Phloroglucinol Attenuates Ultraviolet B-Induced 8-Oxoguanine Formation in Human HaCaT Keratinocytes through Akt and Erk-Mediated Nrf2/Ogg1 Signaling Pathways

Abstract

Ultraviolet B (UVB) radiation causes DNA base modifications. One of these changes leads to the generation of 8-oxoguanine (8- oxoG) due to oxidative stress. In human skin, this modification may induce sunburn, inflammation, and aging and may ultimately result in cancer. We investigated whether phloroglucinol (1,3,5-trihydroxybenzene), by enhancing the expression and activity of 8-oxoG DNA glycosylase 1 (Ogg1), had an effect on the capacity of UVB-exposed human HaCaT keratinocytes to repair oxidative DNA damage. Here, the effects of phloroglucinol were investigated using a luciferase activity assay, reverse transcription-polymerase chain reactions, western blot analysis, and a chromatin immunoprecipitation assay. Phloroglucinol restored Ogg1 activity and decreased the formation of 8-oxoG in UVB-exposed cells. Moreover, phloroglucinol increased Ogg1 transcription and protein expression, counteracting the UVB-induced reduction in Ogg1 levels. Phloroglucinol also enhanced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) as well as Nrf2 binding to an antioxidant response element located in the Ogg1 gene promoter. UVB exposure inhibited the phosphorylation of protein kinase B (PKB or Akt) and extracellular signal-regulated kinase (Erk), two major enzymes involved in cell protection against oxidative stress, regulating the activity of Nrf2. Akt and Erk phosphorylation was restored by phloroglucinol in the UVB-exposed keratinocytes. These results indicated that phloroglucinol attenuated UVB-induced 8-oxoG formation in keratinocytes via an Akt/Erk-dependent, Nrf2/Ogg1-mediated signaling pathway.

Keywords: 8-oxoguanine DNA glycosylase 1; Extracellular signal-regulated kinase; NF-E2-related factor 2; Phloroglucinol; Protein kinase B; Ultraviolet B.

Fig. 1

Phloroglucinol (PG) attenuates UVB-induced 8-oxoguanine (8-oxoG) formation in HaCaT cells. (A) The cells were exposed to 30 mJ/cm² of UVB radiation after treatment with 10 μM PG. The content of 8-oxoG was measured using the 8-OHdG kit. *Significantly different from the control cells (p<0.05); ^#significantly different from the UVB-exposed cells (p<0.05). (B) The formation of 8-oxoG was evaluated under a confocal microscope using avidin-conjugated TRITC dye (Olympus Life Science, Tokyo, Japan); the fluorescence intensity is shown. *Significantly different from the control cells (p<0.05); ^#significantly different from the UVB-exposed cells (p<0.05).

Fig. 2

Phloroglucinol (PG) restores Ogg1 activity in UVB-exposed HaCaT cells. The cells were exposed to 30 mJ/cm² of UVB radiation after treatment with 10 μM PG, incubated for 20 h, and then transfected with FITC-DAB-labeled 8-oxoG for 4 h. The numbers of foci, indicating Ogg1 activity as a function of DAB (FITC quencher) depletion, were analyzed by (A) confocal microscopy and (B) flow cytometry. *Significantly different from the control cells (p<0.05); ^#significantly different from the UVB-exposed cells (p<0.05).

Fig. 3

Phloroglucinol (PG) increases Ogg1 expression at both the mRNA and protein levels. (A) Lipofectamine reagents were used to transfect the cells with plasmid vectors containing the luciferase gene under the control of the Ogg1 promoter. Transfected cells were exposed to 30 mJ/cm² of UVB radiation after treatment with 10 μM PG. Ogg1 gene transcription was examined by Ogg1 gene promoter luciferase assay. *Significantly different from the control cells (p<0.05); ^#significantly different from the UVB-exposed cells (p<0.05). (B) The cells were exposed to 30 mJ/cm² of UVB radiation after treatment with 10 μM PG and incubated for 12 h. The Ogg1 mRNA level was analyzed by RT-PCR. (C) The cells were exposed to 30 mJ/cm² of UVB radiation after treatment with 10 μM PG and incubated for 24 h. Ogg1 protein expression was analyzed by western blotting.

Fig. 4

Phloroglucinol (PG) increases Nrf2 nuclear translocation and binding to the Ogg1 promoter. The cells were exposed to 30 mJ/cm² of UVB radiation after treatment with 10 μM PG. (A) The nuclear proteins were harvested after 24 h of exposure to UVB radiation. The protein levels in the nuclear extracts were analyzed by western blotting using antibodies against Nrf2 and TBP. TBP was used as a nuclear loading control. (B) Confocal image showing the location of Nrf2 (green color). DAPI staining indicates the location of the nucleus (blue color), and the merged image indicates the nuclear localization of Nrf2. (C) Cells were transfected with siNrf2, treated with 10 μM PG, and incubated for 24 h. Western blotting was performed using antibodies against Nrf2, Ogg1, and actin. (D) ChIP analysis with antibodies against Nrf2 and histone 3 and primers for the amplification of the Ogg1 promoter region. The Nrf2 bands reflect Nrf2 binding to the Ogg1 promoter.

Fig. 5

Phloroglucinol (PG) increases Ogg1 expression via an Akt and Erk signaling-dependent, Nrf2-mediated mechanism. (A) After treatment with 10 μM PG for 1 h, the cells were exposed to 30 mJ/cm² UVB radiation and incubated for 6 h. The levels of phosphorylated Akt and Erk were analyzed by western blotting. Total Akt and total Erk2 were the loading controls. (B) Western blot analysis of nuclear Nrf2 expression. The cells were pretreated with 10 μM PG and Akt inhibitor or Erk inhibitor (U0126) for 30 min, exposed to 30 mJ/cm² of UVB radiation, and then incubated for 6 h before processing. TBP protein was the nuclear loading control.

Fig. 6

Phloroglucinol (PG) protects the cells from the effects of UVB exposure through Akt and Erk signaling. (A-D) Cell viability was determined using the MTT method. *Significantly different from the control cells (p<0.05); ^#significantly different from the UVB-exposed cells (p<0.05); **significantly different from the PG+UVB-exposed cells (p<0.05).

Fig. 7

Schematic diagram of the mechanism by which phloroglucinol prevents UVB-induced oxidative damage via the activation of the Nrf2/Ogg1 signaling pathway. Phloroglucinol induces Akt and Erk phosphorylation, and activated Akt and Erk promote Nrf2 translocation to the nucleus. Nrf2 binds to the Ogg1 promoter, thus increasing Ogg1 expression and activity, which ultimately leads to the removal of UVB-induced 8-oxoG.