Article Figures & Data
Figures
- Fig. 1.
Functional expression of TRPV3 proteins in mouse skin keratinocytes and ORS cells of hair follicles. (A) Left panel, Western blot analysis of TRPV3 proteins in human outer root sheath cells, dorsal skin of C57BL/6 mice, and HEK293/TRPV3 stably expressing the TRPV3 as positive controls, and untransfected HEK293 cells as negative control. Right panel, whole-cell patch clamp recordings of HEK293 cells overexpressing hTRPV3 in response to carvacrol alone (300 μM, red bar) or coapplication of 100 μM forsythoside B (blue bar). (B) Immunohistochemistry staining of TRPV3 proteins in mouse skin keratinocytes and hair follicles. Scale bar, 100 μm. (C) TRPV3 current in ORS cells was activated by 300 μM carvacrol (red bar) and inhibited by coapplication of 100 μM forsythoside B (blue bar). The right panel shows current-voltage curves of hTRPV3 channel in response to voltage ramps from −100 to +100 mV before (1), and after 300 μM carvacrol (2), and coaddition of 300 μM carvacrol and 100 μM forsythoside B (3), and washout (4).
- Fig. 2.
Inhibition of TRPV3 by forsythoside B attenuates carvacrol-induced ORS cell death. (A) Measurement of cell death for ORS cells before and after treatment of agonist carvacrol in different concentrations for 12 hours in cell death assay. Scale bar, 100 μm. (B) Summary from (A) for cell death ratio before and after treatment of carvacrol in different concentrations (N.S., no significance; **P < 0.01; ****P < 0.0001, by one-way ANOVA, followed by Dunnett’s test). (C) Measurement of cell death for ORS cells before and after treatment of carvacrol (300 μM) and different concentrations of forsythoside B for 12 hours in cell death assay. Scale bar, 100 μm. (D) Summary of (C) for cell death ratio after treatment of carvacrol (300 μM) and forsythoside B in different concentrations. Data are presented as the mean ± S.D. (N.S., no significance, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by one-way ANOVA, followed by Dunnett’s test.) (E) Western blot analysis of TRPV3 proteins in ORS cells and ORS cells transfected with TRPV3 shRNA. (F) ORS cells and ORS cells transfected with TRPV3 shRNA were treated with 300 μM carvacrol for 24 hours. Cell viability was determined by colorimetric MTT cell viability assay. Data are presented as the mean ± S.D. [n = 6, ****P < 0.0001, by Student’s t test (unpaired).]
- Fig. 3.
Pharmacological activation of TRPV3 by carvacrol inhibits hair growth. (A) Top panel, a schematic drawing for generation of TRPV3 agonist carvacrol-induced hair growth inhibition in C57BL/6 mice. Bottom panel, mice (7–8 weeks) were shaved and given different compounds topically once a day until hair grew out, images of hair growth were recorded every 3 days. Scale bar, 500 μm. (B) Summary from (A) for hair length randomly plucked and measured (n = 8) at different time intervals (9, 12, 15, 18, and 21 day) after topical application of different concentrations of TRPV3 agonist carvacrol (Car) and antagonist forsythoside B. Data are presented as the mean ± S.D. (****P < 0.0001, by two-way ANOVA, followed by Dunnett’s test.) (C) A self-comparison group between the left and right side of the skin after topical application of 191.1 mM carvacrol and vehicle (50% ethanol), respectively, once a day, and images for hair growth were recorded every 3 days. Scale bar, 500 μm. (D) Summary from (C) for comparison of hair length randomly plucked (n = 8) between left (L) and right (R) side of the skin. Data are presented as the mean ± S.D. (****P < 0.0001, by two-way ANOVA, followed by Sidak’s test.)
- Fig. 4.
Inhibition of TRPV3 by forsythoside B attenuates carvacrol-induced hair growth inhibition in mice. (A) Top panel, a schematic drawing for generation of TRPV3 agonist carvacrol-induced hair growth inhibition and treatment with FB in C57BL/6 mice. In bottom panel, mice (7–8 weeks) were shaved and intradermal injections of forsythoside B in different concentrations (0.3, 1, 3 mM in 50 μl) into the dorsal area of skin 30 minutes before topical application of carvacrol (63.7 mM) once a day until hair grew out, images were recorded every 3 days. Scale bar, 500 μm. (B) Summary from (A) for hair length randomly plucked and measured (n = 8) at different time intervals (9, 12, 15, 18, and 21 day) after topical application or coapplication of different concentrations of TRPV3 agonist carvacrol (Car) and antagonist forsythoside B. Data are presented as the mean ± S.D. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by two-way ANOVA, followed by Dunnett’s test.)
- Fig. 5.
Activation of TRPV3 decreases hair follicles, and inhibition of TRPV3 prevents loss of hair follicles induced by carvacrol. (A) Transverse sections (top two rows) and longitudinal sections (bottom row) of dorsal skin tissues were stained with H&E before and after topical application of TRPV3 inhibitor forsythoside B alone or activator carvacrol (Car) in 63.7 or 191.7 mM or combination of Car and FB once a day. Scale bar, 100 μm. The skin tissue sections were prepared on day 10 after shaving. Representative images of skin tissue sections in each group were from eight mice. (B) Summary from (A) for the number of hair follicles before and after topical applications of TRPV3 modulators (carvacrol or forsythoside B) or combination. Data are presented as the mean ± S.D. (N.S., no significance, *P < 0.05; **P < 0.01; ***P < 0.001, by one-way ANOVA followed by Bonferroni’s test.)
- Fig. 6.
A proposed mechanism underlying the death for hair follicle ORS cells and hair loss by TRPV3 activation. TRPV3 channels are expressed in the hair follicle ORS cells. Activation of TRPV3 channel by agonist carvacrol or gain-of-function mutation causes an excessive Ca2+ influx that induces ORS cell death via activation of unidentified Ca2+-dependent kinase. Overactive TRPV3 either by carvacrol (or gain-of-function mutations) causes a time- and dose-dependent reduction of hair shaft elongation and a decrease in hair follicles Probably through the induction of ORS cell death in hair follicles, thus leading to inhibition of hair growth. Pharmacological inhibition of overactive TRPV3 by forsythoside B can reverse hair loss.
Tables
- TABLE 1
Pharmacological activation of TRPV3 by carvacrol causes delay of hair growth in mice Mean ± S.D. n = 8.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by one-way ANOVA, followed by Dunnett’s test
Group Time for Pink Skin Turning to Black Time for Black Skin to Hair Growth Time to Full Hair Growth Day Day Day Control 8.0 ± 0.6 3.0 ± 0.5 9.0 ± 0.4 50% Ethanol 8.0 ± 0.6 3.0 ± 0.7 9.0 ± 0.6 0.3 mM FB 7.0 ± 0.4 2.0 ± 0.5 8.0 ± 0.6 19.1 mM Car 9.0 ± 0.9 4.0 ± 0.7 9.0 ± 0.6 63.7 mM Car 10.0 ± 1.2** 4.0 ± 0.8* 10.0 ± 0.9 191.1 mM Car 11.0 ± 1.8**** 5.0 ± 0.9*** 10.0 ± 1.1** - TABLE 2
Reversal of TRPV3 agonist carvacrol-induced inhibition of hair growth by inhibitor forsythoside B Mean ± S.D. n = 8.*p < 0.05, by one-way ANOVA, followed by Dunnett’s test
Group Time for Pink Skin to Black Time for Black Skin to Hair Growth Time to Full Growth of Hair Day Day Day 63.7 mM Car 10.0 ± 0.9 4.0 ± 0.7 10.0 ± 1.0 Car + 0.3 mM FB 9.0 ± 1.8 4.0 ± 0.7 10.0 ± 1.1 Car + 1.0 mM FB 9.0 ± 1.2 4.0 ± 0.6 9.0 ± 0.8 Car + 3.0 mM FB 7.0 ± 0.7* 3.0 ± 0.5* 9.0 ± 0.6
