Positive regulatory interactions between YAP and Hedgehog signalling in skin homeostasis and BCC development in mouse skin in vivo

Abstract

Skin is a highly plastic tissue that undergoes tissue turnover throughout life, but also in response to injury. YAP and Hedgehog signalling play a central role in the control of epidermal stem/progenitor cells in the skin during embryonic development, in postnatal tissue homeostasis and in skin carcinogenesis. However, the genetic contexts in which they act to control tissue homeostasis remain mostly unresolved. We provide compelling evidence that epidermal YAP and Hedgehog/GLI2 signalling undergo positive regulatory interactions in the control of normal epidermal homeostasis and in basal cell carcinoma (BCC) development, which in the large majority of cases is caused by aberrant Hedgehog signalling activity. We report increased nuclear YAP and GLI2 activity in the epidermis and BCCs of K14-CreER/Rosa-SmoM2 transgenic mouse skin, accompanied with increased ROCK signalling and ECM remodelling. Furthermore, we found that epidermal YAP activity drives GLI2 nuclear accumulation in the skin of YAP2-5SA-ΔC mice, which depends on epidermal β-catenin activation. Lastly, we found prominent nuclear activity of GLI2, YAP and β-catenin, concomitant with increased ROCK signalling and stromal fibrosis in human BCC. Our work provides novel insights into the molecular mechanisms underlying the interplay between cell signalling events and mechanical force in normal tissue homeostasis in vivo, that could potentially be perturbed in BCC development.

Fig 1. YAP activation in the skin of K14-CreER/Rosa-SmoM2 transgenic mice.

H&E histological staining (A) and Immunofluorescence (B) staining of dorsal skin sections of tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic mice detecting GLI2 and YAP. Quantification of % GLI2-YAP co-positive (C), % GLI2 positive (D) and % YAP positive nuclei (E). (F) qPCR quantification of mRNA levels of Thbs, Ctgf, Inhba and Gli2 genes relative to 18S in lysates extracted from the dorsal skin of tamoxifen (control) and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic mice. Basement membranes are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.

Fig 2. Activated ROCK-signalling, increased dermal fibroblast numbers, and dermal fibrosis in the skin of K14-CreER/Rosa-SmoM2 transgenic mice.

(A-G) Immunofluorescence staining and area coverage analysis of dorsal skin tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic littermate mice detecting Fsp1 (A & B) and Vimentin (C), Phalloidin (G), DIAPH3 (H), Thr696-phosphorylated MYPT1 (I & J), Thr18/Ser19-phosphorylated MLC2 (K & L). (D) Masson’s trichrome histological staining of sections through the dorsal neck skin of tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 mice. (E & J) Dual two-photon SHG and monochromatic transmission (Trans; grayscale in merge) images showing collagen (white in single channel, magenta in merged) in tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 skin sections. Area coverage analysis (5 fields/sample from three mice per genotype) of SHG is quantified. Basement membranes and hair follicles are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.

Fig 3. GLI2 activation in the skin of YAP2-5SA-ΔC transgenic mice.

(A) Immunofluorescence staining of dorsal skin sections of YAP2-5SA-ΔC transgenic and wildtype mice detecting GLI2 (green) and YAP (red). Quantification of % YAP-GLI2 co-positive (arrowheads—B), % GLI2 positive (C) and % YAP (D) positive nuclei in the skin sections of YAP2-5SA-ΔC transgenic and wildtype mice. Basement membranes are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.

Fig 4. β-catenin activity mediates GLI2 activation in skin of YAP2-5SA-ΔC mice.

Immunofluorescence stainings of dorsal neck skin sections of P50 of YAP2-5SA-ΔC/K14-creERT/CtnnB1^lox/lox and YAP2-5SA-ΔC/K14-creERT/CtnnB1^-/- littermate mice detecting β-catenin (A, red), and GLI2 (C, green), and YAP (C, red). (B) Genotypic characterization of P50 YAP2-5SA-ΔC/K14-creERT/CtnnB1^lox/lox and YAP2-5SA-ΔC/K14-creERT/CtnnB1^-/- mutant littermate mice. Quantification of % GLI2 (D) and YAP (E) positive nuclei in the skin sections of P50 of YAP2-5SA-ΔC/K14-creERT/CtnnB1^lox/lox and YAP2-5SA-ΔC/K14-creERT/CtnnB1^-/- littermate mice. Basement membranes are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.

Fig 5. Human BCCs exhibit nuclear YAP and β-catenin in association with ROCK signalling activation and increased ECM collagen deposition.

Representative images of immunohistochemical staining (brown) of Gli2 (A), YAP (B), Thr696-phosphorylated MYPT (C) and β-catenin (D) in normal and human BCCs skin samples. (E) Masson’s trichrome histological staining. IHC, Immunohistochemistry. Scale bars = 20 μm.

Fig 6. A model outlining the cross-regulatory interactions between epidermal YAP, ROCK, β-catenin and Hh signalling.

(A) Epidermal SmoM2 activates YAP, ROCK signalling and dermal fibroblasts in the dorsal skin of K14-CreER/Rosa-SmoM2 transgenic mice (based on Figs 1 and 2). (B) Epidermal YAP activates GLI2 mediated by β-catenin activation in the dorsal skin of YAP2-5SA-ΔC transgenic mice (based on Figs 3 and 4). (C) A model outlining the proposed regulatory interactions between epidermal YAP, Hedgehog and ROCK-dependent mechanosignalling to balance skin regeneration based on our findings (red arrows) and on cited studies [25, 27, 29, 34, 39, 40, 45, 47, 48, 63, 64, 65, 66, 69].