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Am J Physiol Heart Circ Physiol 295: H1177-H1181, 2008. First published July 18, 2008; doi:10.1152/ajpheart.91513.2007
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Stimulation of ecto-5'-nucleotidase in human umbilical vein endothelial cells by lipopolysaccharide

R. W. S. Li, Ricky Y. K. Man, Paul M. Vanhoutte, and George P. H. Leung

Department of Pharmacology, The University of Hong Kong, Hong Kong

Submitted 21 December 2007 ; accepted in final form 11 July 2008


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
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The involvement of ecto-5'-nucleotidase (E-5'Nu) in the elevation of extracellular adenosine during inflammation is unclear. In the present study, the effect of lipopolysaccharide (LPS), an inflammation inducer, was investigated on E-5'Nu in human umbilical vein endothelial cells (HUVECs). E-5'Nu activity was enhanced after a 24 h exposure to LPS. This effect was dose dependent, with an EC50 of 1.66 ng/ml. At 10 µM, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 abolished the LPS-induced E-5'Nu activity. However, at 10 µM, the NF-{kappa}B inhibitor ammonium pyrrolidine dithiocarbamate had no effect. LPS upregulated the protein expression but not the messenger RNA expression of E-5'Nu. The inhibition of E-5'Nu by 100 µM {alpha},β-methylene adenosine-5'-diphosphate increased the LPS-induced inflammation, suggesting that E-5'Nu plays a significant role in reducing inflammation, probably through the generation of adenosine. In conclusion, the experiments indicate that LPS upregulates E-5'Nu activity in HUVECs through a PI3K-dependent increase in the abundance of E-5'Nu on cell membranes. Since adenosine is an anti-inflammatory molecule, E-5'Nu upregulation may be crucial in protecting endothelial cells against inflammatory damage.

adenosine; cytokines; inflammation

ECTO-5'-NUCLEOTIDASE (E-5'Nu), also known as CD73, is a 70-kDa membrane-bound glycoprotein that is abundant in the endothelium (18). E-5'Nu extracellularly catalyzes the conversion of purine and pyrimidine ribo- and deoxyribonucleoside monophosphates to their corresponding ribo- and deoxynucleosides. For example, adenosine 5'-monophosphate (AMP) is one of the substrates of E-5'Nu, and adenosine is the corresponding end product (9, 21).

Adenosine exerts its effects by interacting with four receptors known as A1, A2A, A2B, and A3 receptors (25, 27). In addition to its well-known vasodilator and cardioprotective effects (20, 23, 25), adenosine can also serve as an immunosuppressive agent to minimize tissue damage during inflammation (13, 22, 25). The stimulation of A2A receptors downregulates neutrophil functions (25), reduces the recruitment of neutrophil and macrophages to the endothelium (15), and inhibits the production of tumor necrosis factor (TNF)-{alpha} in peripheral mononuclear white blood cells (4).

Endogenous adenosine is increased during inflammation. Since part of the adenosine is derived from the catalytic action of E-5'Nu, it is plausible that E-5'Nu activity is subject to change by inflammation and may play an important role in the regulation of the extracellular adenosine level during inflammation. TNF-{alpha}, which is a proinflammatory cytokine, decreases the expression and activity of E-5'Nu (9), suggesting that less adenosine may be produced to relieve inflammation if E-5'Nu is downregulated. By contrast, another study documented that another proinflammatory cytokine, IFN-{alpha}, upregulates the expression and activity of E-5'Nu in endothelial cells (18). Therefore, the regulation of E-5'Nu in inflammation remains controversial, and the enzyme may be regulated differentially by various inflammatory mediators. Therefore, the present study investigated the effects of lipopolysaccharide (LPS), which is widely used as an inducer of inflammation (1, 19) on E-5'Nu in human umbilical vein endothelial cells (HUVECs).


   MATERIALS AND METHODS
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RESULTS
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Cell culture. HUVECs obtained from American Type Culture Collection (Manassas, VA) were cultured in Ham's Kaighn's Modification F12K medium (Invitrogen, Carlsbad, CA) supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin, 0.1 mg/ml heparin, and 0.05 mg/ml endothelial cell growth supplement. Cells were incubated in 75-cm2 culture flasks at 37°C in an atmosphere containing 5% CO2-95% room air. The medium was changed every 3 or 4 days.

Measurement of E-5'Nu activity. The biochemical assay of E-5'Nu activity was modified from a previously published protocol (26). The assay is based on the measurement of inorganic phosphate, which is generated from the metabolism of AMP (Sigma-Aldrich, St. Louis, MO) by E-5'Nu. Inorganic phosphate forms a colored product after a reaction with ammonium molybdate. In brief, HUVECs were grown to confluence in six-well plates. The medium was removed from each well, and the adherent cells were washed using phosphate-free HEPES-buffered Ringer solution. Reaction buffer (1.5 ml of phosphate-free HEPES-buffered Ringer solution with 1 mM AMP) was added to each well, and the plate was incubated at 37°C for 10 min. The reaction was stopped by 0.3 ml of 30% trichloroacetic acid. Ammonium molybdate (4.5 ml) was then added. After incubation at 45°C for 20 min, the absorbance at 750 nm was measured. The inorganic phosphate production inhibited by 100 µM {alpha},β-methylene adenosine-5'-diphosphate (AOPCP; Sigma-Aldrich), a specific E-5'Nu inhibitor, represented the E-5'Nu activity.

RNA isolation and reverse transcription-polymerase chain reaction. RNA of HUVECs was isolated by use of TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Isolated RNA was reverse transcribed into cDNA using SuperScript First-Strand Synthesis System (Invitrogen). Primers for amplifying E-5'Nu (accession number, NM_002526) were sense 5'-ACCAAGGTTCAGCAGATC-3' (corresponding to nucleotides 244–262) and antisense 5'-CAGTGATTTCATCTTCAAAC-3' (corresponding to nucleotides 624–644), which yielded a PCR product of 401 bp. PCR was conducted in a GeneAmp PCR system 9700 thermocycler (Applied Biosystems, Foster City, CA) with the following parameters: denaturation at 94°C for 30 s, and annealing at 55°C for 1 min and 72°C for 1.5 min. Reactions were carried out for 30 cycles. β-Actin served as an internal control. Primers for amplifying human β-actin (accession number, NM_001101) were sense 5'-ATGGATGATGATATCGCC-3' (corresponding to nucleotides 74–91) and antisense 5'-GTACTTCAGGGTGAGGATGC-3' (corresponding to nucleotides 261–280), which yielded a PCR product of 207 bp. PCR products were loaded onto a 1% agarose gel for analysis, and 0.01% ethidium bromide was added to the gel for visualization of PCR products under ultraviolet illumination. To semiquantify the PCR products of E-5'Nu, the optical density of the E-5'Nu band was normalized to that of β-actin.

Western blot analysis. HUVECs were scraped from 10-cm2 petri dishes and collected by centrifugation at 5,000 rpm for 5 min. Cell pellets were resuspended in lysis buffer (5 mM sodium monophosphate, pH 8) containing a 1:1,000 (vol/vol) dilution of a cocktail of protease inhibitors (Sigma-Aldrich) and were lysed by sonication for 30 s. The protein content of each sample was measured by the Bradford protein assay (Bio-Rad, Hercules, CA). Samples were then resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel, and the proteins were electrotransfered to a polyvinyl difluoride membrane. The membrane was blocked by 5% nonfat dry milk in phosphate-buffered saline (PBS) for 2 h, followed by incubation with a 1:200 (vol/vol) dilution of polyclonal anti-E-5'Nu antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or monoclonal anti-VCAM-1 antibodies (Sigma-Aldrich) at 4°C overnight. Monoclonal anti-β-actin antibodies (Santa Cruz Biotechnology) diluted 1:2,000 (vol/vol) were used as an internal control. After the membrane was washed three times with PBS (10 min each time), the membranes were incubated with a 1:2,000 (vol/vol) dilution of either horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies for E-5'Nu (Santa Cruz Biotechnology) or conjugated goat anti-mouse secondary antibodies for β-actin and VCAM-1 (Santa Cruz Biotechnology) for 1.5 h at room temperature. The membrane was washed three times with PBS and immersed in Western Blot Chemiluminescence Reagent Plus (New Life Science Products, Boston, MA) for 1 min. The light emitted from the membrane was captured on film during an appropriate exposure time. The optical density of E-5'Nu was normalized to that of β-actin.

Determination of IL-8 release by enzyme-linked immunosorbent assays. HUVECs were grown to confluence in 24-well plates. After treatments with Escherichia coli 0111:B4 LPS (10 µg/ml, Sigma-Aldrich) for 24 h, the medium bathing the cells was collected. The amount of IL-8 released from HUVECs was determined by human CXCL8/IL-8 DuoSet ELISA development kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Statistical analyses. Data were expressed as means ± SE. Student's t-test and analysis of variance were used for paired and multiple variants, respectively. A P value < 0.05 was considered as indicative of a statistically significant difference.


   RESULTS
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E-5'Nu activity in HUVECs. The effect of LPS on E-5'Nu activity in HUVECs was time dependent (Fig. 1A). E-5'Nu activity increased after incubation with LPS (10 ng/ml) for 24 h but was not changed at shorter incubation times (3, 6, and 18 h). The effect of LPS on E-5'Nu activity was also dose dependent (Fig. 1B) with an EC50 of 1.66 ± 0.08 ng/ml.


Figure 1
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  		Fig. 1. Effect of LPS on ecto-5'-nucleotidase (E-5'Nu) activity in human umbilical vein endothelial cells (HUVECs). A: HUVECs were treated with 10 ng/ml LPS for 0 (control) to 24 h, and E-5'Nu activities were measured. B: HUVECs were incubated without (control) or with various concentrations of LPS (1 pg/ml to 10 µg/ml) for 24 h, and E-5'Nu activities were measured. Values are means ± SE of 6 sets of experiments performed in triplicate. *P < 0.05 vs. control.

 
Experiments were then carried out to investigate the signaling pathway involved in the LPS-induced E-5'Nu activity. LY-294002 (10 µM; Sigma-Aldrich), a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the LPS-induced E-5'Nu activity (P < 0.05) (Fig. 2). However, ammonium pyrrolidine dithiocarbamate (10 µM; Sigma-Aldrich), a NF-{kappa}B inhibitor, increased the basal activity of E-5'Nu by 17% but had no effect on the LPS-induced increase in E-5'Nu activity (Fig. 3).


Figure 2
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  		Fig. 2. Effect of phosphatidylinositol 3-kinase (PI3K) inhibitor on LPS-induced E-5'Nu activity in HUVECs. HUVECs were treated without (control) or with LPS (10 ng/ml) for 24 h in the absence or presence of LY-294002 (10 µM, a specific PI3K inhibitor). E-5'Nu activities were measured. Values are means ± SE of 4 sets of experiment performed in triplicate. *P < 0.05, LPS vs. control.

 

Figure 3
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  		Fig. 3. Effect of NF-{kappa}B inhibitor on LPS-induced E-5'Nu activity in HUVECs. HUVECs were treated without (control) or with LPS (10 ng/ml) for 24 h in the absence or presence of ammonium pyrrolidine dithiocarbamate (APDC, 10 µM, a specific NF-{kappa}B inhibitor). E-5'Nu activities were measured. Values are means ± SE of 4 sets of experiment performed in triplicate. *P < 0.05, LPS vs. control; ^P < 0.05, APDC vs. control; #P < 0.05, LPS + APDC vs. APDC.

 
Messenger RNA and protein expressions of E-5'Nu. To investigate whether the LPS-induced E-5'Nu activity was due to the upregulation of mRNA and/or protein expression of E-5'Nu, RT-PCR and Western blot analysis were performed, respectively. The mRNA expression of E-5'Nu in HUVECs was not significantly changed after treatment with LPS (Fig. 4, A and B). By contrast, the protein expression of E-5'Nu was increased 2.8-fold (Fig. 4, C and D).


Figure 4
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  		Fig. 4. Effect of LPS on mRNA and protein expressions of E-5'Nu in HUVECs. HUVECs were incubated without (control) or with LPS (10 ng/ml) for 24 h. A: semiquantitative RT-PCR analysis of E-5'Nu mRNA expression in HUVECs with β-actin used as a reference. B: amount of E-5'Nu mRNA normalized to that of β-actin. C: Western blot analysis of E-5'Nu protein expression in HUVECs with β-actin used as a reference. D: amount of E-5'Nu protein normalized to that of β-actin. Values are means ± SE of 3 separate experiments. *P < 0.05 vs. control.

 
E-5'Nu inhibitor on IL-8 release and VCAM-1 expression. The roles of E-5'Nu on IL-8 (a cytokine) release and VCAM-1 (an adhesion molecule) expression were studied. The LPS-induced release of IL-8 was increased by 24% when E-5'Nu in HUVECs was blocked pharmacologically by AOPCP (100 µM, Fig. 5). The LPS-induced release of IL-8 was increased by the same extent with ZM-241385 (1 µM), an adenosine A2A receptor antagonist. However, the effects of AOPCP and ZM-241385 were not additive. Similar to IL-8 release, when E-5'Nu was inhibited by AOPCP, LPS-induced VCAM-1 expression was increased from 3.1- to 4.1-fold (Fig. 6).


Figure 5
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  		Fig. 5. Effect of inhibition of E-5'Nu and adenosine A2A receptors on LPS-induced IL-8 release in HUVECs. IL-8 release was measured in HUVECs treated with LPS (10 ng/ml) for 24 h in the absence (control) or presence of adenosine-5'-diphosphate (AOPCP, 100 µM, an E-5'Nu inhibitor), ZM-241385 (1 µM, an adenosine A2A receptor inhibitor) or both AOPCP and ZM-241385. Values are means ± SE of 4 sets of experiment performed in triplicate. *P < 0.05 vs. control.

 

Figure 6
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  		Fig. 6. Effect of inhibition of E-5'Nu on LPS-induced VCAM-1 expression in HUVECs. HUVECs were treated without (control) or with LPS (10 ng/ml) for 24 h in the absence or presence of AOPCP (100 µM, an E-5'Nu inhibitor). Top: Western blot analysis of VCAM-1 protein expression in HUVECs with β-actin used as a reference. Bottom: amount of VCAM-1 protein normalized to that of β-actin. Values are means ± SE of 3 separate experiments. *P < 0.05 vs. control.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The extracellular level of adenosine is elevated during inflammation, but the mechanism(s) leading to the increased adenosine are not fully understood. The present study shows that E-5'Nu activity is increased after the treatment of LPS, a known inflammation inducer (1, 19). This finding is similar to the observation that E-5'Nu is induced by hypoxia to increase extracellular adenosine, which enhances endothelial barrier function through the activation of adenosine A2B receptors (7). Since inflammation shares similar pathogenesis properties with hypoxia (10), it is not surprising that E-5'Nu activity is also increased during inflammation.

The stimulation of endothelial cells by LPS results in the activation of TNF-receptor-associated factor-6, followed by the activation of several signaling pathways such as NF-{kappa}B and PI3K (6). The present results demonstrate that the LPS-induced E-5'Nu activity is abolished by a PI3K but not by a NF-{kappa}B inhibitor, indicating that LPS-induced E-5'Nu activity is predominantly mediated through a PI3K-dependent pathway. Although the NF-{kappa}B pathway is seemingly not involved in the LPS-induced E-5'Nu activity, the present experiments demonstrate that blockade of NF-{kappa}B increased the basal activity of E-5'Nu. Therefore, it is likely that NF-{kappa}B is a transcriptional factor that may suppress the transcription of the gene encoding E-5'Nu. This notion is supported by studies that have demonstrated that anti-inflammatory effects of methotrexate result from the activation of E-5'Nu through suppression of NF-{kappa}B (14, 16).

The reduction of E-5'Nu activity by TNF-{alpha} involves the loss of E-5'Nu on cell surfaces, suggesting that E-5'Nu activity is proportional to the abundance of E-5'Nu protein on cell surfaces (9). The abundance of E-5'Nu is controlled by transcriptional induction, posttranslational modification, rate of protein endocytosis, or rate of delivery to the cell surface (2). In the present study, an elevation of E-5'Nu activity occurred only if HUVECs were incubated with LPS for at least 24 h. Such a slow response implies that the increased E-5'Nu activity likely is due to an increased presence of E-5'Nu. Consistent with this suggestion, the present Western blot results demonstrated a LPS-mediated increased HUVECs E-5'Nu protein expression. Since the mRNA expression of E-5'Nu in HUVECs was not significantly increased after treatment with LPS, the increased E-5'Nu abundance on the HUVECs cell membrane is not due to transcriptional regulation. The involvement of posttranslational modification or protein endocytosis remains to be investigated.

Adenosine can inhibit the LPS-induced release of proinflammatory cytokines and expression of adhesion molecules such as IL-8 and VCAM-1 (3, 12) through the activation of endothelial adenosine A2A receptors (3, 24). Therefore, the upregulation of E-5'Nu, which is supposed to increase the extracellular adenosine level, can be regarded as a self-protective mechanism that attenuates tissue damage during inflammation (22). A role of E-5'Nu in reducing inflammation can be deduced from the present observation that AOPCP (an E-5'Nu inhibitor) increased the LPS-induced release of IL-8 and ICAM-1 expression in HUVECs. Our data are consistent with a previous observation that E-5'Nu knockout mice lose the innate mechanism to attenuate tissue inflammation (11). Moreover, another study suggested that the upregulation of E-5'Nu by IFN-{alpha} stimulation, shown to increase the generation of adenosine, could be one of the body's own defense mechanisms against inflammation (18). Furthermore, the anti-inflammatory effects of methotrexate and sulfasalazine are diminished by specific E-5'Nu inhibitors (16, 17). Taken in conjunction with the earlier findings, the present results allow the conclusion that E-5'Nu significantly contributes to the limitation of an inflammation response. The anti-inflammatory role of E-5'Nu is likely to be mediated through the production of adenosine. This notion is supported by our observation that ZM-241385 (an adenosine A2A receptor antagonist) increased the LPS-induced IL-8 release as AOPCP did. More importantly, the effects of ZM-241385 and AOPCP were not additive, indicating that the two inhibitors work in the same pathway, which probably involves adenosine. The source of substrate for E-5'Nu is AMP, which is produced through the breakdown of ATP by nucleoside triphosphate diphosphohydrolase. AMP and ATP are supplied to endothelial cells mainly via polymorphonuclear leukocytes (7), neutrophils (5), and lymphoid tissues (29). In addition, endothelial cells are able to release ATP and AMP locally (2). Nucleoside triphosphate diphosphohydrolase activity was not examined in the present study, since AMP is not a limiting factor for E-5'Nu (8, 18, 28).

In conclusion, the present study demonstrates that E-5'Nu activity in HUVECs is increased by LPS. The upregulation of E-5'Nu activity is due to an increase in E-5'Nu protein, probably through a PI3K-dependent signaling pathway. The increase of E-5'Nu activity may play a significant role in the production of adenosine, which then serves as an anti-inflammatory mechanism to protect the tissues.


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This study is supported by the Research Grant Committee Earmarked Grants of Hong Kong Special Administrative Region (project code, 769607), and Seed Funding for basic research (The University of Hong Kong).


   ACKNOWLEDGMENTS
 
We thank Queenie Ting for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. P. H. Leung, Dept. of Pharmacology, The Univ. of Hong Kong, Rm. 51, 2/F, Laboratory Block, Faculty of Medicine Bldg., 21 Sassoon Rd., Pokfulam, Hong Kong (e-mail: gphleung{at}hkucc.hku.hk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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