Lactate dehydrogenase activity drives hair follicle stem cell activation

Abstract

Although normally dormant, hair follicle stem cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier 1 (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allows them to remain dormant and yet quickly respond to appropriate proliferative stimuli.

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Figure 1: Lactate dehydrogenase activity is enriched in HFSCs.
Figure 2: Ldh activity increases during HFSC activation.
Figure 3: Deletion of Ldha blocks HFSC activation.
Figure 4: Deletion of Mpc1 increases lactate production and accelerates the activation of HFSCs.
Figure 5: Pharmacological inhibition of Mpc1 promotes HFSC activation.
Figure 6: Stimulation of Myc levels promotes HFSC activation.

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Acknowledgements

We would like to acknowledge the significant technical support of M. Neebe, J. Cinkornpumin and A. Liu on this project. We are also particularly grateful to members of the Banerjee laboratory for guidance and development of the Ldh activity assay. A.F. and A.C.W. were supported by a fellowship from the Eli and Edythe Broad Center for Regenerative Medicine at UCLA. A.C.W. and M.G. were supported by a fellowship from the Tumor Cell Biology programme at UCLA (NIH). A.C.W. was also supported by a training grant from CIRM. D.J. was supported by awards from a New Idea Award from the Leukemia Lymphoma Society, the Jonsson Comprehensive Cancer Center, the UCLA Clinical Translational Science Institute UL1TR000124, the Prostate Cancer SPORE at UCLA P50 CA092131, and the Eli & Edythe Broad Center for Regenerative Medicine & Stem Cell Research. N.A.G. is a postdoctoral trainee supported by the UCLA Scholars in Oncologic Molecular Imagining program (NCI/NIH grant R25T CA098010). A.S.K. was supported by a UCLA Dissertation Year Fellowship. H.A.C. was supported by National Institute of General Medical Sciences R01-GM081686 and R01-GM0866465. J.R. was supported by NIH (RO1GM094232). H.R.C. was supported by a Research Scholar Grant, RSG-16-111-01-MPC, from the American Cancer Society and the Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA and Rose Hills Foundation Research Award. W.E.L. was supported by NIH-NIAMS (5R01AR57409), an Impact award from CTSI and the Jonsson Comprehensive Cancer Foundation, and The Gaba Fund through the Eli & Edythe Broad Center of Regenerative Medicine at UCLA.

Author information

A.F., J.S., A.S.K., D.J., M.M., M.G. and D.B. performed experiments. A.F., A.S.K., J.L.Z., N.A.G. performed analysis and compiled data. P.S., D.E. and J.R. provided key reagents essential to the work. T.G. and H.A.C. provided important insight and advice. H.R.C. and W.E.L. provided oversight and were financially responsible for the work. A.F., H.R.C. and W.E.L. were responsible for assembling the figures and writing the manuscript.

Correspondence to Heather R. Christofk or William E. Lowry.

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Competing interests

The use of RCGD423 to promote hair growth is covered by a provisional patent application filed by UC Regents and this technology has been licensed by Carthronix LLC. W.E.L. is a member of the board of advisers and a shareholder of Carthronix LLC. None of the work in this study was supported by Carthronix. The use of UK-5099 to promote hair growth is covered by a separate provisional patent filed by UC Regents with W.E.L. and H.R.C. as inventors.

Integrated supplementary information

Supplementary Figure 1 Validation of key reagents and assays.

a, top, IHC with antibody recognizing specifically Ldha (same as used in Fig. 1a). bottom, IHC with antibody recognizing multiple isoforms of Ldh protein. Scale bars indicate 20 micrometers. b, the sorting strategy employed to isolate two populations of cells from the bulge. This particular sort was used to isolate the protein samples shown by western blot in Fig. 1b. c, Validation of colorimetric Ldh enzyme activity assay. The highest Ldh enzyme activity was observed in HFSC bulge and in the muscle. Activity indicated by purple stain; pink color is nuclear fast red counterstain. In absence of substrate lactate there was no detectable activity (purple stain). Right, Additional validation of colorimetric Ldh enzyme activity assay. Enzyme activity inhibited by treating skin with HCl before addition of staining solution with substrate lactate. No Ldh activity (purple stain) detected. Skin in which enzyme activity is not inhibited by Hydrochloric Acid (HCl) shows highest Ldh enzyme activity in HFSC bulge and in the muscle. Scale bars indicate 50 micrometers.

Supplementary Figure 2 Validation of hair cycle stage.

a, Analysis of RNA-seq data to validate that HFSCs in telogen-anagen transition were in fact in such a transition. The telogen-anagen transition is known to be driven by Shh (Gli factors are targets) and Wnt (Lef1, Axin, Ccnd1 are targets) signaling, and correlate with increased proliferation (Ki-67 and Pcna). In addition, Sox4 was previously identified as a regulator of the telogen-anagen transition. n = 3 mice per timepoint. Shown as mean ± s.e.m. Paired t-test was performed, P < 0.05. b, staining for Ki-67 marks dividing cells during various stages of the hair cycle. Brackets indicate the HFSC niche. Scale bars indicate 100 micrometers.

Supplementary Figure 3 Long term deletion of Ldha in HFSCs.

a, K15-CrePR;Ldhafl/fl animals treated with Mifepristone during telogen (day 50) were allowed to develop for 6 months. None of the K15-CrePR;Ldhafl/fl mice showed complete hair regrowth, compared to control animals that all grew their hair coats back completely. Images are representative of at least 12 animals per genotype. b, Histological examination of the long term K15-CrePR;Ldhafl/fl mice showed that Ldha-null HFSCs remained in telogen while WT HFSCs went through anagen and then returned to telogen. This is apparent from thick sections (50 micron, right) that show an increased number of club hairs in the WT relative to Ldha-null follicles. Scale bars indicate 100 micrometers (left), and 20 micrometers (middle and right). c, IHC for HFSC marker Sox9 showed that deletion of Ldha from HFSCs does not affect their presence in the bulge even after 6 months. In addition, IHC and Ldh activity assay demonstrate that the deletion of Ldha was sustained. Because of the mosaicism of the deletion, in some portions of K15-CrePR;Ldhafl/fl skin Ldha was not deleted. Shown on the bottom row is tissue from hair bearing skin in the K15-CrePR;Ldhafl/fl mice where Ldha was still expressed, showing that new hair growth in K15-CrePR;Ldhafl/fl mice was due to lack of deletion of Ldha caused by the mosaic approach used to mediate Cre recombination. Scale bars indicate 20 micrometers. d, To determine how various signaling pathways previously linked to the hair cycle are affected by loss of Ldha in HFSCs, we performed IHC for markers that indicate activity of these pathways in telogen and telogen-anagen transition. Note that pStat5 appears to be suppressed in normal telogen-anagen transition, and this does not seem to occur in Ldha-null HFSCs. pStat1 and pStat3 did not seem to be affected by loss of Ldha. Expression of Gli3, a target of Shh signaling, is typically induced in an activated hair germ derived from HFSCs, but Ldha-null HFSCs do not make an active hair germ. Activation of the Wnt pathway is indicated by nuclear localization of β-catenin, and very little nuclear β-catenin was detected in Ldha-null HFSCs. Scale bars indicate 6 micrometers.

Supplementary Figure 4 Long term deletion of Mpc1 in HFSCs.

a, Six months after initiation of deletion of Mpc1 in HFSCs (K15-CrePR;Mpc1fl/fl), mice lacking Mpc1 show no deleterious effects as measured by the hair cycle (left), pathology (middle, H and E), or staining for HFSCs (right, Sox9). Scale bars indicate 100 micrometers in middle panel, and 50 micrometers in right panel. Images are representative of at least 12 animals per genotype. b, To demonstrate that the deletion of Mpc1 promotes proliferation specifically in HFSCs, we used K15-CrePR;Ldhafl/fl mice bearing a lox-stop-lox-Tomato allele to look at K15 + HFSCs and proliferation with and without Mpc1 deletion (left). In addition, we took advantage of the ires-GFP within the Lgr5-CreER allele to stain for Ki-67 and GFP and look for co-localization with and without Mpc1 deletion (right). White brackets denote bulge area. Scale bars represent 20 micrometers. c, Deletion of Mpc1 in mice bearing the Lgr6-CreER allele shows no premature induction of the hair cycle. d, Ldh activity assay on sorted HFSCs from either control or Lgr6-CreER mediated Mpc1 deletion mice showed increased activity in cells lacking Mpc1. n = 6 mice per genotype pooled from 2 independent experiments. Shown as mean ± s.e.m. Paired t-test was performed, P < 0.05.

Supplementary Figure 5 Stimulation of Jak-Stat signaling and the hair cycle.

RCGD423 was applied topically to shaved mice at day 50. 48 hours after treatment, the skin was harvested and prepared for IHC. IHC with the indicated antibodies demonstrates relative activity of Stat signaling in vehicle versus RCGD423 treated skin. Scale bars indicate 20 micrometers.

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Flores, A., Schell, J., Krall, A. et al. Lactate dehydrogenase activity drives hair follicle stem cell activation. Nat Cell Biol 19, 1017–1026 (2017). https://doi.org/10.1038/ncb3575

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