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Peroxisome Proliferator–Activated Receptor-γ−Mediated Signaling Regulates Mitochondrial Energy Metabolism in Human Hair Follicle Epithelium

2018; Elsevier BV; Volume: 138; Issue: 7 Linguagem: Inglês

10.1016/j.jid.2018.01.033

1523-1747

Yuval Ramot, Majid Alam, Attila Oláh, Tamás Bı́ró, L. Ponce, Jérémy Chéret, Marta Bertolini, Ralf Paus,

Hair Growth and Disorders

Peroxisome proliferator–activated receptors (PPARs) are ligand-activated transcription factors belonging to the family of nuclear hormone receptors. They include three different isoforms; namely PPAR-α, PPAR-β/δ, and PPAR-γ, and have many important roles in the regulation of a large number of physiological processes, including cell proliferation, differentiation, and inflammatory responses (Sertznig et al., 2008Sertznig P. Seifert M. Tilgen W. Reichrath J. Peroxisome proliferator-activated receptors (PPARs) and the human skin: importance of PPARs in skin physiology and dermatologic diseases.Am J Clin Dermatol. 2008; 9: 15-31Crossref PubMed Scopus (99) Google Scholar). Due to their prominent expression in human skin and its appendages, there is a growing interest in PPARs in human skin biology and pathology (Dozsa et al., 2016Dozsa A. Mihaly J. Dezso B. Csizmadia E. Keresztessy T. Marko L. et al.Decreased peroxisome proliferator-activated receptor gamma level and signalling in sebaceous glands of patients with acne vulgaris.Clin Exp Dermatol. 2016; 41: 547-551Crossref PubMed Scopus (18) Google Scholar, Ramot et al., 2015Ramot Y. Mastrofrancesco A. Camera E. Desreumaux P. Paus R. Picardo M. The role of PPARgamma-mediated signalling in skin biology and pathology: new targets and opportunities for clinical dermatology.Exp Dermatol. 2015; 24: 245-251Crossref PubMed Scopus (57) Google Scholar, Ruzehaji et al., 2016Ruzehaji N. Frantz C. Ponsoye M. Avouac J. Pezet S. Guilbert T. et al.Pan PPAR agonist IVA337 is effective in prevention and treatment of experimental skin fibrosis.Ann Rheum Dis. 2016; 75: 2175-2183Crossref PubMed Scopus (60) Google Scholar, Wallmeyer et al., 2015Wallmeyer L. Lehnen D. Eger N. Sochorova M. Opalka L. Kovacik A. et al.Stimulation of PPARalpha normalizes the skin lipid ratio and improves the skin barrier of normal and filaggrin deficient reconstructed skin.J Dermatol Sci. 2015; 80: 102-110Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, Yin and Smith, 2016Yin K. Smith A.G. Nuclear receptor function in skin health and disease: therapeutic opportunities in the orphan and adopted receptor classes.Cell Mol Life Sci. 2016; 73: 3789-3800Crossref PubMed Scopus (20) Google Scholar). PPAR-γ, the most widely investigated subtype, is expressed in the epidermis and the hair follicles (HFs), and controls skin barrier permeability, inhibits keratinocyte proliferation, and promotes epidermal terminal differentiation (Ramot et al., 2015Ramot Y. Mastrofrancesco A. Camera E. Desreumaux P. Paus R. Picardo M. The role of PPARgamma-mediated signalling in skin biology and pathology: new targets and opportunities for clinical dermatology.Exp Dermatol. 2015; 24: 245-251Crossref PubMed Scopus (57) Google Scholar). We have previously shown that agonistic PPAR-γ modulators may exert protective functions on keratin 15+ epithelial progenitor cells in human HFs, while they inhibit hair growth by inducing catagen and inhibiting the proliferation of hair matrix keratinocytes (Ramot et al., 2014Ramot Y. Mastrofrancesco A. Herczeg-Lisztes E. Biro T. Picardo M. Kloepper J.E. et al.Advanced inhibition of undesired human hair growth by PPARgamma modulation?.J Invest Dermatol. 2014; 134: 1128-1131Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Recently, PPAR-γ−mediated signaling has also been implicated in the regulation of mitochondrial energy metabolism, that is, in adipose tissue and the brain (Chaturvedi and Flint Beal, 2013Chaturvedi R.K. Flint Beal M. Mitochondrial diseases of the brain.Free Radic Biol Med. 2013; 63: 1-29Crossref PubMed Scopus (320) Google Scholar, Hock and Kralli, 2009Hock M.B. Kralli A. Transcriptional control of mitochondrial biogenesis and function.Annu Rev Physiol. 2009; 71: 177-203Crossref PubMed Scopus (465) Google Scholar). However, it remains to be studied whether PPAR-γ stimulation impacts on the mitochondrial biology of human HF keratinocytes in situ, which are known to be key players in HF energy metabolism (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 (51) Google Scholar). Therefore, we have asked whether the selective (agonistic) PPAR-γ modulator, N-acetyl-GED-0507-34-Levo (N-acetyl-GED) (Ramot et al., 2014Ramot Y. Mastrofrancesco A. Herczeg-Lisztes E. Biro T. Picardo M. Kloepper J.E. et al.Advanced inhibition of undesired human hair growth by PPARgamma modulation?.J Invest Dermatol. 2014; 134: 1128-1131Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), can modulate mitochondrial properties, using microdissected, organ-cultured human scalp HFs as a physiologically and clinically relevant assay system (Langan et al., 2015Langan E.A. Philpott M.P. Kloepper J.E. Paus R. Human hair follicle organ culture: theory, application and perspectives.Exp Dermatol. 2015; 24: 903-911Crossref PubMed Scopus (106) Google Scholar). In order to screen whether PPAR-γ stimulation can lead to changes in mitochondria-related genes, we have re-analyzed our previously executed genome-wide microarrays (data are accessible through Gene Expression Omnibus series accession number GSE109009), performed on two independent sets of organ-cultured HFs from a female patient's scalp, treated with 0.01 mM N-acetyl-GED for 6 hours (Ramot et al., 2014Ramot Y. Mastrofrancesco A. Herczeg-Lisztes E. Biro T. Picardo M. Kloepper J.E. et al.Advanced inhibition of undesired human hair growth by PPARgamma modulation?.J Invest Dermatol. 2014; 134: 1128-1131Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Using more permissive selection criteria (P < 0.05, >2-fold, equidirectional changes in both patients), four genes involved in the control of mitochondrial function were found to be up-regulated (Supplementary Table S1 online), in line with the hypothesis of a role for PPAR-γ stimulation in mitochondrial function. To further test the possible role of PPAR-γ in mitochondrial function, human anagen VI HFs were microdissected from normal scalp skin that was obtained after written informed consent from two healthy patients, as described previously (Ramot et al., 2011Ramot Y. Tiede S. Biro T. Abu Bakar M.H. Sugawara K. Philpott M.P. et al.Spermidine promotes human hair growth and is a novel modulator of human epithelial stem cell functions.PLoS One. 2011; 6: e22564Crossref PubMed Scopus (50) Google Scholar), adhering to Helsinki guidelines, and under a license from the ethics committee of the University of Münster (reference no.: 2015-602-f-S). The HFs were organ-cultured for 6 days with vehicle or 0.01−1 mM N-acetyl-GED (concentrations were chosen based on the previously identified optimal dose [Ramot et al., 2014Ramot Y. Mastrofrancesco A. Herczeg-Lisztes E. Biro T. Picardo M. Kloepper J.E. et al.Advanced inhibition of undesired human hair growth by PPARgamma modulation?.J Invest Dermatol. 2014; 134: 1128-1131Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]), with change of culture media every 48 hours. To confirm a possible role for N-acetyl-GED in mitochondrial function, we investigated expression of four genes that are known to be key players in mitochondrial biology, namely MTCO1, PGC1α, TFAM, and SLC25A3 (Ramot et al., 2011Ramot Y. Tiede S. Biro T. Abu Bakar M.H. Sugawara K. Philpott M.P. et al.Spermidine promotes human hair growth and is a novel modulator of human epithelial stem cell functions.PLoS One. 2011; 6: e22564Crossref PubMed Scopus (50) Google Scholar, Vidali et al., 2014Vidali S. Knuever J. Lerchner J. Giesen M. Biro T. Klinger M. et al.Hypothalamic-pituitary-thyroid axis hormones stimulate mitochondrial function and biogenesis in human hair follicles.J Invest Dermatol. 2014; 134: 33-42Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Quantitative RT-PCR analyses revealed that there was a significant and concentration-dependent stimulation of PGC1α transcription, while for the other genes, there was a strong trend toward up-regulation of mRNA levels following the treatment (Figure 1a). To further dissect the stimulating effect of N-acetyl-GED on key mitochondrial elements, we also analyzed their expression at the protein level. N-acetyl-GED significantly increased immunoreactivity for MTCO1, a key enzyme of the respiratory chain (Knuever et al., 2012Knuever J. Poeggeler B. Gaspar E. Klinger M. Hellwig-Burgel T. Hardenbicker C. et al.Thyrotropin-releasing hormone controls mitochondrial biology in human epidermis.J Clin Endocrinol Metab. 2012; 97: 978-986Crossref PubMed Scopus (27) Google Scholar, Vidali et al., 2016Vidali S. Chéret J. Giesen M. Haeger S. Alam M. Watson R.E.B. et al.Thyroid hormones enhance mitochondrial function in human epidermis.J Invest Dermatol. 2016; 136: 2003-2012Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) (Figure 1b). This increase was observed in both the outer root sheath (ORS) and hair matrix keratinocytes. A very similar effect was also evidenced for the protein expression of TFAM, a key transcription factor for mitochondrial DNA synthesis (Knuever et al., 2012Knuever J. Poeggeler B. Gaspar E. Klinger M. Hellwig-Burgel T. Hardenbicker C. et al.Thyrotropin-releasing hormone controls mitochondrial biology in human epidermis.J Clin Endocrinol Metab. 2012; 97: 978-986Crossref PubMed Scopus (27) Google Scholar, Vidali et al., 2016Vidali S. Chéret J. Giesen M. Haeger S. Alam M. Watson R.E.B. et al.Thyroid hormones enhance mitochondrial function in human epidermis.J Invest Dermatol. 2016; 136: 2003-2012Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) (Figure 1c). A slightly different effect was observed when we checked the protein expression of VDAC1, a reliable marker for mitochondrial mass in general (Sorgato and Moran, 1933Sorgato M. Moran O. Channels in mitochondrial membranes: knowns, unknowns, and prospects for the future.Crit Rev Biochem Mol Biol. 1933; 18: 127-171Google Scholar, Vidali et al., 2014Vidali S. Knuever J. Lerchner J. Giesen M. Biro T. Klinger M. et al.Hypothalamic-pituitary-thyroid axis hormones stimulate mitochondrial function and biogenesis in human hair follicles.J Invest Dermatol. 2014; 134: 33-42Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In the lower concentration (0.01 mM), there was a slight decrease in the protein expression. However, as with the other mitochondrial markers, the two higher concentrations led to significant up-regulation of immunoreactivity in both the ORS and matrix keratinocytes (Figure 1d). As complementary evidence, we also tested the effects of N-acetyl-GED on isolated human ORS keratinocytes. Interestingly, we found that 6-hour treatments by using the same, noncytotoxic (non-cytotoxicity determined by MTT and CyQUANT assays, Supplementary Figure S1a−S1d online) concentrations (i.e., 0.01, 0.1, and 1 mM) of N-acetyl-GED were able to significantly up-regulate expressions of all the tested "mitochondrion-relevant" genes (i.e., MTCO1, TFAM, PGC1α, VDAC1, and SLC25A3) in the cells of the investigated donor compared to the vehicle-treated group (Figure 2a). Thus, results of this pilot experiment revealing more prominent actions on "pure" ORS keratinocyte cultures than in intact HFs (Figure 1a) invite the hypothesis that, within the HFs, ORS keratinocytes may be the primary targets of N-acetyl-GED−mediated PPAR-γ activation. Furthermore, to obtain independent, indirect proof of the influence of PPAR-γ stimulation on the mitochondrial actions, we also measured ATP release of ORS keratinocytes upon the above 6-hour N-acetyl-GED -treatments (for details, see the Supplementary Materials and Methods online). This showed that N-acetyl-GED concentration-dependently increased the amount of ATP released by cultured human ORS keratinocytes, highlighting again that it is indeed likely to positively regulate mitochondrial activity in these cells (Figure 2b). N-acetyl-GED has been shown before to induce catagen and decrease hair matrix keratinocyte proliferation (Ramot et al., 2014Ramot Y. Mastrofrancesco A. Herczeg-Lisztes E. Biro T. Picardo M. Kloepper J.E. et al.Advanced inhibition of undesired human hair growth by PPARgamma modulation?.J Invest Dermatol. 2014; 134: 1128-1131Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), while the current study shows that N-acetyl-GED promotes mitochondrial energy metabolism. These findings are in agreement with the concept that the HF mainly engages in aerobic glycolysis and does not rely predominantly on mitochondria-dependent glucose metabolism (Philpott and Kealey, 1990Philpott M.P. Kealey T. Metabolic studies on isolated hair follicles: Hair follicles engaged in aerobic glycolysis and do not demonstrate the glucose fatty acid cycle.J Invest Dermatol. 1990; 96: 875-879Crossref Scopus (26) Google Scholar, Williams et al., 1993Williams R. Philpott M.P. Kealey T. Metabolism of freshly isolated human hair follicles capable of hair elongation: a glutaminolytic, aerobic glycolytic tissue.J Invest Dermatol. 1993; 100: 834-840Abstract Full Text PDF PubMed Scopus (46) Google Scholar). These data also raise the question whether the catagen-promoting impact of N-acetyl-GED may be dependent on the promotion of catagen-/terminal differentiation-associated processes in the hair bulb, which are more reliant on oxidative phosphorylation. These preliminary results suggest that, similar to its effects in other tissues, PPAR-γ−mediated signaling is a player in regulating the energy metabolism of human scalp HFs by enhancing mitochondrial function, most probably primarily in the ORS keratinocytes. Next, it deserves to be evaluated whether this modulation of mitochondrial biology read-out parameters by PPAR-γ modulators in human skin shown here can be translated into clinically beneficial effects (e.g., anti-HF aging). Attila Oláh: http://orcid.org/0000-0003-4122-5639 Ralf Paus has received basic research grant from PPM Services S.A., Morbio Inferiore, Switzerland. Yuval Ramot has received travel support from PPM Services S.A. Majid Alam, Leslie Ponce, Jérémy Chéret, and Marta Bertolini were or are employee of Monasterium Laboratory, Münster, Germany. The authors are grateful for the excellent technical assistance of Janine Jakobs and Erika Hollósi. This study was supported in part by a basic research grant from PPM Services S.A., Morbio Inferiore, Switzlerland; Monasterium Laboratory, Skin and Hair Research Solutions GmbH, Münster, Germany; a Hungarian research grant (NRDIO 121360), and an National Institute for Health Research Biomedical Research Centre grant (Inflammatory Hair Diseases Programme, Lead: RP). Download .pdf (.24 MB) Help with pdf files Supplementary Data