Mitochondrial reactive oxygen species promote epidermal differentiation and hair follicle development

Abstract

Proper regulation of keratinocyte differentiation within the epidermis and follicular epithelium is essential for maintenance of epidermal barrier function and hair growth. The signaling intermediates that regulate the morphological and genetic changes associated with epidermal and follicular differentiation remain poorly understood. We tested the hypothesis that reactive oxygen species (ROS) generated by mitochondria are an important regulator of epidermal differentiation by generating mice with a keratinocyte-specific deficiency in mitochondrial transcription factor A (TFAM), which is required for the transcription of mitochondrial genes encoding electron transport chain subunits. Ablation of TFAM in keratinocytes impaired epidermal differentiation and hair follicle growth and resulted in death 2 weeks after birth. TFAM-deficient keratinocytes failed to generate mitochondria-derived ROS, a deficiency that prevented the transmission of Notch and β-catenin signals essential for epidermal differentiation and hair follicle development, respectively. In vitro keratinocyte differentiation was inhibited in the presence of antioxidants, and the decreased differentiation marker abundance in TFAM-deficient keratinocytes was partly rescued by application of exogenous hydrogen peroxide. These findings indicate that mitochondria-generated ROS are critical mediators of cellular differentiation and tissue morphogenesis.

Fig. 1

Deletion of TFAM in the epidermis results in progressive loss of hair follicles and decreased life span. (A) Representative images of control and TFAM cKO mice at P3, P6, and P9 showing lack of hair development in TFAM cKO mice (representative of 12 mice per genotype per day). (B) Western blot analysis of footpad skin lysates from control and TFAM cKO mice demonstrating loss of TFAM and Cox1 (which is encoded by the mitochondrial genome) protein (representative of three independent Western blots). (C) Images of skin sections from control and TFAM cKO mice stained with hematoxylin and eosin (H&E), demonstrating progressive loss of hair follicles in TFAM cKO epidermis (representative of three mice per genotype per day). (D) Immunofluorescence staining of control and TFAM cKO hair follicles for cleaved caspase 3, demonstrating onset of catagen (representative of two mice per genotype). (E) Back skin from control and TFAM cKO mice (P15) stained with toluidine blue, demonstrating an epidermal barrier defect in TFAM cKO mice (representative of three mice per genotype). (F) Kaplan-Meier survival analysis of control and TFAM cKO mice demonstrating reduced survival of TFAM cKO mice (n = 12 mice per genotype).

Fig. 2

TFAM-deficient epidermis displays reduced abundance of differentiation markers and increased proliferation compared with control epidermis. (A) Representative Western blot analysis of epidermal lysates from control and TFAM cKO mice at P9. Graph represents relative normalized keratin 10 or keratin 14 protein amounts ± SEM. n = 7 mice per genotype. *P < 0.05, **P < 0.01. (B) Skin sections from control and TFAM cKO mice at P6 stained for the terminal differentiation markers loricrin, involucrin, or keratin 10 or the basal marker keratin 14 (representative of two mice per genotype). (C) Control and TFAM cKO back skin stained with Oil Red O, demonstrating a loss of sebaceous glands (representative of two mice per genotype). (D) Skin sections from control and TFAM cKO mice at P6, taken after a BrdU pulse, were stained for BrdU incorporation to determine epidermal proliferation rates (representative of three mice per genotype). (E) Quantification of epidermal proliferation rates as assessed by percentage of epidermal basal cells per skin section that stained positive for BrdU incorporation. Graph shows means ± SEM. n = 3 mice per genotype. **P < 0.01.

Fig. 3

Altered cellular metabolism and ROS concentrations in TFAM cKO primary keratinocytes. (A) Oxygen consumption rates of control and TFAM cKO keratinocytes. Graph shows means ± SEM. n = 8 independent keratinocyte pools. **P < 0.01. (B) Cellular superoxide concentrations. Control and TFAM cKO keratinocytes were treated with hydroethidine, and concentrations of its oxidation product 2-hydroxyethidium (2-OH-E+) were measured by high-performance liquid chromatography (HPLC). Graph shows means relative to control ± SEM. n = 5 independent keratinocyte pools. **P < 0.01. (C) Cellular H₂O₂ concentrations measured by DCF-DA fluorescence. Graph shows means relative to control ± SEM. n = 4 independent keratinocyte pools. **P < 0.01. (D and E) Primary keratinocytes from control or TFAM cKO mice were labeled with either [¹³C]glucose (D) or [¹³C]glutamine (E). ¹³C enrichment in cellular citrate pools was analyzed by mass spectrometry. Graph shows means ± SD. n = 3 independent keratinocyte pools. **P < 0.01.

Fig. 4

Primary epidermal keratinocytes from TFAM cKO mice display impaired differentiation in vitro. (A) Western blot analysis of cellular lysates from untreated or CaCl₂-treated control and TFAM cKO mouse keratinocytes (representative of three independent experiments). (B) Western blot analysis of cellular lysates from control and TFAM cKO mouse keratinocytes treated with CaCl₂ and galactose. TFAM cKO keratinocytes were treated with the indicated amounts of galactose oxidase (GAO) (representative of three independent experiments). Graphs represent mean fold protein induction over GAO (0 U/ml) ± SEM. *P < 0.05, **P < 0.01. (C) Western blot analysis of cellular lysates from control keratinocytes left untreated or treated with CaCl₂ in the presence or absence of TPP or MVE (representative of three independent experiments). (D) Western blot analysis of cellular lysates from control keratinocytes left untreated or treated with CaCl₂ in the presence of either EUK134 or dimethyl sulfoxide (DMSO) as vehicle control (representative of three independent experiments. (E) H&E-stained sections taken from 12-day human organotypic keratinocyte raft cultures treated with DMSO or EUK134 from day 0 (representative of two independent experiments). (F) Western blot analysis of differentiation markers in human organotypic keratinocyte raft cultures (representative of two independent experiments).

Fig. 5

Mitochondrial ROS are required for transduction of Notch signals during keratinocyte differentiation. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of epidermal lysates from control and TFAM cKO mice at P9, demonstrating reduced expression of Notch target mRNAs. Graph shows means relative to control mRNA expression ± SEM. n = 4 mice per genotype. *P < 0.05, **P < 0.01. (B) Notch/RBP-J luciferase reporter activity in lysates of control and TFAM cKO keratinocytes treated with CaCl₂ for 0 or 48 hours. Graph shows means relative to control undifferentiated ± SEM. n = 3 independent keratinocyte pools. **P < 0.01. (C) qRT-PCR analysis of keratin 1 or keratin 10 mRNA abundance in control and TFAM cKO keratinocytes cultured at the indicated CaCl₂ concentrations. Graphs show means relative to control undifferentiated ± SEM. n = 3 independent keratinocyte pools. **P<0.01. (D) qRT-PCR analysis of keratin 1 or keratin 10 mRNA abundance in control or TFAM cKO keratinocytes after infection with adenovirus encoding GFP (control) or adenovirus encoding the NICD. Graphs show means relative to control cells with control infection ± SEM. n = 3 independent keratinocyte pools. **P < 0.01.

Fig. 6

Mitochondrial ROS generation is required for β-catenin activation in the epidermis. (A) Skin sections from control and TFAM cKO mice at P6 stained for β-catenin (representative of two mice per genotype). (B) Fold induction of Axin2 mRNA or TCF/LEF luciferase reporter in control or TFAM cKO keratinocytes after treatment with Wnt3a or control treatment. For qRT-PCR experiments, control or Wnt3a-conditioned medium was used for treatment. For luciferase experiments, adenoviral infection with either Wnt3a or GFP (control)–encoding virus was used. Graphs show means relative to control cells with control treatment ± SEM. n = 3 independent keratinocyte pools. *P ≤ 0.05, **P < 0.01. (C) Fold induction of Axin2 mRNA or TCF/LEF luciferase reporter in control or TFAM cKO keratinocytes after treatment with LiCl or NaCl as control. Graphs show means relative to control cells with NaCl treatment ± SEM. n = 3 independent keratinocyte pools. *P < 0.05, **P < 0.01. (D and E) Nonreducing or reducing Western blot analysis of (D) control or TFAM cKO keratinocytes infected with adenovirus encoding either Wnt3a or GFP as control or (E) control keratinocytes treated with NaCl or LiCl. Oxidized and reduced forms of NXN are indicated on nonreducing blots. Graphs represent fold increase in oxidized NXN relative to fold increase in reduced NXN and show the means of three experiments ± SEM. *P < 0.05. (F) Back skin sections from P7 TFAM cKO mice stained with H&E. Mothers of litters received daily injections of either LiCl or NaCl beginning at P0 (representative of four mice per treatment).