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Progenitor-derived endothelin controls dermal sheath contraction for hair follicle regression

Nature Cell Biology volume 25, pages 222–234 (2023)Cite this article

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Abstract

Substantial follicle remodelling during the regression phase of the hair growth cycle is coordinated by the contraction of the dermal sheath smooth muscle, but how dermal-sheath-generated forces are regulated is unclear. Here, we identify spatiotemporally controlled endothelin signalling—a potent vasoconstriction-regulating pathway—as the key activating mechanism of dermal sheath contraction. Pharmacological blocking or genetic ablation of both endothelin receptors, ETA and ETB, impedes dermal sheath contraction and halts follicle regression. Epithelial progenitors at the club hair–epithelial strand bottleneck produce the endothelin ligand ET-1, which is required for follicle regression. ET signalling in dermal sheath cells and downstream contraction is dynamically regulated by cytoplasmic Ca2+ levels through cell membrane and sarcoplasmic reticulum calcium channels. Together, these findings illuminate an epithelial–mesenchymal interaction paradigm in which progenitors—destined to undergo programmed cell death—control the contraction of the surrounding sheath smooth muscle to orchestrate homeostatic tissue regression and reorganization for the next stem cell activation and regeneration cycle.

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Fig. 1: DS cells express receptors of the endothelin signalling smooth muscle contraction pathway.
Fig. 2: Activation of endothelin signalling contracts DS cells and pharmacological inhibition impairs DS contraction and follicle regression.
Fig. 3: Genetic ablation of either endothelin receptor does not impair contraction and regression.
Fig. 4: Genetic dual ablation of both endothelin receptors in the DS impairs follicle regression.
Fig. 5: Spatiotemporal endothelin expression and DS contraction during follicle regression.
Fig. 6: Endothelin-1 from ORS progenitors is required for follicle regression.
Fig. 7: ET-1 regulates DS contraction through cytoplasmic Ca2+ signalling.

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Data availability

Raw and analysed RNA-seq data supporting the findings of this study are available at the Gene Expression Omnibus (GEO) repository under accession code GSE215133. Previously published RNA-seq data that were reanalysed here are available at the GEO (GSE77197 and GSE136996). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Sarah Millar for discussions and comments on the manuscript; David Pollock and Ilse Daehn for sharing the Ednra-, Ednrb- and Edn1-targeted mouse lines and Elena Ezhkova for the K14-creERT2 line; Jill Gregory for support with figure illustrations; and the personnel at the ISMMS Flow Cytometry, Microscopy, Genomics and Mouse Genetics CoREs for technical assistance. The ISMMS Microscopy CoRE was supported by NIH Shared Instrumentation grant IS10RR026639. N.H. was supported by training grant T32GM007280 from NIH/NIGMS; and T32HHD075735 from NIH/NIDCR and F30AR070639 from NIH/NIAMS. N.S. was supported by fellowship of the Training Program in Stem Cell Research from the New York State Department of Health (NYSTEM-C32561GG). M.Y. was supported by the World Premier International Research Center Initiative (MEXT), JSPS KAKENHI (17H06095 and 22H04918) and AMED (JP21zf0127005). M.R. was supported by grants from NIH/NIAMS (R01AR071047, R01AR073259, R01AR077593 and R01AR079475) and New York State Department of Health (NYSTEM-C32561GG).

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Author notes

  1. These authors contributed equally: Pieter Martino, Raghava Sunkara.

Authors and Affiliations

  1. Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Pieter Martino, Raghava Sunkara, Nicholas Heitman, Martina Rangl, Alexia Brown, Nivedita Saxena, Laura Grisanti & Michael Rendl

  2. Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Pieter Martino, Raghava Sunkara, Nicholas Heitman, Martina Rangl, Alexia Brown, Nivedita Saxena, Laura Grisanti & Michael Rendl

  3. Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Nicholas Heitman, Nivedita Saxena & Michael Rendl

  4. Division of Nephrology, University of Utah Health, Salt Lake City, UT, USA

    Donald Kohan

  5. International Institute for Integrative Sleep Medicine, University of Tsukuba, Tsukuba, Japan

    Masashi Yanagisawa

  6. Department of Dermatology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Michael Rendl

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Contributions

P.M., R.S., N.H. and M.R. designed the experiments and the overall study. P.M. and M.R. wrote the manuscript. P.M., R.S., N.H., A.B., N.S. and L.G. performed the experiments. D.K. and M.Y. assisted with data analysis and writing the manuscript. M.R. supervised the study.

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Correspondence to Michael Rendl.

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Extended data

Extended Data Fig. 1 Validation of DS, DP and DF isolation strategy during the growth-to-regression transition.

a, Immunofluorescence for pH3 and CASP3* on P16 Tbx18H2BGFP;Crabp1-GFP; Lef1-RFP reporter mouse back skin (n = 2 mice). P13 anagen back skin served as a positive control for pH3. In the hair bulb, proliferation is decreasing and apoptotic cells are beginning to appear, indicating the growth-to-regression transition. Scale bars, 50 µm. b, Immunofluorescence for aSMA, PDGFRA and ITGA8 on P16 Tbx18H2BGFP;Crabp1-GFP;Lef1-RFP reporter mouse back skin (n = 2 mice). Scale bars, 50 µm. c, Immunofluorescence for ITGA8, aSMA, and GFP on P16 Tbx18H2BGFP;Crabp1-GFP;Lef1-RFP reporter mouse back skin, demonstrating colocalization in the dermal sheath (n = 2 mice). Scale bars, 50 µm. d, Immunofluorescence for GFP and RFP on fresh frozen and fixed P16 Tbx18H2BGFP;Crabp1-GFP;Lef1-RFP reporter mouse back skin, both demonstrating colocalization in the DP (n = 2 mice). Scale bars, 50 µm.

Extended Data Fig. 2 Analysis of DP, DS, and DF transcriptomes at the growth-to-regression transition.

a, Principal components analysis of P16 DS, DP, and DF bulk RNA-sequencing data. b, DF, DP and DS RPKM expression levels of Wif1, Acta2 and Lum at regression onset (n = 2, data are mean with individual data points). c, Gene ontology terms enriched in the DS population. ‘Muscle contraction’ is the top enriched GO term for the DS. P < 0.05, Fisher’s exact test. d, Transcription factors, adhesion/ECM molecules, ligands, receptors, and enzymes that are part of the gene signatures of the DS, DP and DF populations. e, Expression of endothelin receptors across cell types from previously published transcriptome data at P520,32(n = 2, data are mean with individual data points).

Source data

Extended Data Fig. 3 Endothelin signalling contracts DS cells in a dose-dependent manner.

a, Endothelin-1 (ET-1) triggers DS contraction and surface area reduction in DS cells grown on Matrigel in a dose-dependent manner. Scale bars, 50 µm. b, Quantification of cell surface areas during contraction time course (0, 30, and 60 minutes). 24, 20, 21, 18 and 19 cells for control, 100 pM, 1 nM, 10 nM, 100 nM and 1uM ET-1, respectively; n = 3 independent experiments. **P = 0.001 (30 min) and 0.002 (60 min) for 1 nM ET-1. ***P = 0.00027 (30 min) and 8.65 × 10−5 (60 min) for 10 nM ET-1, 1.24 × 10−5 (30 min) and 2.5 × 10−5 (60 min) for 100 nM ET-1, and 2.53 × 10−5 (30 min) and 1.14 × 10−5 (60 min) for 1 µM ET-1. Data are mean ± s.d. and statistical significance was determined by one-way ANOVA with post-hoc Tukey’s HSD for multiple comparison testing.

Source data

Extended Data Fig. 4 Endothelin signalling functionally contracts DS cells independent of proliferation.

a, Endothelin-1 (ET-1) triggers contraction and surface area reduction in DS cells grown on matrigel, both in the absence or presence of proliferation inhibition with Mitomycin-C. Scale bars, 50 µm. b, Quantification of cell surface areas during contraction time course. N = 19, 20, 19 cells for control, ET-1, Mitomycin C + ET-1, respectively, from two independent experiments. **P = 0.0015 (5 min) for Mitomycin C + ET-1; ***P = 0.0002 (5 min), 0.0001 (10 min), 1.42 × 10−5 (20 mins) and 1.12 × 10−5 (30 mins) for ET-1; 0.0002 (10 min), 3.59 × 10−5 (20 min) and 61.90 × 10−5 (30 min) for Mitomycin C + ET-1. Data are mean ± s.d. and statistical significance was determined by one-way ANOVA with post-hoc Tukey’s HSD for multiple comparison testing.

Source data

Extended Data Fig. 5 Topical application of endothelin receptor antagonists impedes HF regression with concentrated and local effects.

a, Schematic of the experimental design for topical application of endothelin receptor antagonists BQ123 and BQ788 (‘BQ’) or vehicle control (DMSO), harvesting of the back skins, and imaging strategy. b, Whole-mount immunofluorescence for K14 from the centre of the application area (Zone 1). Most follicles failed to regress in regions treated with BQ123 + BQ788. Scale bars, 50 µm. c, Whole-mount immunofluorescence for K14, LEF1, and DAPI showing a stalled follicle from Zone 1 and a regressed follicle from Zone 3 of BQ123 + BQ788 treated back skin. Scale bars, 50 µm. d, Whole-mount immunofluorescence for K14 at the edge (Zone 2) and just outside of (Zone 3) the application area, demonstrating a progressive decline in stalled follicles towards the periphery. Scale bars, 50 µm. e, Quantification of stalled HFs from each of the three zones. 1200 follicles per zone for vehicle control; 938, 841, and 1087 follicles for Zone 1, 2, and 3, respectively, in BQ123 + BQ788 treated regions; n = 4 mice). *P = 0.0228 (Zone 3), ***P = 1.69 × 10−10 (Zone 1) and 2.42 × 10−7 (Zone 2), unpaired two-tailed Student’s t-test. Data are mean ± s.d. with individual data points.

Source data

Extended Data Fig. 6 Topical application of endothelin receptor antagonists impedes follicle regression during the second hair cycle.

a, Schematic of the experimental design for topical application of endothelin receptor antagonists BQ123 and BQ788 (‘BQ’) or vehicle control (DMSO), harvesting of the back skins, and imaging strategy. b, Whole-mount immunofluorescence for K14 and DAPI in DMSO (vehicle control) and BQ123 + BQ788 treated back skin. Scale bars, 200 µm. c, Quantification of stalled HFs observed in DMSO (vehicle control) and BQ123 + BQ788 treated back skins. 924 follicles for DMSO and 544 follicles for BQ123 + BQ788, n = 3 mice. **P = 0.0031, unpaired two-tailed Student’s t-test. Data are mean ± s.d. with individual data points. d, DS-specific Ednra ablation at P17. Low magnification image of the follicle from the high magnification image in Fig. 3b. e, DS-specific Ednrb ablation at P17. Low magnification image of the follicle from the high magnification image in Fig. 3E. Scale bars, 50 µm.

Source data

Extended Data Fig. 7 Stalling follicles, but not regressing follicles, in dcKO back skin lack both ETA and ETB receptors.

a, Schematic of DS-specific Ednra and Ednrb double conditional genetic ablation (dcKO) in the skin. b, Immunofluorescence for K14 and both ETA/ETB in control P17 back skins. Endothelin receptors are expressed in the DS. c, Immunofluorescence for K14 and both ETA/ETB in DS-specific dcKO P17 back skin. Only stalling follicles lack both receptors ETA and ETB in the DS. Scale bars, 50 µm. n = 4 mice. Green arrows = presence of ETA and/or ETB in the DS. White arrowheads = lack of both ETA and ETB in the DS.

Extended Data Fig. 8 Patterns of endothelin ligand expression during regression.

a, Edn3 mRNA expression across cell types from previously published transcriptome data at P520,32(n = 2, data are mean with individual data points). b, Edn1 smFISH in a follicle transitioning from early-to-mid regression (n = 3 mice). While only very diffuse expression of Edn1 is detectable throughout most of the ORS, the progenitors located in the bottleneck region of the follicle exhibited very high focal expression of Edn1, corresponding to the site of known DS contraction. Scale bars, 50 µm. c, Fire LUT conversion of ET-1 immunofluorescence signal from Fig. 5d highlights the high focal expression of ET-1 in the bottleneck region of regressing follicles. White arrows indicate the bottleneck region. d, Schematic of ET-1 expression during different stages of regression.

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Extended Data Fig. 9 ET-1 ablation in ORS progenitors by mid-regression.

Immunofluorescence for K14, ET-1 and CASP3* in P17 back skins from Edn1 cKO and control (n = 4 mice per condition). A marked reduction in both ET-1 and CASP3* in the K14+ ORS progenitors of the HFs in cKO back skins was observed compared to control back skins. Scale bars, 50 µm. b, Insets showing K14 and ET-1 colocalization in regressing HF from control back skin, with high focal expression of ET-1 in the bottleneck region. In Edn1 cKO back skins, some HFs exhibit complete ET-1 ablation while other HFs only exhibit partial ET-1 ablation in the K14+ ORS progenitors. Scale bars, 50 µm.

Extended Data Fig. 10 ET-1-induced DS contraction depends on dynamically regulated cytoplasmic Ca2+.

a, Images of Fluo8 levels in DS cells from Fig. 7b including intermediate timepoints (5, 10, 20 mins) during the 30-minute ET-1 or vehicle control (PBS) exposure; n = 2 independent experiments. Scale bars, 50 µm. b, Images of tdT-labelled DS cells at 0, 5, 10, 20 and 30 minutes of ET-1 or vehicle control (PBS) exposure following a 1 h preincubation in calcium channel blockers (50 µM NNC 55-0396 + 50 µM Diltiazem + 10 µM Ryanodine + 10 µM Xestospongin C), ML7 (200 µM), or vehicle control (DMSO); n = 2 independent experiments. Endothelin-mediated DS contraction is abrogated by either blocking of calcium channels or by inhibition of MLCK activity. Scale bars, 50 µm.

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Martino, P., Sunkara, R., Heitman, N. et al. Progenitor-derived endothelin controls dermal sheath contraction for hair follicle regression. Nat Cell Biol 25, 222–234 (2023). https://doi.org/10.1038/s41556-022-01065-w

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