Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair Growth-Promoting Effect of SFRP1 Silencing in Healthy Human Hair Follicles Ex Vivo

TL;DR

This study shows that silencing SFRP1 promotes hair growth, but combining it with DKK1 silencing does not improve the effect in human hair follicles.

Contribution

The study reveals that DKK1 and SFRP1 have compensatory regulation, and combined knockdown does not enhance hair growth benefits.

Findings

SFRP1 knockdown prolonged anagen and increased hair matrix keratinocyte proliferation.

DKK1 knockdown had no effect on hair growth, and combined knockdown did not enhance SFRP1's benefits.

Compensatory regulation between DKK1 and SFRP1 highlights Wnt-signaling complexity in hair growth.

Abstract

Androgen receptor (AR) signaling plays a key role in male pattern baldness. We investigated whether targeting Dickkopf 1 (DKK1) and Secreted frizzled-related protein 1 (SFRP1), two AR-regulated genes, offers a novel therapeutic strategy for hair loss. AR expression was validated in freshly frozen human scalp hair follicles (HFs). AR knockdown was induced in human HFs using AR spherical nucleic acid (SNA). DKK1 and SFRP1 siRNA treatment were performed in HEK293 cells, human dermal papilla cells (hDPC), and human HFs ex vivo. Functional effects of single and combined DKK1 and SFRP1 knockdown were analyzed in human HFs ex vivo by quantitative (immuno)histomorphology. AR knockdown decreased SFRP1 and DKK1 expression. We found reciprocal mRNA upregulation between DKK1 and SFRP1 following their siRNA knockdown in HEK293 and hDPC. We therefore applied a single and combined treatment of DKK1 and SFRP1 siRNA in HFs ex vivo. SFRP1 knockdown prolonged anagen, increased hair matrix keratinocyte proliferation, reduced apoptosis, and increased DKK1 levels in HFs ex vivo, whereas DKK1 knockdown had no effect, and combined knockdown did not enhance SFRP1’s benefits. The culture-dependent compensatory regulation of SFRP1 and DKK1 underscores Wnt-signaling complexity in hair growth and strengthens the rationale for SFRP1 based therapies in anagen maintenance and hair loss.

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Full text

1. Introduction

Male pattern hair loss (MPHL), also known as male pattern androgenetic alopecia (mpAGA), is the most prevalent type of hair loss in men [1], affecting approximately 80% of individuals by the age of 70 [2,3]. The human hair cycle typically consists of three phases: anagen (growth), catagen (regression), and telogen (rest). In healthy individuals up to 90% of the scalp hair follicles (HFs) are in the anagen phase, during which they produce thick, pigmented terminal hair shafts [4]. In contrast, in HFs of mpAGA-affected scalp regions the anagen phase is significantly shortened and the telogen phase is prolonged, resulting in an increased lag period before the generation of a new hair shaft [5,6,7,8]. Furthermore, mpAGA-affected HFs undergo a miniaturization process, during which terminal HFs progressively develop into intermediate and/or vellus HFs that form thinner and less pigmented hair shafts [9,10]. Thus, therapeutic strategies to address mpAGA-affected HFs should prolong anagen/delay catagen, promote the telogen-to-anagen transition, or prevent HF miniaturization [11].

Excessive AR activation caused by elevated local concentrations of dihydrotestosterone (DHT) in human dermal papilla cells (hDPC) of androgen-sensitive HFs is one of the key mechanisms underlying mpAGA pathophysiology [5,12,13,14,15,16]. Accumulation of DHT is driven by the increased activity of 5α-reductase which catalyzes NADH-mediated double bond reduction of free testosterone [17,18,19]. In addition to direct production within HFs, androgens, including DHT, can also be synthesized locally in the skin indicating the HF could also be subjected to this external androgens present in the skin micromilieu [20]. However, their specific contribution in mpAGA has yet to be fully clarified. The 5α-reductase inhibitor Finasteride is one of the two FDA-approved drugs for mpAGA [2,5]. While this drug prevents disease progression, adverse effects are observed in about 2% of patients that can persist even after therapy discontinuation [21,22,23]. Thus, alternative strategies are currently being evaluated to target this pathway [24].

Increasing evidence indicates that AR activation modulates Wnt/β-catenin signaling, a key pathway involved in HF regeneration, growth, and anagen phase duration in healthy and mpAGA-affected human scalp HFs [25,26,27]. Indeed, AR activation downregulates β-catenin expression and activity in murine adult skin [28] and upregulates the Wnt/β-catenin pathway antagonist Dickkopf 1 (DKK1) in balding hDPCs [29]. Furthermore, activated AR signaling in hDPCs from mpAGA patients induces expression of the Wnt antagonist GSK-3β, resulting in reduced epithelial HF stem cell differentiation [30]. In line with these observations, AR stimulation in an androgen-responsive hDPC led to a decreased WNT5 and WNT10b expression and an increased DKK1 expression [31]. Moreover, cyclosporine, a drug promoting hair regrowth and hypertrichosis [32], promotes anagen maintenance also via suppression of the Wnt inhibitor Secreted frizzled-related protein 1 (SFRP1) [33]. Modulation of Wnt signaling by SFRP1 [34], which is expressed specifically in the dermal papilla (DP) [33], to promote hair regrowth has also been targeted with non-drug compounds [35]. Thus, AR- and Wnt signaling, and their interplay, play crucial roles in hair cycle regulation, highlighting their importance as therapeutic targets for the management of mpAGA.

Currently, RNA interference (RNAi)-based therapies, which can be delivered locally [36], are being developed for cosmeceutical and pharmacological applications, showing clinical benefit in several dermatological indications [37]. A DKK1-targeting siRNA, enhancing Wnt pathway activation, has recently been shown to significantly improve hair regeneration in mice in vivo [38], and two further RNAi-based therapeutics targeting the AR are currently being developed, namely asymmetric small interfering RNA (asiRNA) and self-assembled micelle inhibitory RNA (SAMiRNA). The asiRNA-based AR-targeting approach demonstrated effective gene knockdown in a murine mpAGA model in vivo as well as in human HFs ex vivo. AR knockdown by asiRNA induced hair growth promotion in mice, as well as anagen maintenance and an increase in hair bulb size in human HFs ex vivo [39]. The SAMiRNA was interrogated in a small 60-subject double-blind clinical study and although a positive efficacy signal was identified, the effect was not significant [40]. Thus, to achieve more satisfactory clinical results, RNAi-based approaches for promoting hair growth in mpAGA patients require further research and development.

Here, we investigated whether combinational strategies may be more effective in modulating hair growth. Specifically, by silencing AR in human HFs ex vivo, we identified SFRP1 as a Wnt pathway inhibitor, whose expression is directly modulated by AR signaling. Silencing DKK1 or SFRP1 in HEK293 or hDPCs in vitro resulted in the reciprocal up-regulation of the other molecule, depending on the cell type. Consequently, we applied both single and combined DKK1 and SFRP1 siRNA treatments to human HFs ex vivo and observed an increase in DKK1 transcript abundance upon SFRP1 knockdown. Finally, we investigated the effects of the dual silencing of DKK1 and SFRP1 on healthy human HF function ex vivo, without additional AR stimulation. We found that only SFRP1 siRNA treatment effectively prolonged anagen in HFs ex vivo, whereas DKK1 siRNA and combined DKK1 and SFRP1 siRNA treatments had no functional effects. These findings underscore the importance of fine-tuning targeted approaches to modulate Wnt signaling to achieve optimal benefits for hair growth in both healthy individuals and mpAGA patients.

2. Results

2.1. AR Knockdown Effectively Reduces mRNA Expression of the Wnt Pathway Modulators SFRP1 and DKK1 in Human HFs Ex Vivo

AR mRNA expression has been frequently reported for DP cells, whereas AR protein expression in other HF compartments, including the outer root sheath (ORS), is still controversial [41]. Thus, to be able to effectively evaluate AR silencing in our HF experiments, we initially investigated the localization of AR protein in organ cultured healthy HFs by in situ hybridization (ISH) and immunostaining after ex vivo culture for 24 h. Overall, low levels of AR mRNA were detected but a positive signal was seen mainly in the DP, DP stalk (DPst), and dermal cup (DC), and to a lesser extent in germinative and pre-cortical hair matrices (gHM and pcHM), whilst almost no signal was detected in the proximal ORS (Figure 1a). Qualitative immunofluorescence analysis demonstrated AR protein expression in the nucleus of DP fibroblasts from human HFs, obtained from three independent donors (Figure 1b), while AR protein levels were low in the proximal ORS, gHM and pcHM (Figure 1b). Thus, the AR is mainly expressed in the DP, and to a lesser extent also in the ORS and HM of human healthy HFs.

AR knockdown was validated by qPCR after application of 10 µM, 25 µM or 50 µM AR SNA or control SNA for six days, in the absence of additional testosterone or DHT stimulation, resulting in a significant reduction of more than 50% in AR mRNA levels when all tested concentrations were pooled (Figure 2a). Given the comparable results across the tested doses, all further experiments were performed with the lowest concentration of AR SNA (10 µM). Quantitative immunofluorescence confirmed the significant reduced AR expression in the DP and ORS, as well as a trend toward a lower percentage of AR^+^ cells in the DP (Figure 2b,c). Functionally, AR silencing in the absence of testosterone or DHT resulted in reduced mRNA levels of the Wnt inhibitor DKK1 (Figure 2d), confirming an AR-dependent modulation of Wnt signaling in human HFs ex vivo. In addition, lower mRNA expression of SFRP1 was also detected after AR silencing in human HFs (Figure 2d).

After confirming this, we proceeded to silence AR expression in human HFs ex vivo using spherical nucleic acids (SNAs), which consist of a nanoparticle core densely coated with nucleic acids, serving as an effective tool for delivering nucleic acids into cells [42].

Thus, our investigation confirmed that DKK1 expression is inhibited by AR also in human HFs ex vivo and identified SFRP1 as an additional AR target gene.

2.2. Downregulation of SFRP1 or DKK1 Following siRNA Treatment Induces Reciprocal Gene Expression in HEK293 and HDPCs In Vitro

We hypothesized that RNAi-mediated suppression of the Wnt pathway inhibitors DKK1 and SFRP1, resulting in disinhibition/activation of the Wnt signaling pathway, would represent a clinically safer strategy compared to the upregulation of a Wnt activator such as LEF1, which would directly activate the Wnt pathway. Therefore, we first confirmed the spatial expression patterns of SFRP1 and DKK1 in human HFs ex vivo. SFRP1 expression was in line with previously published results [32], with high SFRP1 mRNA levels detected in the DP, DPst, and DC and lower levels present in the HM, ORS, and pre-cortex of healthy HFs ex vivo (Figure S1a). SFRP1 protein expression was predominantly found in the DP, gHM and pcHM, but also in the ORS of three independent donors (Figure S1b). DKK1 mRNA was mainly detected in the CTS and distal ORS, with no transcripts being present in the DP of HFs ex vivo (Figure S1c). Analysis of DKK1 protein was consistent with the mRNA findings, as it was primarily detected in the CTS and distal ORS. However, low levels of DKK1 protein were also present in the DC, but not the DP of HFs of three independent donors (Figure S1d). These findings show different expression patterns for the two Wnt target genes DKK1 and SFRP1 in healthy human HFs ex vivo.

Next, we validated the knockdown efficacy of DKK1 and SFRP1 by siRNA in vitro in HEK293 cells and hDPCs. Treatment with DKK1 and SFRP1 siRNA reduced the respective mRNA levels to 68% and 41% in HEK293 cells and 20% and 0% in hDPCs, relative to treatment with scrambled siRNA (Figure 3a,b). Interestingly, downregulation of DKK1 caused an upregulation of SFRP1 mRNA in HEK293 cells (Figure 3a), whereas in hDPCs, downregulation of SFRP1 induced enhanced DKK1 mRNA expression (Figure 3b).

Thus, these data indicate a differential interplay between DKK1 and SFRP1 across various cell types and suggest that dual targeting of these molecules may produce synergistic effects.

2.3. Knockdown of SFRP1 Induces Upregulation of DKK1 mRNA and Protein in Healthy Human HFs Ex Vivo

Considering the different expression patterns of DKK1 and SFRP1 between distinct HF compartments, and their opposed compensatory regulation in two cell systems in vitro, we next investigated whether the simultaneous knockdown of DKK1 and SFRP1 might hold potential for additive or even synergetic effects on HF function. Sole applications of the respective siRNA as well as their combined application resulted in reduced DKK1 and SFRP1 mRNA levels. Consistent with and validating our results obtained in hDPCs (Figure 3b), DKK1 mRNA was upregulated by SFRP1 siRNA treatment, whereas there was no reciprocal effect of DKK1 knockdown on SFRP1 mRNA (Figure 3c). To confirm these findings at the protein level, ELISA was performed on the HF supernatant obtained at the end of the HF culture. Secretion of DKK1 and SFRP1 protein was significantly reduced by the respective siRNA treatment in male and female HFs (Figure 3d). Combined siRNA treatment induced a trend toward reduced DKK1 levels, and significantly decreased SFRP1 release into the medium, when compared to scrambled siRNA control (Figure 3d). However, SFRP1 siRNA treatment resulted in increased DKK1 protein secretion at the end of the HF cultures, and DKK1 siRNA significantly reduced SFRP1 protein release (Figure 3d). Analysis of the Wnt target genes LEF1 and AXIN2 following single and combined DKK1 and SFRP1 siRNA treatments revealed that LEF1 mRNA expression tended to increase with single SFRP1 siRNA treatment and with combined SFRP1 and DKK1 siRNA treatment, whereas AXIN2 expression was only increased by DKK1 siRNA treatment in one out of three donors (Figure S2). This differential regulation of two additional Wnt target genes following SFRP1 and DKK1 knockdown further underscores the complex interplay of Wnt signaling in human HFs ex vivo.

These findings provide additional evidence of a delicate and complex interaction in the regulation of SFRP1 and DKK1 expression at the transcriptional and post-transcriptional levels.

2.4. Double Knockdown of the Androgen Receptor Target Genes DKK1 and SFRP1 Does Not Potentiate the Hair-Growth-Promoting Effect of SFRP1 in Healthy Human Hair Follicles Ex Vivo

Finally, we investigated the consequences of DKK1 and SFRP1 knockdown on HF function ex vivo. Neither single treatment with DKK1 or SFRP1 siRNA, nor their combined treatment, affected hair shaft elongation (Figure 4a). While DKK1 siRNA had only a minor positive effect on anagen prolongation, SFRP1 siRNA effectively increased the number of female and male HFs in anagen, which was also accompanied by a decreased hair cycle score, significantly increased hair matrix keratinocyte proliferation, and a trend towards a reduced apoptosis (Figure 4b–f). Although a modest anagen-prolonging effect was observed, HFs treated with dual silencing of DKK1 and SFRP1 exhibited a higher number of catagen-phase follicles compared to those treated with scramble control (Figure 4c), which is also reflected by a slight increase in the hair cycle score (Figure 4d).

Taken together, these data reveal that dual targeting of two, apparently synergetic, Wnt modulators does not result in an enhanced beneficial effect on hair growth promotion ex vivo. In addition, SFRP1 is further confirmed as an interesting, and more therapeutically compelling target for anagen maintenance.

3. Discussion

Elevated AR signaling is a molecular driver of mpAGA and reduced AR signaling via Finasteride-mediated inhibition of 5-alpha reductase is one of the two FDA-approved treatments for mpAGA [2]. Direct AR targeting with pharmacological antagonists [2] has shown clinically meaningful impact on improving the appearance of patients. Yet, these therapeutics are associated with side effects [2], that could be avoided by developing non-systemic therapeutic strategies. To this purpose, AR silencing techniques have been tested, which did not yet result in the expected outcome [40]. While suboptimal delivery may have contributed to the lack of efficacy so far, further understanding of the downstream biology of AR signaling is required to develop alternative, more effective therapeutics for mpAGA.

In our study, we pursued this goal by identifying novel AR target genes, notably the Wnt inhibitor SFRP1, which may play a role in modulating hair growth, using an ex vivo HF culture system. We also examined the interplay between SFRP1 and DKK1, another AR-target gene known to modulate Wnt signaling, in both in vitro and ex vivo HF models. Lastly, we assessed whether dual targeting of these two AR target genes, in conjunction with Wnt signaling inhibitors, could potentiate their individual hair-growth–promoting effects in HF organ culture.

Our data demonstrates that AR silencing in human HFs ex vivo leads to decreased transcription of the Wnt modulators DKK1 and SFRP1. Interestingly, when one of these genes was silenced in vitro, a compensatory expression mechanism was observed, which led to the reciprocal up-regulation of the other molecules, in a cell-type dependent manner. Indeed, also in human HFs ex vivo, silencing of SFRP1 led to increased DKK1 expression. Additionally, knockdown of SFRP1 prolonged anagen and significantly increased hair matrix keratinocyte proliferation, while neither knockdown of DKK1 nor double knockdown of the two genes had any effect on the hair cycle or HF function. These findings indicate that, by targeting the compensatory expression mechanisms between DKK1 and SFRP1, the beneficial effects of SFRP1 on hair growth promotion are not further enhanced.

Based on those findings, along with the fact that AR activation mediates the suppression of Wnt/β-catenin signaling in mpAGA [26,28,29,30,31], the potential of targeting Wnt signaling as an alternative or in addition to targeting AR signaling for mpAGA treatment is becoming increasingly evident. This strategy is reinforced by genetic and transcriptional evidence linking Wnt signaling components to the condition [43,44]. Additionally, several Wnt signaling modulators are being investigated preclinically or are already marketed as hair growth promoting agents, including the SFRP1 antagonist WAY-316606 [32] and the cosmetic SFRP1 inhibitors Bioscalin or KY19382. KY19382 acts as a Wnt/β-catenin signaling activator via inhibition of the interaction between CXXC-type zinc finger protein 5 (CXXC5) and Dishevelled (Dsh) [45]. Moreover, several studies have demonstrated the efficiency of natural or synthetic Wnt-activating compounds as an adjuvant strategy against hair loss [46,47], further supporting the specific therapeutic targeting of Wnt signaling for mpAGA treatment, also facilitated in our study.

Therefore, we focused on the Wnt inhibitors DKK1 and SFRP1, which were also reduced upon AR knockdown. While DKK1 is a known AR target gene [29], this has not been described for SFRP1. Instead, SFRP1 is known to inhibit AR expression [12,27]. The clinical significance of DKK1 and SFRP1 in hair loss progression in mpAGA patients is supported by the findings that both genes are upregulated in mpAGA-affected hDPC [29,48,49,50], and that they trigger the anagen-to-catagen transition in human HFs [32,35,51]. Furthermore, DKK1 reduces the size of the HF bulb, which directly affects the width of the hair shaft [52]. Notably, the role of SFRP1 in hair growth has been extensively described in the literature, in human and murine models with its therapeutic inhibition shown to promote hair growth [32,34,53]. These studies underscore the relevance of targeting DKK1 and SFRP1 for hair growth promotion, and prompted us to assess whether their combined knockdown could accelerate the effect of SFRP1 inhibition alone

Silencing of DKK1 resulted in upregulation of SFRP1 in HEK293 cells, while silencing of SFRP1 resulted in upregulation of DKK1 in hDPCs and human HFs ex vivo. It is known that Wnt signaling can be up- or downregulated in response to the same stressor depending on the model system [54], which could explain the differences between HEK293 cells, hDPCs and human HFs ex vivo.

To our knowledge, no compensatory expression mechanism or direct reciprocal regulatory loop between DKK1 and SFRP1 has been previously reported that could explain the observed differences in expression patterns or on the distinct functional outcomes between the three treatments. However, SFRP1 inhibition enhances Wnt/β-catenin signaling and, therefore, it is plausible that Wnt antagonists such as DKK1 are transcriptionally induced as part of a compensatory negative feedback mechanism [55]. This would serve to buffer the increased Wnt activity resulting from SFRP1 loss and thereby contribute to the restoration of pathway homeostasis. Additionally, previous data have shown that SFRP1 knockdown also increased LEF1 expression, which, unlike DKK1, functions as an anagen-promoting factor [56]. In line with this, we found increased LEF1 mRNA expression following SFRP1 knockdown, and thus most likely also enhanced β-catenin–LEF/TCF transcriptional activity. This, in turn, could drive the upregulation of DKK1, and explain the observed prolongation of anagen. Furthermore, SFRPs also interact with Wnt-unrelated molecules that play a role in the hair cycle, including fibronectin, receptor activator for nuclear factor kappa B (NF-κB) ligand (RANKL), bone morphogenetic protein (BMP)/Tolloid and Unc5H3 [57]. In summary, our data suggest that SFRP1 may play a more diverse role than DKK1 in modulating the hair cycle, as reflected in our findings. Nevertheless, these interpretations require experimental confirmation. Particularly the pronounced effects of single SFRP1 knockdown on the hair cycle and hair matrix keratinocyte proliferation, in contrast to the minimal effects observed with single DKK1 knockdown or combined SFRP1/DKK1 knockdown, remain difficult to explain.

In human HFs ex vivo, only the knockdown of SFRP1 substantially prolonged anagen and significantly increased or reduced hair matrix proliferation or apoptosis, respectively. Although we hypothesized that the double knockdown of both genes would result in a more profound synergistic effect on HF function, it did not augment the observed effects and even reduced the positive effect of SFRP1 knockdown. One provocative explanation for these observations could be drawn from a recent article describing a role of SFRP proteins as Wnt ligand carriers or inhibitors, depending on their abundance relative to Wnt ligands [58]. Only at a high concentration of SFRPs, Wnt molecules become sequestered and thus unavailable for receptor binding. We can, therefore, hypothesize that knocking down SFRP1 results in an expression level that is sufficient for its role as a Wnt carrier, thereby further increasing Wnt pathway activity. This provides a plausible explanation for the enhanced ability of SFRP1 to promote anagen.

Contrary to SFRP1 silencing, the single knockdown of DKK1, as well as the combined knockdown of DKK1 and SFRP1 had only a minor effect on anagen maintenance and did not affect hair matrix keratinocyte proliferation or apoptosis. Of note, mouse data demonstrate that treatment with a DKK1-targeting siRNA during anagen induction significantly accelerated the hair cycle but did not prolong anagen [38]. Since DKK1 siRNA did not prolong anagen in our model either, it is possible that its knockdown does not maintain anagen but rather accelerates re-entry into the hair cycle. In line with this, Wnt/β-catenin signaling is a potent driver of the telogen-to-anagen transition [59].

Moreover, the discrepancy between results obtained from murine and human HFs could also be explained by the low expression of DKK1 in healthy human HFs ex vivo demonstrated here, suggesting that DKK1 plays a less significant role in clinically healthy anagen VI HFs from the occipital scalp region than in mpAGA-affected HFs, where its expression is upregulated [49,51,55]. This aligns with the fact that DKK1 is an androgen-responsive gene, but occipital HFs are androgen-independent [3,5].

We acknowledge several limitations in our study, including the small number of human donors and the lack of in-depth mechanistic investigations into how different Wnt modulators influence each other’s function and expression. Although this interaction is highly interesting and of broad biological relevance, it was beyond the scope of the present work. Additionally, we analyzed only clinically healthy occipital HFs, which may mechanistically differ from HFs affected by mpAGA. Thus, our data warrants further investigation, particularly in the context of mpAGA management and the development of RNAi-based therapeutic strategies for hair growth promotion. Additional research on the relationship of Wnt regulators in the context of androgen sensitive versus androgen insensitive human HFs is needed to advance our understanding of the molecular pathophysiology of mpAGA. Specifically, it remains to be determined whether the compensatory mechanism between DKK1 and SFRP1 reported here also occurs in fronto-temporal, androgen-sensitive mpAGA HFs.

In conclusion, our findings reveal intricate regulatory mechanisms governing AR and Wnt signaling in healthy human HFs. The insights from this study underscore the complexity behind therapeutic approaches targeting both AR, and Wnt signaling inhibition for anagen maintenance and hair growth promotion. This compensatory behavior between DKK1 and SFRP1 transcripts has, to our knowledge, not been described before and may have wider implications in biology. Furthermore, our results confirmed previous observations by underscoring the potential of SFRP1 as a therapeutic target for hair loss management.

4. Materials and Methods

4.1. Hair Follicle Donor Information

This study was conducted in accordance with the Declaration of Helsinki principles. Scalp anagen VI HFs were obtained from 12 healthy donors (Table 1) after informed written patient consent and Ethics Committee approval (Monasterium Laboratory Biobank approval 2019-297-f-S; study plan 2020-954-f-S, University of Münster 2015-602-f-S, and Comité de Bioética de la Universidad Fernando Pessoa Canarias (03 (22 June 2020))).

4.2. HEK293 Cell Culture and siRNA Treatment

HEK293 STF cells (CRL-3249™, ATCC, Manassas, VA, USA) were cultivated at 37 °C and 5% CO_2_ in assay medium comprised of 80% DMEM F12 Medium, (30-2006™, ATCC, Manassas, VA, USA) and, 20% FBS (ThermoFisher, Carlsbad, CA, USA) supplemented with 200 µg/mL G-418 (ThermoFisher, Carlsbad, CA, USA; 2 mL in 500 mL media). Then, 6.0 × 105 HEK293 cells/well were seeded in a 6-well plate and transfected with 25 nM DKK1 targeting, SFRP1 targeting and NTC siRNA oligos using Dharmafect 2 reagent (Horizon Discovery, Lafayette, CO, USA) as described by the manufacturer. The cells were incubated at 37 °C for 48 h. Then the cells were harvested to test the target gene mRNA expressions. All siRNAs were purchased from Dharmacon (DKK1 siRNA target: 5’-CUGUGAUUGCAGUAAAUUA-3’, SFRP1 siRNA target: 5’-AGAAGAUGGUGCUGCCCAA-3’, or NTC siRNA: 5’-UAGCGACUAAACACAUCAA-3’).

4.3. Dermal Papilla Fibroblast Culture and siRNA Treatment

Primary hDPCs and cell culture media were obtained from PromoCell (Heidelberg, Germany). In compliance with the Human Tissue Act (2004), all PromoCell cells were isolated with an explicit informed consent of the donors and/or their next-of-kin. Second to sixth passages of subcultures were seeded in a 6-well plate and then cultivated at 37 °C and 5% CO_2_. DharmaFECT 2 (Horizon Discovery, Lafayette, CO) was used to transfect cells, as per the manufacturer’s protocol (DharmaconTM). Briefly, 4 μL of DharmaFECT 2 was combined with 25 nM of siRNA in a volume of 400 μL Opti-mem media (GIBCO, Waltham, MA, USA) and incubated for 20 min. The complexes of DharmaFECT 2 and siRNAs were then added directly to each well with a total volume of 2 mL. The cells were incubated at 37 °C for 48 h. Then the cells were harvested to test the target gene mRNA expressions. All siRNAs were purchased from Dharmacon (DKK1 siRNA target: 5’-CUGUGAUUGCAGUAAAUUA-3’, SFRP1 siRNA target: 5’-AGAAGAUGGUGCUGCCCAA-3’, or NTC siRNA: 5’-UAGCGACUAAACACAUCAA-3’).

4.4. Hair Follicle Organ Culture and Treatment

Human amputated HFs were micro-dissected from follicular unit extraction (FUE) or from scalp skin samples obtained from facelift surgery (Table 1) as previously described [60,61,62]. In short, HFs were cultured in a minimal medium consisting of William’s E medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 2 mM of L-Glutamine (Gibco), 10 ng/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/mL insulin (Sigma-Aldrich), and 1% penicillin/streptomycin (Gibco) at 37 °C and 5% CO_2_ [63,64]. After quality control, anagen VI HFs were randomly assigned to the following experimental groups for up to 5–8 days of culture: (1) control spheric nucleic acid (SNA; 10, 25 and 50 µM) or AR SNA (10, 25 and 50 µM) or (2) scrambled/NTC (non-template control) siRNA (10 µM), DKK1 siRNA (10 µM), SFRP1 siRNA (10 µM) or DKK1 (10 µM) + SFRP1 (10 µM) siRNA. At the end of the culture, HFs were embedded in an OCT cryomatrix (Fisher Scientific, Waltham, MA, USA) and sectioned with a cryostat (Leica, Wetzlar, Germany), and consecutive 6-μm sections of each amputated HF were collected and stored at −80 °C. All siRNAs were purchased from Dharmacon (DKK1 siRNA target: 5’-CUGUGAUUGCAGUAAAUUA-3’, SFRP1 siRNA target: 5’-AGAAGAUGGUGCUGCCCAA-3’, or NTC siRNA: 5’-UAGCGACUAAACACAUCAA-3’).

4.5. Hair Follicle Elongation/Hair Shaft Production

To determine hair shaft growth, each HF was measured from the end of the connective tissue sheath (CTS) to the end of the distal ORS at days 0, 4, 5, 6, and 8 in culture with a digital light microscope at 50× magnification (VHX900; Keyence Corporation, Osaka, Japan), and affiliated software [60,61,62].

4.6. Immunohistochemistry, Immunofluorescence Microscopy, and Quantitative (Immu-No)histomorphometry

Characterization of AR, SFRP1, and DKK1 expression was performed on 6 µM cryosections, and AR and SFRP1 expression were evaluated by immunofluorescence. Sections were fixed in 4% paraformaldehyde (PFA), permeabilized with 0.1–0.5% Triton X-100, and blocked in 10% normal goat serum (NGS) prior to overnight incubation at 4 °C with primary antibodies against AR (1:50; monoclonal rabbit-anti-AR; clone: ERP1535(2), abcam, Cambridge, UK) [41] or SFRP1 (1:200; polyclonal rabbit-anti-SFRP1, abcam) [32]. Secondary antibody incubation employed either a rhodamine-labeled goat anti-rabbit antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA) or an Alexa Fluor 488-labeled goat anti-rabbit antibody (1:400, Invitrogen, Waltham, MA, USA) and was performed at room temperature (RT) for 45 min.

Immunohistochemistry was performed to detect DKK1 expression. Sections were fixed in 4% PFA and endogenous peroxidase activity was quenched by incubation with 3% H_2_O_2_ for 15 min at RT. To reduce background staining, sections were blocked with 10% NGS for 1 h at RT, followed by sequential incubation with avidin and biotin blocking solutions (15 min each). Sections were then incubated overnight at 4 °C with the primary antibody against DKK1 (1:200; monoclonal mouse anti-DKK1, clone 2 A5, Novus Biologicals). After washing, sections were incubated with a biotinylated goat anti-mouse secondary antibody (1:200, Jackson ImmunoResearch) for 45 min at RT. Antigen–antibody complexes were visualized using the Avidin–Biotin Complex–Peroxidase (VEC-PK-6100, Vector) for 30 min at RT, followed by development with the AEC substrate kit (SK-4200, Vector) according to the manufacturer’s instructions.

To stain apoptotic and proliferating cells, a Ki-67/TUNEL double staining was performed using the ApopTag^®^ Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore, Burlington, MA, USA) combined with a Ki-67 staining, as described previously [60,62,65]. Briefly, after fixation with 4% PFA and pre-treatment with the ApopTag solutions for the TdT-Enzyme, cryosections were blocked with goat normal serum and incubated over night at 4 °C with a mouse anti-Ki-67 antibody (1:800 in PBS; Cell Signalling Technology, Danvers, MA, USA). Ki-67 antibody was detected with a goat anti-mouse IgG rhodamine secondary antibody (1:200; Jackson ImmunoResearch) and apoptotic cells were detected with a Fluorescent-labeled Anti-Digoxigenin antibody.

Counterstaining with DAPI was performed during every immunofluorescence staining protocol to visualize nuclei, while with Hematoxylin and Eosin staining during the histochemistry protocol according to routine histochemical methods. Images were taken using a Keyence fluorescence microscope (BZ9100; Osaka, Japan), maintaining a constant set exposure time throughout imaging for further analysis [35]. Staining immunoreactivity or the number of positive cells were counted in the corresponding reference areas depicted in the different figures. Analyses were carried out with ImageJ 1.54p software (National Institutes of Health, Bethesda, MD, USA).

For the histochemical visualization of melanin, the Masson–Fontana staining was performed as previously described [65,66,67].

4.7. Hair Cycle Staging and Scoring

Microscopic hair cycle staging was performed at the end of the culture based on morphology, Masson–Fontana histochemistry and Ki-67/TUNEL immunostaining, as previously described [60,61]. The hair cycle score was calculated using a standardized, arbitrary score (anagen = 100; catagen = 200; early catagen = 300; mid-catagen = 400) [68]. Thus, a lower score means that the HFs are more established in anagen, and a higher score means that the HFs are more progressed to catagen.

4.8. In Situ Hybridization (ISH)

ISH was performed with the RNAscope^®^ multiplex Fluorescent Reagent Kit V2 following the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA, USA) using the following RNAscope™ Probes: Hs-AR-C2 (NM_000044.3, target region 5604–6660 bp), Hs-SFRP1-C2 (NM_003012.4, target region 401–1971 bp), Hs-DKK1-C3 (NM_012242.2, target region 229–1523 bp), PPIB (positive control; NM_000942.4, target region 139–989 bp), and DapB (negative control, EF191515, target region 414–862 bp) [69,70].

4.9. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

For HEK293 and DP qPCR experiments, RNA was purified from cell pellets using TRIzol (Thermo Fisher, Carlsbad, CA, USA) according to the manufacturer’s suggested protocol. cDNA synthesis was performed with 400 ng total RNA according to the manufacturer’s suggested protocol (Superscript kit 11752–250, ThermoFisher, Carlsbad, CA, USA). qPCR reactions were set up according to the TaqMan gene expression master mix protocol (4369016, ThermoFisher, Carlsbad, CA) with 2 µL cDNA and 10 µL total reaction volume using TaqMan probes (ThermoFisher, Carlsbad, CA, USA) run on the QuantStudio 12K Flex Real Time PCR System (Thermo Fisher, Carlsbad, CA, USA) using standard TaqMan settings in a 384-well format. For HFs, total RNA was extracted from 2–3 amputated HFs using the Arcturus^®^ PicoPure^®^ RNA Extraction Kit following the manufacturer’s instructions (Thermo Fisher), then RNA purity and concentration were determined using the Nanodrop ND-1000 assay (Fisher Scientific). For the qPCR protocol, 200–300 ng of mRNA was reverse-transcribed to cDNA using the Tetro cDNA synthesis kit (Bioline, Heidelberg, Germany) according to the manufacturer’s protocols. Quantitative PCR was run in triplicate using TaqMan Fast Advanced Master Mix Product Insert and gene Expression Assay transcripts on the Quantstudio3 Real-Time PCR system, plus associated software (Applied Biosystems; [69]). The following gene TaqMan Expression Assay probes were used: Id: Hs00171172_m1 (AR), Hs00610060_m1 (SFRP1), HS00183740_m1 (DKK1), Hs99999905_m1 (GAPDH), and Hs99999907_m1 (B2M) (Applied Biosystem). The amount of the transcripts was normalized to those of the housekeeping gene (GAPDH or B2M) using the ΔΔCT method.

4.10. ELISA

Detection of secreted DKK1 and SFRP1 was measured on pooled culture medium (duplicate) collected at the end of the culture medium, using the Human Dkk-1 Quantikine ELISA kit (R&D; DKK100B) and the Human SFRP1 ELISA kit (Abcam; ab277082) respectively, following the manufacturer’s instructions. For the ELISAs, the culture medium was diluted in a 1:1 ratio in culture medium and absorbances were measured at 450 nm on a plate reader (GloMax^®^ Discover System, Promega, Madison, WI, USA). The concentrations were determined based on internal standards. Standard curves were generated using linear regression in GraphPad Prism 9, and the unknown DKK1 or SFRP1 concentrations in the samples were interpolated accordingly.

4.11. Statistical Analysis

Statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). Normal distribution of the data was assessed using the D’Agostino and Pearson omnibus normality test. For normally distributed data, multiple group comparison was performed by one-way ANOVA, followed by Tukey’s multiple comparison test, while two-group comparisons were performed using an unpaired Student’s t-test. If the data did not follow a normal distribution, multiple group comparison was carried out using the Kruskal–Wallis test with Dunn’s multiple comparison test, and two-group comparisons were performed using the Mann–Whitney U-test. Data are presented as mean ± SEM. p-values < 0.05 were considered statistically significant.