Synergistic Up-Regulation of Vascular Endothelial Growth Factor (VEGF) Expression in Macrophages by Adenosine A2AReceptor Agonists and Endotoxin Involves Transcriptional Regulation via the Hypoxia Response Element in the VEGF Promoter

Oct 27, 2006Molecular biology of the cell

Adenosine A2A receptor activators and endotoxin together increase VEGF production in immune cells through low-oxygen response control

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Abstract

Adenosine A2A receptor (A2AR) agonists and the TLR4 agonist LPS may synergistically enhance macrophage VEGF expression.

TLR4 agonist LPS and A2AR agonists NECA and CGS21680 increase VEGF transcription in macrophages.
The hypoxia response element (HRE) in the VEGF promoter is critical for the transcriptional activation observed.
LPS treatment raises HIF-1alpha mRNA levels through an NF-kappaB-dependent mechanism, while NECA increases these levels via an A2AR-dependent pathway.
LPS can induce luciferase expression from a HIF-1alpha promoter independently of A2AR.
The increase in HIF-1alpha mRNA due to NECA appears to occur after transcription, suggesting a post-transcriptional regulatory mechanism.
Deletion of NF-kappaB-binding sites from the VEGF promoter does not alter VEGF promoter activity, indicating NF-kappaB's indirect role in VEGF transcription.

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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.

Synergistic Up-Regulation of Vascular Endothelial Growth Factor (VEGF) Expression in Macrophages by Adenosine A2AReceptor Agonists and Endotoxin Involves Transcriptional Regulation via the Hypoxia Response Element in the VEGF Promoter

Abstract

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