title: Protocatechuic acid induces antioxidant/detoxifying enzyme expression through JNK-mediated Nrf2 activation in murine macrophages.
abstract: Protocatechuic acid (PCA) is a main metabolite of anthocyanins, whose daily intake is much higher than that of other polyphenols. PCA has biological effects, e.g., it induces the antioxidant/detoxifying enzyme gene expression. This study was aimed at defining the molecular mechanism responsible for PCA-induced over-expression of glutathione (GSH) peroxidase (GPx) and GSH reductase (GR) in J774 A.1 macrophages. New evidence is provided that PCA increases GPx and GR expression by inducing C-JUN NH(2)-terminal kinase (JNK)-mediated phosphorylation of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). RNA and proteins were extracted from cells treated with PCA (25 μM) for different time points. Quantitative real-time polymerase chain reaction and immunoblotting analyses showed a rapid increase in mRNA (>60%) and protein (>50%) for both the enzymes. This was preceded by the up-regulation of Nrf2, in terms of mRNA and protein, and by its significant activation as assessed by increased Nrf2 phosphorylation and nuclear translocation (+60%). By using specific kinase inhibitors and detecting the activated form, we showed that JNK was the main upstream kinase responsible for Nrf2 activation. Convincing evidence is provided of a causal link between PCA-induced Nrf2 activation and increased enzyme expression. By silencing Nrf2 and using a JNK inhibitor, enzyme enhancement was counteracted. Finally, with the ChIP assay, we demonstrated that PCA-activated Nrf2 specifically bound ARE sequences in enzyme gene promoters. Our study demonstrates for the first time that PCA improves the macrophage endogenous antioxidant potential by a mechanism in which JNK-mediated Nrf2 activation plays an essential role. This knowledge could contribute to novel diet-based approaches aimed at counteracting oxidative injury by reinforcing endogenous defences.
authors.affiliation: Department of Veterinary Public Health and Food Safety, Italian National Institute of Health, Rome, Italy.
authors.forename: Rosaria
authors.initials: R
authors.lastname: Varì
authors.forename: Massimo
authors.initials: M
authors.lastname: D'Archivio
authors.forename: Carmelina
authors.initials: C
authors.lastname: Filesi
authors.forename: Simona
authors.initials: S
authors.lastname: Carotenuto
authors.forename: Beatrice
authors.initials: B
authors.lastname: Scazzocchio
authors.forename: Carmela
authors.initials: C
authors.lastname: Santangelo
authors.forename: Claudio
authors.initials: C
authors.lastname: Giovannini
authors.forename: Roberta
authors.initials: R
authors.lastname: Masella
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry; D020868:Gene Silencing; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D005980:Glutathione Reductase / Q000235:genetics / Q000378:metabolism; D062385:Hydroxybenzoates / Q000494:pharmacology*; D008658:Inactivation, Metabolic; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000302:isolation & purification / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000032:analysis; D015854:Up-Regulation
chemical_list: D000975:Antioxidants; D002843:Chromatin; D062385:Hydroxybenzoates; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C009091:protocatechuic acid; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase; D048031:JNK Mitogen-Activated Protein Kinases