The GPR30 agonist G-1 promotes hair growth via Wnt/Hedgehog signaling in mice
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Jul 01, 2025 version files 3.55 MB
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Figure_1.xlsx
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Figure_2.xlsx
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Figure_3.xlsx
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Figure_4.xlsx
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Figure_5.xlsx
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Figure_6.xlsx
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README.md
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Supplementary_Figure_1.xlsx
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Supplementary_Figure_2.xlsx
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Abstract
Background: GPR30 is a membrane-associated receptor involved in rapid, non-genomic estrogen signaling. Estrogen significantly influences hair growth and susceptibility to hair loss, with differences primarily driven by hormonal factors. While estrogen’s role in regulating hair follicle cycling is recognized, its precise molecular mechanisms remain unclear. This study investigates the role of GPR30 in hair follicle biology and evaluates its potential as a therapeutic target for estrogen-mediated hair loss disorders.
Methods: The GPR30 selective agonist G-1 was administrated to female Gpr30-deficient mice with a C57BL/6J background and human hair follicle dermal papilla cell, and the effects on hair growth and the molecular signaling were evaluated.
Results: We demonstrate that GPR30 is abundantly expressed in mouse skin, particularly during the anagen phase of the hair follicle cycle, implicating it in hair growth regulation. Activation of GPR30 using the selective agonist G-1 in mouse skin and human dermal papilla cells significantly upregulated Wnt/Hedgehog signaling, which are key pathways promoting hair growth. These effects were absent in Gpr30-deficient mice or in those administered a GPR30 antagonist, confirming the essential role of GPR30 in estrogen-mediated regulation of hair follicle activity.
Conclusions: Our findings underscore the importance of GPR30 in modulating hair growth and suggest that GPR30, along with its selective agonists, holds promise as a novel therapeutic target for treating hair loss disorders and other estrogen-responsive conditions.
https://doi.org/10.5061/dryad.xwdbrv1qj
Files and variables
File: Figure_1.xlsx
GPR30 was abundantly expressed in the skin.
(A) Gpr30 mRNA expression in 7-week-old mouse tissues was determined using qRT-PCR (n = 3). Sub. Gland, submandibular gland; s. i., small intestine; bone.m, bone marrow; epi, epididymal; peri, perirenal; sub, subcutaneous; WAT, white adipose tissues; BAT, brown adipose tissues. (B) Gpr30 mRNA expression in female mice during hair follicle cyclic phases was determined using qRT-PCR (n = 4). (C) Gpr30 mRNA expression in male mice during hair follicle cyclic phases was determined using qRT-PCR (n = 4). Internal control: 18S rRNA expression. **p < 0.01 (Tukey’s multiple comparison test). Results are presented as the means ± SE. N.S., not significant.
File: Figure_2.xlsx
GPR30 selective agonist G-1 promotes hair growth.
(A) and (B) Dose-dependent effects of estrogen, G-1, and minoxidil on hair growth in wild-type female mice (A, n = 3–10; B, n = 5–10). Scale bar = 1 cm. (C) and (D) Effects of G-1 on hair growth in wild-type female mice (C, n = 10) and Gpr30-deficient female mice (D, n = 10). Scale bar = 1 cm. **p < 0.01, *p < 0.05 (two-way ANOVA with the Bonferroni post hoc test). Representative photographs were taken on Day 1 and Day 17 post-estrogen treatment, and on Day 12 following treatment with G-1 or minoxidil. These experiments were performed using an independent application trial from three biological replicates. Results are presented as means ± SE.
File: Figure_3.xlsx
RNA sequencing analysis shows activation of Wnt/Hedgehog signaling by G-1 stimulation in the skin.
(A) Volcano plot showing the significance and magnitude of differences in the relative abundances of variable genes in the skin of female mice with or without G-1 treatment (n = 7). (B) Heat map of the top 30 variable gene profiles in the skin of female mice with or without G-1 treatment (n = 7). (C) KEGG enrichment analysis related to molecular function in the skin of wild-type female mice with or without G-1 treatment (n = 7). P-values were adjusted using the false discovery rate (FDR).
File: Figure_4.xlsx
Pathway analysis revealed activation of the Wnt/Hedgehog signaling pathway following G-1 stimulation in the skin.
(A and B) KEGG pathway analysis of Wnt or Hedgehog signaling-related factors in the skin of wild-type female mice with or without G-1 treatment (n = 7).
File: Figure_5.xlsx
G-1 promotes Wnt/Hedgehog signaling via GPR30 in the skin.
(A) Beta diversity as shown via principal component analysis (PCA) based on genes from KEGG in the skin of wild-type and Gpr30-deficient female mice with or without G-1 treatment (n = 3). Compositional similarity was compared using PERMANOVA. (B) Heat map of Wnt/Hedgehog-related gene profiles in the skin of wild-type and Gpr30-deficient female mice with G-1 treatment (n = 3). P-values were adjusted using the false discovery rate (FDR). (C) mRNA expression levels of Wnt/Hedgehog signaling-related genes in the skin of wild-type and Gpr30-deficient female mice measured by qRT-PCR (n = 3–4). Internal controls: 18S rRNA expression. *p < 0.05 (Mann–Whitney U test). All data are presented as the means ± SE.
File: Figure_6.xlsx
G-1 promotes Wnt/Hedgehog signaling in the human primary DPCs.
(A) mRNA expression levels of Wnt signaling-related genes in human hair follicle dermal papilla cells (hDPCs) stimulated with G-1 with or without G-36 measured by qRT-PCR (n = 6). Internal controls: RPLP0 expression. (B) Activation of the Wnt signaling-related protein β-Catenin in hDPCs stimulated with G-1, with or without G-36, was measured using Western blotting (n = 4). Internal controls: β-actin expression. (C) mRNA expression levels of Hedgehog signaling-related genes in hDPCs stimulated with G-1, with or without G-36, measured using qRT-PCR (n = 6). Internal controls: RPLP0 expression. (D) Expression levels of Hedgehog signaling-related proteins in hDPCs stimulated with G-1, measured using western-blotting (n = 8). Internal controls: β-actin expression. *p < 0.05, **p < 0.01 (Tukey–Kramer’s post-hoc test). All data are presented as the means ± SE.
File: Supplementary_Figure_1.xlsx
GPR30 mRNA expression profiles in hair cells.
Gpr30 mRNA expression was analyzed in hair cells of postnatal day 5 mice (n = 2) using the database (http://www.hair-gel.net/). All data are presented as the means. These experiments were performed with independent cell populations isolated from fluorescent reporter mouse lines across three biological replicates. Epi, epidermis; ORS, outer root sheath; Mx, matrix; TAC, transit amplifying progenitors; Mc, melanocytes; DF, dermal fibroblasts; DP, dermal papilla; Neg, mixed negative cells; HF-SC, hair follicle stem cell precursors; HF-ORS, hair follicle outer root sheath; ZZ-DP, zigzag dermal papilla; G/AA-DP, guard/awl and auchene dermal papilla; G-DP, guard dermal papilla; AA/ZZ-DP, awl and auchene/ zigzag dermal papilla.
File: Supplementary_Figure_2.xlsx
Schematic overview of the structure and expression of the Gpr30 gene.
(A) Gpr30 gene-knockout (Gpr30-/-) mice were generated using the CRISPR/Cas9 system in wild-type C57BL/6 zygotes. Coding exon regions are depicted as black boxes and labeled accordingly. The guide RNA (gRNA) and protospacer adjacent motif (PAM) are represented by light blue and purple boxes, respectively. (B) Mouse genotypes were identified by PCR using specific primers to differentiate between wild-type and mutant alleles of Gpr30. (C) Expression of the Gpr30 gene was analyzed in skin samples (n = 4 tissues). Internal control: 18S rRNA expression. All data are presented as the means ± SE.
File: Supplementary_Figure_3.xlsx
GPR30 selective agonist G-1 does not promote hair growth in male mice.
Effects of G-1 (100 nmol) on hair growth in wild-type male mice (n = 10). Scale bar = 1 cm. (two-way ANOVA with the Bonferroni post hoc test). Representative photographs were taken on Days 1 and 17 following G-1 treatment. These experiments were performed using independent application trials from three biological replicates. Results are presented as means ± SE.
File: Supplementary_Figure_4.xlsx
RNA sequencing analysis showed irrelevant BMP and FGF signaling following G-1 stimulation in the skin.
Heat map of BMP and FGF-related gene profiles in the skin of female mice with or without G-1 treatment (n = 7). P-values were adjusted using the false discovery rate (FDR).
Access information
All data generated or analyzed during this study are included in this published article and its Supplementary Material files or are available from the corresponding authors upon reasonable request. The RNA sequencing dataset has been deposited in the DNA Data Bank of Japan with accession numbers DRA020109, DRA021397, E-GEAD-908, and E-GEAD-1078. All data relevant to this study were deposited in the Dryad database (DOI:10.5061/dryad.xwdbrv1qj).