The dynamic nucleocytoplasmic localization of Foxp1 in response to oxidative stress.

<p><b>A:</b> Identification by mass spectrum analysis of phosphorylation at Foxp1 S468 sensitive to 2-hour H₂O₂ stimulation in CHO cells. <b>B:</b> Alignment of Foxp1 N-terminal residues across multiple species revealed a conserved nuclear localization motif of RXXRS (boxed). <b>C:</b> 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. <b>D:</b> Representative images showing defective nuclear localization of the Foxp1NLSm-EGFP fusion protein (green) following transient transfection into HaCat cells. <b>E:</b> 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. <b>F:</b> 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. <b>G:</b> Quantification of the relative Foxp1 levels by gray scale in (F, n = 3).</p

<i>Foxp1</i> deficiency augments the proportion of S-phase HFSC at anagen phase.

<p><b>A:</b> IHC with anti-CD34 (red) and anti-BrdU (green) staining of hair follicles following 4-day BrdU pulse-chase in the <i>Foxp1</i>^<i>fl/fl</i> (WT) and <i>K14-Cre; Foxp1</i>^<i>fl/fl</i> (<i>cKO</i>) 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>B:</b> Quantification of the number of BrdU⁺ cells in the bulges of (A). The <i>cKO</i> 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. <b>C:</b> 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. <b>D:</b> Quantification of the percentages of LRC in the bulges of (C). Few label-retaining cells (LRC) were detected in the bulges of <i>Foxp1 cKO</i> mice. n = 4; *, p<0.05. <b>E</b>: NAC treatment and BrdU injection once a day from P23 to P26 enhanced cell proliferation of HFSCs in WT early anagen. Scale bar, 50μm. <b>F:</b> Quantification of the frequency of BrdU⁺ cells in HFSCs in (E). *, p<0.05. <b>G-H:</b> Western blotting demonstrated a decrease of Foxp1 (G) and p19^ARF (H) protein levels within <i>cKO</i> hair follicles at anagen (P23). <b>I:</b> Down-regulation of <i>p19</i>^<i>ARF</i> transcripts within <i>cKO</i> anagen (P23) hair follicles relative to the WT as determined by qRT-PCR. <b>J:</b> S15 phosphorylated-p53 protein level was relatively decreased within <i>cKO</i> anagen (P23) hair follicles.</p

Foxp1 is translocated from the nucleus to cytoplasm of HFSCs at phases from anagen to catagen accompanying with rise of ROS levels.

<p><b>A:</b> 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>B:</b> Representative dot plot of FACS for HFSCs identified by CD34⁺/Integrin α6⁺ at early telogen (P49). Abbreviations: HFSCs, hair follicle stem cells. <b>C:</b> 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). <b>D:</b> Quantification of (B) indicates a progressive increase in ROS levels in HFSCs from telogen to catagen. *, p<0.05; **, p<0.01.</p

Foxp1 tunes ROS level through protein interaction with Trx-1.

<p><b>A:</b> Anti-Trx1 IHC confirming extensive expression of Trx1 in HFs at anagen (P23), catagen (P40) and telogen (P55). <b>B:</b> Interaction of Foxp1 and Trx-1 endogenous proteins in anagen HFs as determined by Co-IP of protein lysates. <b>C:</b> 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. <b>D:</b> Colocalization of Trx1-RFP and Foxp1-EGFP protein within the nuclei (blue, DAPI) of HaCat cells. <b>E:</b> 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. <b>F:</b> 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.</p

Misexpression of <i>Pknox2</i> in Mouse Limb Bud Mesenchyme Perturbs Zeugopod Development and Deltoid Crest Formation

<div><p>The TALE (Three Amino acid Loop Extension) family consisting of Meis, Pbx and Pknox proteins is a group of transcriptional co-factors with atypical homeodomains that play pivotal roles in limb development. Compared to the in-depth investigations of Meis and Pbx protein functions, the role of Pknox2 in limb development remains unclear. Here, we showed that <i>Pknox2</i> was mainly expressed in the zeugopod domain of the murine limb at E10.5 and E11.5. Misexpression of <i>Pknox2</i> in the limb bud mesenchyme of transgenic mice led to deformities in the zeugopod and forelimb stylopod deltoid crest, but left the autopod and other stylopod skeletons largely intact. These malformations in zeugopod skeletons were recapitulated in mice overexpressing <i>Pknox2</i> in osteochondroprogenitor cells. Molecular and cellular analyses indicated that the misexpression of <i>Pknox2</i> in limb bud mesenchyme perturbed the <i>Hox10-11</i> gene expression profiles, decreased <i>Col2</i> expression and Bmp/Smad signaling activity in the limb. These results indicated that <i>Pknox2</i> misexpression affected mesenchymal condensation and early chondrogenic differentiation in the zeugopod skeletons of transgenic embryos, suggesting <i>Pknox2</i> as a potential regulator of zeugopod and deltoid crest formation.</p></div