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. 2013 Dec;35(6):2255-71.
doi: 10.1007/s11357-013-9521-3. Epub 2013 Apr 12.

Role of p38MAPK and oxidative stress in copper-induced senescence

Emmanuelle Boilan  1 , Virginie Winant, Elise Dumortier, Jean-Pascal Piret, François Bonfitto, Heinz D Osiewacz, Florence Debacq-Chainiaux, Olivier Toussaint
Affiliations

Role of p38MAPK and oxidative stress in copper-induced senescence

Emmanuelle Boilan et al. Age (Dordr). 2013 Dec.

Abstract

In the present work, we indicate that copper is involved in the senescence of human diploid fibroblasts and we describe mechanisms to explain it. Using different techniques, we show for the first time an accumulation of copper in cells during replicative senescence. This accumulation seems to be co-localized with lipofuscin. Second, we observed that an incubation of cells with copper sulfate induced oxidative stress, antioxidant response and premature senescence. Antioxidant molecules reduced the appearance of premature senescence. Third, we found that Nrf2 transcription factor was activated and regulated the expression of genes involved in antioxidant response while p38(MAPK) regulated the appearance of premature senescence.

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Figures

Fig. 1
Fig. 1
Accumulation of copper in RS of WI-38 HDFs. a Intracellular Cu assay in young and senescent WI-38 HDFs using a QuantiChrom Cu assay kit. The results expressed in nanomole of Cu2+/milligramme of protein are given as mean ± S.D. from three independent experiments. Statistical analysis: Student’s t test. ***P < 0.001. b Intracellular Cu visualization in young and senescent WI-38 HDFs using Coppersensor-1. The results are representative of three independent experiments. Magnification, ×100. c Intracellular Cu detection in young and senescent WI-38 HDFs using Howell’s rubeanic acid method. Magnification, ×100. The results are representative of three independent experiments. d Intracellular Cu and lipofuscin detection in young and senescent WI-38 HDFs using TEM + EDX and Z-contrast imaging analysis. Magnification for image 1: ×7,000; for image 2: ×6,000; for image 3: ×37,000; image 4: ×17,000; image 5: ×13,000 and image 6: ×40,000. The graph represents the percentages of total Cu atoms counts inside and outside granules counted by EDX. Counts for carbon was used as atoms reference. e Lipofuscin detection in young and senescent WI-38 HDFs using the Ziehl–Neelsen modified method. Magnification, ×100
Fig. 2
Fig. 2
Treatment with sublethal concentration of CuSO4 increases intracellular copper. a Experimental design and time frame of CuSO4 incubation. b Intracellular Cu assay in WI-38 HDFs just after the end and 72 h after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h using a QuantiChrom copper assay kit. The results expressed in nanomole of Cu2+/milligramme of protein are given as mean ± S.D. from three independent experiments. Statistical analysis: Student’s t test. ns non-significant (P > 0.05); ***P < 0.001. c Intracellular Cu visualization WI-38 HDFs just after the end and 72 h after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h using Coppersensor-1. The results are representative of three independent experiments. d Intracellular Cu detection in WI-38 HDFs just after the end and 72 h after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h using the rubeanic acid method. The results are representative of three independent experiments. Magnification, ×100. e Lipofuscin detection in WI-38 HDFs just after the end and 72 h after the end of treatment with 0 (CTL) or 500 μM CuSO4 for 16 h using the Ziehl–Neelsen modified method. Magnification, ×100
Fig. 3
Fig. 3
Treatment with sublethal concentration of CuSO4 induces premature senescence. The markers of senescence in WI-38 HDFs at 72 h after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h are presented. a Morphology of WI-38 HDFs at 72 h after the end of the treatment with 0 (CTL) or 500 μM of CuSO4 for 16 h using optical microscope with phase contrast. Magnification, ×63. b Percentage of cells positive for SA-βgal at 72 h after the end of the treatment with 0 (CTL) or 500 μM of CuSO4 for 16 h. The results are expressed in percentage of blue cells and are given as mean ± S.D. from three independent experiments. c Percentage of cellular proliferative potential corresponding at incorporation of [3H]-thymidine in cells at 72 h after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h. The results are expressed in percentage compared to values for control cells (100 %) and are given as mean ± S.D. from three independent experiments. d mRNA abundance of senescence-associated genes using RT-PCR in cells at 72 h after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h. The results are expressed in fold induction of values for control cells (1.0) and are given as mean ± S.D. from three independent experiments. Statistical analysis: Student’s t test. ns nonsignificant (P > 0.05); *0.05 > P > 0.01; **0.01 > P > 0.001 and ***P < 0.001
Fig. 4
Fig. 4
Treatment with sublethal concentration of CuSO4 induces oxidative stress. a Production of ROS in WI-38 HDFs at just after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h using DCF-DA dye. The results are expressed in induction fold of values for control cells and are given as mean ± S.D. from three independent experiments. Statistical analysis: Student’s t test, *0.05 > P > 0.01. WI-38 HDFs treated with 500 μM of H2O2 for 2 h were used as positive control. b Immunofluorescence detection of 8-oxodG just after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h. TO-PRO-3 dye was used to detect nuclei. c Immunofluorescence detection of Nrf2 just after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h. TO-PRO-3 dye was used to detect nuclei. d DNA binding of Nrf2 in WI-38 HDFs just after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h using TransAM ELISA kit (Active Motif). The results are expressed as relative DNA binding activity of Nrf2 in comparison with control cells and are given as mean ± S.D. from three independent experiments. Statistical analysis: Student’s t test, ns nonsignificant (P > 0.05); *0.05 > P > 0.01; **0.01 > P > 0.001. WI-38 HDFs treated with 500 μM H2O2 for 2 h were used as positive control. e Immunofluorescence detection of HO-1 and NQO1 just after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h. TO-PRO-3 dye was used to detect nuclei
Fig. 5
Fig. 5
CuSO4 induces premature senescence through oxidative stress. a Production of ROS in WI-38 HDFs just after the end of the treatment with 0 (CTL) or 500 μM CuSO4 and with 50 mM mannitol or not for 16 h using DCF-DA dye. The results are expressed in fold of values for control cells and are given as mean ± S.D. from three independent experiments. Statistical analysis: Student’s t test, n.s. nonsignificant (P > 0.05); *0.05 > P > 0.01; ***P < 0.001. WI-38 HDFs treated with 500 μM of H2O2 for 2 h were used as positive control. b Western blot analysis of Hsp70, HO-1, NQO1 (1) and Nrf2 (2) just after the end of the treatment with 0 (CTL) or 500 μM CuSO4 and with 50 mM mannitol or not for 16 h. Ten microgrammes of protein from whole cell homogenate (1) or from the nuclear or cytoplasmic fractions (2) are used for the analysis. α-Tubulin and lamin-B were used as control loading. WI-38 HDFs treated with 500 μM of H2O2 for 2 h were used as positive control. c Percentage of SA-βgal positive in WI-38 HDFs at 72 h after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h and with antioxidant (50 mM mannitol or 100 μM trolox) or not. The results are expressed in percentage and are given as mean ± S.D. from three independent experiments. The numbers in the chart represent ratios between CTL and CuSO4-treated cells and are given as mean ± S.D. from three independent experiments. Statistical analysis: Student’s t test. **0.01 > P > 0.001; ***P < 0.001. The symbol Δ represents a statistical analysis between ratios. d Percentage of cellular proliferative potential (incorporation of [3H]-thymidine) in WI-38 HDFs at 72 h after the end of the treatment with 0 (CTL) or 500 μM CuSO4 for 16 h with antioxidant (50 mM mannitol or 100 μM trolox) or not. The results are expressed in percentage (CTL = 100 %) and are given as mean ± S.D. from three independent experiments. The numbers in the chart represent ratios between CTL and CuSO4-treated cells and are given as mean ± S.D. from three independent experiments. Statistical analysis: Student’s t test. *0.05 > P > 0.01; **0.01 > P > 0.001. The symbol Δ represents a statistical analysis between ratios
Fig. 6
Fig. 6
Phosphorylation of p38MAPK is implicated in CuSO4-induced premature senescence. a Western blot analysis of Phospho-p38MAPK just after the end of the treatment with 0 (CTL) or 500 μM CuSO4 and with 50 mM mannitol or not for 16 h. α-Tubulin was used as control loading. WI-38 HDFs treated with 500 μM H2O2 for 2 h were used as positive control. b Western blot analysis of Hsp70, HO-1, NQO1 (1) and Nrf2 (2) just after the end of the treatment with 0 (CTL) or 500 μM CuSO4 and with 20 μM of p38MAPK inhibitor SB203580 or not for 16 h. Ten microgrammes of proteins from whole cell homogenate (1) or from the nuclear or cytoplasmic fractions (2) are used for the analysis. α-Tubulin and lamin-B were used as control loading. c DNA binding of Nrf2 just after the end of the treatment with 0 (CTL) or 500 μM CuSO4 and with 20 μM of p38MAPK inhibitor SB203580 or not for 16 h using TransAM ELISA kit. The results are expressed as relative DNA binding of Nrf2 in comparison with control cells and are given as mean ± S.D. from three independent experiments. Statistical analysis: Student’s t test, n.s. non-significant (P > 0.05); *0.05 > P > 0.01. d Percentage of SA-βgal positive and percentage of cellular proliferative potential (incorporation of [3H]-thymidine) in WI-38 HDFs at 72 h after the end of the treatment with 500 μM CuSO4 or not and with 20 μM of p38MAPK inhibitor SB203580 or not for 16 h. The results are expressed in percentage and are given as mean ± S.D. from three independent experiments. The numbers in the chart represent ratios between CTL and CuSO4-treated cells and are given as mean ± S.D. from three independent experiments. Statistical analysis was carried out with the Student’s t test. *0.05 > P > 0.01; **0.01 > P > 0.001; ***P < 0.001. The symbol Δ represents a statistical analysis between ratios
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