Adenosine A2A receptor is a unique angiogenic target of HIF-2alpha in pulmonary endothelial cells

Abstract

Hypoxia, through the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha (HIFs), induces angiogenesis by up-regulating a common set of angiogenic cytokines. Unlike HIF-1alpha, which regulates a unique set of genes, most genes regulated by HIF-2alpha overlap with those induced by HIF-1alpha. Thus, the unique contribution of HIF-2alpha remains largely obscure. By using adenoviral mutant HIF-1alpha and adenoviral mutant HIF-2alpha constructs, where the HIFs are transcriptionally active under normoxic conditions, we show that HIF-2alpha but not HIF-1alpha regulates adenosine A(2A) receptor in primary cultures of human lung endothelial cells. Further, siRNA knockdown of HIF-2alpha completely inhibits hypoxic induction of A(2A) receptor. Promoter studies show a 2.5-fold induction of luciferase activity with HIF-2alpha cotransfection. Analysis of the A(2A) receptor gene promoter revealed a hypoxia-responsive element in the region between -704 and -595 upstream of the transcription start site. By using a ChIP assay, we demonstrate that HIF-2alpha binding to this region is specific. In addition, we demonstrate that A(2A) receptor has angiogenic potential, as assessed by increases in cell proliferation, cell migration, and tube formation. Additional data show increased expression of A(2A) receptor in human lung tumor cancer samples relative to adjacent normal lung tissue. These data also demonstrate that A(2A) receptor is regulated by hypoxia and HIF-2alpha in human lung endothelial cells but not in mouse-derived endothelial cells.

Fig. 1.

Effect of hypoxia, HIF stabilizers, and adenoviral HIF-1α and HIF-2α on adenosine A_2A receptor steady-state mRNA levels. (A) Primary HLMVECs were exposed to air (21% O₂), fetal/physiologic hypoxia (3% O₂), or severe hypoxia (0% O₂) for 24 h. Total RNA was isolated, and 15 μg were loaded per well. Blots were probed with cDNA for human A_2A receptor, A_2B receptor, and 28S rRNA, in that order. (B) Primary HLMVECs were exposed to hypoxia for different time periods. Whole-cell lysates were made, and a total of 100 μg was resolved on a 4–15% gel. Blots were probed with adenosine A_2A antibody followed by β-actin antibody after stripping the blot. (C) Primary HLMVECs and primary HCAECs from 2 different donors were treated with 200 μM DFO, 1 mM DMOG, or 200 μM CoCl₂ for 24 h. Blots were probed with cDNA from human A_2A, followed by stripping and then probing with 28S rRNA. (D) Primary HLMVECs and HPAECs were transduced with 15 pfu per cell of either Ad.mutHIF-1α, Ad.mutHIF-2α, or Ad.LacZ. Twenty-four hours after transduction, total RNA was isolated, and 15 μg was loaded per well. Blots were probed with cDNA for human A_2A receptor, followed by 28S. The age of the cell donor is indicated in years (y). The order of presentation of the groups shown in A, C, and D has been rearranged from the same original Northern blot. (E) Primary HLMVECs were transfected with siRNA targeted against HIF-1α, HIF-2α, or the nontargeting control. Twenty-four hours after transfection, cells were exposed to severe hypoxia (0% O₂) or air (21% O₂) for an additional 24 h. After exposure, total RNA was isolated and real-time RT-PCR performed by using Taqman primers and probes for adenosine A_2A receptor. ∗, Statistical difference from the control expression in air. Δ, Statistical difference from the Control, siControl, and siHIF-1α samples in hypoxia.

Fig. 2.

Identification of HIF-2α response element-binding site in the adenosine A_2A receptor promoter. (A) Luciferase assay showing A_2A receptor promoter activity in HLMVECs and HEK293 cells. ∗, Statistical difference from baseline controls. †, Statistical difference from empty vector activity. (B) Sequence of the A_2A receptor promoter showing response elements in bold and primers used for ChIP PCR amplification as underlined. (C) HLMVECs were exposed to air (Nx:21% O₂) or hypoxia (Hx:1% O₂) for 6 h, after which cells were fixed, chromatin immunoprecipitated with anti-HIF-2α antibody or the control nonspecific antibody, and DNA isolated from the bound complexes. DNA from ChIP was PCR amplified by using specific primers corresponding to the adenosine A_2A receptor response element present in the R5 promoter. As a positive control, the PGK-1 hypoxia-inducible factor response element was also PCR-amplified.

Fig. 3.

Adenosine A_2A receptor activation promotes cellular proliferation, migration, and angiogenic activity in primary HLMVECs. (A) Serum-starved cells were treated with varying concentrations of the adenosine A_2A receptor agonist CGS-21680 for 24 h in the presence of [³H]thymidine at 1 μCi/mL. (B) Cells were transduced with 10 pfu per cell Ad.LacZ or Ad.A_2A receptor. Cells were then serum starved for 24 h and incubated with [³H]thymidine at 1 μCi/mL for an additional 24 h. After [³H]thymidine incorporation, cells were washed and incorporated radioactivity determined in cell lysates. ∗, Statistically significant difference from the nontransduced control cells. Δ, Statistical difference from the Lac-Z transduced controls. (C) Nontransduced, Ad.A_2A receptor-transduced, or Ad.LacZ-transduced primary HLMVECs (100,000 per well in a 24-well plate) were plated on fibronectin-coated inserts in serum-free medium. Positive represents nontransduced cells incubated in complete EBM-2 medium containing growth factors and serum. After incubation for 24 h, the lower sides of the inserts were stained with crystal violet and scanned for photography (Magnification: 1×). Photographs were taken of randomly selected fields, and the number of cells per field was quantitated. ∗, Statistically significant difference from the nontransduced and Ad.LacZ-transduced control cells. (D) Primary HLMVECs (100,000 per well in a 24-well plate) were plated on Matrigel (BD Biosciences) in the presence or absence of the A_2A receptor agonist, CGS-21680 (1 μM). After incubation for 4 h, fields were randomly selected and photographed (Magnification: 10×). Representative picture shows angiogenic tube formation. Branch points were calculated from 11 random fields from each group and plotted. ∗, Statistically significant difference from control cells.

Fig. 4.

Adenosine A_2A receptor expression is increased in lung tumor samples at different stages. (A) Human lung cancer expression panels (I and IV) containing reverse-transcribed cDNA from the different tumor stages were probed with real-time Taqman primers and probes for adenosine A_2A receptor and CD31. The A_2A expression data were normalized to CD31 expression. The different tumor stages were grouped together and plotted after log transformation. ∗, Statistical difference from the normal lung tissue using a 2-sample t test. (B) Normalized A_2A expression data were plotted against the different stages of tumors. ANOVA was used to compare the groups, followed by pairwise comparisons of normal tissue versus each of the other groups using Dunnett's procedure. ∗, Statistically different from the normal lung tissue. (C) CC1–CC4 show significant positive CD31 (PECAM-1; pinkish-red) immunostaining in the bronchovascular bundles (CC1–CC3) and parenchyma (CC3 and CC4) of normal (atelectatic) lung tissue obtained at surgery. Staining of venous and/or lymphatic vessel endothelium (CC1, arrow) and arterial vessel endothelium (CC2 and CC3, arrowheads) was variable but notably positive, as was the capillary endothelium (CC3 and CC4). Immunostaining for adenosine receptor A_2A (brown) was largely absent from these tissues. By contrast, lung cancer specimens from both of these patients (CT1, CT2, CT3, and CT4) stained markedly positive, especially in the tumor stroma. In these regions, CD31-positive-staining cells, likely endothelial cells and/or tumor macrophages, coimmunostained positively for adenosine receptor A_2A or, in some cases, A_2A-positive cells were immediately adjacent to those staining positively for CD31 (Magnification: 40×).