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Keywords:

  • Hair;
  • Hair follicles;
  • Stem cells;
  • TACE/ADAM17;
  • SOX9;
  • Alopecia

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Hair follicles (HFs) are equipped with stem cell niches that allow regeneration. Tumor necrosis factor-α converting enzyme (TACE), also known as A disintegrin and metalloproteinase 17, is a proteolytic enzyme that regulates a variety of cell surface molecules including TNF-α, via ectodomain shedding. We found TACE expression on mouse HFs and conditionally depleted it in cells that expressed sex-determining region Y-related high-mobility-group box 9 (SOX9) transcription factor, an HF stem cell transcription factor (Taceflox/flox-Sox9-Cre, hereafter, “Tace/Sox9”). Tace/Sox9 mice were born with brittle hair with prolonged anagen phase. They underwent diffuse, progressive, and ultimately whole-body hair loss by 20 weeks old. Tace/Sox9 HFs lacked CD34+ bulge cells as demonstrated via immunofluorescence microscopy and flow cytometry. Real-time PCR revealed downregulation of transcription factors Sox9, Lhx2, and Gata3 and upregulation of Lef1. In vitro colony-forming capacity was abolished in Tace/Sox9 keratinocytes, and HFs exhibited increased proliferation in situ, collectively demonstrating that Tace/Sox9 mice failed to establish the bulge niche and to maintain “stemness” of HF stem cells. Epidermal growth factor receptor (EGFR) signaling was impaired in Tace/Sox9 keratinocytes, and mice depleted of Egfr in SOX9-expressing tissues exhibited hair phenotype nearly identical to Tace/Sox9 mice, demonstrating EGFR signaling as a pathway downstream of TACE in HF homeostasis. This study provides mechanistic implication for human TACE-deficiency and for hair abnormality caused by EGFR inhibitors. STEM CELLS2012;30:1781–1785


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Hair follicles (HFs) are equipped with independent stem cell niches that allow hair regeneration [1, 2]. HF stem cells reside in the bulge area [3] and are characterized by the expressions of CD34, integrin α6 [4], and keratin 15 [5]. HF stem cells not only give rise to HFs but also give rise to sebaceous glands and interfollicular epidermis during wound healing [6].

Cells that express sex-determining region Y-related high-mobility-group box 9 (SOX9) transcription factor exist in the whisker pad and pelage hair epithelial placodes during embryonic development [7]. Conditional depletion of Sox9 led to severe proliferative hair defects attributed to the failure to establish the HF niche [7]. Nowak et al. [8] showed that the absence of early SOX9+ HF stem cells blocked morphogenesis of HFs and sebaceous glands, indicating that SOX9+ HF stem cells gave rise to all skin epithelial lineages.

Tumor necrosis factor-α converting enzyme (TACE) or A disintegrin and metalloproteinase 17 (ADAM17) is a proteolytic enzyme [9] that was originally found to release membrane-bound pro-TNFα [10, 11]. TACE is now known to have pleiotropic functions through its ability to cleave a variety of substrates including epidermal growth factor receptor (EGFR) ligands, membrane-bound cytokines and its receptors, and others [12]. TACE deficiency is embryonic lethal [13], and conditionally depleting TACE on monocytes/macrophages protects against septic shock [14].

Our interest in TACE, as well as in HFs, prompted us to generate mice in which TACE was conditionally depleted in HFs. We herein characterize mice that were conditionally depleted of TACE in SOX9-expressing cells [15], which developed progressive alopecia with total body hair loss.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Tace/Sox9 Mice Develop Total Body Hair Loss

Immunofluorescence staining in 8-week-old wild-type C57BL/6 mouse skin revealed TACE expression in the basal layer of the epidermis and in HFs, with accentuation in the bulge area (Fig. 1A). To investigate the role of TACE in HFs, we sought to deplete it in epidermis. Fate mapping of SOX9-expressing lineages by crossing Sox9-Cre mice to CAG-CAT-Z reporter mice [16] revealed diffuse β-galactosidase staining in HFs and interfollicular epidermis (Fig. 1B), confirming that all epithelial components in the epidermis were derived from SOX9-expressing cells [8]. We crossed Sox9-Cre mice with Taceflox/flox mice [15] and obtained mice in which TACE was absent in SOX9-expressing lineages (Tace/Sox9 mice). Tace/Sox9 mice had short, brittle hair throughout their first anagen (Fig. 2A, right upper panel), mimicking a recently reported human disease with ADAM17 mutation [17]. A normal hair cycle was not evident thereafter, and mice began to lose hair around 6 weeks of age. Shaving trunk fur of 6-week-old mice in hair-bearing skin revealed that the majority of HFs in Tace/Sox9 mice were in prolonged anagen phase (Fig. 2B). Tace/Sox9 mice lost virtually all body hair by 20 weeks old (Fig. 2A, right lower panel).

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Figure 1. TACE expression in hair follicles. (A): Frozen skin sections from WT mice stained for TACE and CD34. Nuclei were visualized with Hoechst. Arrowheads depict TACE staining within the bulge area. (B): β-Galactosidase staining in skin sections from SOX9-fate mapped mice with LacZ as reporter gene. Scale bars = 50 and 200 μm (A, B). Abbreviations: SG, sebaceous gland; Sox9, sex-determining region Y-related high-mobility-group box 9; TACE, tumor necrosis factor-α converting enzyme; WT, wild type.

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Figure 2. Progressive hair loss in Tace/Sox9 mice. (A): Hair phenotype of Tace/Sox9 mice at indicated time points. (B): Areas of hair follicles in anagen were visualized by shaving back skin of 6-week-old WT and Tace/Sox9 mice. (C): H&E skin section from WT and Tace/Sox9 mice. Scale bars = 200 μm in P8 and P21 and 100 μm in P90 sections. (D, E): Skin from 3-week-old WT or Tace/Sox9 mice were engrafted onto nude mice. (F): H&E sections of WT and Tace/Sox9 skin grafts from (D). Asterisks depict sebaceous glands. Scale bar = 100 μm. Abbreviations: POD, postoperative days; Sox9, sex-determining region Y-related high-mobility-group box 9; Tace, tumor necrosis factor-α converting enzyme; WT, wild type.

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Histological analysis of skin sections from Tace/Sox9 mice revealed misaligned HFs with enlarged bulbs at P8. At around P21, Tace/Sox9 mice exhibited sparse distribution of HFs, which finally resulted in aberrant HFs that morphologically lacked normal hair shaft, inner root sheath, and outer root sheath (Fig. 2C). In contrast, sebaceous hyperplasia was evident (Fig. 2C, lower panels).

To ensure that hair loss did not attribute to skin inflammation that Tace/Sox9 mice start to develop 6 weeks after birth (data not shown), we engrafted skin from 3-week-old mice onto nude mice. Tace/Sox9 skin grafts appeared grossly uninflamed. Histological analyses revealed no abnormal leukocyte infiltration, including the perifollicular area. Skin grafts nevertheless failed to outgrow hair, denying inflammation as a major cause for hair loss in Tace/Sox9 mice. In contrast to HF morphogenesis, sebaceous gland formation was supported, indicating that sebocyte differentiation does not critically require TACE.

Failure of HF Bulge Niche Establishment in Tace/Sox9 Mice

Histology suggested that Tace/Sox9 HFs lacked the bulge (Fig. 3A, upper panels). To confirm this, we stained frozen skin sections for CD34, a well-established bulge marker [4]. Indeed, HFs in Tace/Sox9 mice lacked CD34-expressing HF structures (Fig. 3A, lower panels). Flow cytometry also revealed that Tace/Sox9 epidermis contained negligible amounts of CD34+ integrin αmath image bulge cells [18] at all confirmed ages, even before the onset of alopecia (Fig. 3B). Colony-forming assay [19] revealed that colony-forming capacity of Tace/Sox9 keratinocytes was markedly impaired (Fig. 3C, 3D). Tace/Sox9 HFs exhibited enhanced proliferation (Ki-67 staining, Fig. 3E), but precocious apoptosis was not evident via caspase-3 staining (data not shown).

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Figure 3. Tace/Sox9 mice fail to establish the bulge niche. (A): H&E sections (top) and frozen sections stained for CD34 (bottom) in WT or Tace/Sox9 mouse skin. Scale bar = 25 μm. (B): Flow cytometry of keratinocytes stained for CD34 and integrin α6. (C, D): Colony-forming assay using epidermal cells from 3-week-old mice. Scale bar = 500 μm. (E): Ki-67 staining in frozen skin sections. Scale bar = 100 μm. (F): mRNA expression of transcription factors analyzed via real-time PCR. *, p < .05; **, p < .005; ***, p < .0001. Abbreviations: Sox9, sex-determining region Y-related high-mobility-group box 9; Tace, tumor necrosis factor-α converting enzyme; WT, wild type.

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To attempt to mechanistically characterize this phenotype, real-time PCR was performed for transcription factors with established importance during HF morphogenesis. Sox9 and Lhx2, former required for HF stem cell maintenance [7, 8] and latter to maintain stem cell characters [20], were significantly downregulated, providing potential explanation for the failure to establish the bulge niche and for enhanced proliferation of HFs. Gata3, which is highly expressed in outer root sheath and inner root sheath during anagen [21], was decreased, and Lef1, whose expression in HF stem cells enhances differentiation to IRS [22], was increased. In aggregate, these data demonstrate that CD34+ bulge niche failed to establish in Tace/Sox9 mouse HFs, and that loss of TACE impacted expression of transcription factors that are required to maintain stem cell characters. TACE is also likely involved in subsequent HF morphogenesis, but determining this would require studies using mice with inducible Cre-alleles.

EGFR ligands are major TACE substrates [12]. Skin grafts derived from EGFR-null mice exhibit aberrant hair differentiation [23] and EGFR inhibitors in humans cause persistent hair growth [24]. Because of these similarities to Tace/Sox9 mice, we evaluated EGFR signaling. Silencing Adam17 via siRNA in keratinocytes in vitro resulted in decreased levels of phosphorylated EGFR (p-EGFR) and (p-ERK) (Fig. 4A). Analysis of epidermal keratinocytes isolated from Tace/Sox9 mice also revealed decreased p-EGFR but not p-ERK (Fig. 4B), suggesting that the ERK pathway was compensated in vivo, and that Tace/Sox9 phenotype was independent of ERK. To determine in vivo effects of EGFR depletion, we generated Egfr/Sox9 mice by crossing Sox9-Cre mice to Egfrflox/flox mice [25]. Egfr/Sox9 mice exhibited hair phenotype nearly identical to that of Tace/Sox9 mice (Fig. 4C, 4D), suggesting EGFR, among other possible mechanisms, as an important signaling pathway downstream of TACE in maintaining HF homeostasis.

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Figure 4. TACE functions through EGFR signaling in hair follicle homeostasis. (A): Cultured keratinocytes (Pam212) were treated with either mock or Adam17 siRNA and evaluated for p-EGFR and p-ERK expression via Western blot analysis. (B): Keratinocytes isolated ex vivo from WT and Tace/Sox9 mice were analyzed for p-EGFR and ERK via Western blot. (C): Gross hair phenotype of Egfr/Sox9 mice. (D): H&E staining of skin sections from mice in (C). Scale bar = 100 μm. Abbreviations: p-ERK, phosphorylated extracellular signal-related kinase; p-EGFR, phosphorylated epidermal growth factor receptor; Sox9, sex-determining region Y-related high-mobility-group box 9; Tace, tumor necrosis factor-α converting enzyme; WT, wild type.

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CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Conditional ablation of TACE in SOX9-expressing cells resulted in failure to establish the bulge niche and in enhanced HF proliferation, leading to progressive and complete hair loss. Aberrant expression of transcription factors and impaired EGFR signaling downstream of TACE contributed to this phenotype, but due to the wide spectrum of TACE substrates, partial contribution of other pathways cannot be denied at this point. Nevertheless, these findings not only reveal a novel element in HF biology but also reveal a new biological aspect of TACE in stem cell maintenance. These findings also provide mechanistic implications in human TACE deficiency as well as skin/hair conditions that occur during EGFR inhibitors usage.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

We thank Showbu Sato for technical assistance. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Research for Prevention and Treatment of Immune/Allergic Diseases from the Ministry of Health, Labor and Welfare of Japan, Keio Gijuku Academic Development Funds, and the Keio University Kanrinmaru Project.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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SC_11-0857_sm_suppldata.pdf104KMaterials and Methods

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