ABSTRACT
AIMS: The loss of nuclear factor E2-related factor 2 (Nrf2) has been shown to protect against atherogenesis in apoE-deficient mice. The mechanism by which Nrf2 deficiency affords atheroprotection in this model is currently unknown, but combined systemic and local vascular effects on lesion macrophages have been proposed. We investigated the effect of bone marrow-specific loss of Nrf2 on early atherogenesis in low-density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice, and assessed the effect of Nrf2 on cellular accumulation of modified LDLs and the expression of inflammatory markers in macrophages. METHODS AND RESULTS: The effect of bone marrow-specific loss of Nrf2 on atherogenesis was studied using bone marrow transplantation of wild-type (WT) or Nrf2(-/-) bone marrow to LDLR(-/-) mice. Mice transplanted with Nrf2(-/-) bone marrow and fed a high-fat diet for 6 weeks exhibited significantly larger atherosclerotic lesions than WT bone marrow transplanted mice. Moreover, in thioglycollate-elicited Nrf2(-/-) macrophages, the uptake of acetylated and malondialdehyde-modified LDLs was increased in comparison with WT controls, with the concomitant increase in the expression of scavenger receptor A and toll-like receptor 4. In addition, the expression of pro-inflammatory monocyte chemoattractant protein-1 and interleukin-6 were increased in Nrf2(-/-) vs. WT macrophages. CONCLUSION: Nrf2 deficiency specific to bone marrow-derived cells aggravates atherosclerosis in LDLR(-/-) mice. Furthermore, the loss of Nrf2 in macrophages enhances foam cell formation and promotes the pro-inflammatory phenotype.
Subject(s)
Atherosclerosis/etiology , Macrophages/physiology , NF-E2-Related Factor 2/physiology , Animals , Chemokine CCL2/genetics , Cholesterol/metabolism , Female , Mice , Mice, Inbred C57BL , Receptors, LDL/physiology , Receptors, Scavenger/analysisABSTRACT
Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells.
Subject(s)
Adenocarcinoma/therapy , Antioxidant Response Elements , Ganciclovir/pharmacology , Genetic Therapy/methods , Lung Neoplasms/therapy , Oxidative Stress/physiology , Thymidine Kinase/metabolism , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma of Lung , Animals , Cell Line, Tumor , Ganciclovir/pharmacokinetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Kelch-Like ECH-Associated Protein 1 , Lentivirus/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Male , Mice , Mice, Nude , Mutation , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Thymidine Kinase/biosynthesis , Thymidine Kinase/genetics , Xenograft Model Antitumor AssaysABSTRACT
A central mechanism in cellular defence against oxidative or electrophilic stress is mediated by transcriptional induction of genes via the ARE (antioxidant-response element), a cis-acting sequence present in the regulatory regions of genes involved in the detoxification and elimination of reactive oxidants and electrophiles. The ARE binds different bZIP (basic-region leucine zipper) transcription factors, most notably Nrf2 (nuclear factor-erythroid 2-related factor 2) that functions as a transcriptional activator via heterodimerization with small Maf proteins. Although ARE activation by Nrf2 is relatively well understood, the mechanisms by which ARE-mediated signalling is down-regulated are poorly known. Transcription factor BACH1 [BTB (broad-complex, tramtrack and bric-a-brac) and CNC (cap'n'collar protein) homology 1] binds to ARE-like sequences, functioning as a transcriptional repressor in a subset of ARE-regulated genes, thus antagonizing the activator function of Nrf2. In the present study, we have demonstrated that BACH1 itself is regulated by Nrf2 as it is induced by Nrf2 overexpression and by Nrf2-activating agents in an Nrf2-dependent manner. Furthermore, a functional ARE site was identified at +1411 from the transcription start site of transcript variant 2 of BACH1. We conclude that BACH1 is a bona fide Nrf2 target gene and that induction of BACH1 by Nrf2 may serve as a feedback-inhibitory mechanism for ARE-mediated gene regulation.
Subject(s)
Basic-Leucine Zipper Transcription Factors/biosynthesis , Fanconi Anemia Complementation Group Proteins/biosynthesis , NF-E2-Related Factor 2/physiology , Response Elements/physiology , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Isothiocyanates , Nitro Compounds/pharmacology , Oleic Acids/pharmacology , Sulfoxides , Thiocyanates/pharmacologyABSTRACT
The nuclear receptor vitamin D receptor (VDR) is known to associate with three vitamin D response element (VDREs)-containing regions within the CDKN1A (p21) gene region. Here we show in MDA-MB453 breast cancer cells that the natural VDR ligand 1alpha,25-dihydroxyvitamin D(3) causes cyclical transcription factor binding and chromatin looping of distal VDREs to the transcription start site (TSS) of the p21 gene, leading to cyclical accumulation of the p21 mRNA. At the chromatin level, association of the mediator protein MED1 precedes both the peaks of VDR binding to VDREs and phosphorylated RNA polymerase (p-Pol II) to the TSS. The loss of co-repressor NCoR1-histone deacetylase (HDAC) 3 complex and inhibitory chromatin looping from VDREs to the TSS are also initial events followed by increased acetylation of histone 3 at lysine 9 at the TSS prior to initiation of transcription. Simultaneous to VDR and p-Pol II peaks, chromatin loops between VDREs and the TSS are formed, and the lysine demethylase LSD1 and the histone acetyltransferase CBP are enriched in both regions. This is followed by a moderate peak in p21 transcript accumulation, repeated in cycles of 45-60 min. The transcript accumulation pattern is disturbed by siRNA inhibition of the mediator protein MED1, LSD1, NCoR1, or various HDACs, whereas CBP appears unnecessary for the response. Inhibition of MED1, HDAC4, or LSD1 by siRNA also attenuates ligand-induced chromatin looping. In conclusion, 1alpha,25-dihydroxyvitamin D(3) regulates p21 transcription by inducing cyclical chromatin looping that depends on both histone deacetylation and demethylation.
