Ecto-5'-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia

Abstract

Under conditions of limited oxygen availability (hypoxia), multiple cell types release adenine nucleotides in the form of ATP, ADP, and AMP. Extracellular AMP is metabolized to adenosine by surface-expressed ecto-5'-nucleotidase (CD73) and subsequently activates surface adenosine receptors regulating endothelial and epithelial barrier function. Therefore, we hypothesized that hypoxia transcriptionally regulates CD73 expression. Microarray RNA analysis revealed an increase in CD73 and ecto-apyrase CD39 in hypoxic epithelial cells. Metabolic studies of CD39/CD73 function in intact epithelia revealed that hypoxia enhances CD39/CD73 function as much as 6 +/- 0.5-fold over normoxia. Examination of the CD73 gene promoter identified at least one binding site for hypoxia-inducible factor-1 (HIF-1) and inhibition of HIF-1alpha expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible CD73 expression. Studies using luciferase reporter constructs revealed a significant increase in activity in cells subjected to hypoxia, which was lost in truncated constructs lacking the HIF-1 site. Mutagenesis of the HIF-1alpha binding site resulted in a nearly complete loss of hypoxia-inducibility. In vivo studies in a murine hypoxia model revealed that hypoxia-induced CD73 may serve to protect the epithelial barrier, since the CD73 inhibitor alpha,beta-methylene ADP promotes increased intestinal permeability. These results identify an HIF-1-dependent regulatory pathway for CD73 and indicate the likelihood that CD39/CD73 protects the epithelial barrier during hypoxia.

Figure 1

Induction of epithelial CD73 by hypoxia. (a) Confluent T84 monolayers were exposed to normoxia (pO₂ 147 torr, 18 hours) or hypoxia (pO₂ 20 torr, 18 hours). Total RNA was isolated, and CD73 mRNA levels were determined by RT-PCR using semiquantitative analysis (increasing cycle numbers, as indicated). As shown, β-actin transcript was determined in parallel and used as an internal standard. (b) Confluent T84 monolayers were exposed to indicated periods of hypoxia (with or without actinomycin D [Act. D], as indicated), monolayers were washed, surface proteins were biotinylated, and cells were lysed. CD73 was immunoprecipitated with mAb 1E9 and resolved by SDS-PAGE, and resultant Western blots were probed with avidin-peroxidase. Representative experiments of three are shown in each case.

Figure 2

Functional increase in CD73 surface activity by hypoxia. (a) Epithelial monolayers were exposed to indicated periods of hypoxia and washed, and surface CD73 activity was determined by HPLC analysis of E-AMP conversion to E-ADO (black bars). To determine specificity, a similar analysis was performed in the presence of the CD73 inhibitor APCP (white bars). Data are derived from five to seven monolayers in each condition, and results are expressed as mean percent E-AMP conversion ± SEM. The inset is a representative HPLC tracing demonstrating peak resolution of E-AMP and E-ADO. mAu, milli-absorbance unit. (b) Confluent T84 monolayers were subjected to hypoxia for 24 hours and treated with 2 mM EDTA for 5 minutes, followed by incubation in cell culture media with normo-calcium in the presence of indicated concentrations of 5′-AMP. TER was monitored over time, and the results shown represent the percent recovery of TER over 2 hours relative to no 5′-AMP. Also shown are plots of monolayers coincubated with the adenosine A_2B receptor antagonist alloxazine (allox; 10 μM). Data are mean ± SEM from three separate experiments. *P < 0.025 compared with normoxia.

Figure 3

Induction of functional CD39 by hypoxia. (a) Confluent T84 monolayers were exposed to normoxia (pO₂ 147 torr, 18 hours) or hypoxia (pO₂ 20 torr, 18 hours). Total RNA was isolated, and CD39 mRNA levels were determined by RT-PCR using semiquantitative analysis (increasing cycle numbers, as indicated). As shown, β-actin transcript was determined in parallel and used as an internal standard. (b) More quantitative real-time PCR was employed to directly compare hypoxia-inducibility of CD39 and CD73. Data were calculated relative to internal control genes (β-actin) and are expressed as fold increase over normoxia ± SEM at each indicated time. Results are derived from two experiments in each condition. (c) Epithelial monolayers were exposed to indicated periods of hypoxia and washed, and surface CD39 activity was determined by HPLC analysis of E-ATP conversion to E-AMP. Data are derived from five to seven monolayers in each condition, and results are expressed as mean percent E-AMP conversion ± SEM.

Figure 4

CD73 luciferase reporter assays. (a) The orientation of the HIF-1 and CREB binding sites in the CD73 luciferase reporter constructs and the location of truncations used for transient transfections (see Methods for details). WT, wild-type. (b) Confluent BAE monolayers were transiently transfected with plasmids expressing sequence corresponding to full-length CD73 (pGL2^2.0NT, bp –1902 to +63) or to the 5′ truncations pGL2^1.1NT (bp –993 to +63), pGL2^0.57NT (bp –518 to +63), or pGL2^0.15NT (bp –92 to +63), as well as with the SV40 promoter upstream from the luc reporter gene. Twelve hours later, cells were exposed to hypoxia or normoxia for 48 hours and assessed for luciferase activity. All transfections were normalized to cotransfected β-galactosidase activity. Data are mean ± SEM from three separate experiments.*P < 0.01, significantly different from normoxia; **P < 0.025, significantly different than other hypoxia conditions.

Figure 5

Role of HIF-1 and CREB in CD73 hypoxia-inducibility. (a) Confluent BAE monolayers were exposed to mock treatment (Ctl), HIF-1α sense oligonucleotides (S), or HIF-1α antisense oligonucleotides (AS) for 48 hours. Total protein was solubilized, and HIF-1α expression was examined by Western blot. Nx, normoxia. (b) Confluent BAE monolayers were transiently transfected with plasmids expressing sequence corresponding to truncations at the 5′ end (pGL2^0.57NT, bp –518 to +63) in the presence or absence of HIF-1α sense or antisense oligonucleotides. Twelve hours later, cells were exposed to hypoxia for 48 hours and assessed for luciferase activity. Data are mean ± SEM from three separate experiments. **P < 0.01. (c) Confluent BAE monolayers were transiently transfected with plasmids expressing sequence corresponding to truncations at the 5′ end (pGL2^0.57NT, bp –518 to +63) or plasmids encoding HIF-1 or CRE mutations, as indicated. Twelve hours later, cells were exposed to hypoxia or normoxia for 48 hours and assessed for luciferase activity. All transfections were normalized to cotransfected β-galactosidase activity. Data are mean ± SEM from three separate experiments. *P < 0.025 compared with the corresponding nonmutated control; **P < 0.01 compared with normoxia.

Figure 6

Role of hypoxia-induced CD73 in vivo. BL/6/129 mice were gavaged with vehicle (PBS) or APCP (2 mg/100 g body weight, based on previous work in rats [ref. 27]) in combination with permeability tracer (60 mg/100 g body weight of FITC-labeled dextran, mol wt 4,000 daltons, as indicated) and exposed to ambient hypoxia (8% O₂, 92% N₂) or ambient room air for 4 hours. (a) Small-intestine hypoxia was monitored by localization of EF5 relative to the normoxia control. Also shown is a stain control (primary antibody omitted). (b) Total RNA was isolated from mucosal scrapings (enriched with epithelial cells), and CD73 mRNA levels were determined by RT-PCR using semiquantitative analysis (increasing cycle numbers, as indicated). As shown, β-actin transcript was determined in parallel and used as an internal standard. (c) Serum analysis of FITC concentration was performed as an indicator of intestinal permeability. Data are mean permeability ± SEM pooled from four to six animals per condition. *P < 0.025 compared with normoxia in vehicle controls; **P < 0.05 for APCP treatment compared with animals exposed to vehicle only.