sequentially probed with an anti-pp63 antibody, stripped and then reprobed with an anti-p63 antibody. Shown are representative data from two independent experiments with similar results. (e) A proposed model for SC differentiation to TA cells through p63 phosphorylation. SCs with high proliferative potential express high p63 levels with low phosphorylation (p63^hipp63^lo). During homoeostasis and wound repair, SCs exit from the SC state and differentiate into TA cells with increased levels of p63 phosphorylation (p63^hipp63^hi), ultimately leading to the loss of proliferative potential and terminal differentiation due to the loss of p63 (5–7). P, phosphorylated serine residues at positions 66/68 of the ΔNp63α isoform.

While full-thickness wounds in mice heal by both contraction and re-epithelialization, human wounds heal primarily by re-epithelialization (8). To investigate whether re-epithelialization itself is accompanied by an expansion of p63^hipp63^hi cells, we utilized a surface injury model of wound healing (9). Consistent with the full-thickness wound model, the frequency of p63^hipp63^hi cells dramatically increased among all p63-positive cells within the first week after the injury (Fig. 2c, S2). Accordingly, relative pp63 levels were increased in wounds compared to adjacent uninjured epidermis by Western blot (Fig. 2d). These data indicate that re-epithelialization is accompanied by an expansion of epidermal progenitors with high p63 phosphorylation.

Conclusions

Our studies show that wound repair of the epidermis involves an expansion of epidermal progenitors with high p63 phosphorylation. By analogy to homoeostatic conditions (Fig. 1) (6), these progenitors with high pp63 levels likely represent TA cells. However, the signals that regulate p63 phosphorylation need to be determined to further assess the functional significance of this post-translational modification of p63 during epidermal SC differentiation to TA cells (Fig. 2e). Notably, activation of c-myc promotes pathogenesis of chronic wounds caused by enhanced SC differentiation (10–13). As p63 and c-myc share common regulatory pathways such as the Wnt/β-catenin signalling pathway (14,15), elucidation of their cooperative roles should promote our understanding of SC fate decisions in the epidermis. As such, it is intriguing to speculate that strategies to modulate p63 phosphorylation therapeutically may help to improve progenitor cell-related pathologic conditions of the epidermis.

Supplementary Material

figure

Figure S1. Proliferation and characterization of epidermal progenitors in response to full-thickness wounds in mice.

Figure S2. Expansion of p63^hipp63^hi epidermal progenitors in response to surface injury in mice.

text

Data S1. Supplemental Materials and Methods.