Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jan;18(1):14-23.
doi: 10.1091/mbc.e06-07-0596. Epub 2006 Oct 25.

Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2A receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter

Madhuri Ramanathan  1 , Grace Pinhal-Enfield, Irene Hao, Samuel Joseph Leibovich
Affiliations

Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2A receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter

Madhuri Ramanathan et al. Mol Biol Cell. 2007 Jan.

Abstract

Macrophages are an important source of vascular endothelial growth factor (VEGF). Adenosine A2A receptor (A2AR) agonists with Toll-like receptor (TLR) 2, 4, 7, and 9 agonists synergistically induce macrophage VEGF expression. We show here using VEGF promoter-luciferase reporter constructs that the TLR4 agonist Escherichia coli lipopolysaccharide (LPS) and the A2AR agonists NECA and CGS21680 synergistically augment VEGF transcription in macrophages and that the HRE in the VEGF promoter is essential for this transcription. We examined whether LPS and/or NECA induce HIF-1alpha expression. HIF-1alpha mRNA levels were increased in LPS-treated macrophages in an NF-kappaB-dependent manner; NECA strongly increased these levels in an A2AR-dependent manner. LPS induced luciferase expression from a HIF-1alpha promoter-luciferase construct in an A2AR-independent manner. Further stimulation with NECA did not increase HIF-1alpha promoter activity, indicating that the A2AR-dependent increase in HIF-1alpha mRNA is post-transcriptional. LPS/NECA treatment also increased HIF-1alpha protein and DNA binding levels. Deletion of putative NF-kappaB-binding sites from the VEGF promoter did not affect LPS/NECA-induced VEGF promoter activity, suggesting that NF-kappaB is not directly involved in VEGF transcription. Taken together, these data indicate that LPS/NECA-induced VEGF expression involves transcriptional regulation of the VEGF promoter by HIF-1alpha through the HRE. HIF-1alpha is transcriptionally induced by LPS and post-transcriptionally up-regulated in an A2AR-dependent manner.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Schematic representation of the mouse VEGF promoter-luciferase reporter construct and its derivative plasmids containing deletions of specific cis-elements from the VEGF promoter.
Figure 2.
Figure 2.
Coligation of TLR4 and A2ARs increases steady state levels of VEGF mRNA. Murine peritoneal macrophages were treated with LPS (100 ng/ml), NECA (1 μM), a combination of LPS and NECA, or hypoxia for 4, 8, 12, or 16 h. At the end of the incubation period, total RNA was isolated and subjected to TaqMan real-time PCR analysis as described in Materials and Methods. VEGF mRNA levels were normalized to the corresponding levels of endogenous cyclophilin D mRNA, and data were calculated as fold expression relative to the untreated control group. Hypoxia, a known inducer of VEGF expression, served as a positive control. Results reported are means ± SD for duplicate samples from at least three independent experiments.
Figure 3.
Figure 3.
Synergistic up-regulation of VEGF gene expression by LPS and NECA occurs at the transcriptional level and requires the hypoxia response element (HRE) within the VEGF promoter. RAW 264.7 cells were transiently transfected with (A) pVEGF-Luc, a luciferase reporter constructed by cloning a 1.3-kb fragment (−963 to +404) containing the promoter region of the mouse VEGF gene into the promoterless luciferase vector (pGL3-basic), (B) pVEGF-Luc or a series of luciferase reporter constructs each containing a VEGF promoter fragment lacking an individual cis-acting element, or (C) pHRE-Luc or pGL3-promoter vector. Cells were cotransfected with Renilla luciferase reporter plasmid phRL-TK to normalize for transfection efficiency. Transfectants were stimulated with LPS (100 ng/ml), NECA (1 μM), a combination of LPS and NECA, or hypoxia. After 24-h incubation, cells were lysed and assayed for firefly and Renilla luciferase activities. Hypoxia, a known inducer of VEGF expression, served as a positive control. Data were calculated as fold induction relative to the untreated control group. Results reported here are means ± SD for duplicate samples from at least three independent experiments.
Figure 4.
Figure 4.
LPS and NECA treatment of macrophages increases the steady state levels of HIF-1α mRNA. (A) Murine peritoneal macrophages were treated with LPS (100 ng/ml), NECA (1 μM), a combination of LPS and NECA, or hypoxia for 4, 8, 12, or 16 h. Total RNA was then isolated and subjected to TaqMan real-time PCR analysis as described in Materials and Methods. HIF-1α mRNA levels were normalized to the corresponding levels of endogenous cyclophilin D mRNA, and data were calculated as fold expression relative to the untreated control group. (B) Time course of VEGF and HIF-1α mRNA induction after the treatment of macrophages with LPS and NECA. (C) Murine peritoneal macrophages were treated with LPS (100 ng/ml), NECA (1 μM), a combination of LPS and NECA, or hypoxia for 12 h with or without BAY11-7085 (5 μM), or ZM241385 (1 μM). Total RNA was then isolated and subjected to TaqMan real-time PCR analysis as described above. Results reported are means ± SD for duplicate samples from at least three independent experiments.
Figure 5.
Figure 5.
Induction of the HIF-1α exon I.2 promoter by LPS and NECA. RAW264.7 cells were transfected with pHXN1aLuc, a luciferase reporter plasmid containing the murine HIF-1α exon I.2 promoter. Cells were cotransfected with Renilla luciferase reporter plasmid phRL-TK to normalize for transfection efficiency. Transfectants were treated with LPS (100 ng/ml), NECA (1 μM), a combination of LPS and NECA, or hypoxia for 20 h. In some cases, cells were treated with the A2AR antagonist ZM241385 (1 μM). Cells were lysed and assayed for firefly and Renilla luciferase activities. Data were calculated as fold induction relative to the untreated control group. Results reported here are means ± SD for duplicate samples from at least three independent experiments.
Figure 6.
Figure 6.
Treatment of macrophages with LPS and NECA leads to increased HIF-1α DNA binding activity. RAW 264.7 cells were treated with LPS (100 ng/ml) (L), NECA (1 μM) (N), a combination of LPS and NECA (L/N), or hypoxia (H). After incubation for 12 h, nuclear extracts were prepared. (A) For Western blot analysis, 50 μg protein were electrophoresed on SDS-PAGE, transferred to nitrocellulose membranes, and immunostained with a rabbit anti-HIF-1α polyclonal antibody. As a loading control, blots were immunostained with a rabbit anti-nucleoplasmin (NPM) antibody. (B) HIF-1α DNA-binding activity was quantified as described in Materials and Methods. HIF-1α bound to HIF-1–specific double-stranded oligonucleotides was detected by an ELISA-based assay using an mAb against HIF-1α. Hypoxia-induced samples were included as a positive control. Results are expressed as the fold increase of the absorbance at 450 nm over untreated control. Results reported here are means ± SD for duplicate samples from at least three independent experiments.
Figure 7.
Figure 7.
LPS/NECA-induced activation of the VEGF promoter-luciferase construct and the HRE reporter construct is blocked by the A2AR antagonist ZM241385, but not by the NF-κB inhibitor BAY11-7085. RAW 264.7 cells were transiently transfected with (A) pVEGF-Luc or (B) pHRE-Luc. Cells were cotransfected with Renilla luciferase reporter plasmid phRL-TK to normalize for the transfection efficiency. Transfectants were stimulated with LPS (100 ng/ml), NECA (1 μM), a combination of LPS and NECA, or hypoxia either in the presence or absence of BAY11-7085 (5 or 10 μM) or ZM241385 (1 μM). After 24-h incubation, cells were lysed and assayed for firefly and Renilla luciferase activities. Hypoxia, a known inducer of VEGF expression, served as a positive control. Data were calculated as fold induction relative to the untreated control group. Results reported here are means ± SD for duplicate samples from at least three independent experiments.
Figure 8.
Figure 8.
Inhibitor of NF-κB BAY11-7085 down-regulates LPS-induced TNF-α expression but not LPS/NECA-induced VEGF expression in murine peritoneal macrophages. Murine peritoneal macrophages were stimulated with LPS (100 ng/ml), NECA (1 μM), a combination of LPS and NECA, or hypoxia either in the presence or absence of BAY11-7085 (1 or 5 μM) or ZM241385 (1 μM). After 24-h incubation, conditioned media were harvested and assayed for (A) TNF-α and (B) VEGF protein levels by ELISAs. MTT assays were used to normalize the cytokine levels to cell viability. Data were calculated as fold induction relative to the untreated control group. Results reported here are means ± SD for duplicate samples from at least three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aderem A., Ulevitch R. J. Toll-like receptors in the induction of the innate immune response. Nature. 2000;406:782–787. - PubMed
    1. Akira S., Takeda K. Toll-like receptor signalling. Nat. Rev. Immunol. 2004;4:499–511. - PubMed
    1. Albina J. E., Mastrofrancesco B., Vessella J. A., Louis C. A., Henry W. L., Jr, Reichner J. S. HIF-1 expression in healing wounds: HIF-1alpha induction in primary inflammatory cells by TNF-alpha. Am J. Physiol. Cell Physiol. 2001;281:C1971–C1977. - PubMed
    1. Blouin C. C., Page E. L., Soucy G. M., Richard D. E. Hypoxic gene activation by lipopolysaccharide in macrophages: implication of hypoxia-inducible factor 1alpha. Blood. 2004;103:1124–1130. - PubMed
    1. Cho M., Hunt T. K., Hussain M. Z. Hydrogen peroxide stimulates macrophage vascular endothelial growth factor release. Am J. Physiol. Heart Circ. Physiol. 2001;280:H2357–H2363. - PubMed

Publication types

MeSH terms

LinkOut - more resources

-