Thyroid hormone signaling controls hair follicle stem cell function

Abstract

Observations in thyroid patients and experimental animals show that the skin is an important target for the thyroid hormones. We previously showed that deletion in mice of the thyroid hormone nuclear receptors TRα1 and TRβ (the main thyroid hormone-binding isoforms) results in impaired epidermal proliferation, hair growth, and wound healing. Stem cells located at the bulges of the hair follicles are responsible for hair cycling and contribute to the regeneration of the new epidermis after wounding. Therefore a reduction in the number or function of the bulge stem cells could be responsible for this phenotype. Bulge cells show increased levels of epigenetic repressive marks, can retain bromodeoxyuridine labeling for a long time, and have colony-forming efficiency (CFE) in vitro. Here we demonstrate that mice lacking TRs do not have a decrease of the bulge stem cell population. Instead, they show an increase of label-retaining cells (LRCs) in the bulges and enhanced CFE in vitro. Reduced activation of stem cells leading to their accumulation in the bulges is indicated by a strongly reduced response to mobilization by 12-O-tetradecanolyphorbol-13-acetate. Altered function of the bulge stem cells is associated with aberrant activation of Smad signaling, leading to reduced nuclear accumulation of β-catenin, which is crucial for stem cell proliferation and mobilization. LRCs of TR-deficient mice also show increased levels of epigenetic repressive marks. We conclude that thyroid hormone signaling is an important determinant of the mobilization of stem cells out of their niche in the hair bulge. These findings correlate with skin defects observed in mice and alterations found in human thyroid disorders.

© 2015 Contreras-Jurado et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Figures

FIGURE 1:

TR inactivation does not lead to SC depletion in hair bulges. (A) Double immunofluorescence of SC markers keratin 15 (K15), CD34, and Sox9 in the bulges of dorsal skin of WT and TR KO mice. Nuclei were counterstained with DAPI (blue), and merge images are shown. (B) Confocal images of α6 integrin, CD34, and K15 expression in whole mounts of tail skin in both groups. (C) Transcript levels of the indicated SC markers determined in telogen epidermis of WT and TR KO animals. Results are expressed relative to the values obtained in WT mice. (D) Flow cytometry analysis of surface levels of CD34 and α6 integrin in WT and KO mice. Subpopulations of CD34+ cells expressing high and low levels of α6 integrin were calculated. Data are means ± SD. *p < 0.05, **p < 0.01.

FIGURE 2:

LRC quantification in whole mounts of tail epidermis. (A) WT and TR KO neonatal mice (six mice/group) were injected with BrdU, and LRCs were identified by immunofluorescence after 75 d. (B) BrdU labeling intensity in the bulges of untreated WT and TR-deficient mice was quantified, and the number of LRCs with high, medium, and low label intensity is shown. High intensity represents nuclei in which the labeled area is >55 μm2; medium intensity, >42 but <55 μm2; and low intensity, >28 but <42 μm2. **p < 0.01.

FIGURE 3:

Enhanced colony formation efficiency in TR-deficient mice (A) Representative images of CFE of keratinocytes isolated from adult WT and TR KO mice plated at two different concentrations (5 × 104 and 25 × 104 cells/well). (B) Quantification of the size and number of colonies obtained in both genotypes. Data are means ± SD. **p < 0.01.

FIGURE 4:

LRCs in the bulges of TR KO mice are enriched in trimethylated histone 3 at lysines 9 and 27. (A) Neonatal mice were injected with BrdU, and after the chase period, whole mounts of tail epidermis from WT and TR KO mice were used for double immunofluorescence with H3K9me3 and BrdU antibodies. (B) Quantification of the label intensity of H3K9me3 in the bulges, hair germs, and LRCs (BrdU-positive cells in the bulges). (C) Representative images obtained as in A with BrdU and H3K27me3 antibodies. (D) Quantification of H3K27me3 in the bulges, hair germs, and LRCs. (E) Double immunofluorescence images of BrdU and acetyl histone 4 (AcH4). (F) Quantification of AcH4 in the bulges, hair germs, and LRCs of WT and TR KO mice. Data are means ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 5:

TR deletion blocks LRC mobilization. (A) Neonatal mice were labeled with BrdU, and 68 d later, tails were topically treated with TPA as described in Materials and Methods. Representative LRC images of BrdU immunofluorescence in whole mounts of tail epidermis from vehicle-treated (control) and TPA-treated WT and TR KO mice. (B) Number of LRCs in which the label occupied an area >28 μm2 was scored in control and TPA-treated mice. ANOVA followed by Bonferroni test was used to analyze differences among groups. (C) Percentage of LRCs mobilized upon TPA treatment in both genotypes. Data are means ± SD. **p < 0.01 ***p < 0.001.

FIGURE 6:

Smad signaling is aberrantly activated in the follicles of TR KO mice. (A) Double immunofluorescence images of K14 and pSmad1,5,8 in the skin of control and TPA-treated WT and TR KO mice, showing increased Smad phosphorylation in the hair follicles of TR-deficient mice. (B) Double immunofluorescence of nuclear β-catenin and K5 in the same groups. Hair follicles are marked by a white line, and brackets indicate the location of the bulges (Bu) and the sebaceous gland (Sg). Bars, 150 μm.

FIGURE 7:

Reduced expression of β-catenin targets in TR-deficient mice. (A) Transcript levels of the β-catenin target genes Cyclin D1 and c-Myc determined in epidermis of control and TPA-treated WT and TR KO animals. Results are expressed relative to the Gus mRNA levels. Data are means ± SD. **p < 0.01, ***p < 0.001. (B) Double immunofluorescence of K5 and activated c-Myc in the skin of control and TPA-treated WT and TR KO mice.

FIGURE 8:

Reduced Smad signaling in the hair follicles of hyperthyroid mice. (A) Representative double immunofluorescence images of K14 and pSmad1,5,8 in the follicles of control mice and mice treated for 30 d with thyroid hormones (hyperthyroid). Arrows indicate the presence of cells positive for the phosphorylated Smad. (B) K5 and nuclear β-catenin detected by double immunofluorescence in control and hyperthyroid hair follicles. β-Catenin–positive cells are shown with arrows. Bu, bulge; SG, sebaceous gland. Bars,150 μm.