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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.

Introduction

Recognized as the most prevalent form of clinical hair loss, androgenetic alopecia (AGA) significantly impacts both the physical appearance and overall psychological well-being [1]. AGA is primarily driven by multiple factors that disrupt the natural growth cycle of hair follicles (HFs), leading to their miniaturization [2]. Currently, treatments for AGA include topical therapies, oral medications, and surgical interventions [3]. However, optimal outcomes are not always achieved, and adverse effects are occasionally reported, which may lead to the discontinuation of treatment [4]. Recently, the activation of miniaturized HFs through the modulation of signaling pathways is considered as a promising and innovative strategy for the management of hair loss [5].

The growth cycle of HFs is tightly regulated by the precise temporal and spatial secretion of norepinephrine, a neuropeptide released by the sympathetic nervous system [6]. Situated between the vessels and HFs, the sympathetic nerves are closely associated with the arrector pili muscles (APMs) in the dermis [7]. These nerves innervate the bulge region, known as the niche of hair follicle stem cells (HFSCs), promoting APM contraction and HFSC activation. Recently, Shwartz et al. have shown that norepinephrine (NE) secretion from sympathetic nerves stimulates hair growth in mice by activating adrenergic β2 receptors (ADRB2) [8]. In the absence of ADRB2 activation, HFSCs were observed to enter a state of deep quiescence, resulting in the failure of APM formation and subsequent hair regeneration [9]. While NE is recognized as a potent α-adrenergic receptor agonist, its activity on β-adrenergic receptors is relatively limited [10]. In contrast, isoproterenol (ISO) was approved for the treatment of angio-cardiopathy in 1986 and widely utilized as a vasoactive drug for its selective agonistic effects on β-adrenergic receptors [11]. Although the mechanism by which ISO promotes HF growth has not been extensively investigated, there is a growing need for further research into its therapeutic potential.

The PI3K/AKT signaling pathway plays a crucial role in cellular proliferation and migration, glucose metabolism, cytoskeletal reorganization, and angiogenesis [12]. Glycogen synthase kinase 3β (GSK3β), an evolutionarily-conserved serine/threonine kinase, is integral to glucose metabolism [13]. Normally, GSK3β acts as a negative regulator by inhibiting the activity of β-catenin. Upon activation of the PI3K/AKT pathway, GSK3β is inactivated through phosphorylation, leading to the accumulation of β-catenin in the cytoplasm [14]. Recognized as a key regulator of hair follicular fate determination, the β-catenin subsequently activates the nucleus genes involved in cell division and growth regulation, such as c-myc and Cyclin D1 [15]. Previous studies have demonstrated that β-catenin promoted hair regeneration by activating downstream genes that regulate the differentiation of HFSCs [16]. In contrast, inhibition of Wnt/β-catenin pathway by its antagonist DKK1 prevents HFSCs from differentiating into various follicular components, thereby suppressing hair growth [17].

In this study, the genetic differences between AGA miniaturized HFs and the normal HFs were identified. Notably, the expression of the PI3K/AKT signaling pathway was observed to be suppressed in miniaturized HFs, accompanied by reduced ADRB2 expression and impaired activation of cell population enriched in HFSC. Additionally, it was proposed that ISO facilitates the activation and proliferation of cell population enriched in HFSC via the PI3K/AKT/β-catenin signaling pathway. Our findings provide insight into the crosstalk between ADRB2 and the PI3K/AKT/β-catenin signaling pathway in the regulation of stem cell activity.

Methods

Statement: the work has been reported in line with the ARRIVE guidelines 2.0

Sample collection

Hair follicle samples from AGA or normal patients were anonymously collected with approval from the Medical Ethics Committee of Southern Medical University as part of an exempt protocol. During hair transplantation procedures, discarded follicular tissue samples were obtained from the occipital region and bald frontal areas. All specimens were derived from male patients aged 35 to 42 years. None of the participants had used finasteride or minoxidil prior to sample collection.

Animals

3 weeks old C57BL/6J female mice and 5 weeks old BALB/c-nude mice were purchased from the Experimental Animal Centre at Southern Medical University. In vivo experiments, depilated C57BL/6 mice were randomly received daily treatments with PBS or ISO, n = 4. (Eight random numbers are generated using Excel and assigned to each mouse. No animal anaesthesia was used in this study. The random numbers were then sorted by size, with the first 4 mice being the control group and the last 4 being the treatment group). To relieve the mice, the rapid cervical dislocation method was performed correctly by a well-trained person to sacrifice the mice, and no anesthetic was used during the animal experiments. Photographs of the skin was conducted daily, and dorsal skin were collected at each treatment time point. Skin samples for H&E staining and immunohistochemistry were fixed in 4% paraformaldehyde at 4 °C for 24 h, while those designated for qPCR analysis were stored at -80 °C. Ethical approval for all experimental procedures was obtained from the Experimental Animal Centre of Southern Medical University. The sample size was decided following the “3R principle” (reduce, replace, optimize). The work has been reported in line with the ARRIVE guidelines 2.0.

Hair follicle organ culture

Hair follicles in the anagen stage were micro-dissected from discarded occipital scalp samples under a dissecting microscope, as described in the previous article [18]. Individual HF was placed in the 24-well culture plate with Williams E medium supplemented with 10 ng/ml hydrocortisone, 10 µg/ml insulin, 10 U/ml penicillin, and 2 mM l-glutamine in the presence of ISO or PBS. The HFs were photographed using an inverted microscope and measured for relative length and morphology every other day for 6 days. HFs were harvested after treatment for H&E staining.

Cell Preparation and culture

Neonatal murine epidermal cells and dermal cells were harvested from C57BL/6J mice [19]. Briefly, the skin was mechanically removed by tweezers for digestion with 0.1% Dispase at 37℃ for 1 h. Then the skin specimen was separated into epidermis and dermis by forceps. The epidermis was fragmented and digested using 0.025% trypsin at 37 °C for 10 min, while the dermis was treated with 0.2% collagenase at 37℃ for 1 h. Following enzymatic digestion, the reaction was terminated by adding an equal volume of 10% fetal bovine serum in DMEM. The cell suspension was then passed through 70 μm strainers. After centrifugation and subsequent washing steps, neonatal murine epidermal and dermal cells were successfully isolated. As described previously [20], cell population enriched in HFSC were isolated from dissected human individual HFs samples. In brief, the bulge region was dissected and treated with 0.1% dispase for 45 min. Under a stereomicroscope, the dermal sheath was removed. The rest tissue was further digested with 0.025% trypsin at 37 °C for 10 min. Digestion was halted by adding an equal volume of 10% FBS, and the cell suspension was filtered through a 70 μm strainer. After centrifugation at 300×g for 5 min, the cells were plated onto six-well plates pre-coated with 10 µg/mL human fibronectin and cultured in keratinocyte serum-free medium (K-SFM) supplemented with 10 µM Y-27,632. The cells were maintained in a 37 °C incubator with 5% CO₂.

Hair regeneration in vivo

Briefly, the trunk skin of neonatal C57BL/6J mice was mechanically separated and digested with 0.1% Dispase at 37 °C for 1 h. The skin specimen was then divided into epidermis and dermis using forceps. The epidermis was minced and digested in 0.025% trypsin at 37 °C for 10 min and the dermis was minced and digested in 0.2% collagenase (Sigma-Aldrich, St. Louis, MO, United States) at 37 °C for 1 h. After digestion, an equal volume of 10% FBS in DMEM was added to terminate the reaction, and the samples were filtered through 70 μm strainers. Following centrifugation and washing, murine neonatal epidermal cells and dermal cells were obtained. For in vivo implantation, five experimental groups were established for this study as follows: (1) a mixture of natal-murine epidermal cells with and pre-prepared natal-murine dermal cells (positive control), (2 and 3) a mixture of cell population enriched in HFSC with or without ISO and pre-prepared natal-murine dermal cells, (4) pre-prepared natal-murine epidermal cells alone (negative control) and (5) pre-prepared natal-murine dermal cells alone (negative control). Unless specified otherwise, for each intracutaneous injection, 1.5 × 106 dermal cells and 1.0 × 105 epidermal cells or cell population enriched in HFSC were suspended in 50 µL of DMEM and injected into the hypodermis of BALB/c-nude mice using a 29-gauge needle, forming a visible bleb. After 3 weeks, the skin at the injection site was excised. The number of hair follicles formed was quantified by microscopic photography and morphometry.

RNA sequencing analysis

Total mRNA from the hair follicle stem cells was extracted using a TRIzol kit (Invitrogen). RNA quality was verified on an Agilent 2100 BioAnalyzer (Agilent Technologies, CA, USA) and examined using RNase-free agarose gel electrophoresis. After total RNA extraction, mRNA was isolated and purified using oligomeric (dT) magnetic beads (Invitrogen, USA). Thereafter, the mRNA was fragmented into short sequences using fragmentation buffer and reverse transcribed into double-stranded cDNA. The QiaQuick PCR extraction Kit (Qiagen, Venlo, Holland) was used to purify the cDNA fragments, repair base ends, and connect the Illumina sequencing adapters. Then, the sizes of the ligated products were determined using agarose gel electrophoresis. The products were amplified by using PCR and sequenced using the Illumina NovaSeq 6000 by Gene Denovo Biotechnology Co. (Guangzhou, China). Finally, the FastQC tool was used to assess sequence quality and filter out the low-quality reads. Data processing was performed using the DESeq2 package in R software (GSE36169). Gene expression with a change in p<0.05 and log2|FC| >1 were included in subsequent analyses. The identified differentially expressed genes were visualized by volcano plot, and KEGG pathway enrichment was used to identify the critical signaling pathways.

Quantitative real-time polymerase chain reaction

Total RNA was extracted from hair follicles samples or cells using TRIzol (Takara, Tokyo, Japan) and then reverse-transcribed to complementary DNA (cDNA) with PrimeScript® RT reagent (Takara) according to the manufacturer’s instructions. Subsequent qRT‒PCR was performed with SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara) in a Light Cycle Roche 480 II Real-time PCR system (Roche, Basel Switzerland). The primer sequences are listed in Supplementary Table S1. The expression levels of target genes were normalized to those of GAPDH. The relative expression level was calculated using the 2-Ct method.

Clinical study

This study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University (NFEC-2021-349). Sixty-eight men were recruited in the department of Plastic and Aesthetic Surgery in Nanfang Hospital of Southern Medical University (China) from 2021 to 2022. Participants who were diagnosed as AGA with type III to V according to the Norwood-Hamilton classification by the clinicians. Subjects were asked to apply 1 mL of 10ng/ml isoproterenol solution daily for 6 months. Written informed consent was obtained from all cases prior to enrollment. The privacy rights of the patients were well protected. Photography and dermoscopic assessments were conducted at baseline and at 3-month, and 6-month follow-up to evaluate hair growth. Hair counts were performed in triplicate by three trained observers in a blinded manner to minimize bias.

Statistical analysis

Statistical analyses were conducted by GraphPad Prism 8 software. Quantifiable data were displayed as the mean ± standard deviation (SD). Statistical significance was analyzed by Student’s t test or one-way analysis of variance (ANOVA). A difference of p < 0.05 was considered significant.

Results

Inhibition of PI3K/AKT signaling and dysfunction of adrenergic activation occurred in the pathogenesis of AGA

To elucidate the pathogenesis of AGA, follicular unit extraction (FUE) was performed to collect miniaturized follicles from AGA patients and normal follicles from the occipital region of healthy individuals (used for eyebrow transplantation). Thorough transcriptome sequencing was conducted on two groups: the AGA group (miniaturized follicles, including the bulge region) and the control group (CON, normal follicles, including the bulge region). Significant differences in gene expression were identified between the groups (Fig. 1A, S1A-D), with notable alterations in genes related to hair cycle, hair follicle development and hair follicle morphogenesis (Fig. 1B). The PI3K/AKT signaling pathway was markedly down-regulated in AGA follicles (Fig. 1C). KEGG analysis indicated that ADRB2 activation could inhibit apoptosis via the PI3K/AKT signaling (Fig S1E), linking adrenergic signaling to cell growth. Quantitative reverse transcription polymerase chain reaction (qPCR) analysis confirmed significantly reduced ADRB2 mRNA expression in AGA follicles compared to controls (Fig. 1D). Immunofluorescence staining further revealed the decreased expression of tyrosine hydroxylase (TH), marker of sympathetic nerves, Cytokeratin 15 (CK15), marker of HFSCs, and ADRB2 in AGA follicles (Fig. 1E-G). These findings suggested that the suppression of PI3K/AKT signaling in AGA follicles may be associated with reduced adrenergic activation via ADRB2.

Fig. 1
figure 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

ISO induced the hair growth of murine hair follicles

To investigate the effects of ADRB2 activation on the hair cycle, in vivo experiments were performed using the β-adrenergic receptor agonist ISO on telogen-phase C57BL/6 mice. PBS or ISO (with concentration gradients based on Shwartz’s study [8]) was topically applied to the dorsal skin, followed by H&E staining for skin histological sections for follicular analysis (Fig. 2A). After six days of ISO treatment, greying of the depilated skin indicated the initiation of hair regrowth, whereas control skin remained pink (Fig. 2B). Histological analysis confirmed a significant increase in hair numbers in ISO-treated mice (Fig. 2C-D). Subcutaneous ISO injections (Fig. 2E) similarly accelerated hair regrowth and increased follicle density in treated mice (Fig. 2F-I). qPCR analysis revealed elevated mRNA levels of the proliferation marker Ki67, CK15, and ADRB2 in ISO-treated skin compared to controls (Fig. 2J). Collectively, these findings indicated that ISO may promote hair follicle growth on the back of mice, and ISO intervention can up-regulate the expression of cell proliferation index (Ki67), hair follicle stem cell index (CK15), and ADRB2 in the back tissue of mice.

Fig. 2
figure 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

ISO promoted growth of AGA hair follicles in organ culture

To further investigate whether ISO promotes hair growth under pathological conditions, the effects of exogenous ISO on AGA HFs were examined using an ex vivo organ culture model. Isolated HFs of AGA patients were divided into groups treated in medium (as a control) with or without varying doses of ISO. Hair shaft and follicle length were measured every other day (Fig. 3A). ISO was found to enhance hair shaft elongation in a dose-dependent manner, with concentration of 10− 10g/ml ISO showing the most pronounced effect compared to other concentrations and the control (Fig. 3B-C). While control follicles remained in the telogen phase, ISO-treated follicles transitioned to the anagen phase with normal structure preserved (Fig. 3D-E). Next, the impact of exogenous ISO on follicular cell proliferation was evaluated. The results indicated a significantly higher number of Ki67-positive cells in ISO-treated HFs compared to controls (Fig. 3F-G) Ki67-positive proliferating cells were predominantly located in the outer root sheath (ORS) and the hair matrix region adjacent to the dermal papilla, consistent with areas of active progenitor cell proliferation and transit amplification. Since the bulge stem cells are responsible for new hair generation, the potential of ISO to promote the differentiation of cell population enriched in HFSC into progenitor cells in AGA follicles was examined. ISO treatment resulted in significantly enhanced fluorescence intensity of CK15 and CD200 signals within the bulge region, suggestive of elevated expression levels in HFSCs and progenitor populations. Furthermore, the proportion of Ki67-positive cells in the bulge region was also markedly increased in the ISO-treated group (Fig. 3H-I). In summary, these findings suggested that ISO may promote hair growth by inducing the transition of cell population enriched in HFSC from dormancy to activation.

Fig. 3
figure 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

ISO enhanced the proliferation of cell population enriched in HFSC

To elucidate the potential mechanisms regulating activation of HFSCs during hair growth induction, a strategy was employed to characterize the effects of ISO on cell population enriched in HFSC in primary culture. Cell population enriched in HFSC were isolated from the human HFs as previously described [20]. Different concentrations of ISO were then applied to the cell population enriched in HFSC to assess its impact on stem cell activity. Initially, the Live/Dead Kit was utilized to assess the non-toxic effects of varying ISO concentrations on cell population enriched in HFSC (Fig. 4A and E). Subsequently, to evaluate the effect of ISO on HFSC proliferation, we performed crystal violet staining to assess overall cell growth, followed by immunofluorescence staining for the proliferation marker Ki67 and the stem cell marker CK15 (Fig. 4B-D and F-G). The results demonstrated that the proliferation of cell population enriched in HFSC increased after the ISO treatment (Fig. 4F-G). The positive effect was inhibited by the ADRB2 receptor blocker, propranolol. Furthermore, A reconstitution assay [19] was conducted to evaluate the capacity of ISO-treated cells to regenerate HFs (Fig. 4H). In the negative control groups, which were grafted with natal-murine dermal cells or natal-epidermis cells alone, almost no new hair formation was observed (Fig. 4I). In contrast, de novo hair shafts were induced at the recipient sites three weeks post-implantation in the positive control group grafted with a mixture of natal-murine dermal cells and natal-epidermis cells. Besides, both the cell population enriched in HFSC cultured with standard medium or ISO could also induce HFs. A significant increase in HFs was observed in the ISO-treated group compared to the untreated group. Histological examination revealed the presence of de novo HFs in both the positive control and experimental groups (Fig. 4J). Collectively, these findings indicated that ISO might enhance the proliferation of cell population enriched in HFSC.

Fig. 4
figure 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

ISO activated the cell population enriched in HFSC via PI3K/AKT/β-catenin signaling pathway

To further validate the molecular mechanism by which ISO activates cell population enriched in HFSC, the expression of gene in the PI3K/AKT signaling pathway was assayed in ISO-treated cells. qPCR analysis of ISO-treated cells revealed an upregulation of PI3K and AKT, along with decreased of GSK3β, a key target of the PI3K/AKT signaling pathway (Fig. 5A-C). This effect was inhibited by the ADRB2-inhibitor propranolol or the PI3K pathway inhibitor LY-294. β-catenin, a critical regulator of hair regeneration and stem cell differentiation [21], was also implicated. Normally, GSK3β suppresses β-catenin expression; however, suppression of GSK3β leads to β-catenin accumulation and activation of its gene regulatory functions [14]. qPCR analysis demonstrated that ISO treatment increased β-catenin expression in cell population enriched in HFSC, along with the upregulation of c-myc and Cyclin D1 (CCND1), genes associated with cell division and growth regulation (Fig. 5A-C). Further analysis with LY-294 showed that inhibition of the PI3K pathway significantly reduced the expression of hair growth gene factor IGF-1 (Fig. 5C). To further validate the molecular mechanism by which ISO activates HFSCs, Western blot analysis of ISO-treated HFSCs revealed an upregulation of phosphorylated PI3K and AKT, along with increased phosphorylation of GSK3β (Fig. 5D-E, G-H). This effect was inhibited by the ADRB2-inhibitor propranolol or the PI3K pathway inhibitor LY-294 (Fig. 5F, I). Further analysis with LY-294 showed that inhibition of the PI3K pathway significantly reduced the expression of β-catenin and the hair growth downstream marker Lef-1 (Fig. 5J-K). Together, these findings confirmed that ISO activated the PI3K/Akt/β-catenin signaling pathway in cell population enriched in HFSC, thereby regulating hair cell growth.

Fig. 5
figure 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

ISO enhanced hair regrowth in AGA patients

To assess the clinical feasibility of ISO for AGA treatment, a clinical study was conducted to evaluate its efficacy. Following treatment with 10− 8g/ml ISO, significant increases in average hair count, density and diameter were observed in AGA patients compared to baseline (Fig. 6A-E) (Table 1). No severe adverse events were reported throughout the treatment period. These results indicated that ISO might offer therapeutic benefits in AGA patients.

Fig. 6
figure 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

Table 1 Hair growth relevant parameters at baseline, 3 and 6 months

Discussion

Hair follicle stem cells are critical for sustaining hair regeneration and preserving the homeostasis of the stem cell niche [22, 23]. The pathogenesis of hair loss is primarily driven by the depletion or irreversible quiescence of the stem cell pool [24]. The sympathetic nervous system is crucial to regulate the homeostasis and function of HFSCs [25]. Activation of the sympathetic nerves triggers the release of norepinephrine (NE), which acts on the adrenergic-β2 receptor (ADRB2) receptor of HFSCs [8]. Such process upregulates the expression of the hair growth-promoting factor Sonic hedgehog (Shh) while downregulating the dormancy factors Foxp1 and Fgf18. Eventually, HFSC proliferation and differentiation are stimulated, enabling the progression of the hair growth cycle. However, as a strong adrenergic receptor α-agonist, studies showed the limitation of adrenergic receptor β-agonistic effect of NE [26]. Here, we demonstrated that isoproterenol (ISO), a selective β-adrenergic receptor agonist, enhanced HFSC proliferation. This is achieved through activation of ADRB2, which stimulates the PI3K/AKT/β-catenin signaling (Fig. 7).

Fig. 7
figure 7

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

In atrophic HFs of AGA patients, the sympathetic nerve growth was found to be suppressed, accompanied by the downregulation of PI3K/AKT signaling pathway and corresponding reduction expression of ADRB2. Furthermore, topical application of ISO was shown to accelerate hair growth in telogen-phase mice and enhance cell proliferation in human hair follicle organ cultures and HFSC cell cultures. ADRB2 activation has been shown to be essential for hair development and maintenance [27]. In neonatal mice, suppression of ADRB2 expression delays hair growth and maturation, while deletion of ADRB2 results in arrest of the hair growth cycle [8]. Furthermore, ADRB2 is involved in cellular signaling by interacting with pathways such as PI3K and MAPK, regulating cellular functions and behaviors [28]. To simulate both superficial (epidermal/dermal interface) and deep perifollicular exposure routes to comprehensively assess ISO’s pharmacodynamic range, we initially applied topical ISO to assess local permeability and surface follicle penetration, and subsequently performed subcutaneous injections to ensure adequate delivery to the lower follicular units and stem cell niches, especially in deeper dermal regions. Our results showed that topical ISO at 10⁻⁸ g/ml was sufficient to induce pigmentation and hair shaft elongation in most telogen follicles, whereas subcutaneous ISO at the same concentration elicited a more robust and earlier transition to anagen, possibly due to more consistent tissue penetration (Fig. 2).

In the in vitro culture of ISO-treated cell population enriched in HFSC, a significant improvement in cell proliferation was observed, suggesting that ISO may promote hair regeneration by activating cell population enriched in HFSC. Notably, ISO at optimal concentrations significantly enhanced cell proliferation, whereas higher doses showed no significant effect, aligning with the results from follicular organ culture. Consistent with previous findings, Botchkarev et al. observed that the isoproterenol promoted the progression of the hair cycle from anagen III to anagen IV in early anagen organ cultures [7]. Similarly, Kong et al. reported that NE effectively stimulated keratinocyte proliferation in organotypic skin cultures, thereby facilitating hair growth [29]. In our study, PI3K-AKT signaling was found to be suppressed during the pathogenesis of AGA, accompanied by reduced expression of HFSC markers. In vitro treatment with ISO improved the delayed hair regeneration of miniaturized AGA follicles and promoted the proliferation of cell population enriched in HFSC. These findings aligned with previous studies. The PI3K/AKT/β-catenin signaling pathway was identified by Wang et al. as essential for maintaining the HF cycle and facilitating post-injury regeneration [30]. Liu et al. demonstrated that activation of PI3K/AKT/β-catenin pathway significantly increased HF density in rabbits [31]. Jin et al. reported that stimulation of the PI3K/AKT/β-catenin pathway of HFSCs promoted their activation and enhancing hair regeneration in mice [32]. Salvador et al. further confirmed that activation of the PI3K/AKT/β-catenin pathway is a pivotal event in hair regeneration, regulating the follicle cell cycle and proliferation [33]. In conclusion, these results supported that ISO activated the PI3K/AKT/β-catenin signaling pathway through ADRB2, highlighting the crosstalk within the mechanisms underlying in the pathogenesis of hair loss.

To assess the clinical feasibility of ISO for AGA treatment, we conducted a clinical study, yielding promising results in hair count, density, and diameter following ISO treatment. These findings highlight the PI3K/AKT/β-catenin signaling pathway, mediated by ADRB2 activation, as a potential therapeutic target for hair loss treatment. While ISO has been widely used in acute cardiovascular care at systemic doses [11], its long-term safety profile for dermatological or regenerative indications remains insufficiently studied, particularly in the context of chronic topical or percutaneous application. We recognize the potential risk of systemic absorption, especially with repeated use or compromised skin barrier integrity leading to off-target cardiovascular effects such as tachycardia, arrhythmias, or hypotension. Future studies are warranted to optimize the therapeutic protocol for ISO in hair loss management.

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.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request. The data (RNAseq) supporting the results reported in the paper could be archived in NCBI Gene Bank (GSE36139/DOI: 10.1126/scitranslmed.3003122). All additional files are included in the manuscript.

Abbreviations

AGA:

Androgenetic alopecia

HFSCs:

Hair follicle stem cells

ADRB2:

Adrenergic β2 receptor

SHH pathway:

Sonic Hedgehog pathway

ISO:

Isoproterenol

HFs:

Hair follicles

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Acknowledgements

The authors declare that they have not use AI-generated work in this manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (Grant Nos. 82102353), the Natural Science Foundation of Guangdong Province (Grant Nos. 2021A1515012150), the Guangdong Provincial Regional Joint Fund (Grant Nos.2020A1515110037), the Science and Technology Program of Guangzhou (Grant No. 202102020155, and K30617055)

Author information

Authors and Affiliations

Authors

Contributions

JZ, JP and ZF performed all the experiments and prepared the figures and tables. JZ, JP and HW provided with the statistical assistance. JZ, ZF, LW and YM performed the clinical study. JZ wrote the first draft of the manuscript. RC and YC revised the manuscript for important intellectual content. RC and ZH contributed to the conception and design of the study. All authors contributed to manuscript revision, read and approved the submitted version.

Corresponding authors

Correspondence to Yu Chai, Zhiqi Hu or Ruosi Chen.

Ethics declarations

Ethics approval and consent to participate

(1) Title of the approved project: A study of the therapeutic effect of isoproterenol on androgenetic alopecia; Name of the institutional approval committee: Medical Ethical Committee of Southern Medical University; (3) Approval number: NFEC-2021-349; (4) Date of approval: 2022-11-03. The patients provided their written informed consent for participation in the study and the use of samples. Animal study: (1) Title of the approved project: Isoproterenol intervention in C57/BL6 mice in vitro; Name of the institutional approval committee: Animal Care and Use Committee, Southern Medical University; (3) Approval number: IACUC-LAC-20210318002; (4) Date of approval: 2021-09-15.

Consent for publication

The patients / participants provided their written informed consent to participate in this study

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Zhang, J., Peng, J., Fan, Z. et al. Therapeutic potential of isoproterenol in androgenetic alopecia: activation of hair follicle stem cells via the PI3K/AKT/β-Catenin signaling pathway. Stem Cell Res Ther 16, 306 (2025). https://doi.org/10.1186/s13287-025-04418-y

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