Olfactory receptor OR2AT4 regulates human hair growth

Olfactory receptors are expressed by different cell types throughout the body and regulate physiological cell functions beyond olfaction. In particular, the olfactory receptor OR2AT4 has been shown to stimulate keratinocyte proliferation in the skin. Here, we show that the epithelium of human hair follicles, particularly the outer root sheath, expresses OR2AT4, and that specific stimulation of OR2AT4 by a synthetic sandalwood odorant (Sandalore®) prolongs human hair growth ex vivo by decreasing apoptosis and increasing production of the anagen-prolonging growth factor IGF-1. In contrast, co-administration of the specific OR2AT4 antagonist Phenirat® and silencing of OR2AT4 inhibit hair growth. Together, our study identifies that human hair follicles can engage in olfactory receptor-dependent chemosensation and require OR2AT4-mediated signaling to sustain their growth, suggesting that olfactory receptors may serve as a target in hair loss therapy.

Interestingly, several ORs are also expressed in human epidermis 19,20 , including OR2AT4, whose selective activation by the synthetic sandalwood odorant (Sandalore ® ) promotes human epidermal keratinocyte migration and proliferation in vitro and wound re-epithelialization ex vivo 20 . Sandalore ® -induced Ca2 + signaling could be blocked in OR2AT4-transfected Hana3A cells when this was co-applied at equimolar concentrations with the potent competitive OR2AT4 antagonist in presence of Sandalore ® , Phenirat ®20 . Given the intimate connections between hair growth and wound healing [21][22][23][24] , we hypothesized that this OR might also impact on human hair growth. This hypothesis was investigated by immunohistology, qRT-PCR, western blot, microarray, phospho-kinase assay, and gene silencing in healthy, organ-cultured human scalp hair follicles (HFs) 25 .
The present study shows that human HFs express a specific OR, namely, OR2AT4. The activation of this OR by its specific agonist, Sandalore ® , prolongs anagen maintenance ex vivo by decreasing hair matrix keratinocytes apoptosis and increasing the production of IGF-1 in the outer root sheath (ORS). The anagenprolonging effect mediated by Sandalore ® is OR2AT4 dependent, as confirmed by co-administration of Sandalore ® with the OR2AT4 competitive antagonist, Phenirat ® , as well as the specific knock-down of OR2AT4 in human HFs. Taken together, we show that human HFs can engage in chemosensation and that the specific activation of OR2AT4 is required to sustain HF growth.

Results
Human HFs express OR2AT4. Immunofluorescence microscopy, qRT-PCR, and western blot analysis revealed that human scalp HFs in the anagen VI stage of the hair cycle 26,27 express OR2AT4 at the transcript and protein level (Figs. 1a, 2a-f). Interestingly, OR2AT4 protein was predominantly expressed by suprabulbar keratinocytes of the proximal ORS (Figs. 1a, 2c), while hair matrix keratinocytes also expressed low-level OR2AT4 protein (Figs. 1a, 2b), both in healthy scalp skin in situ 27   and in amputated microdissected anagen HFs ex vivo 25,26 (Fig. 1a). Of note, OR2AT4 expression was downregulated during spontaneous, apoptosisdriven HF regression (catagen) 26,27 (Fig. 2d-g). Thus, using the primary antibody employed here 20 , intrafollicular OR2AT4 expression is strikingly restricted to defined epithelial HF compartments and is hair cycle dependent.
Next, we examined two key growth factors that control the anagen-catagen transformation during human HF cycling, i.e., catagen-promoting TGF-β2 and anagen-maintaining IGF-1; these growth factors are prominently produced by those proximal ORS keratinocytes 28-33 that express OR2AT4 maximally. This analysis revealed a significant decrease in TGF-β2 ( Supplementary Fig. 3a) and a significant increase of IGF-1 (Fig. 1d) protein expression in the proximal ORS after long-term Sandalore ® treatment ex vivo. The co-administration of OR2AT4 antagonist, Phenirat ® , significantly reversed the Sandalore ® -induced intrafollicular upregulation of IGF-1 (Fig. 1d) but did not affect TGF-β2 expression ( Supplementary Fig. 3a).
Anagen-prolonging effect of Sandalore ® is OR2AT4 specific. Subsequently, we selectively silenced OR2AT4 by siRNA administration to organ-cultured human scalp HFs ex vivo 32,34,35 , as documented by significantly reduced intrafollicular OR2AT4 mRNA and protein expression (Fig. 4a, b). Despite the presence of excess ligand (Sandalore ® ), OR2AT4 knock-down significantly promoted catagen induction compared to HFs treated with scrambled oligos (Fig. 5a), decreased IGF-1 protein expression (Fig. 5b), and enhanced hair matrix keratinocyte apoptosis (Fig. 5d, e). Instead, hair matrix keratinocyte proliferation (Fig. 5c) or TGFβ2 protein expression in the ORS ( Supplementary Fig. 4a) remained unaffected. These data show that the Sandalore ® -induced hair growth stimulation documented above is indeed OR2AT4 dependent, rather than due to off-target effects of this synthetic odorant and that OR2AT4 signaling is required for anagen maintenance.
Sandalore ® -mediated HF response involves different pathways. Microarray analysis independently confirmed anti-apoptotic effects of Sandalore ® (Fig. 6, Supplementary Fig. 5, and Supplementary Data 1), since transcripts of pro-apoptotic genes were significantly downregulated (e.g., TP53AIP1: -10.27×), while antiapoptotic genes were significantly upregulated (e.g., FGF-2: +7.83×) in HFs treated short term with Sandalore ® (6 h, Supplementary Fig. 5a, b and Supplementary Data 1). Interestingly, an additional microarray analysis of organ-cultured scalp HFs in which OR2AT4 had been knocked down ex vivo showed that transcription of the IFI6 (G1P3) gene, whose silencing increases keratinocyte apoptosis 36 , was downregulated by administering OR2AT4 siRNA for 6 h, compared to scrambled oligonucleotidetreated HFs ( Supplementary Fig. 6a, b and Supplementary Data 1). This corresponds well to our observation that OR2AT4 silencing increases apoptosis of HF matrix keratinocytes (Fig. 5d, e) and further underscores the importance of continued OR2AT4 stimulation by as yet unknown endogenous ligands to suppress apoptosis in the hair matrix of human anagen HFs.
Intriguingly, the strongest transcriptional upregulation (77.6× increase) was seen for dermcidin, a potent antimicrobial peptide with broad bactericidal activities that reportedly is only produced by sweat gland epithelium in human skin 40,41 . However, quantitative immunohistomorphometry confirmed that dermcidin protein is also upregulated by Sandalore ® in the epithelium of human scalp HFs (Supplementary Fig. 7a and Table 1). This demonstrates that human HFs also express dermcidin and raises the fascinating question whether OR2AT4 may act as a chemosensory receptor for selected bacterial metabolites, in response to which intrafollicular dermcidin production may be upregulated to manage the complex HF-microbiome 42,43 .
While IGF-1 signaling is known to be involved in olfactory bulb development and function [54][55][56] , the current study reveals that IGF-1 expression/secretion in human epithelial tissue is also controlled by OR-mediated signaling and demonstrates that IGF-1 production underlies an OR2AT4-controlled chemosensory regulation.

Discussion
Collectively, these data show that the growth, cyclic transformation, epithelial cell apoptosis, and IGF-1 production of a dynamic human (mini-)organ, i.e., scalp HFs 29 , underlies an ORdependent chemosensory control. Thus, human HFs can "smell" in the sense that they recruit the evolutionarily oldest and Moreover, we identify one specific OR, namely, OR2AT4, whose stimulation with a synthetic agonist (Sandalore ® ) 20 and whose selective silencing profoundly impacts on human hair growth ex vivo primarily via regulating expression and secretion of the key hair growth-promoting factor, IGF-1 ( Fig. 8 and Supplementary Discussion 2). However, while IGF-1-mediated signaling is required for human hair growth promotion by Sandalore ® (Fig. 1e), our phospho-kinase activity and gene expression profiling results suggest that additional pathways (e.g., p38a/ERK1/2/ MSK1/2, HB-EGF/EGF-R, and FGF-7 pathways (Fig. 6, Supplementary Figs. 5, 6, and Supplementary Data 1)) are involved that deserve further exploration [57][58][59][60][61] . Perhaps most intriguingly, our silencing data suggest that OR2AT4-mediated signaling is required for maintaining human scalp HFs in anagen and for suppressing keratinocyte apoptosis in the hair matrix (Fig. 5d, e). This begs the question: What are the endogenous intrafollicular OR2AT4 ligands in human HFs? The endogenous ligands for human ORs remain to be definitively clarified, and those for OR2AT4 are unknown. Candidates include molecules with Sandalore ® -like structure, short-chain fatty acids 13 , and-namely, in view of our dermcidin results ( Supplementary Fig. 7a and Table 1)-metabolites of resident HF microbiota 42,43 .
Taken together, our ex vivo data suggest that olfactotherapy by topically applied cosmetic OR2AT4 ligands like Sandalore ® may promote human hair growth by prolonging anagen and inhibiting premature catagen development (e.g., in androgenetic alopecia and telogen effluvium).
Thus, using scalp HFs as accessible and tractable model organs and by selectively targeting OR2AT4, our study reveals an important, translationally relevant frontier in the OR-dependent chemosensory physiology of peripheral human tissues.

Methods
Human samples. Temporal and occipital human scalp skin was obtained from healthy donors (38-69 years old) undergoing routine face-lift surgery after informed consent and ethical approval (University of Muenster, no. 2015-602-f-S). No sample size calculation was performed. Number of three different donors was used due to the small availability of the tissue used in the study. This number of three was used in many previous studies, given statistical significance.
Tissue specimens. Scalp skin samples were either cut into small pieces (4 mm), embedded into OCT, and frozen in liquid nitrogen, or processed for HF microdissection 25 .
HF organ culture. Human scalp samples were obtained 1 day after face-lifting procedure (i.e., after overnight transport from collaborating surgeons) and used at the same day for microdissecting human anagen VI scalp HFs. The HF microdissection technique employed for setting up the classical Philpott assay 25,26,62 used in the current study, removes all perifollicular tissue with the sole exception of the HF's dermal sheath, and thus does not contain any other skin appendage structures (e.g., eccrine gland elements) 25 . Microdissected human scalp HFs were cultured at 37°C with 5% CO 2 in a minimal media of William's E media (WEM, Gibco, Life Technologies) supplemented with 2 mM of L-glutamine (Gibco), 10 ng/ml hydrocortisone (Sigma-Aldrich), 10 μg/ml insulin (Sigma-Aldrich), and 1% penicillin/streptomycin mix (Gibco) 25,26,62 . After microdissection, the HFs were first incubated in WEM for 24 h for re-equilibration. HFs after quality control (fully pigmented and presence in anagen VI phase) were randomly allocated to the different experimental groups.
For the IGF-1 neutralizing antibody experiments, IGF-1 neutralizing antibody (1 µg/ml, ab9572, Abcam) was added 30 min before adding Sandalore ® to the corresponding groups. Culture medium was replaced every second day and after 6 days. HFs were then embedded in cryomatrix (Fisher Scientific), and snap frozen in liquid nitrogen for (immuno-)histology.
SiRNA transfection-knockdown OR2AT4 in organ-cultured HFs. Human anagen VI HFs were transfected using a commercial siRNA reagent system (Sc-45064, Santa Cruz) following the manufacturer's instructions 32,34,35 . Briefly, stock solutions (10 µM) of siRNA OR2AT4 (gift from Prof. Hanns Hatt 20 ) and siRNA control (scrambled oligo) were prepared using RNAse-free water. HF transfection was performed 24 h after microdissection for 6 h using either 100 mM OR2AT4 siRNA or control scramble siRNA. After 24 h of incubation with fresh WEM medium, HFs were collected per group in RNA later and stored at 4°C for further RNA extraction and qRT-PCR analysis or immediately frozen in liquid nitrogen and stored at −80°C for microarray analysis. Finally, fresh WEM medium was replaced every second day and after 5 days of culture, HFs were snap frozen in OCT for further quantitative (immuno-)histomorphometry analysis.
Histology. For histochemical visualization of melanin, Masson-Fontana staining was performed on frozen sections. Melanin was stained as brown dots 26 .
For TGFβ2 (Sc-90, Santa Cruz) and IGF-1 (Sc-1422, clone G-17, Santa Cruz 31,32 ), tissue cryosections were fixed in acetone and endogenous peroxidase activity was blocked with 3% of H 2 O 2 (Merck Milipore). This step was followed by an avidin-biotin blocking step (SP2001, Vectorlabs) and a preincubation with TNB buffer (Tris HCl+NaCl+Casein). The corresponding primary antibody was   incubated at 4°C overnight (1/1000 for TGFβ2 and 1/250 for IGF-1). Secondary antibody incubation was performed at RT for 45 min before using the Tyramide signal amplification kit (NEL700001KT, Perkin Elmer). Counterstaning with DAPI was performed to visualize nuclei. To stain apoptotic and proliferating cells, we used the apoptag kit (Merck Milipore) following the manufacturer's protocol followed by Ki-67 staining 25,26,33,63 . Primary antibody was incubated overnight (Ki-67, M7240 Clone: MIB-1, DAKO, 1/20) after the TdT-enzyme step. The secondary antibody was incubated for 45 min at RT after the fluorescent-labeled anti-Digoxigenin step of the apoptag kit. Counterstaning with DAPI was performed to visualize nuclei. Negative controls were performed by omitting the primary antibody. Images were taken using a Keyence fluorescence microscope BZ9100 (Osaka, Japan) maintaining a constant set exposure time throughout imaging for further analysis.
Quantitative reverse transcriptase-PCR. Total RNA was isolated from whole microdissected HFs using RNeasy Mini Kit (Quiagen) following the manufacturer's instructions described in the manufacturer's protocol. RNA purity and concentrations were determined using the Nanodrop ND-1000 assay (Fisher Scientific). Reverse transcription of the RNA into cDNA was performed using the TetrocDNA Synthesis Kit (Bioline), according to the manufacturer's instructions. RNA concentrations were adjusted between 50 to 500 nM for each sample set to allow further quantification comparison between samples and experiments after qRT-PCR. Controls were performed using the housekeeping gene GAPDH. Real-time quantitative polymerase chain reaction (qRT-PCR) was run in triplicate using TaqMan Fast Advanced Master Mix Product Insert and gene Expression Assay transcripts (Id: Hs01060665_g1 for ACTB, Hs02758991_g1 for GAPDH, and Hs02339277_s1 for OR2AT4, Applied Biosystem) on the qTower2.2 thermocycler. Real-time quantification plots and Ct values were collected and stored by the qPCRsoft2.1 software. The amount of the transcripts was normalized to those of the housekeeping gene using the ΔΔCT method using EXCEL.
Whole-genome microarray analysis. RNA isolation, sample processing, and microarray analyses (Agilent Technologies), as well as statistical evaluation, were performed by Arrows Biomedical GmbH (Muenster, Germany). Expressional alteration was considered to be significant only when ≥1.8-fold and equidirectional changes were observed in at least three of four patients (independent experiments). An additional analysis has been performed using 5-fold and equidirectional changes in the four different donors (independent experiments) in order to identify the top up and downregulated genes.
Human phospho-kinase array. In order to gauge which signaling pathways are regulated by the specific stimulation of OR2AT4, we performed a phospho-kinase array 9 . Total protein was isolated from whole microdissected HFs using a specific buffer from the Human Phospho-Kinase Array (ARY003B, R&D System), following the manufacturer's protocol. Briefly, protein extracts were diluted and incubated overnight with the Human Phospho-Kinase Array. The array was  Fig. 6 Microarray-based analysis of genes related to anagen-prolonging pathways after stimulation with Sandalore ® (500 µM). a, b Venny diagrams 65 show the upregulated and downregulated genes (cut-off: fold change >−1.8 or >+1.8 and equidirectional changes). White squares indicate genes upregulated and downregulated in at least three of four donors (independent experiments). The heatmap shows the list and the expression level of the most upregulated and downregulated genes related to the different pathways regulated after OR2AT4 activation (cut-off: fold change >−1.8 or >+1.8 and equidirectional changes) in at least three of four donors (independent experiments) (c). Green: apoptosis related, orange: dermcidin related, and violet: IGF related washed to remove unbound proteins, followed by incubation with a cocktail of biotinylated detection antibodies. Streptavidin-HRP and chemiluminescent detection reagents were applied, and a signal was produced at each capture spot corresponding to the amount of phosphorylated protein bound.
Statistical analyses. All data are expressed as mean ± SEM (and variance is different between the groups) and were analyzed by one-way ANOVA or Kruskall-Wallis test and Dunn's multiple comparisons test as post hoc test when more than two groups were compared or Student's t-test or Mann-Whitney test when only two groups were compared (GraphPad Prism 6, GraphPad Software, San Diego, CA, USA) after performing d'Agostino and Pearson omnibus normality test. P < 0.05 was regarded as significant.

Data availability
The data discussed in this publication are available from the authors and the microarray data have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE102887.  8 Proposed mechanism of action of OR2AT4 activation by Sandalore ® and (unknown) endogenous ligand(s) in human hair follicle epithelium. The activation of OR2AT4 at the cell surface of outer root sheath keratinocytes (ORS KCs; location: see green cells in the central HF cartoon) by endogenous ligands and/or Sandalore ® upregulates the expression of genes and kinases involved in programmed cell death, thus preventing intrafollicular apoptosis (e.g., by phosphorylation of PRAS40 preventing its interaction with mTOR1, upregulation of NF-κB pathway) or downregulates key players in the apoptotic machinery (e.g., dephosphorylation of p53, downregulation of Bad). In parallel, OR2AT4 activation by exogenous (Sandalore ® ) or endogenous ligands (e.g., metabolites of the HF microbiome) induces the upregulation of PAPPA that cleave the IGFBP4/IGF1 complex to release IGF-1 (pink arrows). The released IGF-1 triggers the activation of IGF-1R on the same cell (autocrine signaling, purple arrow) or on hair matrix keratinocytes (HM KCs; orange "cell") (paracrine signaling, orange arrow). The activation of IGF-Rs on HM keratinocytes then induces signaling cascades (e.g., PI3K/AKT and/or p38a/ERK1/2/ MSK1/2) that activate different transcription factors and particularly CREB, which results in an anti-apoptotic effect and prolonged anagen phase in human HFs. P phosphorylation, green square gene upregulation, red square gene downregulation, green circle phosphorylation, red circle dephosphorylation