Lightening up the UV response by identification of the arylhydrocarbon receptor as a cytoplasmatic target for ultraviolet B radiation - PubMed (original) (raw)
. 2007 May 22;104(21):8851-6.
doi: 10.1073/pnas.0701764104. Epub 2007 May 14.
Claudia Schäfer, Christian Calles, Thorsten Bernsmann, Thorsten Bernshausen, Melanie Wurm, Ulrike Hübenthal, Jason E Cline, Hossein Hajimiragha, Peter Schroeder, Lars-Oliver Klotz, Agneta Rannug, Peter Fürst, Helmut Hanenberg, Josef Abel, Jean Krutmann
Affiliations
- PMID: 17502624
- PMCID: PMC1885591
- DOI: 10.1073/pnas.0701764104
Lightening up the UV response by identification of the arylhydrocarbon receptor as a cytoplasmatic target for ultraviolet B radiation
Ellen Fritsche et al. Proc Natl Acad Sci U S A. 2007.
Abstract
UVB radiation-induced signaling in mammalian cells involves two major pathways: one that is initiated through the generation of DNA photoproducts in the nucleus and a second one that occurs independently of DNA damage and is characterized by cell surface receptor activation. The chromophore for the latter one has been unknown. Here, we report that the UVB response involves tryptophan as a chromophore. We show that through the intracellular generation of photoproducts, such as the arylhydrocarbon receptor (AhR) ligand 6-formylindolo[3,2-b]carbazole, signaling events are initiated, which are transferred to the nucleus and the cell membrane via activation of the cytoplasmatic AhR. Specifically, AhR activation by UVB leads to (i) transcriptional induction of cytochrome P450 1A1 and (ii) EGF receptor internalization with activation of the EGF receptor downstream target ERK1/2 and subsequent induction of cyclooxygenase-2. The role of the AhR in the UVB stress response was confirmed in vivo by studies employing AhR KO mice.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
UVB irradiation causes translocation of the AhR into the nucleus and induces transcription of the AhR-dependent gene CYP1A1. (A) HaCaT cells were transiently transfected with a GFP-coupled AhR and irradiated with 10 mJ/cm2 UVB or 30 J/cm2 UVA. Although UVB irradiation translocated the AhR into the nucleus, UVA had no effect on AhR compartmental distribution. (Scale bar, 20 μm.) (B) AhR immunoprecipitation and Western blotting of HaCaT nuclear extracts that were UVB (10 mJ/cm2) or sham irradiated show an accumulation of AhR protein after irradiation. (C) Real-time RT-PCR analyses reveal an inhibition of UVB-induced CYP1A1 mRNA induction after treatment with the competitive AhR-inhibitor 3′methoxy-4′nitroflavone (MNF) or in AhR knockdown HaCaTs (AhR KO), whereas vector or AhR nonsilencing (n.s.) transduced cells were not impaired in CYP1A1 response. ∗∗, P < 0.01.
Fig. 2.
AhR controls epidermal growth factor receptor (EGFR) internalization and downstream signaling after UVB irradiation. (A) UVB irradiation (10 mJ/cm2) led to EGFR internalization with a disappearance from the cell membranes (arrow in sham control) and paranuclear accumulation (arrow in UVB irradiation) after 30 min. AhR knockdown (KO) prevented EGFR internalization (arrow indicates EGFR at the cell membrane; N indicates nuclei). (Scale bar, 20 μm.) (B) Western blot analyses of the EGFR downstream target ERK1/2 revealed UVB-induced ERK1/2 phosphorylation that is partially AhR-dependent because it is antagonized by AhR knockdown. Cells transduced with nonsilencing AhR shRNA (AhR n.s.) showed no effect on ERK1/2 phosphorylation compared with the vector controls. (C) Real-time RT-PCR demonstrated an inhibition of the UVB-induced COX-2 mRNA induction in AhR KO HaCaT cells compared with the vector or AhR n.s. transduced HaCaTs. (D) COX-2 Western blot shows a reduction of COX-2 protein induction after UVB irradiation in AhR KO cells compared with the vector and n.s. controls. ∗∗, P < 0.01.
Fig. 3.
Tryptophan (Trp) is the chromophore for UVB and the precursor for the photoproduct formylindolo(3,2-b)carbazole (FICZ) that activates the AhR. Trp starvation (4 h) in trp-free medium abolished CYP1A1 mRNA inducibility (A) and EGFR internalization (B) after UVB irradiation (10 mJ/cm2) in HaCaT cells that were reconstituted after introduction of 1 mM Trp 1 h before irradiation. (Scale bar, 20 μm.) ∗∗, P < 0.01.
Fig. 4.
UVB irradiation leads to an intracellular formation of FICZ. Identification of the Trp-photoproduct FICZ by HPLC-MS: Trp-starved cells were incubated with 1 mM [13C1115N2]Trp (labeled with ∗) 1 h before irradiation with 60 mJ/cm2 UVB. Control cells were sham irradiated. After 10 min, cells were harvested, and cell extracts were prepared for HPLC-MS-MS analyses. The mass analysis revealed the occurrence of [13C15N]FICZ (arrow) with the expected mass of 305.3.
Fig. 5.
FICZ causes AhR-dependent signaling. AhR-proficient and -deficient HaCaT cells were treated with FICZ (100 nM) for the indicated times. (A) Immunocytochemical analyses demonstrated an AhR-dependent EGFR internalization 30 min after FICZ treatment. (B and C) Real-time RT-PCR and Western blot analyses showed AhR dependent COX-2 mRNA at 4 h (B) and COX-2 protein expression at 6 h (C) after exposure to FICZ. ∗∗, P < 0.01.
Fig. 6.
AhR KO mice show a decreased UVB responsiveness. Dorsal skin of wild-type and AhR KO C57BL/6 mice was shaved 24 h before exposure. Mice were irradiated dorsally with a single exposure of UVB (20 min 40 sec; 600 J/cm2). Twelve hours after irradiation, mice were euthanized, and skin samples were excised. RT-PCR analyses indicate an AhR dependence of Cyp1a1 and Cox-2 mRNA induction in UVB-irradiated mouse skin.
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