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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biochem Biophys Res Commun. Author manuscript; available in PMC Oct 24, 2009.
Published in final edited form as:
PMCID: PMC2583397
NIHMSID: NIHMS70651

TNF-alpha upregulates the A2B adenosine receptor gene: the role of NAD(P)H oxidase 4

Abstract

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor–alpha (TNF-α) is released during vascular injury, and contributes to lesion formation by affecting VSMC proliferation. Recently, an A2B adenosine receptor (A2BAR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response, including the release of TNF-α Here, we show a regulatory loop by which TNF-α upregulates the A2BAR in VSMC in vitro and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A2BAR, and Nox inhibitors dampen the effect of TNF-α. Hence, our study is the first to show that signaling associated with vascular disease, such as Nox4, is also able to upregulate the tissue protecting A2BAR.

Keywords: A2B adenosine receptor, vascular smooth muscle cells, tumor necrosis factor-alpha, NAD(P)H oxidase 4

INTRODUCTION

Tumor necrosis factor-alpha (TNF-α) is a potent pro-inflammatory cytokine generated by many types of cells in response to inflammation. TNF-α contributes to atherosclerotic lesion formation, in part by stimulating VSMC to proliferate [1]. Adenosine is a potent autocrine and paracrine signaling molecule generated or released from cells under stress [2], and has been shown to reduce the release of TNF-α from cells stimulated by an immune response [3, 4], as its signaling is generally tissue protective [5]. Adenosine elicits its effects through interaction with one or more of the four adenosine receptors: A1AR, A3AR, A2AAR and A2BAR [6, 7].

Inhibitory effects of adenosine on TNF-α release at baseline or in response to bacterial lipopolysaccharide (LPS) are modulated via the A2ARs [8, 9], and furthermore, in a regulatory loop fashion, A2BAR gene expression is upregulated by TNF-α signaling [1012]. The specific molecules involved in the signaling downstream of TNF-α that induce A2BAR upregulation are unknown. Adenosine signaling through the A2BAR has been shown to inhibit smooth muscle cell proliferation [13, 14], and stimulation of A2BAR post-infarction prevents remodeling of cardiac tissues [15]. Thus, as TNF-α is upregulated in atherosclerotic plaques and can induce expression of the A2BAR, and adenosine is released from cells under stress [1, 2, 5, 11, 12] and can inhibit VSMC proliferation [13, 14], we sought to understand the signaling pathway involved in TNF-α-induced upregulation of A2BAR in the context of VSMC.

One signaling mechanism downstream of TNF-α binding its receptor is the generation of reactive oxygen species (ROS) [16]. In the vasculature, NAD(P)H oxidase (Nox) enzymes are responsible for the majority of ROS production [17], and in VSMC Nox4 seems to be the dominant isoform expressed [18]. Recently, ROS generated by Nox4 were found to be required for VSMC to maintain their fully differentiated phenoptype [19]. Nox complexes are made up of multiple proteins, consisting of heme redox components, a 6 transmembrane protein with a carboxy terminus that binds FAD and NAD(P)H moieties to generate ROS [20], and a second membrane-bound component, p22phox [21, 22]. TNF-α stimulation was shown to upregulate p22phox in rat VSMC [23], and this component is required for Nox4 activity [24]. In addition, TNF-α specifically upregulates Nox4 in human aortic SMC and the authors suggest that Nox4 is the cause of ROS in the context of inflammatory conditions [25].

In this paper, we sought to elucidate the mechanism of TNF-α-induced upregulation of the A2BAR and propose that the generation of ROS by TNF-α-activated Nox4 regulates factors that induce expression of the A2BAR.

MATERIAL & METHODS

Vascular smooth muscle cell culture conditions

Aortic VSMC were isolated from neonatal Sprague-Dawley rats, as described previously [26, 27], and were seeded at a density of 1.5 × 105 cells per well of a 6-well plate; all experiments were performed with cells in their first passage. VSMC were cultured in VSMC growth medium containing DMEM, 10% bovine calf serum (BCS), 1 mM sodium pyruvate, 1 mM nonessential amino acids, 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Invitrogen, Carlsbad, CA).

Measurement of A2BAR mRNA expression

Total RNA from aortic VSMC was prepared with Trizol® (Invitrogen, Carlsbad, Ca) according to the manufacturer’s instructions [28]. One µg of RNA was used to make complementary DNA (cDNA), using the Superscript II kit (Invitrogen, Carlsbad, CA, cat# 18064-22) according to manufacturer’s instructions. A2BAR mRNA was quantified using ABI Gene Array TaqMan® primers (ABI, Foster City, CA, cat# Rn00567696_m1), and 18s rRNA (cat# 4319413E) with the TaqMan® Gene Expression Master Mix (ABI, Foster City, CA, cat# 4370048), using the ABI 7300 Real-Time PCR System. In experiments with inhibitors, cells were treated with the inhibitor at the indicated concentrations, with reapplication 24 hours later. When inhibitors were use in conjunction with another stimulus (Ad-β-gal, Ad-Nox4, Ad-DN-Nox4 adenoviruses, or TNF-α, inhibitors were incubated with cells for 1 hour before the stimulus was added.

Adenoviral transduction

Adenoviruses Ad-β-gal, Ad-NAD(P)H oxidase 4 (Nox4) and Ad-DN-Nox4 were a generous gift from Dr. Barry Goldstein, Thomas Jefferson University [29], and were used at a multiplicity of infection (MOI) of 50. In promoter construct transfection assays, adenoviral particles were added first and 3–6 hours later, the medium was replaced, followed by DNA transfection as described below.

Human growth hormone quantification

Three to four days post-transfection of plasmid using FuGENE 6®, 200 L of media was collected and quantification of human growth hormone (hGH) secreted into media was determined using the hGH ELISA kit® (Roche, Indianapolis, IN cat# 1585878) according to manufacturer’s instructions. Absorbance was read with a µQuant microplate reader (Bio-Tek Instruments, Winooski, VT).

β-Galactosidase enzymatic assay

β-Galactosidase (β-gal) assays were performed to quantify the amount of enzyme in the aorta of A2BAR-knockout/β-gal-knock-in (A2BAR-β-gal) mice [27]. Organs were snap frozen, ground and directly mixed with PMI solution (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, 50 mM β-mercaptoethanol (American Bioanalytical, Natick, MA, cat# AB01340) and 0.2 mg 2-nitrophenyl-β-D-galactopyranoside (ONPG, Sigma-Aldrich, St. Louis, MO, cat# N1127)) at a concentration of 150 µl PMI per 3 mg of protein. The solution was incubated in a 37°C water bath for 1–3 hours and stopped by addition of 0.5 mL 1M Na2CO3. Solution was analyzed on a spectrophotometer at 420 nm (O.D. typically in the range of 0.2 to 0.7).

In vivo TNF-α injection into mice

All mice used were bred in our facilities or purchased from Jackson Laboratories (Bar Harbor, ME). C57BL/6J wild-type mice (cat# 000664) were used as controls against the A2BAR-β-gal knockout mice. Mice were injected intraperitoneally with 150 µg/kg of recombinant mouse TNF-α and 16 hours after injection organs were isolated and subjected to β-galactosidase enzymatic activity assay, as described above, or subjected to RNA preparation.

Statistical analysis

Statistical significance was determined by a two-tailed Student’s t-test for unpaired variables assuming equal variance. Data were considered significant at p ≤ 0.05.

RESULTS AND DISCUSSION

Effect of an inflammation-inducing agent and TNF-α on A2BAR mRNA and promoter activity in vitro and in vivo

In addition to our findings describing the induciblity of the A2BAR gene in proliferating VSMC by the transcription factor B-Myb [30], a recent paper by Kong et al. identified HIF-1α as responsible for the induction of A2BAR expression in endothelial cells under hypoxic conditions [31]. In the context of atherosclerosis, proliferation and hypoxia are stress responses, and A2BAR activation has been shown to be protective in these instances [14, 31]. Inflammation also plays a role in atherosclerosis [32], and as the A2BAR has been shown to also have a protective effect in regard to inflammatory stress [33], we sought to investigate the influence of the inflammatory response on A2BAR gene expression. This was pursued by first using bacterial lipopolysaccharide (LPS) as an inflammation-inducing agent [34]. Figure 1A illustrates that LPS significantly increases A2BAR mRNA levels. LPS binds to toll-like receptors and elicits a signaling pathway that causes the release of inflammatory cytokines, such as TNF-α [27]. As shown in Figure 1B, TNF-α mimicked the effects seen with LPS. To investigate the A2BAR gene promoter activity directly, −5.81-hGH [30] was transfected into VSMC and effects of LPS and TNF-α were quantified. The A2BAR gene promoter activity was similarly increased when cells were treated with LPS and TNF-α (Figure 1C).

Figure 1
Upregulation of A2BAR mRNA in response to inflammatory stimuli. A. Rat aortic VSMC were serum starved prior to treatment with vehicle (control) or bacterial lipopolysaccharide (LPS). Total RNA was collected after 48 hours of exposure and subjected to ...

TNF-α upregulation of A2BAR mRNA in vivo

To examine the above effect in vivo, we utilized the A2BAR-β-gal mouse, in which the endogenous A2BAR gene promoter is driving the gene encoding for β-galactosidase (β-gal) [27]. According to previous results, the expression of the A2BAR gene in the vasculature is uneven, with very few patches in the aorta and a greater abundance in the mesenteric artery [27]. To determine if TNF-α upregulates A2BAR expression in vivo, mice were injected with recombinant mouse TNF-α, and 16 hours later the artery was isolated and protein lysate subjected to an assay for β-gal activity. Figure 2A illustrates that TNF-α significantly increases the endogenous A2BAR gene promoter by nearly 1.5-fold in vivo. Similarly, wild-type mice injected with TNF-α showed a significant induction of A2BAR mRNA in the artery, matching the expression pattern seen in the A2BAR-β-gal mouse (Figure 2B). These results show that the in vitro effects seen with A2BAR expression in cultured vascular smooth muscle cells and promoter construct activity are mimicked in the context of the mouse in vivo.

Figure 2
TNF-α-induced A2BAR mRNA upregulation in vivo. Mice were injected with PBS (Vehicle) or 150 g/kg TNF-α (TNF) and organs collected 16 hours post injection. A. Mesenteric artery from A2BAR-β-gal mice were snap frozen, pulverized, ...

Nox4 can mimic the effect of TNF-α on A2BAR mRNA and promoter activity

One downstream effect of TNF-α binding to its receptor on vascular smooth muscle cells is the activation and upregulation of NAD(P)H oxidase 4 (Nox4) [25]. In VSMC, Nox4 activity can be increased upon stimulation [17]. Consistent with previously published findings, exposure to TNF-α increased levels of ROS in VSMC [35], and addition of H2O2 alone induced activation of the A2BAR gene promoter (Supplementary Figure 1A & B). To determine if Nox4 can mimic the effect of TNF-α, we used an adenovirus to over-express Nox4 [29] in primary VSMC. Overexpression of Nox4 significantly increased the level of A2BAR mRNA as compared to control adenovirus (Figure 3).

Figure 3
Over-expression of Nox4 upregulates A2BAR mRNA. Primary aortic VSMC were serum starved, then transduced with adenovirus over-expressing NAD(P)H oxidase 4 (Nox4), a dominant-negative Nox4 (DN-Nox4) or control virus (Ad-β-gal). Total RNA was collected ...

Additionally we used an adenovirus that over-expresses a dominant-negative form of Nox4 that lacks the FAD binding domain (DN-Nox4) [29]. As compared to the wild-type, the dominant-negative form does not induce expression of A2BAR mRNA. Additionally, A2BAR mRNA expression in cells transduced with Ad-DN-Nox4 was significantly lower compared to cells transduced with Ad-β-gal (Figure 3). The data indicate that activity of Nox4 induces A2BAR mRNA expression. When the −5.81-hGH A2BAR gene promoter construct is transfected into VSMC over-expressing Nox4, the promoter activity is increased in the same pattern as that seen at the mRNA level (Supplementary Figure 2). The responses to over-expression of Nox4 mimic those observed with application of TNF-α, suggesting that Nox4 might be a component of the TNF-α-mediated regulation of A2BAR expression.

Effect of Nox inhibitors on A2BAR mRNA

To test if Nox activation mediates TNF-α effects on A2BAR gene expression, we used Nox inhibitors. Apocynin is a plant-derived chemical that functions as an inhibitor of Nox enzyme activity by preventing cytosolic components of the Nox complex from interacting with the membrane-bound components, thus rendering the enzyme inactive [36]. Diphenylene iodonium (DPI) is reduced by the flavin moieties in various ROS-generating enzymes, and subsequently forming a covalent bond with the flavin or adjacent amino acids within the Nox enzyme, thus rendering it inactive [37]. Primary aortic VSMC were pretreated with inhibitors for 1 hour followed by application of TNF-α, as detailed in Materials and Methods. Figure 4 illustrates that both apocynin and DPI reduced TNF-α-induced A2BAR mRNA expression, suggesting that the activation of Nox4 is upstream of A2BAR gene expression in this mechanism.

Figure 4
Inhibition of Nox4 activity prevent TNF-α-induced upregulation of A2BAR mRNA. VSMC were pretreated with vehicle (control), 500 uM apocynin or 2 uM DPI for 1 hour followed by addition of 20 ng/mL recombinant mouse TNF-α. The latter was ...

In vascular injury or in the formation of an atherosclerotic plaque, secretion of TNF-α from leukocytes and macrophages recruited to the inflamed area contributes to lesion formation, in part, by stimulating VSMC to proliferate and migrate into the lumen [1, 38, 39]. Adenosine is an autocrine and paracrine signaling molecule generated or released from cells under stress [2]. It has been shown to inhibit the release of TNF-α from cells stimulated by an immune response [3,4], which supports the conclusion that its signaling is generally tissue protective [5]. Adenosine inhibits TNF-α release from monocytes and macrophages by signaling through the A2AAR or A2BAR, both at baseline and in response to bacterial LPS [8, 40]. Furthermore, in a regulatory-loop fashion, A2BAR gene expression has been shown to be upregulated by TNF-α in astroglial and intestinal endothelial cells [10, 11]. Adenosine signaling through the A2BAR has also been found to inhibit smooth muscle cell proliferation [13, 14], and stimulation of A2BAR post-infarction prevents remodeling of cardiac tissues [15].

Considering that TNF-α induces expression of the A2BAR in some cell types [10, 11], and adenosine is released from cells under stress [1, 2, 5], which can inhibit VSMC proliferation [13, 14], we examined whether TNF-α affects A2BAR expression in VSMC. We found that TNF-α stimulated A2BAR gene promoter activity and increased mRNA expression in cultured cells (Figure 1), as well as in in vivo mouse models (Figure 2).

TNF-α stimulation can generate ROS by activating Nox enzymes [41]. Additionally, TNF-α signaling upregulates Nox4 gene expression in human aortic SMC, which is responsible for the generation of ROS under inflammatory conditions in these cells [25]. In accordance with this mechanism, when Nox4 was over-expressed, A2BAR mRNA and gene promoter activity were increased (Figure 3). Additionally, inhibitors of Nox activity prevented TNF-α-induced upregulation of A2BAR mRNA (Figure 4).

A study with endothelial cells showed that Nox enzymes are involved in TNF-α-induced expression of E-selectin, VCAM-I, and ICAM-I, which are adhesion molecules that aid in the recruitment of leukocytes to the inflamed or injured area [42]. The A2BAR-β-gal knockout mouse showed an increase in some of these same adhesion molecules [27], suggesting that the A2BAR has effects that oppose those of the TNF-α-induced-Nox activity in the regulation of these adhesion molecules. Future studies will focus on exploring this intriguing regulatory link.

Supplementary Material

02

ACKNOWLEDGEMENTS

We thank Dr. Barbara Schreiber and the cell core for preparation of smooth muscle cell cells and for insight, as well as Dr. Xu Yong for initial assistance with qPCR. This work was supported by National Institutes of Health (NIH) Public Health Services Grant HL13262. KR is an Established Investigator with the American Heart Association.

Footnotes

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