Fig 1.
Foxp1 is translocated from the nucleus to cytoplasm of HFSCs at phases from anagen to catagen accompanying with rise of ROS levels.
A: Foxp1 distribution among distinct populations of hair follicles. Foxp1 was localized within nuclei from anagen I to III, exported to cytoplasm from catagen I to catagen V, and progressively relocalized into the nuclei at catagen VI and telogen. Lower panel was the high power view of upper panel. Scale bars: 50 μm. Blue, DAPI; red, anti-Foxp1. Abbreviations: Ep, epidermis; IFE, interfollicular epidermis; Bu, bulge; HG, hair germ; DP, dermal papillae; ORS, outer root sheath; IRS, inner root sheath; HF: hair shaft. B: Representative dot plot of FACS for HFSCs identified by CD34+/Integrin α6+ at early telogen (P49). Abbreviations: HFSCs, hair follicle stem cells. C: Histograms of DCFDA fluorescence intensities of HFSCs at telogen (P20), early anagen (P24), late anagen (P27) and catagen (P40) (n = 6, 6, 7, and 6, respectively). D: Quantification of (B) indicates a progressive increase in ROS levels in HFSCs from telogen to catagen. *, p<0.05; **, p<0.01.
Fig 2.
The dynamic nucleocytoplasmic localization of Foxp1 in response to oxidative stress.
A: Identification by mass spectrum analysis of phosphorylation at Foxp1 S468 sensitive to 2-hour H2O2 stimulation in CHO cells. B: Alignment of Foxp1 N-terminal residues across multiple species revealed a conserved nuclear localization motif of RXXRS (boxed). C: Linear schematics of Foxp1 depicting mutations (in blue) of the RXXRS motif and S468. Zinc-finger (ZF), leucine-zipper (LZ) and forkhead domains (FHD) are indicated by boxes. In Foxp1N, RDTR is mutated to HDTG, leading to loss of function of NLS; in Foxp1(S468A), A is substituted for S, leading to loss of phosphorylation at S468. D: Representative images showing defective nuclear localization of the Foxp1NLSm-EGFP fusion protein (green) following transient transfection into HaCat cells. E: Representative images showing defective nuclear export of the Foxp1(S468A)-EGFP fusion protein in transfected HaCat cells following one-hour stimulation with 500 μM H2O2. Green, EGFP fluorescence; DAPI, blue; scale bar: 10 μm. F: 293T cells were transfected with Foxp1 or Foxp1(S468A) expression constructs. Western blot was conducted to evaluate the relative level of Foxp1 protein in cytoplasm or nucleus following one-hour stimulation with 500 μM H2O2. G: Quantification of the relative Foxp1 levels by gray scale in (F, n = 3).
Fig 3.
Foxp1 deficiency augments the proportion of S-phase HFSC at anagen phase.
A: IHC with anti-CD34 (red) and anti-BrdU (green) staining of hair follicles following 4-day BrdU pulse-chase in the Foxp1fl/fl (WT) and K14-Cre; Foxp1fl/fl (cKO) mice (P20-P23). The upper panel showed the timing of BrdU injection and sectioning. Abbreviations: Bu, bulge; HG, hair germ; DP, dermal papillae. Scale bars: 25 μm. B: Quantification of the number of BrdU+ cells in the bulges of (A). The cKO hair follicles at P24 displayed extensive BrdU+ cells in the hair germ and bulge cells, whereas the WT controls had few BrdU+ cells in the identical regions (n = 3,4). *, p<0.05. C: IHC for hair follicles at P55 following 28-day BrdU pulse-chase. The upper panel showed the timing of BrdU injection and sectioning. Scale bars: 75 μm. D: Quantification of the percentages of LRC in the bulges of (C). Few label-retaining cells (LRC) were detected in the bulges of Foxp1 cKO mice. n = 4; *, p<0.05. E: NAC treatment and BrdU injection once a day from P23 to P26 enhanced cell proliferation of HFSCs in WT early anagen. Scale bar, 50μm. F: Quantification of the frequency of BrdU+ cells in HFSCs in (E). *, p<0.05. G-H: Western blotting demonstrated a decrease of Foxp1 (G) and p19ARF (H) protein levels within cKO hair follicles at anagen (P23). I: Down-regulation of p19ARF transcripts within cKO anagen (P23) hair follicles relative to the WT as determined by qRT-PCR. J: S15 phosphorylated-p53 protein level was relatively decreased within cKO anagen (P23) hair follicles.
Fig 4.
Deficiency of Foxp1 in hair follicles impairs ROS accrual at anagen and catagen.
A: Representative histograms of DCFDA mean fluorescence intensities (MFI) in HFSCs from Foxp1fl/fl (WT) and K14-Cre; Foxp1fl/fl (cKO) mice at telogen (P20), early anagen (P24), late anagen (P37) and catagen stages (P40) (n, 6–8). B: Quantification of the MFIs of (A) reveal decreased ROS levels in anagen and catagen in cKO mice compared to controls. NS, no significance; *, p<0.05. C: Flowchart depicting NAC treatment from P23 to P27 and sectioning for dorsal P45 HFs in mice. D: NAC treatment decreased ROS levels in HFSCs from both the WT and Foxp1 cKO mutant mice as determined by DCFDA staining/flow cytometry. E: Comparative dorsal views of the back hair follicles at P45 of cKO and WT mice as in (C). F: HE histological analysis for hair follicles at P45 as depicted in the flowchart of (C, n = 3–4).
Fig 5.
Foxp1 tunes ROS level through protein interaction with Trx-1.
A: Anti-Trx1 IHC confirming extensive expression of Trx1 in HFs at anagen (P23), catagen (P40) and telogen (P55). B: Interaction of Foxp1 and Trx-1 endogenous proteins in anagen HFs as determined by Co-IP of protein lysates. C: Interaction of Foxp1 and Trx-1 following ectopic expression of Foxp1-His and Trx1 in transfected HeLa cells. Cell lysates were immunoprecipitated by anti-Trx1 antibody and detected by anti-His antibody. D: Colocalization of Trx1-RFP and Foxp1-EGFP protein within the nuclei (blue, DAPI) of HaCat cells. E: Flow cytometry of DCFDA-stained HEK293T cells following transient transfection of the indicated constructs (2 μg Foxp1 and/or 2 μg Trx1 expressing vector) indicated that Foxp1 releases inhibition of Trx-1-mediated ROS accrual. F: Model for the mechanism by which Foxp1 regulates redox homeostasis during hair cycling. Foxp1 is located within nuclei under conditions of low oxidative stress. Foxp1 suppresses the function of the Trx1 protein in decreasing ROS levels, and then imposes cell cycle arrest through p19/p53 axis. Foxp1 is exported into the cytoplasm when the ROS levels approach a high threshold.