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Differential regulation of HIF-1alpha isoforms in murine macrophages by TLR4 and adenosine A(2A) receptor agonists.

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  • 1. Department of Cell Biology and Molecular Medicine and The Cardiovascular Research Institute, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
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    • Ramanathan M 1
    (1 author)

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Journal of Leukocyte Biology, 28 May 2009, 86(3):681-689
https://doi.org/10.1189/jlb.0109021 PMID: 19477908 PMCID: PMC2796622

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Abstract 

Adenosine A(2A)R and TLR agonists synergize to induce an "angiogenic switch" in macrophages, down-regulating TNF-alpha and up-regulating VEGF expression. This switch involves transcriptional regulation of VEGF by HIF-1, transcriptional induction of HIF-1alpha by LPS (TLR4 agonist), and A(2A)R-dependent post-transcriptional regulation of HIF-1alpha stability. Murine HIF-1alpha is expressed as two mRNA isoforms: HIF-1alphaI.1 and -I.2, which contain alternative first exons and promoters. HIF-1alphaI.2 is expressed ubiquitously, and HIF-1alphaI.1 is tissue-specific. We investigated the regulation of these isoforms in macrophages by TLR4 and A(2A)R agonists. HIF-1alphaI.1 is induced strongly compared with HIF-1alphaI.2 upon costimulation with LPS and A(2A)R agonists (NECA or CGS21680). In unstimulated cells, the I.1 isoform constituted approximately 4% of HIF-1alpha transcripts; in LPS and NECA- or CGS21680-treated macrophages, this level was approximately 15%, indicating a substantial contribution of HIF-1alphaI.1 to total HIF-1alpha expression. The promoters of both isoforms were induced by LPS but not enhanced further by NECA, suggesting A(2A)R-mediated post-transcriptional regulation. LPS/NECA-induced expression of HIF-1alphaI.1 was down-regulated by Bay 11-7085 (NF-kappaB inhibitor) and ZM241385 (A(2A)R antagonist). Although VEGF and IL-10 expression by HIF-1alphaI.1-/- macrophages was equivalent to that of wild-type macrophages, TNF-alpha, MIP-1alpha, IL-6, IL-12p40, and IL-1beta expression was significantly greater, suggesting a role for HIF-1alphaI.1 in modulating expression of these cytokines. A(2A)R expression in unstimulated macrophages was low but was induced rapidly by LPS in a NF-kappaB-dependent manner. LPS-induced expression of A(2A)Rs and HIF-1alpha and A(2A)R-dependent HIF-1alpha mRNA and protein stabilization provide mechanisms for the synergistic effects of LPS and A(2A)R agonists on macrophage VEGF expression.

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J Leukoc Biol. 2009 Sep; 86(3): 681–689.
Published online 2009 May 28. https://doi.org/10.1189/jlb.0109021
PMCID: PMC2796622
PMID: 19477908

Differential regulation of HIF-1α isoforms in murine macrophages by TLR4 and adenosine A2A receptor agonists

Madhuri Ramanathan,* Wenting Luo,* Balázs Csóka,† György Haskó,† Dmitry Lukashev,‡ Michail V. Sitkovsky,‡ and Samuel Joseph Leibovich*,1
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Abstract

Adenosine A2AR and TLR agonists synergize to induce an “angiogenic switch” in macrophages, down-regulating TNF-α and up-regulating VEGF expression. This switch involves transcriptional regulation of VEGF by HIF-1, transcriptional induction of HIF-1α by LPS (TLR4 agonist), and A2AR-dependent post-transcriptional regulation of HIF-1α stability. Murine HIF-1α is expressed as two mRNA isoforms: HIF-1αI.1 and -I.2, which contain alternative first exons and promoters. HIF-1αI.2 is expressed ubiquitously, and HIF-1αI.1 is tissue-specific. We investigated the regulation of these isoforms in macrophages by TLR4 and A2AR agonists. HIF-1αI.1 is induced strongly compared with HIF-1αI.2 upon costimulation with LPS and A2AR agonists (NECA or CGS21680). In unstimulated cells, the I.1 isoform constituted ~4% of HIF-1α transcripts; in LPS and NECA- or CGS21680-treated macrophages, this level was ~15%, indicating a substantial contribution of HIF-1αI.1 to total HIF-1α expression. The promoters of both isoforms were induced by LPS but not enhanced further by NECA, suggesting A2AR-mediated post-transcriptional regulation. LPS/NECA-induced expression of HIF-1αI.1 was down-regulated by Bay 11-7085 (NF-κB inhibitor) and ZM241385 (A2AR antagonist). Although VEGF and IL-10 expression by HIF-1αI.1−/− macrophages was equivalent to that of wild-type macrophages, TNF-α, MIP-1α, IL-6, IL-12p40, and IL-1β expression was significantly greater, suggesting a role for HIF-1αI.1 in modulating expression of these cytokines. A2AR expression in unstimulated macrophages was low but was induced rapidly by LPS in a NF-κB-dependent manner. LPS-induced expression of A2ARs and HIF-1α and A2AR-dependent HIF-1α mRNA and protein stabilization provide mechanisms for the synergistic effects of LPS and A2AR agonists on macrophage VEGF expression.

Keywords: endotoxin, VEGF, TNF-α, inflammation, transcription factors, alternative activation

Introduction

HIF-1 is a transcription factor involved in the expression of a number of oxygen-regulated genes such as VEGF [1, 2], erythropoietin [3], NO synthase 2 [4, 5], and glycolytic enzymes [6]. It is made up of two subunits, HIF-1α and HIF-1β (aryl hydrocarbon receptor nuclear translocator) [3]. Although HIF-1β is constitutively expressed, HIF-1α expression is regulated primarily by oxygen levels [7,8,9]. HIF-1α is believed to be constitutively transcribed and translated [9, 10]. However, under normoxic conditions, the HIF-1α protein is hydroxylated, recognized as a substrate for ubiquitination, and subsequently targeted for proteasomal degradation [11,12,13]. Hypoxia prevents this hydroxylation, thus blocking the proteasomal degradation of HIF-1α. Stabilized HIF-1α then dimerizes with HIF-1β and translocates to the nucleus, where it induces the activation of genes containing HREs in their promoters [14,15,16,17,18]. Increased expression of HIF-1α protein has been reported, even under normoxic conditions, by signaling pathways other than hypoxia [19,20,21,22,23,24,25]. For example, HIF-1α has been found to be induced in tumor cell lines by a variety of growth factors and hormones, including insulin, insulin-like growth factor, angiotensin II, and epidermal growth factor [25,26,27,28]. We have shown recently that in murine peritoneal macrophages, as well as in the RAW 264.7 macrophage-like cell line, coactivation of TLR4 and adenosine A2AR pathways results in increased expression of HIF-1α [29]. We have shown previously that this coactivation of TLR4 and A2AR results in an angiogenic switch in macrophages and RAW 264.7 cells that strongly up-regulates VEGF expression and simultaneously down-regulates TNF-α expression, constituting an M1- to M2-like switch [30, 31]. This up-regulation of VEGF is induced at the transcriptional level, and the HRE in the VEGF gene promoter is essential for VEGF expression via this pathway [29]. Costimulation of RAW 264.7 macrophages with a TLR4 agonist (LPS) and an A2AR agonist (CGS21680 or NECA) also induced increased luciferase expression from a HRE-luciferase reporter construct, suggesting that involvement of HIF-1α, and HIF-1α mRNA and protein as well as HIF-1α DNA-binding activity were strongly induced by LPS/NECA treatment but not by either of these agonists alone [29]. Wenger et al. [32, 33] reported that mouse HIF-1α can be expressed as two mRNA isoforms—HIF-1αI.1 and -I.2—containing two alternative first exons, and that two different promoters drive the transcription of these isoforms. The exon I.2 promoter is a housekeeping-type promoter, whereas the exon I.1 promoter shows tissue-specific regulation [32, 33]. Subsequent studies have shown that in TCR-activated T cells, the HIF-1αI.1 isoform is selectively up-regulated [34]. Deletion of the I.1 isoform was sufficient to markedly enhance the expression of inflammatory cytokines by activated T cells, suggesting that this isoform may function as a negative regulator of T cells and may attenuate their activation [35]. In this study, we examined the regulation of these two isoforms of mouse HIF-1α in macrophages by LPS and A2AR agonists. We also examined the contribution of the HIF-1αΙ.1 isoform to the synergistic up-regulation of VEGF and down-regulation of TNF-α induced by LPS and A2AR agonists in macrophages by using mice specifically engineered to lack the HIF-1α I.1 isoform.

MATERIALS AND METHODS

Reagents

NECA and CGS21680 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bay 11-7085 was purchased from Axxora, LLC (San Diego, CA, USA), and ZM241385 was purchased from Tocris-Cookson (Bristol, UK). Purified Escherichia coli LPS free of TLR2 agonists was a gift of Dr. Stefanie Vogel (University of Maryland, College Park, MD, USA).

Animals

C57Bl/6J mice (female, 7–8 weeks) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice lacking the HIF-1α I.1 isoform were backcrossed with C57Bl/6J mice for more than 10 generations and are congenic with C57Bl/6J mice. The animal experimentation protocols were approved by the NJMS Animal Care and Use Committee (Newark, NJ, USA).

Cell culture

Murine peritoneal macrophages were harvested from C57Bl/6J mice (7–8 weeks) as described previously [36]. Mice were injected i.p. with 2.5 ml thioglycolate broth, and 4 days later, peritoneal macrophages were harvested. RAW 264.7 (murine macrophage-like cell line) cells were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured as a monolayer in RPMI-1640 medium (Cellgro, Mediatech Inc., Herndon, VA, USA) supplemented with 10% FBS (Gemini Bio-Products, Calabasas, CA, USA), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Irvine Scientific, Santa Ana, CA, USA). The cells were incubated at 37°C in a humidified incubator in 5% CO2 and 95% air. All experiments were performed at a concentration of 1 × 106 cells/ml medium. Cells were plated 20–24 h prior to stimulation. The medium used for stimulation contained 1% FBS, and cells were stimulated with LPS (100 ng/ml), NECA (1 μM), CGS21680 (1 μM), a combination of LPS and NECA and LPS and CGS21680, or hypoxia (1% O2). For incubation of cells under hypoxic conditions, cultures were placed in a hypoxia chamber (Billups-Rothenberg, Del Mar, CA, USA), which was filled with a gas mixture of 1% O2, 5% CO2, and 94% N2. The sealed chamber was then placed in a 37°C incubator.

Extraction of RNA and real-time RT-PCR

Mouse peritoneal macrophages were plated in 60 mm dishes (4×106 cells/dish). Following overnight incubation, cells were treated with various reagents as indicated in the figure legends. RNA was isolated using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer’s protocol for isolation of total RNA from animal cells. cDNA was synthesized from 1 μg total RNA using TaqMan RT reagents (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Real-time PCRs were performed in an ABI Prism 7500 sequence detector using 1/10 vol each cDNA reaction and TaqMan Universal PCR Master Mix. The TaqMan gene expression assays for murine A2AR (Mm00802075_m1) and murine cyclophilin D (Mm00835365_g1) were purchased from Applied Biosystems. Isoform-specific primers and probes for TaqMan real-time PCR for murine HIF-1α Ι.1 and -I.2 mRNA isoforms were designed using Primer Express® 2.0 software (Applied Biosystems) in such a way that the amplicons contained murine HIF-1α exon I.1-exon 2 junction (GenBank Accession Numbers Y09086 and Y09085) and murine HIF-1α exon I.2-exon 2 junction (GenBank Accession Numbers Y13656 and Y09085), respectively. The primers and probes were synthesized at the Molecular Resource Facility at NJMS-University of Medicine and Dentistry of New Jersey (Newark, NJ, USA). 5′-Ends of the probes were labeled with the fluorescent dye FAM, and 3′-ends were coupled to the quencher molecule Black Hole Quencher™ dye-1. Sequences of primers and probes are presented in Table 1. TaqMan assays used to measure total HIF-1α mRNA levels have been described previously [29]. The real-time PCR reactions were carried out using the manufacturer’s protocol for absolute quantification. For each sample, expression levels for the transcripts of interest were normalized to that of endogenous cyclophilin D, and data were calculated as fold expression relative to the average of the untreated control group. Prior studies have indicated that cyclophilin D expression levels show minimal variation under the various activation conditions used in these experiments [29].

TABLE 1.

Primers and Probes Used for TaqMan Real-Time PCR

AssayHIF-1α isoform I.1
Forward primer5′-AATACATTTTCTCTGCCAGTTTTCTG-3′
Reverse primer5′-TTGCTGCATCTCTAGACTTTTCTTTT-3′
Probe
5′-FAM-CGTGTTAGAGCAATTAAAGGATGAGTTCTGAACGTC-BHQ-3′
AssayHIF-1α isoform I.2
Forward primer5′-CACCGATTCGCCATGGA-3′
Reverse primer5′-TTTCTTTTCGACGTTCAGAACTCAT-3′
Probe5′-FAM-CGGCGGCGAGAACGAGAAGAAA-BHQ-3′

Plasmids

pH1030Luc (a luciferase reporter construct containing the promoter of the murine HIF-1α I.1 isoform) and pHXN1aLuc (a luciferase reporter construct containing the promoter of the murine HIF-1α I.2 isoform) were kindly provided by Dr. Roland Wenger (University of Zurich, Switzerland). phRL-TK (Renilla luciferase reporter plasmid) was purchased from Promega Corp. (Madison, WI, USA).

Transient transfections and reporter assays

All plasmids were prepared using the PhoenIX Midiprep kit (Qbiogene, Carlsbad, CA, USA) and were electrophoresed to confirm that they were in the supercoiled form. RAW 264.7 cells were transiently transfected using Superfect (Qiagen, Valencia, CA, USA) as described previously [37]. Cells were cotransfected with phRL-TK (Promega Corp.) to normalize for transfection efficiency. Following 18 h incubation, the transfectants were resuspended in fresh medium and plated into six-well plates at a density of 0.625 × 106 cells/well, which were allowed to adhere for 6–7 h at 37°C. The medium was then changed to RPMI 1640 with 1% FBS, and the cells were stimulated as follows: LPS (100 ng/ml), NECA (1 μM), LPS/NECA, or hypoxia. Following 22 h incubation, cells were lysed with passive lysis buffer (Promega Corp.), and luciferase assays were performed using the dual-luciferase assay kit (Promega Corp.) following the manufacturer’s protocol. Luciferase light units were measured using an Lmax luminiscence microplate reader (Molecular Devices, Sunnyvale, CA, USA), using a dual injector system. Firefly luciferase light units were normalized to Renilla luciferase light units to normalize for transfection efficiency. The results reported are representative of at least three independent experiments.

Relative abundance of the two HIF-1α isoforms in macrophages stimulated with LPS and A2AR agonists

Total RNA was extracted from murine peritoneal macrophages stimulated with LPS, NECA, CGS21680, or a combination of LPS and NECA or LPS and CGS21680 for 10 h. The RNA samples were then reverse-transcribed, and exact copy numbers of the two HIF-1α isoforms were determined by TaqMan real-time PCR. A 195-bp DNA fragment encompassing the murine HIF-1α exon I.1-exon 2 junction and a 247-bp DNA fragment encompassing the murine HIF-1α exon I.2-exon 2 junction were PCR-amplified using primers flanking the region used for the TaqMan real-time PCR detection of the isoforms. The sequences of the primers are given in Table 2. The PCR products were quantified accurately and used to generate standard curves for the absolute quantification of individual isoforms by TaqMan real-time PCR.

TABLE 2.

Primers for PCR Amplification of Region Encompassing the Murine HIF-1α Εxon I.1-Exon 2 and Murine HIF-1α Exon I.2-Exon 2 Junction

HIF-1α exon I.1-exon 2
Forward primer5′-TTCCCCTCTCCTGTAAGCAA-3′
Reverse primer5′-GAAGTGGCAACTGATGAGCA-3′
HIF-1α exon I.2-exon 2
Forward primer5′-CGCCTCTGGACTTGTCTCTT-3′
Reverse primer5′-GAAGTGGCAACTGATGAGCA-3′

Expression of VEGF, TNF-α, MCP-1, MIP-2, IL-1β, IL-6, and IL-12p40 by macrophages from mice lacking the HIF-1α I.1 isoform

Macrophages from HIF-1α I.1−/− mice were incubated with RPMI-1640 medium containing 1% FBS, and cells were stimulated with LPS, NECA, CGS21680, a combination of LPS and NECA and LPS and CGS21680, or hypoxia (1% O2), as described above. Conditioned media were harvested 20 h following stimulation and assayed for VEGF content using a Quantikine M murine VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA). TNF-α, MCP-1, MIP-2, IL-1β, IL-6, and IL-12p40 content was assayed by ELISA using kits (R&D Systems), as described by the manufacturer. Samples were assayed in triplicate, and results are expressed as means ± sd.

RESULTS

Coligation of TLR4 and A2ARs causes a more pronounced and sustained increase in the steady-state levels of HIF-1α I.1 than HIF-1α I.2 mRNA

Murine peritoneal macrophages were stimulated with LPS, NECA, CGS21680, a combination of LPS and NECA or CGS21680, or hypoxia for 4, 8, 12, and 18 h. Total RNA was extracted, and TaqMan real-time RT-PCR analyses were performed for the quantification of mRNA levels for the HIF-1α isoforms I.1 and I.2 as well as total HIF-1α using primer/probe sets described in Materials and Methods. Hypoxia did not have any effect on the mRNA levels of both isoforms or of total HIF-1α. NECA, when added alone, did not increase mRNA levels for either isoform, whereas LPS caused a transient 5.8-fold and 3.8-fold peak increase in the mRNA levels for HIF-1α isoforms I.1 and I.2, respectively (Fig. 1, A and B). LPS/NECA treatment, however, caused a significantly higher induction of the HIF-1αI.1 isoform (27.7-fold) than the I.2 isoform at its peak (ninefold) at 8 h. By 12 h, the I.2 isoform mRNA levels dropped to sevenfold; however, the I.1 isoform mRNA levels increased further to 32.8-fold, indicating a strong and sustained induction of this isoform. Levels of total HIF-1α mRNA also peaked (9.9-fold) at 8 h after treatment with LPS/NECA and remained significantly high (9.4-fold) at 12 h, suggesting a significant contribution of the I.1 isoform to total HIF-1α mRNA levels and in sustaining the response (Fig. 1C).

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Steady-state levels of HIF-1α mRNA isoforms in macrophages costimulated with ligands for TLR4 and A2AR. Murine peritoneal macrophages were treated with LPS (100 ng/ml), CGS21680 (1 μM), NECA (1 μM), a combination of LPS and CGS21680 and LPS and NECA, or hypoxia for 4, 8, 12, or 18 h. At the end of the incubation period, total RNA was isolated and subjected to TaqMan real-time PCR using specific primers and probes for (A) HIF-1αI.1 isoform, (B) HIF-1αI.2 isoform, and (C) total HIF-1α. The mRNA levels for the HIF-1α isoforms and total HIF-1α were normalized to the corresponding levels of endogenous cyclophilin D mRNA, and data were calculated as fold expression relative to the untreated control group. Results reported are means ± sd for duplicate samples from a representative of at least three independent experiments.

LPS/NECA-induced expression of the HIF-1αI.1 isoform is more sensitive than the HIF-1αI.2 isoform to the NF-κB inhibitor Bay 11-7085 and the A2AR antagonist ZM241385

Murine peritoneal macrophages were stimulated with LPS, NECA, or a combination of LPS and NECA for 10 h in the presence or absence of the NF-κB inhibitor Bay 11-7085 or the A2AR antagonist ZM241385, which is a relatively specific A2AR antagonist at nanomolar concentrations (inhibitor constant in vitro for mouse A2AR of 1–10 nM) [38] but at a concentration of 1 μM, is also likely to inhibit A2BRs. Total RNA was extracted, and TaqMan real-time RT-PCR analyses were performed for the quantification of mRNA levels for the HIF-1αI.1 and -I.2 isoforms. LPS/NECA-induced expression of both isoforms of HIF-1α was inhibited by Bay 11-7085 and ZM241385 but to different degrees (Fig. 2, A and B). There was a 50% and a 71% decrease in LPS/NECA-induced expression of the I.1 isoform at concentrations of 2.5 μM and 5 μM Bay 11-7085, respectively. LPS/NECA-induced expression of isoform I.2, however, was not affected by 2.5 μM and was down-regulated only 35% by 5 μM Bay 11-7085. ZM241385 caused a 92.7% inhibition of LPS/NECA-induced expression of the I.1 isoform, whereas expression of isoform 1.2 was only down-regulated partially (54.7%). ZM241385 had little effect on the transient induction of either isoform by LPS alone. Taken together, these data suggest that LPS/NECA-induced expression of the I.1 isoform is highly dependent on activation of NF-κB as well as A2AR signaling, whereas expression of the 1.2 isoform is much less dependent on these mechanisms.

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Effect of NF-κB inhibitor Bay 11-7085 and the A2AR antagonist ZM241385 on LPS/NECA-induced expression of HIF-1αI.1 and -I.2 isoforms. Murine peritoneal macrophages were treated with LPS, NECA, or a combination of LPS and NECA for 10 h in the presence or absence of the NF-κB inhibitor Bay 11-7085 or the A2AR antagonist ZM241385. Total RNA was extracted, and TaqMan real-time RT-PCR analyses were performed for the quantification of mRNA levels for (A) HIF-1α I.1 and (B) HIF-1α I.2 isoforms. The mRNA levels for HIF-1α isoforms were normalized to the corresponding levels of endogenous cyclophilin D mRNA, and data were calculated as fold expression relative to the untreated control group. Results reported are means ± sd for duplicate samples from a representative of at least three independent experiments.

LPS induces up-regulation of the HIF-1αI.1 and -I.2 isoforms at the transcriptional level, whereas A2AR agonists act at the post-transcriptional level to augment LPS-induced expression

To determine whether synergistic up-regulation of HIF-1α isoforms I.1 and I.2 by LPS and NECA occurs at the transcriptional level, RAW 264.7 cells were transiently transfected with luciferase reporter plasmids pH1030Luc or pHXN1aLuc, containing the promoter regions of isoforms I.1 and I.2, respectively. The transfectants were treated with LPS, NECA, a combination of LPS and NECA, or hypoxia in the presence or absence of Bay 11-7085 or ZM241385. In control (untreated) transfectants, luciferase expression induced from the I.2 promoter was 10 times higher than that from the I.1 promoter (Fig. 3, A and B). LPS alone caused 2.4- and 3.3-fold induction of I.1 and I.2 promoters, respectively, suggesting that LPS up-regulates expression of both of the isoforms of HIF-1α at the transcriptional level. Costimulation of transfectants with LPS and NECA did not alter the activity of either promoter significantly beyond that observed with LPS alone, suggesting that A2AR signaling contributes to up-regulation of both isoforms of HIF-1α at the post-transcriptional rather than the transcriptional level. Similarly, treatment with Bay 11-7085 ZM241385 did not affect the activity of the promoters. As mentioned earlier, ZM241385 is a relatively specific A2AR antagonist at nanomolar concentrations but at the concentration tested here (1 μM), is also likely to inhibit A2BRs. Thus, the lack of effect of this inhibitor clearly indicates that adenosine receptor signaling does not play a role in the transcriptional activation of HIF-1α isoforms in these cells. Hypoxia had only a minor effect on the promoter activities of both of the isoforms (data not shown).

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Regulation of HIF-1αI.1 and -I.2 isoform promoter activities by LPS and NECA. RAW 264.7 cells were transiently transfected with (A) pH1030Luc (a luciferase reporter construct containing the promoter of the murine HIF-1α I.1 isoform) or (B) pHXN1aLuc (a luciferase reporter construct containing the promoter of the murine HIF-1α I.2 isoform). Cells were cotransfected with phRL-TK to normalize for transfection efficiency. Transfectants were stimulated with LPS (100 ng/ml), NECA (1 μM), or a combination of LPS and NECA in the presence or absence of the NF-κB inhibitor Bay 11-7085 or the A2R antagonist ZM241385. After 24 h incubation, cells were lysed and assayed for firefly and Renilla luciferase activities. Results are presented as relative luciferase units (RLU). Note that the y-axis scales are different in A and B. Results reported here are means ± sd for duplicate samples from a representative of at least three independent experiments.

The abundance of the HIF-1αI.1 isoform is increased relative to HIF-1αI.2 in macrophages stimulated with LPS and NECA

As the HIF-1α I.1 isoform was induced much more strongly by LPS/NECA than the I.2 isoform, but the basal I.2 promoter activity was 10 times stronger than the I.1 promoter activity, we compared the relative abundance of these two HIF-1α mRNA isoforms in macrophages. Absolute copy numbers were determined by TaqMan real-time PCR analysis, using engineered cross-exon-defined cDNA fragments for HIF-1αI.1 and -I.2 isoforms. Serial dilutions of defined concentrations of these fragments were prepared and used to construct PCR standard curves, which were then used to analyze the various reverse-transcribed mRNA samples from the treated macrophage cultures, providing their absolute molar concentrations. From these concentrations, the absolute copy numbers were then calculated (moles×Avogadro’s number, 6.023×1023). Absolute copy numbers are shown in Figure 4A, and copy numbers and relative percentages of each isoform calculated based on these numbers are presented in Table 3. Based on the determination of absolute copy numbers, the HIF-1α I.1 isoform comprised only ~4% of total HIF-1α transcripts in untreated cells. LPS treatment raised the proportion of this isoform to 7.8%. Although NECA or CGS21680 raised the level of the I.1 isoform transcripts only slightly (by ~1%), the I.1 isoform made up ~15% of total HIF-1α mRNA induced by LPS/NECA or LPS/CGS21680. This indicated clearly that although HIF-1α I.2 remains the major isoform in macrophages treated with LPS and A2AR agonists, the higher magnitude of induction of the I.1 isoform makes a significant contribution to the total HIF-1α expression. The relative fold-induction of the two isoforms is shown in Figure 4B.

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Relative abundance of the two HIF-1α isoforms in macrophages stimulated with LPS and A2AR agonists. Murine peritoneal macrophages were treated with LPS (100 ng/ml), NECA (1 μM), CGS21680 (1μM), or a combination of LPS and NECA for 10 h. TaqMan real-time RT-PCR analyses were performed for the quantification of mRNA levels for the HIF-1αI.1 and -I.2 isoforms. Levels of HIF-1α mRNA isoforms were normalized to the corresponding levels of endogenous cyclophilin D mRNA. The data are presented as (A) absolute copy number and (B) fold induction of mRNA expression relative to the untreated control group. Results reported are means ± sd for duplicate samples from a representative of at least three independent experiments.

TABLE 3.

Effects of LPS and A2AR Agonists on the Absolute Copy Number and Percentile Contribution of HIF-1αI.1 and I.2 mRNA Isoforms in Murine Macrophages

HIF-1αI.1
HIF-1αI.2
Copy # (A)a% TotalCopy # (B)a% Total
C1,085,284 ± 18654.1824,880,710 ± 527,85195.8
L7,563,632 ± 65,0137.8189,334,300 ± 79,34092.19
N1,862,354 ± 177,3215.2533,629,565 ± 392,42494.75
CGS1,645,278 ± 123,6144.9331,731,456 ± 321,43295.07
L/N40,034,256 ± 719,73815.05226,000,000 ± 6,560,00084.95
L/CGS38,456,213 ± 648,72114.98218,342,534 ± 5,721,34285.02
aMurine peritoneal macrophages were treated with medium alone as control (C), LPS (L; 100 ng/ml), NECA (N; 1 μM), CGS21680 (CGS; 1 μM), or a combination of LPS and NECA (L/N) or LPS and CGS21680 (L/CGS) for 10 h. Absolute copy numbers of HIF-1αI.1 and -I.2 were determined by TaqMan real-time PCR, using specific engineered cross-exon cDNA fragments. Serial dilutions of defined concentrations of these fragments were prepared and used to construct PCR standard curves, which were then used to analyze the concentrations of each HIF-1α isoform in the reverse-transcribed mRNA samples from the macrophage cultures, providing their absolute molar concentrations. Levels of HIF-1α mRNA isoforms were normalized to the corresponding levels of endogenous cyclophilin D mRNA. From these concentrations, the absolute copy numbers were then calculated (moles×Avogadro’s number, 6.023×1023). Relative percentages of each isoform were then calculated based on these numbers [e.g., % HIF-1αI.1=A×100/(A + B)]. 

Treatment of macrophages with LPS increased the steady-state levels of A2AR mRNA, and A2AR agonists did not affect LPS-induced A2AR mRNA expression

Murine peritoneal macrophages were stimulated with LPS, NECA, CGS21680, or a combination of LPS and NECA or CGS21680 for 2, 4, and 8 h. Total RNA was extracted, and mRNA levels for the A2AR were determined by TaqMan real-time RT-PCR analysis. A2AR expression was strongly induced by LPS, causing a 28-fold increase in the A2AR mRNA levels in 2 h (Fig. 5). Costimulation with NECA or CGS21680 did not significantly alter the A2AR mRNA levels induced by LPS. NECA alone caused a slight (less than threefold) and transient increase. LPS-induced expression of A2AR mRNA was sustained for at least 8 h, showing an ~40-fold increase over untreated control. (Data for CGS21680 not shown.)

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Steady-state levels of adenosine A2AR mRNA in macrophages stimulated with LPS and NECA. Murine peritoneal macrophages were treated with LPS (100 ng/ml), NECA (1 μM), or a combination of LPS and NECA for 2, 4, or 8 h. At the end of the incubation period, total RNA was isolated and subjected to TaqMan real-time PCR using specific primers and probes for murine A2AR. The mRNA levels for A2AR were normalized to the corresponding levels of endogenous cyclophilin D mRNA, and data were calculated as fold expression relative to the untreated control group. Results reported are means ± sd for duplicate samples from a representative of at least three independent experiments.

Effect of lack of the HIF-1α I.1 gene on LPS and A2AR agonist-induced regulation of VEGF, IL-10, TNF-α, MIP-1α, IL-1β, IL-12p40, IL-6, MCP-1, and MIP-2 expression

Murine peritoneal macrophages from HIF-1α I.1−/− and HIF-1αI.1+/+ mice were stimulated with LPS, NECA, CGS21680, a combination of LPS and NECA or CGS21680, or hypoxia for 20 h. Conditioned media were then assayed for their content of VEGF, IL-10, TNF-α, MIP-1α, IL-1β, IL-6, IL-12p40, MCP-1, and MIP-2 by ELISA. Figure 6 compares the levels of these factors produced by macrophages from wild-type versus knockout mice, respectively. VEGF was expressed at low levels by HIF-1αI.1+/+ and HIF-1αI.1−/− macrophages, and its expression was not affected significantly by LPS. NECA and CGS21680 induced VEGF expression strongly by LPS-treated wild-type and knockout macrophages to a similar extent. IL-10 induction by LPS was greater in HIF-1αI.1−/− than in HIF-1αI.1+/+ macrophages, and its expression was increased to a similar extent by CGS21680 or NECA in wild-type and knockout cells. LPS induced TNF-α expression strongly by HIF-1αI.1+/+, and this induction was markedly higher in HIF-1αI.1−/− macrophages. NECA and CGS21680 suppressed LPS-induced TNF-α expression strongly by wild-type and knockout macrophages. A similar pattern was observed for MIP-1α, IL-1β, IL-6, and IL-12p40. MCP-1 and MIP-2 were up-regulated strongly by LPS in wild-type macrophages, but their expression was only increased weakly in knockout cells. Also, LPS-induced MCP-1 and MIP-1α expression was only suppressed weakly by CGS21680 or NECA in both cell types. VEGF expression by HIF-1α I.1−/− macrophages and HIF-1α I.1+/+ mice was increased markedly to a similar extent under hypoxic conditions. None of the other factors were induced by hypoxia in wild-type or knockout macrophages.

An external file that holds a picture, illustration, etc. Object name is zgb0090949250006.jpg

Production of VEGF, TNF-α, and several cytokines by macrophages from wild-type and HIF-1αI.1−/− mice in response to LPS and A2AR agonists. Murine peritoneal macrophages were treated with LPS (100 ng/ml), NECA (1 μM), CGS21680 (1 μM), a combination of LPS and NECA and LPS and CGS21680, or hypoxia (1% O2) for 20 h. At the end of the incubation period, conditioned media were harvested and assayed for VEGF, TNF-α, IL-10, MIP-1α, IL-6, IL-12p40, IL-1β, MCP-1, and MIP-2 content by ELISA. Results reported are means ± sd for triplicate samples from a representative of three independent experiments.

DISCUSSION

Wenger et al. [32, 33] showed that murine HIF-1α can be expressed as two isoforms: a long form (I.2) and a shorter isoform (I.1) containing an alternate first exon, promoter, and 5′-untranslated region. The I.1 isoform is 12 aa shorter than the ubiquitously expressed I.2 isoform. The promoter of the major HIF-1α I.2 isoform shows features of a housekeeping-type promoter, whereas the I.1 isoform is expressed in a tissue-specific manner. The inducible nature of the I.1 isoform was suggested when it was shown to be up-regulated selectively during the late stages of mouse spermiogenesis [39] as well as in TCR-activated CD4+ and CD8+ T cells [34]. Furthermore, deletion of the I.1 isoform was sufficient to markedly enhance the expression of inflammatory cytokines by activated T cells, suggesting that this isoform may function as a negative regulator of T cells and may attenuate their activation [35]. Although the I.1 isoform was shown to play a predominant role in activated T cells, this isoform constitutes <30% of total HIF-1α mRNA in these cells [35].

We have reported previously that in murine peritoneal macrophages, activation of TLR2, -4, -7, and -9, along with the adenosine A2AR, synergistically up-regulates VEGF expression and down-regulates expression of inflammatory cytokines such as TNF-α and IL-12 [31]. This has been termed an “angiogenic switch” and has features of an M1- to M2-like alternative activation pathway [31, 40]. We then showed that the synergistic induction of VEGF gene expression occurs by the transcriptional activation of the VEGF promoter via the HRE [29], and the HRE activation, under normoxic conditions, is brought about by the increased expression of ΗΙF-1α induced by the synergistic action of LPS and NECA. A2AR-mediated, normoxic induction HIF-1 in macrophages was also confirmed recently by Ponti et al. [41], who found that adenosine and A2AR agonists induced a transient, rapid up-regulation of HIF-1α expression in J774A.1 cells at the transcriptional and post-transcriptional levels in a protein kinase C- and PI3K-dependent but iron-independent manner. Our further investigations reveal that the LPS/NECA or LPS/CGS21680-combined treatment of peritoneal macrophages and RAW 264.7 cells causes a strong up-regulation of total HIF-1α mRNA expression, which peaks at 8–12 h. LPS and NECA alone are much weaker inducers, and the combined action of LPS and A2AR agonists (NECA or CGS21680) is required for the strong and sustained induction of HIF-1α expression. As the HIF-1αI.1 and -I.2 isoforms were shown previously to be differentially regulated in activated T cells, thereby modulating the adaptive immune response [34, 35], in the present study, we investigated whether the adenosine A2AR-mediated angiogenic switch of TLR-induced macrophages involves differential regulation of these HIF-1α isoforms. Using isoform-specific TaqMan real-time PCR assays, we showed that both HIF-1α isoforms were up-regulated by coligation of TLR4 and A2AR; however, the I.1 isoform was induced much more strongly than the I.2 isoform. The HIF-1αI.1 isoform constituted only ~4% of the total HIF-1α transcripts in unstimulated cells; however, in LPS/A2AR agonist-treated macrophages, this level was elevated to ~15% of the total, suggesting that under these conditions, the I.1 isoform contributes substantially to the expression of total HIF-1α. As I.1 and I.2 HIF-1α isoforms were shown to display similar transcriptional activity [34], selective activation of the I.1 isoform by this pathway is likely to contribute significantly to LPS/A2AR agonist-induced VEGF expression.

In activated T cells, expression of HIF-1α I.1 was shown to be rapid and independent of de novo protein synthesis and thus, was shown to follow the pattern of immediate early response gene expression [34]. In the present study, LPS was found to up-regulate the expression of both HIF-1α mRNA isoforms rapidly (within 4 h), and NECA or CGS21680 was found to further enhance and sustain the LPS-induced mRNA levels. Comparison of the luciferase activities driven by the promoters of these two isoforms indicated that the basal promoter activity of the HIF-1αI.2 isoform is much stronger than that of the I.1 isoform. Activities of both of the promoters were induced by LPS, but costimulation with NECA did not enhance the promoter activities further, suggesting that A2AR signaling up-regulates the expression of both isoforms at the post-transcriptional level.

To determine the functional significance of the increased relative abundance of the HIF-1αI.1 isoform, macrophages from mice that lack the HIF-1αI.1 isoform were studied for their ability to respond to LPS and the A2AR agonists CGS21680 and NECA. VEGF expression by HIF-1αI.1−/− macrophages in response to LPS, NECA, CGS21680, LPS/NECA, and LPS/CGS21680, as well as to hypoxia, was similar to that of HIF-1αI.1+/+ macrophages, suggesting that the HIF-1αI.1 isoform does not play a crucial role in the regulation of expression of this factor. Similarly, IL-10 expression was unaffected by the absence of the HIF-1αI.1 isoform. LPS induction of TNF-α expression was, however, elevated significantly in HIF-1αI.1−/− macrophages, and the suppression of TNF-α expression by CGS21680 or NECA was similar in wild-type and HIF-1αI.1−/− macrophages. Likewise, LPS-induced expression of MIP-1α, IL-1β, IL-6, and IL-12p40 was markedly greater in HIF-1αI.1−/− macrophages and was suppressed to a similar extent by CGS21680 or NECA. Thus, the HIF-1αI.1 isoform appears to play a significant role in regulating macrophage expression of several inflammatory cytokines, including TNF-α, MIP-1α, IL-1β, IL-6, and IL-12p40, as suggested previously in activated T cells [35]. However, this isoform does not appear to play a significant role in the regulation of VEGF or IL-10 expression. The expression of TNF-α and many inflammatory cytokines in macrophages is generally NF-κB-dependent. Interdependent roles for HIF-1 and NF-κB have been reported previously in the regulation of hypoxic responses [42,43,44]. The results presented here suggest that the HIF-1αI.1 isoform plays a significant role in modulating the NF-κB-dependent induction of inflammatory cytokines and that lack of this isoform results in a markedly more robust expression of these cytokines in response to LPS. The mechanism for this effect is not yet clear. In regard to the mechanism of A2AR-mediated suppression of TNF-α and other inflammatory cytokines, this is also not entirely clear but appears to be regulated post-transcriptionally at the level of mRNA stability and/or at the translational level [31, 45, 46]. This suppressive effect is not affected by the lack of the HIF-1αI.1 isoform.

Expression of HIF-1αI.1 by LPS/NECA was down-regulated strongly by the NF-κB inhibitor Bay 11-7085 and by the A2AR antagonist ZM241385. We, in this study, and others [46] have shown that in macrophages, LPS induces A2AR expression rapidly in a NF-κB-dependent manner. HIF-1α is constitutively expressed in unstimulated cells, but NECA does not induce increased HIF-1α mRNA expression in the absence of LPS stimulation. This suggests that the LPS-induced expression of A2ARs might be an essential step in the LPS/NECA-mediated up-regulation of HIF-1α and thus, of VEGF expression in macrophages. It seems likely that LPS plays a dual role in this synergy, first, by up-regulating the expression of the HIF-1α gene at the transcriptional level in a NF-κB-independent manner and second, by up-regulating expression of A2ARs in a NF-κB-dependent manner, thereby allowing A2AR agonists to trigger the pathway leading to the stabilization of HIF-1α transcripts and protein. The results reported here suggest that the HIF-1αI.1 isoform is a negative regulator of inflammatory cytokine expression not only in T cells (35, 47, 48), but also in myeloid cells. How adenosine receptor activation brings about the stabilization of HIF-1α mRNA and protein is not yet clear and is the subject of future studies. Similarly, how the HIF-1αI.1 isoform, which is preferentially induced by the LPS/A2AR-dependent pathway, modulates the expression of inflammatory cytokines, such as TNF-α, MIP-1α, IL-1β, IL-6, and IL-12p40, is not yet clear and is the subject of further study.

ACKNOWLEDGMENTS

This work was supported by a grant from the U.S. Public Health Service (RO1-GM068636). The reporter plasmids pH1030Luc and pHXN1aLuc were kindly provided by Professor Roland H. Wenger (University of Zurich). The TLR4 agonist-purified E. coli LPS free of TLR2 agonists was kindly provided by Dr. Stefanie Vogel (University of Maryland).

Footnotes

Abbreviations: CGS21680=2-[p-(2-carboxylethyl)-phenylethyl amino]-5′-N-ethyl-carboxamido-adenosine, HIF-1=hypoxia-inducible factor 1, HRE=hypoxia response element, NECA=5′-N-ethyl-carboxamido-adenosine, NJMS=New Jersey Medical School, VEGF=vascular endothelial growth factor

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