Histone Acetyltransferase-Dependent Pathways Mediate Upregulation of NADPH Oxidase 5 in Human Macrophages under Inflammatory Conditions: A Potential Mechanism of Reactive Oxygen Species Overproduction in Atherosclerosis

Abstract

Histone acetylation plays a major role in epigenetic regulation of gene expression. Monocyte-derived macrophages express functional NADPH oxidase 5 (Nox5) that contributes to oxidative stress in atherogenesis. The mechanisms of Nox5 regulation are not entirely elucidated. The aim of this study was to investigate the expression pattern of key histone acetyltransferase subtypes (p300, HAT1) in human atherosclerosis and to determine their role in mediating the upregulation of Nox5 in macrophages under inflammatory conditions. Human nonatherosclerotic and atherosclerotic tissue samples were collected in order to determine the expression of p300 and HAT1 isoforms, H3K27ac, and Nox5. In vitro determinations were done on human macrophages exposed to lipopolysaccharide in the absence or presence of histone acetyltransferase inhibitors. Western blot, immunohistochemistry, immunofluorescence, real-time PCR, transfection, and chromatin immunoprecipitation assay were employed. The protein levels of p300 and HAT1 isoforms, H3K27ac, and Nox5 were found significantly elevated in human atherosclerotic specimens. Immunohistochemistry/immunofluorescence staining revealed that p300, HAT1, H3K27ac, H3K9ac, and Nox5 proteins were colocalized in the area of CD45⁺/CD68⁺ immune cells and lipid-rich deposits within human atherosclerotic plaques. Lipopolysaccharide induced the levels of HAT1, H3K27ac, H3K9ac, and Nox5 and the recruitment of p300 and HAT1 at the sites of active transcription within Nox5 gene promoter in cultured human macrophages. Pharmacological inhibition of histone acetyltransferase significantly reduced the Nox5 gene and protein expression in lipopolysaccharide-challenged macrophages. The overexpression of p300 or HAT1 enhanced the Nox5 gene promoter activity. The histone acetyltransferase system is altered in human atherosclerosis. Under inflammatory conditions, HAT subtypes control Nox5 overexpression in cultured human macrophages. The data suggest the existence of a new epigenetic mechanism underlying oxidative stress in atherosclerosis.

Figure 1

Human atherosclerotic plaques exhibit a significant upregulation of p300, HAT1, H3K27ac, and Nox5 proteins. Note the densitometric analysis of type A-HAT (p300) (a), type B-HAT (HAT1) (b), H3K27ac (e), and Nox5 (f) protein levels analyzed by western blot of atherosclerotic (carotid artery) and nonatherosclerotic (superior thyroid artery) tissues derived from patients undergoing extended endarterectomy. n = 8‐11; ^∗∗P < 0.01, ^∗∗∗P < 0.001. P values were taken in relation to nonatherosclerotic condition. Representative immunoblots depicting the induction of p300 (c), HAT1 (d), H3K27ac (g), and Nox5 (h) proteins in atherosclerotic carotid arteries. Non-athero: nonatherosclerotic tissue; Athero: atherosclerotic tissue.

Figure 2

Comparative reactivity analysis of two different anti-Nox5 antibodies in protein extracts derived from cultured human Mac. Representative immunoblots depicting the Nox5 protein expression in Mac employing sc-67006 antibody (a) or ab191010 antibody (b). Immunogen sequences of sc-67006 antibody (c) and ab191010 antibody (d).

Figure 3

IHC localization of p300, HAT1, H3K27ac, H3K9ac, and Nox5 proteins in the area of infiltrated immune cells/Mac, fibrous cap, and lipid-rich core of a human carotid atherosclerotic plaque. The images ((a) 4x magnification, (b) 10x magnification) show representative IHC staining of 5 μm thick serial sections from a human carotid atherosclerotic plaque for p300, HAT1, H3K27ac, H3K9ac, Nox5, CD45, CD68, and αSMA proteins. The red lesional lipid deposits were detected by Oil Red O staining. The positively stained cells are marked with arrows. Note the localization of Nox5 protein in the area of fibrous cap and lipid-rich core of atherosclerotic plaque expressing markers of infiltrated immune cells (CD45, CD68) and vascular SMCs (αSMA). The images are representative of 4 independent IHC assays.

Figure 4

IF localization of H3K27ac, H3K9ac, and Nox5 proteins in the area of infiltrated immune cells/Mac, fibrous cap, and lipid-rich core of a human carotid atherosclerotic plaque. Representative IF images (5x, 10x, and 20x magnification) of 5 μm thick adjacent frozen sections depicting positively stained cells (Alexa Fluor™ 594) for H3K27ac, H3K9ac, Nox5, CD45, and αSMA in a human carotid atherosclerotic plaque. Note the appearance of specific staining of proteins within fibrous cap, and lipid-rich core of atherosclerotic plaque, especially at a higher magnification (i.e., 20x).

Figure 5

Exposure of cultured human Mac to LPS induces the upregulation of HAT1, H3K27ac, H3K9ac, and Nox5 protein levels in a concentration-dependent manner. Note the densitometric analysis of HAT1 (a), H3K27ac (b), H3K9ac (e), and Nox5 (f) protein levels analyzed by western blot in Mac exposed to increasing concentrations of LPS (0.1-1 μg/mL). n = 3‐4; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. P values were taken in relation to vehicle-treated cells. Representative immunoblots depicting the gradual upregulation of HAT1 (c), H3K27ac (d), H3K9ac (g), and Nox5 proteins (h) in LPS-challenged cells.

Figure 6

LPS-induced histone acetylation-related pathways trigger the upregulation of Nox5 gene and protein expression in Mac. Cultured human Mac were exposed for 24 h to 1 μg/mL LPS in the absence or presence of CPTH2 or C646 (1-5 μmol/L). The mRNA and protein levels were determined by real-time PCR and western blot. Concentration-dependent effects of CPTH2 and C646 on Nox5 mRNA (a, b) and protein (c, d) levels in LPS-exposed cells. Representative immunoblots showing the upregulation of Nox5 protein in LPS-treated cells and the regulatory effects of CPTH2 and C646 pharmacological inhibitors. n = 4; ^∗P < 0.05, ^∗∗P < 0.05, and ^∗∗∗P < 0.001. P values were taken in relation to LPS treatment condition.

Figure 7

Pharmacological inhibition of HAT downregulates the mRNA expression levels of Nox1 (a), Nox2 (b), and Nox4 (c) subtypes in LPS-exposed Mac. n = 3; ^∗P < 0.05, ^∗∗P < 0.05, and ^∗∗∗P < 0.001. P values were taken in relation to LPS treatment condition.

Figure 8

Overexpression of p300 or HAT1 upregulates the promoter activity of human Nox5 gene in Mac. The RAW264.7 cells were transiently transfected with Nox5 gene promoter-luciferase reporter gene constructs in the presence of empty vector or p300/HAT1 expression vectors. Representative agarose gel electrophoresis showing the digestion products of the c1-c8 constructs (a). Schematic depiction of the 5′-deletion mutants of the Nox5 gene promoter used in the cotransfection assays (b). Induction of luciferase level directed by the DNA regulatory elements derived from human Nox5 gene promoter in response to p300 or HAT1 overexpression in cultured Mac (c). n = 4; ^∗∗∗P < 0.001. P values were taken in relation to the corresponding empty vector control.

Figure 9

LPS induces enrichment of p300, HAT1, H3K27ac, and H3K9ac proteins at the proximal promoter region of Nox5 gene in cultured human Mac. Representative ChIP assay followed by agarose gel electrophoresis analysis of PCR products shows that p300 and HAT1 proteins are constitutively located within Nox5 gene promoter in resting Mac in vitro (a). Schematic depiction of the human Nox5 gene promoter (chromosome 15q23) showing the relative positions of the oligonucleotide primer sets spanning the entire proximal promoter region. Positive or negative DNA–p300/HAT1/H3K27ac/H3K9ac interactions are indicated with +/- marks (b). The increased abundance of p300, HAT1, H3K27ac, and H3K9ac proteins within Nox5 gene promoter in LPS-activated human Mac (c, d). n = 3; ^∗∗∗P < 0.001. P values were taken in relation to resting Mac condition.