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Ultraviolet A Radiation-Induced Immediate Iron Release Is a Key Modulator of the Activation of NF-κB in Human Skin Fibroblasts  Olivier Reelfs, Rex M.

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Presentation on theme: "Ultraviolet A Radiation-Induced Immediate Iron Release Is a Key Modulator of the Activation of NF-κB in Human Skin Fibroblasts  Olivier Reelfs, Rex M."— Presentation transcript:

1 Ultraviolet A Radiation-Induced Immediate Iron Release Is a Key Modulator of the Activation of NF-κB in Human Skin Fibroblasts  Olivier Reelfs, Rex M. Tyrrell, Charareh Pourzand  Journal of Investigative Dermatology  Volume 122, Issue 6, Pages (June 2004) DOI: /j X x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Time-course of nuclear factor-κB (NF-κB) DNA-binding activity and steady-state levels of IκBα following ultraviolet A (UVA) radiation-mediated oxidative stress. FEK4 cells were irradiated with a UVA dose of 250 kJ per m2, collected at various times (0.25–12 h) post-irradiation and used to prepare cytoplasmic and nuclear extracts. (a) NF-κB and Oct-1 DNA-binding activities in the nuclear extracts were analyzed by electrophoretic mobility shift assay (EMSA). (b) The corresponding cytoplasmic extracts were assayed by western blot as described in Materials and Methods for the levels of IκBα protein. The cytoplasmic protein heme oxygenase-2 (HO-2) was used as a marker of even loading. “C” indicates Control unirradiated cells. (c) The nuclear extract prepared under (a) at 6 h post-irradiation was analyzed by EMSA using competition and supershift experiments: Extracts were preincubated as follows: with nothing (lane 3), with 50-fold excess of unlabeled wild-type (lane 4) or mutated (lane 5) NF-κB oligonucleotide, with antibodies (lanes 6 and 7), with preimmune serum (lane 8). Probe (lane 1) indicates the reaction without cellular extract. DOC (lane 2) is a cytoplasmic extract of unirradiated cells, treated with DOC and NP40 as described in Materials and Methods. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Effect of ultraviolet A (UVA) radiation on the cellular localization of both p65 and Oct-1 proteins. FEK4 cells grown on coverslips were UVA irradiated with a dose of 250 kJ per m2. At 1 and 6 h post-irradiation, the cells were rinsed, fixed, and processed for immunofluorescent detection of p65 and Oct-1, as described in Materials and Methods. “C” indicates Control unirradiated cells. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Kinetics of measurement of the total cellular nuclear factor-κB (NF-κB) DNA-binding activity following ultraviolet A (UVA) irradiation of FEK4 cells. FEK4 cells were UVA irradiated with a dose of 250 kJ per m2. Total cellular extracts (“Totex”) were prepared at the indicated times post-irradiation and assayed in electrophoretic mobility shift assay as described in Materials and Methods for NF-κB (top panel) and Oct-1 (bottom panel) DNA-binding activity, respectively. “C” indicates Control unirradiated cells. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Effect of Desferal (DF) and hemin treatments on the cellular localization of both p65 and Oct-1 following ultraviolet A (UVA) irradiation of FEK4 cells. FEK4 cells were pre-treated with 10 μM DF or 4 μM hemin as described in Materials and Methods and UVA irradiated with a dose of 250 kJ per m2. At 1 and 6 h post-irradiation, the cells were rinsed, fixed, and processed for immunofluorescent detection of (a) p65 and (b) Oct-1. “C” indicates Control unirradiated cells. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Effect of the modulation of intracellular labile iron pool (LIP) on nuclear factor-κB (NF-κB) DNA-binding activity. (a) FEK4 cells were incubated in the presence of 10 and 100 μM of either Desferal (DF) or salicylaldehyde isonicotinoyl hydrazone (SIH) for 18 h and ultraviolet A (UVA) irradiated with a dose of 250 kJ per m2. NF-κB DNA-binding activity in nuclear extracts prepared at 6 h post-irradiation was analyzed by electrophoretic mobility shift assay as described in Materials and Methods. (b) Cells were treated with Fe-citrate (10 and 50 μM) or hemin (4 and 20 μM) as described in Materials and Methods and then UVA irradiated with a dose of 250 kJ per m2. Nuclear extracts were prepared at 6 h post-irradiation and used for analysis of NF-κB DNA-binding activity. “C” indicates Control unirradiated cells. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Effect of membrane antioxidants butylated hydroxytoluene (BHT) and α-tocopherol acetate on ultraviolet A (UVA)-mediated activation of nuclear factor-κB (NF-κB). FEK4 cells were pre-treated with BHT (25 μM) or α-tocopherol acetate (α-toco) (100 μM) for 18 h and UVA irradiated with a dose of 250 kJ per m2. NF-κB DNA-binding activity in the nuclear extracts was analyzed by electrophoretic mobility shift assay at 6 h post-irradiation. “C” indicates Control unirradiated cells. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

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