Roles of GasderminA3 in Catagen-Telogen Transition During Hair Cycling

Abstract

Hair follicles undergo cyclic behavior through regression (catagen), rest (telogen), and regeneration (anagen) during postnatal life. The hair cycle transition is strictly regulated by the autonomous and extrinsic molecular environment. However, whether there is a switch controlling catagen-telogen transition remains largely unknown. Here we show that hair follicles cycle from catagen to the next anagen without transitioning through a morphologically typical telogen after Gsdma3 mutation. This leaves an ESLS (epithelial strand-like structure) during the time period corresponding to telogen phase in WT mice. Molecularly, Wnt10b is upregulated in Gsdma3 mutant mice. Restoration of Gsdma3 expression in AE (alopecia and excoriation) mouse skin rescues hair follicle telogen entry and significantly decreases the Wnt10b-mediated Wnt/β-catenin signaling pathway. Overexpression of Wnt10b inhibits telogen entry by increasing epithelial strand cell proliferation. Subsequently, hair follicles with a Gsdma3 mutation enter the second anagen simultaneously as WT mice. Hair follicles cannot enter the second anagen with ectopic WT Gsdma3 overexpression. A luciferase reporter assay proves that Gsdma3 directly suppresses Wnt signaling. Our findings suggest that Gsdma3 has an important role in catagen-telogen transition by balancing the Wnt signaling pathway and that morphologically typical telogen is not essential for the initiation of a new hair cycle.

Figure 1. Gsdma3 expression pattern and morphological changes of AE mouse hair follicles during the hair cycle

(a) Gsdma3 expression pattern during the hair cycle. (b) H&E staining of back skins in WT and AE mice. White dashed lines show the DP structures. (c) H&E staining and schematic show morphology of hair follicles at P29. (d) Schematic illustration of the abnormal Hair cycle in AE mice compared with their WT littermates. n>20. AE: Gsdma3 mutant mice; WT: Wild type. Bu: bulge; DP: dermal papilla; ES: epidermal strand; HG: hair germ; HS: hair shaft; SHG: secondary hair germ. Bar=50µm.

Figure 2. Wnt pathway is upregulated in AE mice

Bar charts display fold change of (a) Wnt ligand mRNA expression, (b) Wnt receptors, (c) β-catenin and Lef1, (d) Wnt targets and (e) Wnt inhibitors in WT and AE back skin (n>3) at different time points. (f) Immunofluorescence analysis of Wnt10b and Lef1 expression in WT and AE hair follicles. (g) Immunofluorescence shows the colocalization of nuclear β-catenin with Gsdma3 in the WT and AE hair matrix (arrowheads). n>10. Data are reported as average ± SD. *P<0.05, **P<0.01. AE: Gsdma3 mutant mice; WT: Wild type. DP: dermal papilla. Bars=50µm.

Figure 3. Gsdma3 overexpression restores the telogen phase in AE mice

(a) H&E staining shows the structural changes of AE mouse skin after AdGsdma3 compared to AdRFP injection. (b) Magnification of the hair bulb after viral treatment. (c) Immunofluorescence analysis of Wnt10b, β-catenin, Lef1 expression and BrdU positive cells in AdGsdma3 and AdRFP-injected hair bulbs (white arrow shows nuclear staining). RT-PCR and Western blotting shows the mRNA (d) and protein (e) levels of Wnt10b, β-catenin, Lef1 after AdGsdma3 and AdRFP treatment (n=3). (f) H&E staining shows hair follicles maintain catagen phase after Wnt10b overexpression. Data are reported as average ± SD. *P<0.05, **P<0.01. AE: Gsdma3 mutant mice; WT: Wild type. DP: dermal papilla. Bars=50µm.

Figure 4. WT and AE mouse hair follicles start the second anagen at a similar time

(a) H&E staining shows the progression of hair follicle regeneration from the second anagen onset. (b) Immunofluorescence shows K15 and BrdU expression during hair follicle re-growth (arrows indicate BrdU positive cells). (c–e) Statistical charts show the BrdU positive cells in different hair follicle compartments during hair regeneration (n=20). (f–h) Immunofluorescence staining and statistical charts show the expression of nuclear β-catenin (n=20; white arrows). Data are reported as average ± SD. # no statistical difference, *P<0.05. AE: Gsdma3 mutant mice; WT: Wild type. DP: dermal papilla. Bars=50µm.

Figure 5. Gsdma3 overexpression at anagen onset blocks hair follicle regeneration by repressing Wnt signaling

(a) H&E staining shows the hair follicles (blue arrows) don’t enter anagen after AdWnt10b+AdGsdma3 treatment. (b–d) Statistical charts and immunofluorescence analysis show mRNA and protein level changes after virus transduction. (e) Statistical chart of the different BrdU+ cell numbers between WT and AE mice after AdGsdma3 treatment. (f) Immunofluorescence shows Wnt signaling after AdGsdma3 injection into skin after depilation. (g–h) Statistical charts show Ki67 and BrdU+ cells in depilated skin treated with AdGsdma3. (i–l) Fluorescence and statistical charts show the GFP, mRNA, and protein levels and TOP/FOP ratios in JB6 cells transfected with pEGFP-Gsdma3 or pEGFP-N1 (n=3). *P<0.05. Bar=500µm (a). DP: dermal papilla.

Figure 6. Schematic of hair cycle after Gsdma3 mutation

At mid-catagen phase of WT hair follicles, Gsdma3 inhibits Wnt10b which leads to low proliferation. Combined with high inhibitors and high apoptosis, the hair follicles transit from catagen to telogen. However, after Gsdma3 mutation, Wnt10b is not inhibited leading to high proliferation. Moreover, low apoptosis and high Wnt10b-promotes hair stem cell emigration from the bulge region to keep the ESLS, resulting in a blocked catagen-telogen transition. At anagen onset of WT hair follicles, low inhibitor and high activator promote anagen phase entry. But after Gsdma3 mutation, low inhibitors and high Wnt10b will force catagen stage follicles to directly enter anagen phase. The ESLS may become different lineages including SHG, ORS and IRS.