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Regulation of adenosine receptor engagement by ecto-adenosine deaminase¹

TOMOKO HASHIKAWA^*,, SCOTT W. HOOKER^*, JERZY G. MAJ^*,2, CHRISTOPHER J. KNOTT-CRAIG, MASAHIDE TAKEDACHI, SHINYA MURAKAMI and LINDA F. THOMPSON^*,3

^* Oklahoma Medical Research Foundation, Immunobiology and Cancer Research Program, Oklahoma City, Oklahoma, USA;
Department of Periodontology, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japan; and
Department of Surgery, Oklahoma University Health Sciences Center, Oklahoma City, Oklahoma, USA

³Correspondence: Oklahoma Medical Research Foundation, 825 N.E. 13th St., Oklahoma City, OK 73104, USA. E-mail: Linda-Thompson{at}omrf.ouhsc.edu

SPECIFIC AIMS

CD26 is a multifunctional ecto-enzyme that cleaves dipeptides from the amino terminus of polypeptides that have either L-alanine or L-proline in the penultimate position, transduces activation signals to T cells, and binds to adenosine deaminase (ADA). CD26-transfected Jurkat and Molt 4 clones were established to determine the role of ecto-ADA in controlling extracellular adenosine (Ado) levels and thus adenosine receptor (AR) signaling. Our hypothesis was that if ecto-ADA regulated extracellular Ado levels, then the ADA inhibitor 2'-deoxycoformycin (dCF) should increase the cAMP response to Ado.

PRINCIPAL FINDINGS

1. Jurkat and Molt 4 cells transfected with CD26 bind ecto-ADA
Jurkat and Molt 4 cells were transfected with CD26, and ecto-ADA binding was monitored with a goat anti-ADA antibody. Only transfected cells bound ecto-ADA. ADA binding could be increased such that CD26 became saturated with ADA by incubating the transfectants with an exogenous source of ADA such as a lysate of Jurkat cells. Confocal microscopy revealed that ecto-ADA and CD26 were colocalized on the cell surface of CD26 transfectants.

2. The kinetics of the cAMP response to Ado are different for Jurkat and Molt 4
Jurkat cells express A1R and A2bR; Molt 4 cells express A1R, A2aR, and A2bR. Both cell types exhibited increased concentrations of intracellular cAMP in response to extracellular Ado. The kinetics of the cAMP response differed between the two cell lines, being maximal for Jurkat cells at 1 min but only after 5 min for Molt 4.

3. Ecto-ADA can inhibit the cAMP response of CD26 transfectants to Ado
The ability of ecto-ADA to inhibit the cAMP response of CD26 transfectants to Ado was demonstrated by an increase in cAMP levels in the presence of the potent and specific ADA inhibitor dCF (Fig. 1 ). The degree to which ecto-ADA inhibited cAMP responses depended on the extent to which CD26 was saturated with ADA and the kinetics of the cAMP response. Thus, dCF had little effect on the cAMP response of CD26-transfected Jurkat cells to Ado (Fig. 1a , left bars) unless they were pretreated with an exogenous source of ADA (Jurkat lysate). Although these cells expressed high levels of CD26, when cultured at low density they expressed low levels of ecto-ADA. This, compounded with their rapid cAMP response of 1 min, limited the ability of ecto-ADA to degrade extracellular Ado and influence the cAMP response. Treatment of CD26-transfected Jurkat cells with an exogenous source of ADA, such that CD26 was saturated with ADA, caused a 40% reduction in the cAMP response (Fig. 1a , right bars). Further treatment with dCF increased the cAMP response by 1.3-fold. In contrast, dCF increased the cAMP response of CD26-transfected Molt 4 cells by >twofold, even without exposure to an exogenous source of ADA (Fig. 1b , left bars). When CD26 was saturated with ecto-ADA, the cAMP response was inhibited by >80%; the further addition of dCF increased the cAMP response by 15-fold (Fig. 1b , right bars). These differences between CD26-transfected Jurkat and Molt 4 cells could not be explained by differences in the levels of CD26 or ecto-ADA expression. Rather, the slower kinetics of the cAMP response of Molt 4 cells is a more likely explanation (5 min vs. 1 min for Jurkat cells). ADA enzyme activity assays showed that the ADA activity of CD26-transfected Jurkat cells was similar to that of CD26-transfected Molt 4. However, since the cAMP response of Jurkat cells was much faster than that of Molt 4, there was insufficient time for ecto-ADA on Jurkat cells to inhibit the cAMP response by degrading a significant amount of Ado.

View larger version (38K): [in this window] [in a new window]   Figure 1. Regulation of cAMP responses by ecto-ADA. CD26-transfected Jurkat (a) or Molt 4 (b) were preincubated with the phosphodiesterase inhibitor Ro 20-1724 (10 µM) for 10 min at 37°C, then incubated with or without Jurkat lysate for 30 min at 37°C and stimulated with 20 µM Ado for 1 min (Jurkat) or 5 min (Molt 4) at 37°C in the presence or absence (Ctrl) of 5 µM dCF. Data are the mean ± SD of 4 cAMP determinations and are representative of 3 separate experiments.

4. Ecto-ADA can inhibit the cAMP response of human neonatal thymocytes to Ado
Human thymocytes also expressed CD26 and ecto-ADA. Although the level of CD26 expression was much lower than on transfected Jurkat or Molt 4 cells, CD26 on freshly isolated human neonatal thymocytes was saturated with ecto-ADA. When thymocytes were incubated with Ado at high density (2x10⁸ cells/assay) to mimic the situation in thymic tissue, the cAMP response was inhibited by >95% vs. that under our standard assay conditions (2x10⁶ cells), and this inhibition could be largely reversed by dCF (Fig. 2 ). The ability of ecto-ADA to influence the accumulation of cAMP was aided by the relatively slow kinetics of the cAMP response in thymocytes (maximal at 30 min). These data suggest that ecto-ADA may regulate AdoR-mediated changes in cAMP levels in tissues even with only moderate CD26 expression, as long as the kinetics of the cAMP response are not too fast and there is a source of ADA to bind to CD26. In the case of the thymus, ADA is probably derived from the large proportion of developing thymocytes that die by apoptosis as a consequence of failing selective checkpoints.

View larger version (27K): [in this window] [in a new window]   Figure 2. cAMP responses of human thymocytes. Different numbers of cells (2x106, 2x107, 2x108) were preincubated with 10 µM Ro 20-1724 for 10 min at 37°C, then stimulated with 20 µM Ado for 10 min at 37°C in the presence or absence (Ctrl) of dCF. The concentration of dCF was 5 µM for experiments with 2 x 106 cells and 50 µM for those with 2 x 107 and 2 x 108 cells. Data are mean ± SD of 4 cAMP determinations and are representative of 3 separate experiments.

CONCLUSIONS AND SIGNIFICANCE

The ability of ecto-ADA to regulate cAMP responses to Ado depended on the level of ecto-ADA expression and the kinetics of the cAMP response. For CD26-transfected cells, this was most significant when CD26 expression was high, CD26 was saturated with ecto-ADA, and the kinetics of the cAMP response were relatively slow. However, our data with human neonatal thymocytes suggest that ecto-ADA may regulate AR-mediated changes in cAMP levels even in tissues with only moderate CD26 expression, as long as the kinetics of the cAMP response are relatively slow and there is a source of ADA to bind to CD26. This may occur in other lymphoid tissues such as bone marrow and lymph nodes, in tissues where T cells are activated, leading to CD26 up-regulation, and/or at sites of inflammation. CD26 has a broad tissue distribution including kidney, small intestine, lung, spleen, liver, skeletal muscle, and heart as well as activated T cells. Thus, ecto-ADA has the potential to regulate AR signaling in a variety of tissues.

In our experiments, we added exogenous Ado to initiate AR signaling. In vivo, however, Ado may be generated through the action of another ecto-enzyme, CD73, or ecto-5'-NT. Indeed, recent work by Matsuoka et al. indicates that Ado produced by the action of ecto-5'-NT acts rapidly to engage A2bR on the surface of Xenopus laevis oocytes before it can be degraded by exogenous ADA. Preliminary experiments in our lab with Molt 4 cells transfected with ecto-5'-NT and CD26, however, suggest that Ado produced from AMP by ecto-5'-NT is still subject to degradation by ecto-ADA bound to CD26 (T. Hashikawa, unpublished observation). Thus, we propose that both ecto-5'-NT and ecto-ADA are likely to play important roles in the control of extracellular Ado levels and thus AR engagement (Fig. 3 ). Considering the potent anti-inflammatory action of Ado and the ability of AR to regulate a multitude of important physiologic processes, it is important to define the cell type-specific and developmentally regulated expression of these two enzymes in vivo, as well as to understand how their expression levels change under pathological conditions. Experiments are under way to further exploit transfected Jurkat and Molt 4 cells to explore the relative importance of ecto-5'-NT and ecto-ADA in the regulation of AR signaling.

View larger version (21K): [in this window] [in a new window]   Figure 3. Regulation of adenosine receptor signaling by ecto-ADA bound to CD26. Exogenous Ado or Ado produced from AMP by CD73 can engage cell surface AR and initiate a cAMP response. However, if the cells are CD26+ and there is a source of extracellular ADA to bind to CD26, then ecto-ADA can degrade extracellular Ado and inhibit AR signaling.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0011fje

² Present address: Laboratory of Signal Transduction, Sloan Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

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