Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or VSports app下载. mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2012 Feb 17;287(8):5627-38.
doi: 10.1074/jbc.M111.328120. Epub 2011 Dec 19.

p63 regulates human keratinocyte proliferation via MYC-regulated gene network and differentiation commitment through cell adhesion-related gene network

Ning Wu  1 , Jérome Rollin, Ingrid Masse, Jérôme Lamartine, Xavier Gidrol
Affiliations

p63 regulates human keratinocyte proliferation via MYC-regulated gene network and differentiation commitment through cell adhesion-related gene network (V体育官网入口)

Ning Wu et al. J Biol Chem. .

Abstract

Although p63 and MYC are important in the control of epidermal homeostasis, the underlying molecular mechanisms governing keratinocyte proliferation or differentiation downstream of these two genes are not completely understood. By analyzing the transcriptional changes and phenotypic consequences of the loss of either p63 or MYC in human developmentally mature keratinocytes, we have characterized the networks acting downstream of these two genes to control epidermal homeostasis. We show that p63 is required to maintain growth and to commit to differentiation by two distinct mechanisms. Knockdown of p63 led to down-regulation of MYC via the Wnt/β-catenin and Notch signaling pathways and in turn reduced keratinocyte proliferation. We demonstrate that a p63-controlled keratinocyte cell fate network is essential to induce the onset of keratinocyte differentiation. This network contains several secreted proteins involved in cell migration/adhesion, including fibronectin 1 (FN1), interleukin-1β (IL1B), cysteine-rich protein 61 (CYR61), and jagged-1 (JAG1), that act downstream of p63 as key effectors to trigger differentiation. Our results characterized for the first time a connection between p63 and MYC and a cell adhesion-related network that controls differentiation VSports手机版. Furthermore, we show that the balance between the MYC-controlled cell cycle progression network and the p63-controlled cell adhesion-related network could dictate skin cell fate. .

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Influence of knockdown of MYC or p63 on keratinocyte differentiation. A and B, p63 and MYC expression was measured by qRT-PCR (A) and Western blot (B) after introduction of siRNA oligonucleotide duplexes. siCT is a non-targeting siRNA control, siP63 is an siRNA duplex against all p63 isoforms, and siMYC is an siRNA targeting the MYC oncogene. mRNA expression levels were normalized to siCT (A). Samples used in A and B were extracted 48 h after siRNA transfection. C, cell growth was inspected by Vialight. Error bars, S.D. of six biological replicates; Student's t test was used for statistical analysis. ***, p < 0.001. D, cell proliferation was assessed by EdU incorporation measured by flow cytometry. Three independent experiments were used, and Student's t test was used for statistical analysis. ***, p < 0.001. E, Ki67 staining of HaCaT cells 48 h post-transfection by siRNA. Red, Ki67; Blue, Hoechst. F and G, time course of qRT-PCR (F) and Western blot (G) analysis of human cytokeratin 1 (K1) and cytokeratin 10 (K10) expression in HaCaT cells transfected with siCT, siMYC, or siP63. mRNA expression levels were normalized to day 0. β-Actin was used as a loading control in the Western blot. H, time course of qRT-PCR analysis of K1 and K10 mRNA levels in primary human keratinocytes (PHK), transfected with siCT, siMYC, or siP63. mRNA expression levels were normalized to day 0. Error bars in all qRT-PCR graphs represent S.D. of triplicate samples from one representative experiment.
FIGURE 2.
FIGURE 2.
Shared cell cycle arrest mechanisms in cells treated with siP63 or siMYC. A, cell cycle phase determination analysis performed by flow cytometry. HaCaT cells were treated with siCT (left), siP63 (middle), or siMYC (right). PI, propidium iodide. B, Live/Dead staining of siRNA-transfected HaCaT cells. Red, EthD-1; green, calcein AM. C, genetic networks regulating the cell cycle were generated by IPA. The lists of genes used in IPA were obtained from transcriptome analysis of HaCaT cells treated with siP63 for 48 h. All cell cycle-related genes in siP63 were extracted from the IPA database with a very significant p value (<6.2 × 10−35) to generate this network (see “Experimental Procedures”). Nodes (genes or proteins) in the networks are shown by different shapes (biological functions) and colors (red indicates up-regulated, and green represents down-regulated). Edges are represented as solid or dashed lines to indicate direct and indirect interactions, respectively. D and E, qRT-PCR analysis of cyclin-dependent kinase inhibitors (such as p15, p16, and p21) and MYC in siP63 (D) or in siMYC (E) HaCaT keratinocytes. mRNA expression levels were normalized to siCT, and error bars in qRT-PCR graphs show S.D. of triplicate samples from one representative experiment. Student's t test was used for statistical analysis. *, p < 0.05; **, p < 0.01. F and G, identical to D and E in human primary keratinocytes lacking either p63 (F) or MYC (G).
FIGURE 3.
FIGURE 3.
Knockdown of p63 down-regulates MYC. qRT-PCR (A) and Western blot (B) analysis of MYC expression when p63 was knocked down by siRNA targeting all p63 isoforms in HaCaT cells or primary human keratinocytes (PHK). C, MYC expression in ΔNp63-, TAp63-, or p63-knockdown cells. 48 h post-transfection, MYC expression was measured by qRT-PCR. 18 S rRNA was an endogenous control to normalize the results. D, structure of the MYC promoter region cloned into the luciferase reporter plasmids. The heavy horizontal lines represent the promoter sequences in each reporter plasmid. E, HaCaT cells were cotransfected with siCT or siP63 and the MYC promoter luciferase reporter constructs. Luciferase activities (means ± S.D.) were measured 48 h post-transfection. Error bars, S.D. of five biological replicates. Firefly luciferase luminescence was normalized to an internal control Renilla luciferase. F, schematic representation of the MYC promoter region between −300 and −650 bp (which corresponds to the differences between pDel-3 and pDel-4). Known TF binding sites are indicated by arrows. G, HaCaT cells were cotransfected with control siRNA or all-p63 siRNA and TCF-4, YY-1, JUN, or FOS luciferase reporter plasmids (TFs marked in red in G). Detailed luminescence measurements and analysis can be found under “Experimental Procedures.” Error bars, S.D. of replicates. The Mann-Whitney test was used for statistical analysis in E and G. *, p < 0.05.
FIGURE 4.
FIGURE 4.
The Wnt/β-catenin and Notch pathways are responsible for the MYC down-regulation in p63 knockdown keratinocytes. A, list of modulators of the Notch and β-catenin pathways that were differentially expressed according to siP63 transcriptome analysis. Green arrows, decrease in expression; red arrows, increase compared with siCT-treated cells. B, Western blots of cleaved NICD, total β-catenin, and MYC in siP63 as compared with siCT. C, qRT-PCR analysis of expression of HES1 and HEY1, which are two transcriptional target genes of the Notch signaling pathway. The values represent the mean ± S.D. of three replicate samples from one representative experiment. D and E, MYC expression study at mRNA level (D) and at protein level (E) after activation of the β-catenin or Notch pathway via overexpression of Wnt3 or NICD, respectively. A plasmid expressing GFP was used as a transfection control. Co-transfection of siRNA and plasmids was done by nucleofection using Amaxa (see details under “Experimental Procedures”). Error bars in all qRT-PCR graphs represent the S.D. of triplicates from one representative experiment.
FIGURE 5.
FIGURE 5.
Candidate keratinocyte differentiation effectors identified by comparative siMYC and siP63 transcriptome analysis. A, Venn diagram indicates distribution of differentially expressed genes in siMYC and siP63. 546 genes are common to siMYC and siP63. B, IPA with the list of oppositely expressed genes in the common part of A extracted a genetic network associated with a function of cell migration with a very significant p value (p < 3 × 10−14). The genes in this network are down-regulated in siP63 keratinocytes and up-regulated in siMYC keratinocytes (supplemental Fig. S6). This network was named the KFC network and contains candidate keratinocyte differentiation effectors acting downstream of p63. C and D, expression levels of genes identified in genetic networks validated by qRT-PCR in HaCaT cells (C) and primary keratinocytes (D). Student's t test was used for statistical analysis. *, p < 0.05; **, p < 0.01. Error bars, S.D.
FIGURE 6.
FIGURE 6.
An extracellular matrix genetic network is involved in keratinocyte differentiation. A, validations of siRNA knockdown targeting FN1, MMP13, JAG1, IL1B, or CYR61. Cells were transfected with 10 nm siRNA for 48 h, and then the gene expression levels were quantified by qRT-PCR. The effect of knockdown of each gene was compared with siCT. B and C, time course analysis of K1 (B) and K10 (C) expression were determined by qRT-PCR 9 days post-transfection of HaCaT cells with siRNA against FN1, MMP13, IL1B, JAG1, CYR61, or siCock (mixture of these five siRNAs). D, expression of oncogene hubs in human iPS. hFibr, human fibroblast; e-hiPSC, early human iPS; l-hiPSC, late human iPS; hESC, human embryonic stem cells. Each spot represents expression data from one biological sample. Error bars, S.D.
FIGURE 7.
FIGURE 7.
Schematic representation of the mechanism of commitment of keratinocyte differentiation controlled by p63. p63 controls keratinocyte proliferation and differentiation independently via different genetic networks. p63 regulates the cell cycle in part by regulating MYC expression through both the Wnt/β-catenin and Notch pathways. To trigger the onset of differentiation, p63 controls the KCF network composed of keratinocyte differentiation effectors. Most of these effectors, such as FN1, IL1B, JAG1, and CYR61, are located in the extracellular matrix and are implicated in cell migration.
See this image and copyright information in PMC

References

    1. Fuchs E. (2007) Scratching the surface of skin development. Nature 445, 834–842 - V体育平台登录 - PMC - PubMed
    1. Fuchs E., Raghavan S. (2002) Getting under the skin of epidermal morphogenesis. Nat. Rev. Genet. 3, 199–209 - V体育ios版 - PubMed
    1. Yang A., Schweitzer R., Sun D., Kaghad M., Walker N., Bronson R. T., Tabin C., Sharpe A., Caput D., Crum C., McKeon F. (1999) p63 is essential for regenerative proliferation in limb, craniofacial, and epithelial development. Nature 398, 714–718 - PubMed (V体育ios版)
    1. Mills A. A., Zheng B., Wang X. J., Vogel H., Roop D. R., Bradley A. (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398, 708–713 - PubMed
    1. Senoo M., Pinto F., Crum C. P., McKeon F. (2007) p63 is essential for the proliferative potential of stem cells in stratified epithelia. Cell 129, 523–536 - PubMed

"V体育官网" Publication types

MeSH terms

"VSports在线直播" Associated data