DOI: 10.5061/dryad.xwdbrv1qj
title: The GPR30 agonist G-1 promotes hair growth via Wnt/Hedgehog signaling in mice

description: 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.
description: # The GPR30 agonist G-1 promotes hair growth via Wnt/Hedgehog signaling in
 mice
 [https://doi.org/10.5061/dryad.xwdbrv1qj](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* &lt; 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* &lt; 0.01, **p* &lt; 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* &lt; 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* &lt; 0.05, ***p* &lt; 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/](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).