Enormous effort over the last 3 decades has identified a number of signaling pathways that act on hair follicle stem cells (HFSCs) to promote both quiescence as well as activation (Chan et al., 2004Chan K.S. Sano S. Kiguchi K. Anders J. Komazawa N. Takeda J. et al.Disruption of Stat3 reveals a critical role in both the initiation and the promotion stages of epithelial carcinogenesis.J Clin Invest. 2004; 114: 720-728Crossref PubMed Scopus (311) Google Scholar, DasGupta and Fuchs, 1999DasGupta R. Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation.Development. 1999; 126: 4557-4568Crossref PubMed Google Scholar, Kimura-Ueki et al., 2012Kimura-Ueki M. Oda Y. Oki J. Komi-Kuramochi A. Honda E. Asada M. et al.Hair cycle resting phase is regulated by cyclic epithelial FGF18 signaling.J Invest Dermatol. 2012; 132: 1338-1345Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Ming Kwan et al., 2004Ming Kwan K. 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Cutaneous consequences of inhibiting EGF receptor signaling in vivo: normal hair follicle development, but retarded hair cycle induction and inhibition of adipocyte growth in Egfr(Wa5) mice.J Dermatol Sci. 2010; 57: 155-161Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, Vauclair et al., 2005Vauclair S. Nicolas M. Barrandon Y. Radtke F. Notch1 is essential for postnatal hair follicle development and homeostasis.Dev Biol. 2005; 284: 184-193Crossref PubMed Scopus (109) Google Scholar, Zhang et al., 2006Zhang J. He X.C. Tong W.G. Johnson T. Wiedemann L.M. Mishina Y. et al.BMP signaling inhibits hair follicle anagen induction by restricting epithelial stem/progenitor cell activation and expansion.Stem Cells. 2006; 24: 2826-2839Crossref PubMed Scopus (135) Google Scholar). With respect to intrinsic mechanisms of HFSC regulation, less is known about the cellular metabolism of individual cell types in the epidermis. In general, it has been presumed that somatic cells use mostly the electron transport chain (ETC) to produce energy from pyruvate that was generated by the uptake and processing of glucose, while early embryonic and cancer cells are thought to also rely on production of lactate from pyruvate. We recently demonstrated that HFSCs balance the production of energy through the ETC with the production of lactate as well (Flores et al., 2017Flores A. Schell J. Krall A.S. Jelinek D. Miranda M. Grigorian M. et al.Lactate dehydrogenase activity drives hair follicle stem cell activation.Nat Cell Biol. 2017; 19: 1017-1026Crossref PubMed Scopus (143) Google Scholar). Previous efforts to define metabolic activities in the epidermis focused on measurements of enzyme activities on entire follicles (Hamanaka et al., 2013Hamanaka R.B. Glasauer A. Hoover P. Yang S. Blatt H. Mullen A.R. et al.Mitochondrial reactive oxygen species promote epidermal differentiation and hair follicle development.Sci Signal. 2013; 6: ra8Crossref PubMed Scopus (223) Google Scholar, Kloepper et al., 2015Kloepper J.E. Baris O.R. Reuter K. Kobayashi K. Weiland D. Vidali S. et al.Mitochondrial function in murine skin epithelium is crucial for hair follicle morphogenesis and epithelial-mesenchymal interactions.J Invest Dermatol. 2015; 135: 679-689Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In addition, several studies used transgenic models targeting the entire epidermis (including the follicle) for deletion of ETC components (Hamanaka et al., 2013Hamanaka R.B. Glasauer A. Hoover P. Yang S. Blatt H. Mullen A.R. et al.Mitochondrial reactive oxygen species promote epidermal differentiation and hair follicle development.Sci Signal. 2013; 6: ra8Crossref PubMed Scopus (223) Google Scholar, Kloepper et al., 2015Kloepper J.E. Baris O.R. Reuter K. Kobayashi K. Weiland D. Vidali S. et al.Mitochondrial function in murine skin epithelium is crucial for hair follicle morphogenesis and epithelial-mesenchymal interactions.J Invest Dermatol. 2015; 135: 679-689Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Those studies suggested that genetic blockade of the ETC leads to degeneration of the follicle. It is less clear whether inhibition of ETC complexes, as opposed to genetic ablation of ETC, would affect cell metabolism or fate decisions. In the study by Flores et al., 2017Flores A. Schell J. Krall A.S. Jelinek D. Miranda M. Grigorian M. et al.Lactate dehydrogenase activity drives hair follicle stem cell activation.Nat Cell Biol. 2017; 19: 1017-1026Crossref PubMed Scopus (143) Google Scholar, we deleted Ldha specifically in HFSCs and found that blocking lactate production prevented activation of HFSCs. In addition, deletion of a protein required to transport pyruvate into mitochondria (Mpc1) in HFSCs led to the increase of lactate production and acceleration of HFSC activation (Flores et al., 2017Flores A. Schell J. Krall A.S. Jelinek D. Miranda M. Grigorian M. et al.Lactate dehydrogenase activity drives hair follicle stem cell activation.Nat Cell Biol. 2017; 19: 1017-1026Crossref PubMed Scopus (143) Google Scholar). Therefore, we hypothesized that pharmacological ETC inhibition would promote HFSC activation because of increased production of lactate. To determine whether manipulation of ETC activity could affect HFSC activation, we used topical application of various inhibitors of ETC components during a resting phase of the hair cycle. At postnatal day 50, the HF is in telogen, a resting phase where the stem cells of the follicle are quiescent until the start of the next hair cycle at day 70−80 (Greco et al., 2009Greco V. Chen T. Rendl M. Schober M. Pasolli H.A. Stokes N. et al.A two-step mechanism for stem cell activation during hair regeneration.Cell Stem Cell. 2009; 4: 155-169Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, Muller-Rover et al., 2001Muller-Rover S. Handjiski B. van der Veen C. Eichmuller S. Foitzik K. McKay I.A. et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15Abstract Full Text Full Text PDF PubMed Google Scholar, Paus and Foitzik, 2004Paus R. Foitzik K. In search of the “hair cycle clock”: a guided tour.Differentiation. 2004; 72: 489-511Crossref PubMed Scopus (233) Google Scholar). Rotenone, phenformin, and antimycin A are all established inhibitors of Complex I and Complex III, respectively (Graeber et al., 1976Graeber G.M. Marmor B.M. Hendel R.C. Gregg R.O. Pancreatitis and severe metabolic abnormalities due to phenformin therapy.Arch Surg. 1976; 111: 1014-1016Crossref PubMed Scopus (12) Google Scholar). Animals were shaved at postnatal day 47 and treated with the indicated compounds or vehicle on the shaved area every 48 hours for the indicated duration. After 3−4 treatments (8−12 days), animals treated with ETC inhibitors began to show signs of hair cycle activation macroscopically, judged by pigmentation of the skin in black mice, whereas vehicle-treated mice did not show significant pigmentation for at least 20 days (Figure 1a and Supplementary Figure S1a online). As defined previously, the epidermis of murine skin becomes pigmented upon induction of the hair cycle, which is indicative of the generation of melanocytes injecting pigment (melanin) into the keratinocytes, which go on to make the hair shaft, as well as those in the interfollicular epidermis. Therefore, the induction of pigmentation observed after 8−12 days in ETC inhibitor−treated mice was most likely indicative of hair cycle activation induced by this treatment. To demonstrate that the pigmentation induced by ETC inhibition was in fact due to changes in HFSC activation, tissue was harvested and subjected to pathology. Histological analysis showed that follicles in back skin treated with ETC inhibitors underwent a normal telogen-to-anagen transition (Figure 1b and Supplementary Figure S1b). These findings were in contrast to previous studies showing that transgenic abrogation of the ETC led to HF degeneration (Kloepper et al., 2015Kloepper J.E. Baris O.R. Reuter K. Kobayashi K. Weiland D. Vidali S. et al.Mitochondrial function in murine skin epithelium is crucial for hair follicle morphogenesis and epithelial-mesenchymal interactions.J Invest Dermatol. 2015; 135: 679-689Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To determine whether the hair cycle induction driven by ETC inhibition was typical, we measured the thickness of each layer of skin across different stages of treatment. As shown in Figure 1c, all of the ETC inhibitors increased the thickness of the epidermis, dermis, and particularly the hypodermis, suggesting an expansion of the adipocytes. Analysis of ETC-inhibited skin showed an increase in Ki67 in HFSCs a week after treatment, evidence of HFSC activation in response to ETC inhibition (Figure 1d and Supplementary Figure S1d). To determine whether application of the ETC inhibitors promoted inflammation, which could cloud interpretation of hair cycle data, we assessed various markers of chemokine response and the presence of inflammatory immune cells after treatment. We found no evidence of significant inflammation by these measures in response to ETC inhibition (Supplementary Figure S1d). To determine the effect on cellular metabolism of ETC inhibition by rotenone, phenformin, and antimycin A, we performed two measures of metabolic pathways. First, we quantified lactate dehydrogenase activity on cells isolated from the epidermis treated with ETC inhibitors for 48 hours (Figure 2a). Next, we employed metabolomics on sorted HFSCs with and without treatment for either 48 hours or 10 days. These analyses indicated an increase in Ldh activity and lactate levels, as well as several other glycolytic intermediates, in response to ETC inhibition by rotenone, phenformin, and antimycin A (Figure 2b). This is consistent with our previous data showing that deletion of Mpc1 in HFSCs blocked pyruvate entry into mitochondria, leading to increased levels of lactate (Flores et al., 2017Flores A. Schell J. Krall A.S. Jelinek D. Miranda M. Grigorian M. et al.Lactate dehydrogenase activity drives hair follicle stem cell activation.Nat Cell Biol. 2017; 19: 1017-1026Crossref PubMed Scopus (143) Google Scholar). As mice age, the hair cycle is known to become protracted, such that upon shaving, only portions of the back skin show regrowth of hair within 1−2 months. We treated various batches of aged mice (at least 17 months) for 30 days with ETC inhibitors to determine whether this metabolic manipulation could stimulate the hair cycle even in dormant follicles. We found that topical application of phenformin, rotenone, or antimycin A all led to more complete hair regrowth across the entire back skin on a time course similar to that of younger mice (Supplementary Figure S2a online). As in younger animals, treatment with these ETC inhibitors led to an increase in lactate pool levels, as measured by metabolomics (Supplementary Figure S2b). The results presented here describe a method to promote lactate production and subsequent hair cycle activation. Building on our previous genetic dissection of metabolism and HFSC activation, this work provides a relatively simply route to promote proliferation of HFSCs. Wild-type male and female 49-day-old C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) for all topical experiments. All animals were maintained in University of California Los Angeles Division of Laboratory Medicine−approved pathogen-free barrier facilities and procedures were performed using protocols that adhere to the standards of the University of California Los Angeles Animal Research Committee, Office of Animal Research Oversight, and the National Institutes of Health. Aged animals were acquired from a National Institute on Aging repository. All drugs were suspended in DMSO and aliquoted at approximately fives times the half maximal inhibitory concentration for the purposes of penetrating the epidermal barrier upon in vivo application. Aliquots were then mixed with the appropriate volume of Premium Lecithin Organogel Base (Transderma Pharmaceuticals Inc., Coquitlam, BC, Canada) and topically applied onto the shaved dorsal skin of 49-day-old telogen-stage mice. Two experiment time points were used: acute treatment (two doses across 48 hours) or long-term studies (one dose three times a week for approximately 1.5 weeks). Full-thickness dorsal skin was collected for histological analysis, cryosectioning, RNA isolation, or epidermal stem cell isolation according to well-established FACS protocol. A set of full-thickness skin samples were obtained from post-mortem tissue harvesting, fixed overnight in 4% paraformaldehyde, and then dehydrated for paraffin embedding and slide generation; hematoxylin and eosin staining was performed according to standard protocol. For immunohistochemistry, formalin-fixed, paraffin-embedded tissue slides were cleared and rehydrated through a series of ethanol washes. Antigen retrieval (20 minutes in pressure cooker, microwaved at 100 power) was performed with 10 mM citrate. Slides were incubated in hydrogen peroxide (30 minutes at 4°C) and then blocked with 10% goat serum/0.1% Tween for 1 hour at room temperature. Primary antibodies were added and incubated overnight. The following antibodies were used: rabbit Sox9 1:800 (Abcam, Cambridge, MA; 185667), rabbit Ki67 1:200 (Abcam 16667), phospho-EGFR 1:500 (Abcam 40815), IL-6 1:500 (Abcam 6672), and CD11b 1:250 (550282; BD Pharmingen, San Jose, CA). Sections were washed the following day with 0.1% phosphate buffered saline with Tween and incubated with rabbit secondary horseradish peroxidase−labeled polymer (Dako, Carpinteria, CA) for 1 hour at room temperature, and then quickly washed with 0.1% phosphate buffered saline with Tween and phosphate buffered saline. AEC chromogen (Dako) was used for the colometric development reaction. Slides were then briefly counterstained with hematoxylin, mounted with Faramount Aqueous Mounting Media (Dako), and sealed for subsequent visualization by light microscopy. Hair cycles were assessed macroscopically by photographic documentation. A scale was established to grade hair cycle stage: 1) telogen (pink, white skin when dorsally shaved); 2) pigmentation (broad blue/gray pigment spots/patches on shaved dorsal area); 3) hair growth (dark pigmentation coupled with small patches of fur); and 4) anagen (dark, full patches of fur within dorsal area) in relation to number of doses and elapsed time of treatment. Hematoxylin and eosin−stained images of all treatment conditions were imaged at ×20 magnification to assess morphological changes in control and treated skin. Hair cycle stages were also evaluated by follicular morphology into respective telogen, telogen-to-anagen transition, or anagen categories. Additionally, the epidermis, dermis, and subcutaneous fat layers were independently measured via ImageJ software (National Institutes of Health, Bethesda, MD), with approximately 25 measurements/skin layer/animal. Scale was set at 1.68 micrometer/pixel for global layer measurements. Protein lysate was obtained from cell pellets and resuspended in RIPA buffer with Halt protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA). Staining solutions for lactate dehydrogenase were prepared with Tris buffer (pH 7.4), XTT, nicotinamide-adenine dinucleotide, phenazine methosulfate, substrate (lactate), and reagent-grade water and held at 37°C until use. Solution was added directly onto samples prepared in triplicate across a 96-well plate. A microplate reader held at 37°C measured 457-nm absorbances every 3 minutes across a 3-minute period to assess enzyme kinetics. The experiments were performed as described in Flores et al., 2017Flores A. Schell J. Krall A.S. Jelinek D. Miranda M. Grigorian M. et al.Lactate dehydrogenase activity drives hair follicle stem cell activation.Nat Cell Biol. 2017; 19: 1017-1026Crossref PubMed Scopus (143) Google Scholar. Cells were washed with cold 150 mM ammonium acetate (pH 7.3), 1 ml cold 80% MeOH was added, and 10 nmol D/L-norvaline was added. After mixing and pelleting centrifugation, the supernatant was moved to glass vials, dessicated under vacuum, and resuspended in 70% acetonitrile. Five micrometers of sample were injected onto a Luna NH2 (150 mm × 2 mm; Phenomenex, Torrance, CA) column. Samples were analyzed by an UltiMate 3000RSLC (Thermo Scientific) coupled to a Q Exactive mass spectrometer (Thermo Scientific). The Q Exactive ran with polarity switching (+3.50 kV/−3.50 kV) in full scan mode with an m/z range of 65−975. Separation was performed using 5 mM NH4AcO (pH 9.9) and acetonitrile. The gradient ran from 15% to 90% over 18 minutes, followed by an isocratic step for 9 minutes and reversal to the initial 15% for 7 minutes. Metabolites were quantified with TraceFinder 3.3 (Thermo Fisher Scientific) using accurate mass measurements (≤3 parts per million) and retention times. Bright-field immunohistochemistry, hematoxylin and eosin−stained, and Nuclear Fast Red (Sigma, St Louis, MO)−stained images were captured using an Olympus BX51 light microscope (Olympus, Tokyo, Japan). Data were analyzed and error bars represent standard error of the mean. An unpaired, two-tailed Student t test determined significance, with P < 0.05 considered statistically significant, denoted by asterisks (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). The authors state no conflict of interest. We would like to acknowledge the efforts of technical staff of the Lowry lab, particularly Jessica Cinkornpumin, and of the Coller Lab, particularly David Jelinek. All metabolomics analyses were performed and initially processed by the UCLA Metabolomics Core Facility run by Daniel Braas. This work was supported by a training grant to MM (NSF Graduate Research Fellowship [2015203740]). In addition, this work was supported by both Innovation and GABA Fund awards from the Broad Center for Regenerative Medicine (UCLA) and the National Institutes of Health (DLJ: R01AG040288). Download .pdf (.21 MB) Help with pdf files Supplementary Figures S1 and S2