Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photodamage and carcinogenesis, and an urgent need exists for improved molecular photoprotective strategies different from (or synergistic with) photon absorption. Recent studies suggest a photoprotective role of cutaneous gene expression orchestrated by the transcription factor NRF2 (nuclear factor-E2-related factor 2). Here we have explored the molecular mechanism underlying carotenoid-based systemic skin photoprotection in SKH-1 mice and provide genetic evidence that photoprotection achieved by the FDA-approved apocarotenoid and food additive bixin depends on NRF2 activation. Bixin activates NRF2 through the critical Cys-151 sensor residue in KEAP1, orchestrating a broad cytoprotective response in cultured human keratinocytes as revealed by antioxidant gene expression array analysis. Following dose optimization studies for cutaneous NRF2 activation by systemic administration of bixin, feasibility of bixin-based suppression of acute cutaneous photodamage from solar UV exposure was investigated in Nrf2(+/+) versus Nrf2(-/-) SKH-1 mice. Systemic administration of bixin suppressed skin photodamage, attenuating epidermal oxidative DNA damage and inflammatory responses in Nrf2(+/+) but not in Nrf2(-/-) mice, confirming the NRF2-dependence of bixin-based cytoprotection. Taken together, these data demonstrate feasibility of achieving NRF2-dependent cutaneous photoprotection by systemic administration of the apocarotenoid bixin, a natural food additive consumed worldwide.
Antioxidant gene expression; Bixin; NRF2; Skin photodamage; Systemic photoprotection.
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Figure 1. Bixin upregulates NRF2 signaling and antioxidant defenses in epidermal keratinocytes
(A) For Oxidative Stress RT
2 Profiler ™ PCR Expression Array analysis, HEKs were exposed to bixin (20 μM, 24 h) followed by gene expression analysis; upper panel: scatter blot depiction of bixin-induced gene expression (versus untreated); cut-off lines: threefold up- or down-regulation; the insert shows the chemical structure of bixin; bottom panel: numerical expression changes [n=3, mean ± SD; (p<0.05)]. (B) Bixin (0-20 μM, 0-24 h) increased the protein levels of NRF2 and its target genes as assessed by immunoblot analysis; left panel: dose-response, right panel: time course. (C) HaCaT keratinocytes cotransfected with NQO1-ARE firefly luciferase and Renilla luciferase reporters were treated with bixin (0-40 μM) for 16 h. Dual luciferase activities were measured; data are expressed as means ± SD (* p<0.05, ctrl. vs. bixin treated groups). (D) HaCaT keratinocytes were treated with bixin (20 μM; 0-48 h exposure time), and cell lysates were subjected to immunoblot analysis. (E) HaCaT keratinocytes were treated with bixin (0-40 μM, 24 h), and total cellular glutathione was determined [n=3; means ± SD (* p<0.05, ctrl. vs. bixin groups]. (F) HaCaT keratinocytes were exposed to bixin (20 μM; 1 and 24 h exposure time) followed by dye sensitization (generating 1O 2) and subsequent loading with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA)]. Intracellular oxidative stress was then assessed by flow cytometric determination of DCF fluorescence intensity [means ± SD, n=3; means without a common letter differ (p < 0.05)]. (G) HaCaT keratinocytes were exposed to various anti-oxidants [1 h pretreatment: trolox (1 mM), tiron (500 μM), N-acetyl-L-cysteine (NAC; 10 mM)] followed by addition of bixin (40 μM; 4 h) and NRF2/KEAP1 immunoblot analysis.
Figure 2. Bixin induces KEAP1-C151-dependent NRF2 upregulation and increases
Nrf2 protein half-life (t
1/2) in human keratinocytes. (A-D) HaCaT cells were either left untreated (control; empty bar) or treated with bixin (40 μM, filled bar; 4 h and 16 h), and mRNA was extracted. Relative mRNA levels [ NRF2 (A), KEAP1 (B), GCLM (C), AKR1C1 (D)] as determined by quantitative real-time RT-PCR [means ± SD (* p<0.05, control vs. bixin treated group)]. (E) HaCaT cells were either left untreated or treated with bixin (40 μM, 4h). Cycloheximide (CHX, 50 μM) was added and cells were lysed at the indicated time points followed by immunoblot analysis using NRF2 and GAPDH antibodies. Band intensities were quantified and plotted against the time after CHX treatment to obtain half-life (t 1/2) values. (F) HaCaT cells were cotransfected with plasmids encoding the indicated proteins; 24 h later the cells were then left untreated or treated with either SF (5 μM) or bixin (40 μM) along with MG132 (10 μM) for 4 h. Anti-NRF2 immunoprecipitates were analyzed by immunoblotting with anti-HA antibody detecting ubiquitin-conjugated NRF2. (G) HaCaT cells cotransfected with the plasmids expressing either wild type KEAP1 ( KEAP1-WT) or C151 mutated KEAP1 ( KEAP1-C151S) along with NQO1-ARE firefly luciferase and Renilla luciferase reporters were left untreated or treated with the indicated compounds (16 h). Dual luciferase activities were measured; data are expressed as means ± SD (* p<0.05, Control vs. compound treated groups; # p<0.05, KEAP1-WT vs. KEAP1-C151S group.)
Figure 3. Systemic administration of bixin activates cutaneous NRF2 and NRF2 targets
Nrf2 and +/+ Nrf2 mice; n = 6 per group) received bixin treatment (200 mg/kg; i.p.) or carrier control (corn oil), followed by solar UV (UVB 240 mJ/cm −/− 2) or mock exposure performed 48 h after bixin administration. (A) After UV exposure (24 h), IHC analysis (NRF2, GCLM, AKR1C1) was performed using skin tissue sections; representative tissue from each group is shown (scale bar: 100 μm). (B) Skin tissue lysates from Nrf2 mice were subjected to immunoblot analyses with anti-NRF2, KEAP1, AKR1C1, GCLM, and GAPDH antibodies (n=3, each lane represents an individual mouse). (C-D) Skin prepared from mice as specified in (A) was processed for determination of mRNA levels [ +/+ Gclm (C) and Akr1c1 (D)] using quantitative RT-PCR; means ± SD (* p<0.05, control vs. treatment groups).
Figure 4. Systemic administration of bixin suppresses UV-induced epidermal thickening, apoptosis, and oxidative DNA damage in
Nrf2 mice but not +/+ Nrf2 mice −/−
Nrf2 and +/+ Nrf2 mice; n = 6 per group) received bixin treatment (200 mg/kg; i.p.) or carrier control (corn oil), followed by solar UV (UVB 240 mJ/cm −/− 2) or mock exposure performed 48 h after bixin. (A) After irradiation (24 h), H&E staining and in situ TUNEL analysis visualizing epidermal apoptotic cells were performed [n = 6; representative tissue from each group is shown (scale bar: 100 μm)]. In addition, 8-oxo-dG- and CPD-lesions were visualized by IHC; representative tissue from each group is shown. (B) Epidermal thickness in H&E-stained sections was measured as the distance between the top of the basement membrane and the bottom of the stratum corneum at five randomly selected fields from each mouse specimen. (C) Quantification of TUNEL-positive cells (green fluorescent nuclei) in five random fields per section; 200 × magnification; [means ± SD (* p<0.05, control vs. treatment groups; # p<0.05, UV vs. bixin+UV groups)].
Figure 5. Systemic administration of bixin attenuates UV-induced cutaneous hyperproliferation and inflammation in
Nrf2 mice but not +/+ Nrf2 mice −/−
Mice were treated as detailed in Figs. 3 and 4 followed by IHC analysis for (A) Ki67 and (B) MMP9 (scale bar: 100 μm). (C-D) Skin tissue lysates from bixin/UV-exposed
Nrf2 and +/+ Nrf2 mice were also subjected to immunoblot analyses with anti-p-p65, p65, and GADPH antibodies followed by quantification using densitometry (D). (E) mRNA levels of −/− IL6, TNFa and MMP9 were determined using quantitative RT-PCR. Results are expressed as means ± SD (* p<0.05, control vs. treatment groups; # p<0.05, UV vs. bixin+UV groups).
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Carotenoids / pharmacology*
Cell Survival / drug effects
Cytoprotection / physiology
In Situ Nick-End Labeling
Keratinocytes / drug effects*
Keratinocytes / radiation effects
NF-E2-Related Factor 2 / metabolism*
Oxidative Stress / drug effects
Reverse Transcriptase Polymerase Chain Reaction
Sunlight / adverse effects
Ultraviolet Rays / adverse effects
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