Olfactory receptor OR2AT4 regulates human hair growth

Abstract

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.

Fig. 1

Hair follicles express OR2AT4, which specific stimulation endorses IGF-1-dependent anagen prolongation. a Representative images showing OR2AT4 protein expression (using the previously published OR2AT4 antibody) in proximal outer root sheath and hair matrix keratinocytes of human scalp microdissected hair follicles. b Hair cycle score and staging were evaluated in treated and vehicle HFs after 6 days of culture using Ki-67/TUNEL immunofluorescence and Masson–Fontana histochemistry. Mean ± SEM, n = 16–24 HFs from three donors (independent experiments), Kruskal–Wallis test (P = 0.0923, n.s not significant) and Dunn’s multiple comparisons test as post hoc test, ns not significant, Mann–Whitney test, *P < 0.05. Representative pictures of Masson–Fontana histochemistry in vehicle and treated HFs after 6 days of treatment. c Apoptotic hair matrix keratinocytes were counted in the hair matrix of all treated and vehicle HFs. Representative pictures of Ki67/TUNEL. Mean ± SEM, n = 18–21 HFs from three donors (independent experiments), Kruskal–Wallis (P = 0.005) test and Dunn’s multiple comparisons test as post hoc test, #P < 0.05, ##P < 0.01, ###P < 0.001. d IGF-1 expression was measured in ORS keratinocytes in treated and vehicle HFs. Representative pictures of IGF-1 immunofluorescence. IGF-1 expression was quantified in ORS keratinocytes in treated and vehicle HFs using ImageJ. Mean ± SEM, n = 18–21 HFs from three donors (independent experiments), Kruskal–Wallis (P < 0.001) and Dunn’s multiple comparisons test as post hoc test, ^##P < 0.01, ^###P < 0.001, and Student’s t-test, *P < 0.05. e Hair cycle score and staging were measured in treated and vehicle HFs after 6 days of culture. Representative pictures of vehicle and treated HFs after 6 days of treatment. Mean ± SEM, n = 22–29 HFs from three donors (independent experiments), Kruskal–Wallis test (P = 0.1434) and Dunn’s multiple comparisons test as post hoc test, n.s not significant, and Student’s t-test after performing an iterative Grubbs outlier test, *P < 0.05. CTS connective tissue sheath, DP dermal papilla, HM hair matrix, ORS outer root sheath, IRS inner root sheath, HS hair shaft. Scale bar: 100 µm

Fig. 2

OR2AT4 mRNA and protein expression in human scalp epidermis and hair follicles in situ. a Representative pictures of OR2AT4 immunofluorescence in human scalp epidermis of three different donors (positive control). Red line delineates the dermo-epidermal basement membrane. Cytosolic expression of OR2AT4 in hair matrix and suprabulbar outer root sheath (ORS) keratinocytes. Scale bar: 100 µm. b, c Representative pictures of confocal imaging of OR2AT4 immunofluorescence in human scalp HFs from three different donors (independent experiments). Scale bar: 100 µm. d, e mRNA (normalized against GAPDH) and in situ protein expression of OR2AT4 in anagen and catagen microdissected HF epithelium. Mean ± SEM, n = 3 from nine HFs/donor from three donors (independent experiments), Student’s t-test, *P < 0.05. f, g Western blot analysis and quantitative results of OR2AT4 (normalized against actin) in anagen and catagen microdissected human scalp HFs. Mean ± SEM from nine HFs/donor from two donors (independent experiments). The specific band for OR2AT4 is found around 44 kDa, although the predicted molecular weight of OR2AT4 is 36 kDa. This slight difference can be explained by a post-translational modification (an acetylation site has been identified on the lysine at the position 303 [source: phosphoSitePlus^®]) that would increase the molecular weight. A1-2 indicates anagen HFs and C1-2 indicates catagen HFs from donor 1 and 2. DP dermal papilla, HM hair matrix

Fig. 3

High concentration of Sandalore^® (500 µM) regulates hair matrix keratinocytes apoptosis and intrafollicular OR2AT4 expression. a, b The number of Ki-67+ and TUNEL+ cells in the hair matrix was evaluated in the hair bulb of all treated and vehicle HFs. c Representative pictures of Ki-67/TUNEL staining. Mean ± SEM, n = 19–21 HFs from two donors (independent experiments), unpaired Student’s t-test or Mann–Whitney test, *P < 0.05, **P < 0.01. DP dermal papilla, HM hair matrix. Scale bar: 100 µm. d OR2AT4 protein expression was evaluated using ImageJ in the ORS of Sandalore^®-treated and control HFs after 6 days of culture. e Representative pictures of OR2AT4 expression in the ORS of cultured HFs. Mean ± SEM, n = 12–15 HFs from two donors (independent experiments), Student’s t-test, *P < 0.05. CTS connective tissue sheath, IRS inner root sheath, ORS outer root sheath. Scale bar: 100 µm

Fig. 4

OR2AT4 expression in Scambled oligos or siRNA OR2AT4 Sandalore^®-treated, microdissected human scalp HFs after 6 h of organ culture. a Microdissected, human scalp HFs were treated for 6 h with OR2AT4 siRNA or scrambled oligos, using the intrafollicular gene silencing technique. OR2AT4 mRNA expression was evaluated after 24 h of culture in siRNA or scrambled oligo-transfected HFs. b OR2AT4 protein expression was evaluated using ImageJ in the ORS of siRNA-treated and control HFs after 6 days of culture. Representative pictures of OR2AT4 expression in the ORS of cultured HFs. Mean ± SEM, n = 19–24 HFs from three donors (independent experiments), Student’s t-test, *P < 0.05, ***P < 0.001. CTS connective tissue sheath, IRS inner root sheath, ORS outer root sheath. Scale bar: 100 µm

Fig. 5

The anagen prolongation effect of Sandalore^® is OR2AT4 dependent. a Hair follicle cycle score and staging were performed in HFs treated with siRNA OR2AT4 or scrambled oligos in the presence of Sandalore^®. Representative images of vehicle and treated HFs after 6 days of treatment. Mean ± SEM, n = 16–18 HFs from three donors (independent experiments), Mann–Whitney test, *P < 0.05. b IGF-1 expression was quantified in ORS keratinocytes in treated and vehicle HFs using ImageJ. Representative pictures of IGF-1 immunofluorescence. Mean ± SEM, n = 22–24 HFs from three donors (independent experiments), Mann–Whitney test, *P < 0.05. c, d Ki67+ cells and TUNEL+ cells were counted in the hair matrix of siRNA and control HFs. Representative pictures of Ki67/TUNEL double-staining in the hair bulb of HFs. Mean ± SEM, n = 17–18 HFs from three donors (independent experiments), Mann–Whitney test, *P < 0.05, n.s. not significant. e The number of cleaved caspase-3+ cells (white arrows) in the hair matrix was evaluated in the hair bulb of all HFs treated with OR2AT4-siRNA or scrambled oligos. Representative pictures of cleaved caspase-3 staining. Mean ± SEM, n = 18 HFs from three donors (independent experiments), Student’s t-test, *P < 0.05. CTS connective tissue sheath, DP dermal papilla, HM hair matrix, ORS outer root sheath, IRS inner root sheath. Scale bar: 100 µm

Fig. 6

Microarray-based analysis of genes related to anagen-prolonging pathways after stimulation with Sandalore^® (500 µM). a, b Venny diagrams 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

Fig. 7

OR2AT4 stimulation by Sandalore^® treatment regulates different protein phosphorylation signaling pathways in human HFs ex vivo. Plot showing regulation of the phosphorylation of 45 kinases mediated by Sandalore^® (50 and 500 µM) in cultured microdissected HFs from four different donors (independent experiments). Among those kinases, the most interesting ones underline in green (upregulated) and red (downregulated) rectangles. PRAS40 proline-rich AKT1 substrate 40, S46 phosphorylated p53, p53 phosphoprotein p53, p38α mitogen-activated protein kinases 14, ERK1/2 extracellular signal-regulated kinases1/2, EGFR epidermal growth factor receptor, MSK1/2 mitogen- and stress-activated protein kinase 1/2, PYK2 protein tyrosine kinase 2, Hsp60 heat shock protein 60, JNK1/2/3 c-Jun N-Terminal Protein Kinase 1/2/3, AMPKα1 AMP-activated protein kinase α1, PLCγ phospholipase C γ1, Fgr Feline Gardner-Rasheed proto-oncogene, WNK1 WNK lysine-deficient protein kinase 1 isoform

Fig. 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