Hair Loss Caused by Gain-of-Function Mutant TRPV3 Is Associated with Premature Differentiation of Follicular Keratinocytes

Abstract

Gain-of-function mutations in the TRPV3 gene can cause Olmsted syndrome characterized by palmoplantar and periorificial keratoderma, itch, and hair loss. The mechanism underlying the hair loss remains unclear. In this study, we engineered an Olmsted syndrome mouse model by introducing the point mutation G568V to the corresponding Trpv3 locus in the mice. These mice developed fully penetrant hair loss. The hair loss was associated with premature differentiation of follicular keratinocytes characterized by precocious degeneration of trichohyalin and keratins, increased production of deiminated proteins, elevated apoptosis, and attenuation of transcription regulators (Foxn1, Msx2, Dlx3, and Gata3) known to regulate hair follicle differentiation. These abnormalities occurred in the medial‒proximal region of the inner root sheath and the hair shaft, where Trpv3 is highly expressed, and correlated with an impaired formation of the hair canal and the hair shaft. The mutant Trpv3 mice also exhibited increased proliferation in the outer root sheath, accelerated hair cycle, reduction of hair follicle stem cells, and miniaturization of regenerated hair follicles. Findings from this study suggest that precocious maturation of postmitotic follicular keratinocytes drives hair loss in patients with Olmsted syndrome.

Figure 1.. Trpv3 expression pattern in hair follicles.

(a) In situ hybridization of Trpv3 (pink) in dorsal skins of postnatal (P) 5 and P12 mice. (b - c) Duplex in situ hybridization of Trpv3 (pink) and Gata3 (cyan) in (b) and Trpv3 (pink) and Tchh (cyan) in (c) in dorsal skins of P12 mice. (d) In situ hybridization of TRPV3 (cyan) and GATA3 (pink) in a human fetus scalp hair follicle. (e) Immunofluorescence labeling of FLAG and KRT75 or TCHH (with the AE15 antibody) in dorsal skins of P8 Trpv3-Flag knock-in mice. Nuclei were stained with hematoxylin (blue, a - d) or labeled with DAPI (blue, e). Scale bar = 200 μm (a), 25 μm (b - c), 100 μm (d - e).

Figure 2.. Hair phenotype in Trpv3 knock-in mouse model.

(a - b) Appearance (a) and histology (b) of dorsal skins from Trpv3^+/+, Trpv3^+/G568V and Trpv3^G568V/G568V littermates. (c) Transmission electron microscopy of hair follicles at approximately 500 μm depth. Arrow points to the electron-light gap between the IRS and HS Cu. ORS, outer root sheath; CL, companion layer; He, Henle’s layer; Hu, Huxley’s layer; IRS Cu, inner root sheath cuticle; HS Cu, hair shaft cuticle; Co, cortex; Me, medulla. (d) Quantification of the gap between the IRS and HS Cu in (c). n = 3 mice per genotype. ≥ 18 hair follicles were examined per genotype. Data are presented as mean ± SD. *** P < 0.001 (Two-tailed Student’s unpaired t test). Scale bar = 100 μm (b), 4 μm (c).

Figure 3.. Impaired hair follicle differentiation in Trpv3 knock-in mice.

(a) Immunofluorescence labeling of KRT71 (green), KRT75 (green), KRT82 (red), and hair cortex keratins detected by the AE13 antibody (red) in dorsal skins of P12 Trpv3^+/+ and Trpv3^G568V/G568V littermates. Arrow heads point to the portions of hair follicles positive for KRT71, KRT82, and AE13, respectively. Nuclei were stained with DAPI (blue). (b) Western blotting of KRT71, hair cortex keratins (AE13), and KRT75, and quantifications. Whole length images can be found in Supplementary Figure S6. n = 3 mice per genotype. Data are presented as mean ± SD. * P < 0.05. ** P < 0.005. ns, not significant. One-way ANOVA analysis was followed by Dunnett’s Multiple Comparison Test. Scale bar = 100 μm (a).

Figure 4.. Late differentiation of dorsal hair follicles in P12 Trpv3 knock-in mice.

(a) Illustration of posttranslational modification of TCHH. (b - c) Immunofluorescence labeling of TCHH (red, b) and immunohistochemistry of deiminated proteins (ruby red, c). (d) Quantification of distance between the Auber’s line (point a) and the most proximal signal of deiminated proteins (point b), and ratio of deiminated proteins (point b to c) and nonpermanent portion of the hair follicles (point a to c), as shown in (c). n = 3 mice per genotype. ≥ 10 hair follicles per genotype were examined. Data are presented as mean ± SD. *** P < 0.001 (Two-tailed Student’s unpaired t test). (e) In situ hybridization of Padi3 (pink, left) and Tgm3 (pink, right). (f - g) TUNEL staining and quantification. n = 5 mice per genotype and ≥ 16 hair follicles per genotype were examined. Data are presented as mean ± SD. *** P < 0.001 (Two-tailed Student’s unpaired t test). Scale bar = 200 μm (b - c), 100 μm (e - f).

Figure 5.. Examination of regulators for hair follicle induction and differentiation.

(a - b) In situ hybridization of Axin2 (pink, left) and Ptch1 (pink, right) in (a), and Foxn1 (pink, top left), Msx2 (pink, top right), Dlx3 (pink, bottom left) and Gata3 (pink, bottom right) in (b), in dorsal skins of P12 Trpv3^+/+ and Trpv3^G568V/G568V littermates. For (a - b), nuclei were stained with hematoxylin (blue). Scale bar = 50 μm (a - b).

Figure 6.. Impaired hair regeneration in Trpv3 knock-in mice.

(a) Appearance at indicated age. (b - d) Alkaline phosphatase staining of dorsal skins of P249 littermates (b), and quantification of hair follicle density (c) and anterioposterior orientation (d). A, anterior. P, posterior. Radial axis represents the number of hair follicle in that specific angle. n = 4 mice per genotype and ≥ 20 microscopic fields per genotype were examined. Number of hair follicles evaluated: Trpv3^+/+, 278; Trpv3^+/G568V, 262; Trpv3^G568V/G568V, 283. Data are presented as mean ± SD. *** P < 0.001 (One-way ANOVA analysis was followed by Dunnett’s Multiple Comparison Test). (e) Histology of dorsal skins at indicated postnatal days. (f - g) BrdU labeling and quantification in P12 littermates. n = 5 mice per genotype and ≥ 27 hair follicles per genotype were examined. Data are presented as mean ± SD. *** P < 0.001, ** P < 0.05 (Two-tailed Student’s unpaired t test). (h - j) Immunofluorescence labeling of CD34 (h) and NFATC1 (i), and quantification (j) in P48 littermates. n = 3 mice per genotype and ≥ 17 hair follicles per genotype were examined. Data are presented as mean ± SD. *** P < 0.001 (Two-tailed Student’s unpaired t test). Scale bar = 500 μm (b), 200 μm (e), 100 μm (f), 50 μm (h - i).