Suppression of Wnt/β-catenin signaling by EGF receptor is required for hair follicle development

Abstract

Mice that lack the epidermal growth factor receptor (EGFR) fail to develop a hair coat, but the mechanism responsible for this deficit is not completely understood. Here, we show that EGFR plays a critical role to attenuate wingless-type MMTV integration site family member (Wnt)/β-catenin signaling during postnatal hair follicle development. Genetic ablation of EGFR in mice resulted in increased mitotic activity in matrix cells, apoptosis in hair follicles, and impaired differentiation of epithelial lineages that form hair. EGFR is activated in wild-type hair follicle stem cells marked with SOX9 or NFATc1 and is essential to restrain proliferation and support stem cell numbers and their quiescence. We observed elevated levels of Wnt4, 6, 7b, 10a, 10b, and 16 transcripts and hyperactivation of the β-catenin pathway in EGFR knockout follicles. Using primary keratinocytes, we linked ligand-induced EGFR activation to suppression of nascent mRNA synthesis of Wnt genes. Overexpression of the Wnt antagonist sFRP1 in mice lacking EGFR demonstrated that elevated Wnts are a major cause for the hair follicle defects. Colocalization of transforming growth factor α and Wnts regulated by EGFR in stem cells and progeny indicates that EGFR autocrine loops control Wnts. Our findings define a novel mechanism that integrates EGFR and Wnt/β-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.

FIGURE 1:

Genetic ablation of EGFR kinase increases proliferation, DNA damage, and apoptosis and interferes with differentiation during hair morphogenesis. (A) KI EGFR mice (KI) lack hair coat. (B) H&E-stained KI mouse skin sections reveal aberrant and disoriented hair follicles (HFs). (C) Reduced hair follicle length in KI. (D–I) Immunofluorescence and quantifications. (D, E) Increased mitotic activity (mitosis-specific marker PH3) in KI matrix (Mx) cells. There were 30–75 cells counted per hair follicle. (F, G) Elevated levels of γH2AX (marker of DNA breaks) and activated caspase-3 (Ac-CAS3; apoptosis marker) in KI. (H, I) Reduction of trichohyalin (AE15), an inner root sheath (IRS) and medulla (med) marker, and hair keratin (AE13), a hair shaft (HS) marker of the cortex and medulla in KI. All analyses and pictures were from mice at P7. Graphs contain means with SEM (n ≥ 3 mice; ≥20 hair follicles/mouse). *p value ≤ 0.05. Scale bars: 10 μm.

FIGURE 2:

EGFR maintains hair follicle stem cell numbers and their quiescence during hair follicle development. (A) Immunofluorescence showing detection of pEGFR in WT bulge (Bu) hair follicle stem cells marked by SOX9 or NFATc1 at P7. (B–E) Immunofluorescence and quantifications. (B) Decreased SOX9 (+) cells in KI bulge. (C) NFATc1 (+) hair follicles are reduced in KI. (D) Increased proliferation of SOX9 (+) cells in KI bulge. (E) Colocalization of NFATc1 and Ki67 in KI (denoted by arrows), but not WT bulge. White dashed line marks the epidermal–dermal boundary. Graphs contain means with SEM (n ≥ 3 mice; ≥20 hair follicles/mouse). (B, D) There were 30–75 cells counted per hair follicle. *p value ≤ 0.05. Scale bars: 10 μm.

FIGURE 3:

Loss of EGFR kinase leads to elevated β-catenin signaling during hair morphogenesis. (A, B) qPCR analysis showing increased transcript levels of Wnt/β-catenin target genes (Axin2, Lef1, and Lgr6) and Wnt ligands (Wnt4, 6, 7b, 10a, 10b, and 16) in KI skin. Results are presented as relative to WT and are the mean and SEM of five replicates. qPCR transcripts were normalized to the mean of five housekeeping genes Gapdh, B2M, Actb, GusB, and Tbp. *p value ≤ 0.05. (C) Western blotting reveals elevated expression of Lef1 and β-catenin protein in KI skin. Bands were quantified by densitometry, and Lef1 and β-catenin intensities were normalized to β-actin and presented relative to WT (n = 5). (D) Immunofluorescence showing increased Lef1 protein expression in KI hair follicles. (E) Immunofluorescence showing increased nuclear β-catenin in KI matrix (Mx), dermal papilla (DP), and bulge (Bu) cells (marked by arrows in DP and Bu). All analyses and hair follicle pictures were from mice at P7. Scale bars: 10 μm.

FIGURE 4:

Multiple Wnt transcripts were elevated in KI EGFR hair follicles. Chromogenic ISHs reveal increased Wnt4, Wnt6, and Wnt16 mRNAs (red spots) in KI matrix (Mx) and bulge (Bu), Wnt7b in KI bulge, and Wnt10a and Wnt10b mRNA in KI inner root sheath (IRS) and matrix. Arrows in WT denote in situ staining in bulge. All hair follicle pictures were from mice at P7. Scale bars: 10 μm.

FIGURE 5:

EGFR regulates Wnt/β-catenin signaling in hair follicle stem cells. (A–C) Multiplex fluorescence ISHs and quantifications showing increased transcript expression of Wnt/β-catenin target genes Axin2, Lef1, and Lgr6 in Nfatc1 (+) cells in KI bulge. A representative ISH image is shown to the left of the bar graph. Arrows in A–C denote a nucleus magnified in the inset. (D) Quantifications of multiplex fluorescence ISHs demonstrate elevated Wnt4, 6, 7b, and 16 transcript levels in Nfatc1 (+) cells in KI bulge. All analyses and hair follicle pictures were from mice at P7. Graphs contain means with SEM (n ≥ 3 mice; ≥20 hair follicles/mouse; 25–50 cells counted per hair follicle). KI values are relative to WT (set to 1). *p value ≤ 0.05. Scale bars: 10 μm.

FIGURE 6:

Ligand-induced EGFR activation suppresses nascent Wnt mRNA synthesis. (A) qPCR measurements of Wnt mRNAs in primary mouse keratinocytes isolated from P0 WT skin and treated without (control) or with TGFα (30 ng/ml) for 6 h. The levels of Wnt4, 6, 7b, 10a, 10b, and 16 mRNA were decreased by TGFα in primary mouse keratinocytes. (B) qPCR measurements of 4sU-labeled, newly transcribed Wnt mRNAs isolated from primary mouse keratinocytes treated without (control) or with TGFα (30 ng/ml) for 3 h. c-Myc mRNA levels were measured as positive control for TGFα treatment. Results are presented as relative to control and are the mean and SEM of three replicates. qPCR transcripts were normalized to the housekeeping genes Gapdh and B2m. *p value ≤ 0.05.

FIGURE 7:

Overexpression of the Wnt antagonist sFRP1 rescues hair follicle defects in mice lacking EGFR. (A) H&E sections from KI mice injected with adenovirus (Ad) expressing sFRP1 compared with KI mice injected with adenovirus expressing GFP reveal partial rescue of hair follicle morphology. (B) Western blotting confirms expression of sFRP1 by adenovirus in skin. (C) sFRP1 expression reduces nuclear β-catenin in KI matrix (Mx) and bulge (Bu) cells. (D) Hair follicle length increase in KI treated with sFRP1 adenovirus. (E–O) Immunofluorescence and quantifications. (E) sFRP1 restores hair keratin (AE13) in KI follicles. (F) Decreased mitotic activity (mitosis-specific marker phospho-histone H3, PH3) in KI matrix cells by sFRP1-expressing adenovirus. (G, H) γH2AX (+) and activated caspase-3 (+) (Ac-CAS3) hair follicles are reduced in KI treated with sFRP1 adenovirus. (I, J) sFRP1 expression decreases Axin2 transcripts in Nfatc1 (+) and Sox9 (+) stem cells in KI. (K, L) SOX9 (+) cells in the bulge and NFATc1 (+) hair follicles are rescued by sFRP1 in KI. (M, N) Adenovirus expressing sFRP1 reduces proliferation of SOX9 (+) cells in KI bulge. (O) sFRP1 restores quiescence of NFATc1 (+) cells in KI. Arrows denote NFATc1/Ki67 double-positive cells in KI treated with GFP adenovirus. Mice were treated with adenovirus at P4, and analyses were performed at P7. Graphs contain means with SEM (n ≥ 3 mice; ≥20 hair follicles/mouse). (F, I–K, N) There were 50–150 cells counted per hair follicle. KI values are relative to WT (set to 1). *p value ≤ 0.05. Scale bars: 10 μm.

FIGURE 8:

Conditional deletion of EGFR in skin results in hair follicle abnormalities identical to KI mice. (A) Mice with skin-targeted deletion of EGFR (Egfr^flox/floxK14cre) fail to produce hair coat. (B) H&E-stained P7 skin sections from Egfr^flox/floxK14cre mice demonstrate abnormal and misaligned hair follicles. (C) Loss of hair keratin (AE13), a hair shaft (HS) differentiation marker of the cortex and medulla in Egfr^flox/floxK14cre mice. (D) Immunofluorescence and quantification of mitosis-specific marker phospho-histone H3 (PH3) showing increased mitotic activity in matrix (Mx) cells of Egfr^flox/floxK14cre. (E, F) Elevated levels of γH2AX (marker of DNA breaks) and activated caspase-3 (Ac-CAS3; apoptosis marker) in Egfr^flox/floxK14cre follicles. (G) Immunofluorescence and quantification of hair follicle stem cells marked by SOX9 showing reduced SOX9 (+) bulge (Bu) cells in Egfr^flox/floxK14cre. (H) Immunofluorescence and quantification of hair follicle stem cells marked by NFATc1 showing reduced NFATc1 (+) hair follicles in Egfr^flox/floxK14cre mouse skin sections. (I) Ki67 (proliferation marker) and SOX9 double immunofluorescence and quantification reveals increase in SOX9 (+) bulge cells positive for Ki67 in Egfr^flox/floxK14cre. (J) Loss of quiescence as reflected by the colocalization of NFATc1 and Ki67 in Egfr^flox/floxK14cre. Arrows denote NFATc1/Ki67 double-positive cells in Egfr^flox/floxK14cre bulge. Graphs contain means with SEM (n ≥ 3 mice; ≥20 hair follicles/mouse). (D, G, I) There were 30–75 cells counted per hair follicle. *p value ≤ 0.05. (K) Increased nuclear β-catenin in Egfr^flox/floxK14cre hair follicle matrix (Mx), dermal papilla (DP), and bulge (Bu). Arrow denotes nuclear β-catenin staining in Egfr^flox/floxK14cre bulge. (L) Chromogenic ISHs demonstrate up-regulation of Wnt4 mRNA (red spots) in Egfr^flox/floxK14cre matrix, DP, and bulge. (M) Increased Wnt10b transcript was observed only in Egfr^flox/floxK14cre matrix. Scale bars: 10 μm.

FIGURE 9:

Transcripts for EGFR-regulated Wnts and EGFR ligands are colocalized in hair follicle stem cells and their progeny. Multiplex fluorescence ISHs on WT skin sections at P7 detects cellular colocalization of Tgfα and Nfatc1 with Wnt4 (A), Wnt7b (B), or Wnt16 (C) in bulge (Bu); (D) Tgfα with Wnt4 in matrix (Mx); (E) Tgfα with Wnt10a in inner root sheath (IRS); and (F) Areg with Wnt4 in outer root sheath (ORS). Arrows denote a nucleus magnified in the inset. Scale bars: 10 μm.

FIGURE 10:

EGFR autocrine loops control Wnt signaling. Model of hair follicle cell is presented in which TGFα/EGFR autocrine loop restrains Wnt expression and signaling. In the absence of EGFR kinase (KI), the transcription of Wnt genes increases and drives elevated Wnt/β-catenin signaling (right).