Therapeutic potential of isoproterenol in androgenetic alopecia: activation of hair follicle stem cells via the PI3K/AKT/β-Catenin signaling pathway

Abstract

Background: Androgenetic alopecia (AGA) is characterized by the depletion or dormancy of hair follicle stem cells (HFSCs), leading to hair thinning and miniaturization. Reactivating the dormant HFSCs is a promising therapeutic approach. Adrenergic β2 receptor (ADRB2) activation has been shown to promote hair growth in animal models via the Sonic Hedgehog (SHH) pathway, but its potential for treating clinical AGA patients remains unexamined.

Methods: We investigated the role of the PI3K/AKT signaling pathway in AGA pathogenesis, focusing on the hair follicle-sympathetic nerve axis. The ADRB2 agonist, isoproterenol (ISO), was administered to assess its effects on AGA hair follicle organ culture model and HFSC proliferation. The mechanisms underlying these effects were explored by analyzing the PI3K/AKT/β-Catenin pathway.

Results: Our results showed abnormal PI3K/AKT pathway expression in AGA hair follicles, with associated defects in the hair follicle-sympathetic nerve axis. ISO treatment accelerated AGA hair follicle growth and promoted the proliferation of HFSC. Mechanistically, ISO facilitated the HFSC activation by modulating the PI3K/AKT/β-Catenin pathway.

Conclusions: ISO effectively promotes hair growth in both animal models and AGA patients. ISO stimulating the proliferation of dormant cell population enriched in HFSC. This process was likely mediated by the PI3K/AKT/β-Catenin pathway. These findings provide novel insights into the reactivation of HFSCs and suggest that adrenergic signaling stimulation may be a promising strategy for managing hair loss.

Keywords: Androgenetic alopecia; Clinical trial; Hair follicle stem cell; Hair regrowth; Isoproterenol; PI3K/AKT/β-catenin signaling.

Fig. 1

Inhibition of PI3K/AKT signaling and dysfunction of adrenergic activation occurred in the pathogenesis of AGA. (A) The volcano map showed the differentially expressed genes between the AGA miniaturized HFs and normal HFs. (B) The GO-enrichment analysis revealed that the AGA miniaturized HFs were significantly associated with the processes such as hair cycle, hair follicle development, and hair follicle morphogenesis. (C) The KEGG pathway enrichment bubble plot revealed that the AGA miniaturized HFs are significantly associated with PI3K/AKT signaling pathway. (D) qPCR showed the gene expression levels of ADRB2, PI3K and AKT in the AGA miniaturized HFs and normal HFs. (E, F and G) The fluorescent intensity of TH, CK15 and ADRB2 in the AGA miniaturized HFs and normal HFs were shown by immunofluorescence. Scale bar: 50 μm. n = 5. All data are presented as the means ± standard deviations from at least three independent experiments. Student’s t test. AGA, androgenetic alopecia; HFs, hair follicles. **p < 0.01, ***p < 0.001

Fig. 2

Isoproterenol (ISO) induced the hair growth of murine hair follicles. (A) Process of applying ISO as a topical spray in murine models. n = 4. (B) Gross observation of the trunk skin of C57BL/6 mice at different times treated with different doses of ISO or PBS. (C and D) H&E staining showed the neo-HFs of mice after 10 days treatment. Scale bars: 100 μm. (E) Process of topical injection with 10^− 8g/ml ISO in murine models. (F) Gross observation of the trunk skin of C57BL/6 mice at different times treated with 10^− 8g/ml ISO or PBS after 8 days. n = 4. (G and I) H&E staining showed the neo-HFs of mice after 10 days treatment. Scale bars: 100 μm. (H) Relative pigmentation of murine skin in different treatment groups. (J) The mRNA expression of Ki67, CK15 and ADRB2 in murine skin of different treatment groups was analyzed by qPCR. All data are presented as the means ± standard deviations from at least three independent experiments. Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, NS: not significant

Fig. 3

ISO promoted growth of AGA hair follicles in organ culture. (A) Process of topical applying ISO in AGA hair follicles organ culture. (B) Morphology of hair follicles at baseline and after 6 days of treatment in William E medium with or without different doses of ISO (n = 10). (C) Hair shaft elongation after treatment. (D and E) The morphology and hair cycle of hair follicles after 6 days of treatment. (F and G) The expression of Ki67 in the matrix and ORS of HFs treated with or without different doses of ISO was shown by immunofluorescence. Scale bar: 50 μm. (H and I) The expression of CK15, CD200 and Ki67 in the bulge zone of HFs treated with or without different doses of ISO were shown by immunofluorescence. All data are presented as the means ± standard deviations from at least three independent experiments. Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001, NS: not significant

Fig. 4

ISO enhanced the proliferation of cell population enriched in HFSC. (A and E) Live/dead staining of cell population enriched in HFSC after 24 h of treatment with different doses of ISO. Live and dead cells are shown in green and red, respectively. Scale bars: 100 μm. (B, C and F) Morphology and colony formation assays of cells cultured for 3 days with or without ISO and Propranolol. (D and G) The expression of CK15 and Ki67 in HFs treated with or without ISO and Propranolol were shown by immunofluorescence. (H) Process of regenerating hair follicles in vivo. mDCs and mECs or cell population enriched in HFSC were injected into the hypodermis of BALB/c-nude mice, forming a visible bleb. After 3 weeks, the skin at the injection site was excised. n = 4. (I) The outlook and morphology of the de novo hair follicles. (J) Analysis of the reconstituted hair follicle number among the different groups. All data are presented as the means ± standard deviations from at least three independent experiments. Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001, NS: not significant

Fig. 5

ISO activated the cell population enriched in HFSC via PI3K/AKT/β-catenin signaling pathway. (A-C) qPCR results showed the gene expression of the upstream molecules of PI3K/AKT signaling in cell population enriched in HFSC stimulated by ISO, LY-294 (PI3K signaling pathway inhibitor) or Propranolol (ADRB2 inhibitor). ISO significantly upregulated the gene expression of β-catenin, c-myc and CCND1. Also, LY-294 significantly downregulated the gene expression of IGf-1 induced by ISO. CCND1, Cyclin D1. IGF-1, Insulin like growth factor-1. (D and G) Western blotting analysis showed the protein expression of the upstream molecules of PI3K/AKT signaling in cell population enriched in HFSC stimulated by ISO. (E and H) The cell population enriched in HFSC were treated with ISO for 0, 30, 60, and 90 min and subjected to western blotting analysis to examine the protein expression of PI3K/AKT signaling. (F and I) Western blotting results showed that LY-294 or Propranolol significantly downregulated the protein expression of PI3K/AKT signaling induced by ISO. (J-K) Western blotting results showed that LY-294 significantly downregulated the protein expression of β-catenin and Lef-1 (hair growth development marker) induced by ISO. All data are presented as the means ± standard deviations from at least three independent experiments. Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, NS: not significant

Fig. 6

ISO enhanced hair regrowth in AGA patients. (A) Global photography of the representative AGA patient 1 at baseline and 3 months. (b) Medical tattoo was used to mark the target area, and phototrichogram of the treated area in AGA patient 1 at baseline and 3 months were recorded. Red arrows represented hair regeneration. (C) Global photography of the representative AGA patient 2 at baseline and 6 months. (D) Phototrichogram of the treated area in AGA patient 2. (E) Hair count, hair diameter and terminal hair ratio at baseline, 3 and 6 months. Hair count/diameter improvements at 3 and 6 months were only compared with baseline. n = 68

Fig. 7

Isoproterenol regulated PI3K/AKT/β-catenin signaling pathway to activate hair follicle stem cells