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Cited by 120Epigenetics and DNA MethylationTelomeres, Telomerase, and SenescenceSkin Protection and Aging

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Particulate matter-induced senescence of skin keratinocytes involves oxidative stress-dependent epigenetic modifications

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Ryu, Yea Seong, Kang, Kyoung Ah, Piao, Mei Jing, Ahn, Mee Jung, Yi, Joo Mi, Bossis, Guillaume, Hyun, Young-Min, Park, Chang Ook, Hyun, Jin Won·2019·Experimental & Molecular Medicine
DOI 10.1038/s12276-019-0305-4PMC PMC6802667
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Abstract

Ambient air particulate matter (PM) induces senescence in human skin cells. However, the underlying mechanisms remain largely unknown. We investigated how epigenetic regulatory mechanisms participate in cellular senescence induced by PM with a diameter <2.5 (PM 2.5) in human keratinocytes and mouse skin tissues. PM 2.5-treated cells exhibited characteristics of cellular senescence. PM 2.5 induced a decrease in DNA methyltransferase (DNMT) expression and an increase in DNA demethylase (ten–eleven translocation; TET) expression, leading to hypomethylation of the p16INK4A promoter region. In addition, PM 2.5 led to a decrease in polycomb EZH2 histone methyltransferase expression, whereas the expression of the epigenetic transcriptional activator MLL1 increased. Furthermore, binding of DNMT1, DNMT3B, and EZH2 to the promoter region of p16INK4A decreased in PM 2.5-treated keratinocytes, whereas TET1 and MLL1 binding increased, leading to decreased histone H3 lysine 27 trimethylation (H3K27Me3) and increased H3K4Me3 in the promoter of p16INK4A. PM 2.5-induced senescence involved aryl hydrocarbon receptor (AhR)-induced reactive oxygen species (ROS) production. ROS scavenging dampened PM 2.5-induced cellular senescence through regulation of DNA and histone methylation. Altogether, our work shows that skin senescence induced by environmental PM 2.5 occurs through ROS-dependent the epigenetic modification of senescence-associated gene expression. Our findings provide information for the design of preventive and therapeutic strategies against skin senescence, particularly in light of the increasing problem of PM 2.5 exposure due to air pollution.

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Fig. 1 PM2.5-induced skin cellular senescence. a Human keratinocyte cell lines HaCaT and HEK001 and normal human epidermal keratinocytes (NHEKs) were each treated with 50 μg/mL PM2.5 and evaluated with various assays related to cellular senescence at the indicated time points. b Cells were stained for β-galactosidase activity using SPiDER-βGal working solution and DAPI. c Nuclear fractions were electrophoresed, and the senescence marker p16INK4A was detected by western blot analysis with the corresponding antibodies. TBP is shown as a loading control. d Morphological changes accompanying senescence induced by PM2.5 were observed by microscopy. e Cell proliferation was evaluated using trypan blue dye exclusion assays. f Colonies were counted after trypan blue staining. g Cells were stained for SAHF structure, followed by DAPI staining. The arrows indicate SAHF in senescent cells induced by PM2.5. *Significantly different from untreated cells (p < 0.05)
Fig. 1 PM2.5-induced skin cellular senescence. a Human keratinocyte cell lines HaCaT and HEK001 and normal human epidermal keratinocytes (NHEKs) were each treated with 50 μg/mL PM2.5 and evaluated with various assays related to cellular senescence at the indicated time points. b Cells were stained for β-galactosidase activity using SPiDER-βGal working solution and DAPI. c Nuclear fractions were electrophoresed, and the senescence marker p16INK4A was detected by western blot analysis with the corresponding antibodies. TBP is shown as a loading control. d Morphological changes accompanying senescence induced by PM2.5 were observed by microscopy. e Cell proliferation was evaluated using trypan blue dye exclusion assays. f Colonies were counted after trypan blue staining. g Cells were stained for SAHF structure, followed by DAPI staining. The arrows indicate SAHF in senescent cells induced by PM2.5. *Significantly different from untreated cells (p < 0.05)CC BY · PubMed Central
Fig. 2 P16INK4A transcriptional upregulation during PM2.5-induced cellular senescence correlates with DNA demethylation. The expression of p16INK4A a mRNA and b protein at the indicated time points was assessed by RT-PCR and western blot assays, respectively. c Nuclear fractions were electrophoresed, and DNMT1, DNMT3B, and TET1 were detected by western blotting with the corresponding antibodies. TBP is shown as a loading control. The nuclear location of d DNMT1, e DNMT3B, and f TET1 was determined by confocal microscopy after FITC labeling with the corresponding antibody. g TET1 activity was detected by confocal microscopy after Alexa594 labeling with a 5-hmC antibody
Fig. 2 P16INK4A transcriptional upregulation during PM2.5-induced cellular senescence correlates with DNA demethylation. The expression of p16INK4A a mRNA and b protein at the indicated time points was assessed by RT-PCR and western blot assays, respectively. c Nuclear fractions were electrophoresed, and DNMT1, DNMT3B, and TET1 were detected by western blotting with the corresponding antibodies. TBP is shown as a loading control. The nuclear location of d DNMT1, e DNMT3B, and f TET1 was determined by confocal microscopy after FITC labeling with the corresponding antibody. g TET1 activity was detected by confocal microscopy after Alexa594 labeling with a 5-hmC antibodyCC BY · PubMed Central
Fig. 3 TET1 binds to and demethylates the p16INK4A locus in response to PM2.5. a ChIP-qPCR was performed using primer sets specific for the regions indicated by A (−0.6 kb) to F (+5.6 kb) along the p16INK4A locus. ChIP using antibodies against b DNMT1, c DNMT3B, and d TET1 were analyzed by qPCR. e Quantitative methylation levels of the p16INK4A promoter region normalized to the Alu element. f Bisulfite sequencing analysis of the p16INK4A promoter region. The black circles represent methylated cytosine resides; the white circles represent unmethylated cytosine residues. Cells were transfected with TET1 siRNA and incubated for 24 h. g TET1 and p16INK4A were detected by western blotting with the corresponding antibodies. h β-Galactosidase activity was assessed by confocal microscopy after staining with SPiDER-βGal working solution and DAPI. *Significantly different from untreated cells (p < 0.05)
Fig. 3 TET1 binds to and demethylates the p16INK4A locus in response to PM2.5. a ChIP-qPCR was performed using primer sets specific for the regions indicated by A (−0.6 kb) to F (+5.6 kb) along the p16INK4A locus. ChIP using antibodies against b DNMT1, c DNMT3B, and d TET1 were analyzed by qPCR. e Quantitative methylation levels of the p16INK4A promoter region normalized to the Alu element. f Bisulfite sequencing analysis of the p16INK4A promoter region. The black circles represent methylated cytosine resides; the white circles represent unmethylated cytosine residues. Cells were transfected with TET1 siRNA and incubated for 24 h. g TET1 and p16INK4A were detected by western blotting with the corresponding antibodies. h β-Galactosidase activity was assessed by confocal microscopy after staining with SPiDER-βGal working solution and DAPI. *Significantly different from untreated cells (p < 0.05)CC BY · PubMed Central
Fig. 4 Senescence induced by PM2.5 is regulated via histone methylation of the p16INK4A locus. a EZH2 and MLL1 were detected by western blotting with the corresponding antibodies. b, c The nuclear localization of EZH2 and MLL1 was detected by confocal microscopy after FITC labeling with the corresponding antibodies. ChIP products using antibodies directed against d EZH2, e H3K27Me3, f MLL1, and g H3K4Me3 were analyzed by qPCR. Cells were transfected with MLL1 siRNA and incubated for 24 h. h MLL1 and p16INK4A were detected by western blotting with the corresponding antibodies. i β-Galactosidase activity was assessed by confocal microscopy after staining with SPiDER-βGal working solution and DAPI. The interaction between TET1 and MLL was examined by j immunoprecipitation and by k PLA. *Significantly different from untreated cells (p < 0.05)
Fig. 4 Senescence induced by PM2.5 is regulated via histone methylation of the p16INK4A locus. a EZH2 and MLL1 were detected by western blotting with the corresponding antibodies. b, c The nuclear localization of EZH2 and MLL1 was detected by confocal microscopy after FITC labeling with the corresponding antibodies. ChIP products using antibodies directed against d EZH2, e H3K27Me3, f MLL1, and g H3K4Me3 were analyzed by qPCR. Cells were transfected with MLL1 siRNA and incubated for 24 h. h MLL1 and p16INK4A were detected by western blotting with the corresponding antibodies. i β-Galactosidase activity was assessed by confocal microscopy after staining with SPiDER-βGal working solution and DAPI. The interaction between TET1 and MLL was examined by j immunoprecipitation and by k PLA. *Significantly different from untreated cells (p < 0.05)CC BY · PubMed Central
Fig. 5 PM2.5-induced senescence involves the AhR-ROS signaling pathway. a Nuclear AhR in HaCaT cells and NHEKs was detected by western blotting with the corresponding antibody. b Nuclear AhR in HaCaT cells and NHEKs was detected by confocal microscopy after FITC labeling with an AhR antibody. Quantification of ROS production in c HaCaT cells and d NHEKs by the Amplex® Red kit. *Significantly different from untreated cells (p < 0.05). Cells were transfected with AhR siRNA and incubated for 24 h, and ROS production was quantified in e HaCaT cells and f NHEKs. *Significantly different from untreated cells (p < 0.05). g β-Galactosidase activity was assessed by confocal microscopy after staining with SPiDER-βGal and DAPI. h The ROS level was assessed by confocal microscopy after DCF-DA staining. Nuclear AhR was detected by i western blotting and j confocal microscopy. k β-Galactosidase activity was assessed by confocal microscopy after staining with SPiDER-βGal. l Skin sections were assessed to determine the thickness of the mouse skin epidermis. m Skin sections were stained for keratin-10 and PCNA and counterstained with hematoxylin. *Significantly different from untreated cells (p < 0.05); **significantly different from PM2.5-treated cells (p < 0.05)
Fig. 5 PM2.5-induced senescence involves the AhR-ROS signaling pathway. a Nuclear AhR in HaCaT cells and NHEKs was detected by western blotting with the corresponding antibody. b Nuclear AhR in HaCaT cells and NHEKs was detected by confocal microscopy after FITC labeling with an AhR antibody. Quantification of ROS production in c HaCaT cells and d NHEKs by the Amplex® Red kit. *Significantly different from untreated cells (p < 0.05). Cells were transfected with AhR siRNA and incubated for 24 h, and ROS production was quantified in e HaCaT cells and f NHEKs. *Significantly different from untreated cells (p < 0.05). g β-Galactosidase activity was assessed by confocal microscopy after staining with SPiDER-βGal and DAPI. h The ROS level was assessed by confocal microscopy after DCF-DA staining. Nuclear AhR was detected by i western blotting and j confocal microscopy. k β-Galactosidase activity was assessed by confocal microscopy after staining with SPiDER-βGal. l Skin sections were assessed to determine the thickness of the mouse skin epidermis. m Skin sections were stained for keratin-10 and PCNA and counterstained with hematoxylin. *Significantly different from untreated cells (p < 0.05); **significantly different from PM2.5-treated cells (p < 0.05)CC BY · PubMed Central
Fig. 6 PM2.5-induced ROS regulate DNA and histone methylation status. a DNMT1, DNMT3B, TET1, EZH2, MLL1, and p16INK4A were detected by western blotting with the corresponding antibodies. ChIP products using antibodies against b DNMT1, c DNMT3B, and d TET1, as well as e EZH2 and f MLL1, were analyzed by qPCR. g Skin sections were stained for DNMT3B, TET1, EZH2, MLL1, and p16INK4A and counterstained with hematoxylin. *Significantly different from untreated cells (p < 0.05); **significantly different from PM2.5-treated cells (p < 0.05). h Working model for PM2.5-induced skin cellular senescence. In the presence of PM2.5, the PM2.5-AhR-ROS pathway prompts the binding of TET1 and MLL1 to the p16INK4A locus in place of DNMT and EZH2. This binding induces p16INK4A expression, which participates in the induction of skin cell senescence
Fig. 6 PM2.5-induced ROS regulate DNA and histone methylation status. a DNMT1, DNMT3B, TET1, EZH2, MLL1, and p16INK4A were detected by western blotting with the corresponding antibodies. ChIP products using antibodies against b DNMT1, c DNMT3B, and d TET1, as well as e EZH2 and f MLL1, were analyzed by qPCR. g Skin sections were stained for DNMT3B, TET1, EZH2, MLL1, and p16INK4A and counterstained with hematoxylin. *Significantly different from untreated cells (p < 0.05); **significantly different from PM2.5-treated cells (p < 0.05). h Working model for PM2.5-induced skin cellular senescence. In the presence of PM2.5, the PM2.5-AhR-ROS pathway prompts the binding of TET1 and MLL1 to the p16INK4A locus in place of DNMT and EZH2. This binding induces p16INK4A expression, which participates in the induction of skin cell senescenceCC BY · PubMed Central

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Authors
Ryu, Y. S., Kang, K. A., Piao, M. J., Ahn, M. J., Yi, J. M., Bossis, G., Hyun, Y.-M., Park, C. O., & Hyun, J. W.
Affiliations
  • Jeju National University (KR)
  • Inje University (KR)
  • Centre National de la Recherche Scientifique (FR)
  • Yonsei University (KR)
Corresponding author
Hyun, Jin Won
ORCID
Venue
Experimental & Molecular Medicine
Publisher
Springer Science and Business Media LLC
Published
2019-09
Volume
51
Issue
9
Pages
1-14
ISSN
  • 1226-3613 (ISSN-L)
  • 2092-6413
DOI
10.1038/s12276-019-0305-4
PMC
PMC6802667
OpenAlex
W2976086758
Cited by
119
References
40
Citations by year
2026: 10 · 2025: 21 · 2024: 18 · 2023: 14 · 2022: 20 · 2021: 20 · 2020: 16
FWCI
4.63
Type
journal article
Language
en
Open access
Licence
Locations
  • Experimental & Molecular Medicine (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by)
  • YUHSpace (Yonsei University Medical Library) (submittedVersion, cc-by-nc-nd)
Funders
National Research Foundation of Korea (2017R1A4A1014512)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Telomeres, Telomerase, and Senescence
  • Health Sciences › Medicine › Dermatology › Skin Protection and Aging
Keywords
particulate matter, PM2.5, cellular senescence, keratinocytes, epigenetic modifications, DNA methylation, histone methylation, aryl hydrocarbon receptor, reactive oxygen species, oxidative stress, EZH2, MLL1, TET1, air pollution, skin aging
MeSH terms
Enhancer of Zeste Homolog 2 Protein, DNA (Cytosine-5-)-Methyltransferase 1, DNA Methyltransferase 3B, Animals, DNA (Cytosine-5-)-Methyltransferases, DNA-Binding Proteins, Humans, Promoter Regions, Genetic, Histone-Lysine N-Methyltransferase, Proto-Oncogene Proteins, Skin, Keratinocytes, Cellular Senescence, Reactive Oxygen Species, Receptors, Aryl Hydrocarbon, Oxidative Stress, DNA Methylation, Cyclin-Dependent Kinase Inhibitor p16, Epigenesis, Genetic, Cell Proliferation, Mice, Myeloid-Lymphoid Leukemia Protein, Particulate Matter

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