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RECEPTORS AND SIGNAL TRANSDUCTION
Departments of 1Pharmacology, 2Microbiology and Immunology, and 3Pathology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada
Submitted 17 May 2005 ; accepted in final form 29 March 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CD26; deaminase binding protein; mitogen-activated protein kinases; nucleosides; tumor microenvironment
In cancer, tumorigenesis is often accompanied by a reduction in the expression of DPPIV (9, 46). This reduced DPPIV expression is directly associated with carcinogenesis because inducible gene transduction of DPPIV into melanoma cells has been shown to dramatically reverse the malignant phenotype (80). Decreased levels of DPPIV have also been linked to increased invasion and metastasis (18, 38, 55). In hepatocellular and colorectal carcinomas there is highly variable expression of DPPIV (70, 73, 74), suggesting that local influences may regulate the expression of DPPIV and therefore its participation in tumor progression.
We have recently identified a common tumor metabolite as a regulator of the cell surface expression of DPPIV protein (72). Adenosine, a purine nucleoside known best for its role in energy metabolism, is produced at increased levels in the extracellular fluid of solid tumors (8). This is a consequence of changes in the rates of production and removal of adenosine that are characteristic of hypoxic tissues like those in the poorly vascularized tumor (15, 31, 77). The adenosine that is produced is likely to have widespread actions within the tumor, including suppression of the cell-mediated immune response (33, 34, 40, 43, 44), promotion of angiogenic activity (4), stimulation of the motility of tumor cells (82), and stimulation of tumor cell growth (47, 48). We recently found that adenosine also downregulates the surface expression of DPPIV protein on HT-29 human colorectal carcinoma cells, and that this effect is accompanied by decreases in DPPIV dipeptidase activity, ADA binding, adhesion to fibronectin, and cellular motility (72). The downregulation of cell-surface DPPIV protein occurs at relatively high adenosine concentrations when a single dose of adenosine is used (EC50: 43.3 ± 12.1 µM) but repeated dosing to simulate steady-state levels takes the required dose down to the 10–5 M range (72), which approximates to the concentration present in the tumor extracellular fluid (8). The downregulation of DPPIV protein begins by 12 h following exposure to adenosine, reaches a maximum after 48 h, and is maintained if cultures are replenished with adenosine (72).
Cellular responses to adenosine are usually effected through one or more of four different subtypes (A1, A2A, A2B, and A3) of G protein-coupled adenosine receptors (25), all of which are expressed to some extent on HT-29 cells (M. Mujoomdar and J. Blay, unpublished observations). We therefore anticipated that the downregulation of DPPIV would occur following adenosine occupation of such a receptor subtype(s). However, although we had shown that adenosine acts at the cell surface, extensive studies to implicate a classic adenosine receptor(s) were negative. In contrast, we show here that the adenosine downregulation of DPPIV on HT-29 cells is mediated by a novel mechanism that acts through an increase in tyrosine protein phosphatase activity to cause a decrease in the tyrosine phosphorylation of ERK1/2 mitogen-activated protein kinase (MAPK) that leads to the alteration in DPPIV mRNA and ultimately cell-surface protein. This novel action of adenosine has implications for our ability to manipulate adenosine-dependent events within the solid tumor microenvironment.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture. HT-29 cells were cultured in Dulbecco's modified Eagle's medium (without antibiotics) supplemented with 10% (vol/vol) heat-inactivated newborn calf serum and maintained as stocks in 80-cm2 flasks at 37°C in a humidified atmosphere of 90% air-10% CO2. Cells for use in binding assays and ERK1/2 immunoblot analysis were seeded into 48-well and 6-well plates, respectively, and allowed to adapt to culture for 48 h. Cultures were then changed to medium containing 1% newborn calf serum for a further 48 h, and then treated with drugs or control vehicle for evaluation of changes in DPPIV surface protein expression or ERK1/2 phosphorylation. The coformycin (2.5 µM) or EHNA (20 µM) added in most experiments to reduce the rate of breakdown of exogenous adenosine by cellular ADA did not in themselves affect the basal level of DPPIV expression (data not shown).
RT-PCR.
Semiquantitative RT-PCR was used to evaluate DPPIV mRNA regulation by adenosine. The polymerase chain reaction was conducted in a PTC-100 thermal cycler (MJ Research, Watertown, MA) using standard protocols. The primer sequences used for the PCR were (the product size is given after the reverse primer): DPPIV (forward: 5'-CAAATTGAAGCAGCCAGACA-3'; reverse: 5'-CAGGGCTTTGGAGATCTGAG-3') (354 bp) and GAPDH (forward: 5'-TGGAAATCCCATCACCATCT-3'; reverse: 5'-TAGTGACGGTGGGTCTTCTG-3') (351 bp). The amplification protocols for DPPIV (25 cycles) and GAPDH (26 cycles) were chosen to yield PCR products in the linear range of amplification. The PCR products were visualized on a 1.5% agarose gel containing 0.2 µg/ml ethidium bromide. Band intensity was measured by densitometry and results were normalized to the steady-state expression of GAPDH. Real-time PCR amplification was performed using a Stratagene Mx3000P system (Cedar Creek, TX). DPPIV gene expression was analysed using the manufacturer's software, standardized against GAPDH and normalized to control expression using the 2–
Ct method. Similar results were obtained using different DPPIV primer sequences (data not shown).
Radioantibody binding assay for DPPIV. Monolayer cultures of HT-29 cells in 48-well plates were assayed for cell-surface DPPIV protein, as previously described (72). Briefly, cultures were incubated with anti-DPPIV antibody or isotype control, washed, and the bound antibody was measured using 125I-labeled sheep anti-mouse IgG as tracer. Data are corrected for background binding of isotype antibody, and for cell number. Where antagonists needed to be solubilized using a nonaqueous solvent, we used dimethyl sulfoxide such that the final concentration never exceeded 0.2% (vol/vol). Adenosine at the concentrations used in these studies does not induce apoptosis in HT-29 cells (48, 72). The figures show representative results from at least three separate experiments. Data were evaluated using two-tailed Student's t-test for unpaired data, or when necessary, one-way ANOVA with Tukey-Kramer multiple-comparison posttest.
ERK1/2 immunoblot analysis. Total cellular protein was isolated from HT-29 cells grown to 60–75% confluence in 6-well plates. Cells were rinsed twice with ice-cold PBS and dissolved in lysis buffer composed of 50 mM Tris·HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM NaF, 1x protease inhibitor mix, for 45 min at 4°C. The cell lysates were clarified by centrifugation (10 min at 12,000 g) and quantified by Bradford protein assay according to the manufacturer's instructions. Twenty micrograms of protein extract per lane were separated by SDS-PAGE using 10% gels and electroblotted to nitrocellulose. Blots were blocked with 5% skimmed milk in Tris-buffered saline with 0.1% Tween 20 for 1 h at room temperature. Blots were then probed overnight at 4°C with anti-phospho-ERK1/2 antibody at a 1 µg/ml concentration followed by incubation with sheep anti-mouse IgG-HRP-conjugated secondary antibody for 1 h at room temperature. Protein expression was detected using an enhanced chemiluminescence detection system. To confirm equal sample loading, the blots were stripped and reprobed with anti-ERK1/2 (nonphosphorylated) mAb, followed by sheep anti-rabbit IgG-HRP secondary mAb.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50% at 12 h after exposure (Fig. 1B). The downregulation of DPPIV by adenosine therefore involves regulation at the mRNA level and is not simply a consequence of altered trafficking of mature protein to or from the cell surface.
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We used selective adenosine receptor antagonists in an attempt to dissect out the receptor pathway. Despite many efforts, we were unable to block DPPIV downregulation. Representative results are shown in Table 1. It should be noted that in these and other data, there is substantial variation in the apparent DPPIV expression in untreated cultures. This is due in part to the declining specific radioactivity with time of the 125I-labeled secondary antibody used to track the surface immunoreactive DPPIV, but also to different levels of DPPIV expression at different times in culture. DPPIV is a differentiation marker for intestinal epithelium, and increases in parallel with acquisition of a slightly more differentiated phenotype as the HT-29 cells approach confluence. Adenosine promptly downregulated DPPIV beginning 12 h after exposure (72) irrespective of time in culture, showing that its action is not simply one of hindering this differentiation process.
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7.5 pmol/h in the culture format used here), the steady-state concentration (in the face of constant metabolism) of adenosine would reach only 0.21 ± 0.02 µM (M. Mujoomdar and J. Blay, unpublished observations). This is insufficient to perturb baseline DPPIV levels (72) or to interfere with antagonism. To test whether simultaneous inhibition of more than one receptor subtype might abrogate adenosine-evoked DPPIV downregulation, we treated HT-29 cells with a combination of DPCPX, CSC, alloxazine, and MRS-1523 at concentrations reflecting their relative inhibitory potency in other systems and at the highest addition that could be used without incurring excessive cytotoxicity (cell death due to the antagonists ranged from 1.6 to 8.7% in these experiments). Figure 2A shows the result of this approach. There was absolutely no diminution of the effect of adenosine, even though this combination is sufficient to entirely block the effect of adenosine on CXCR4 chemokine receptor expression in these cells (60). Consistent with the failure of adenosine receptor antagonists to block the adenosine response, we found no effect of the broadly selective agonists NECA and R-PIA on DPPIV expression, at concentrations as high as 30 µM (Fig. 2B). Taken together, these data indicate that adenosine downregulates DPPIV by a mechanism not involving the conventional adenosine receptor subtypes.
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Phospholipase C (PLC) activation, which has been observed in a minority of responses to adenosine (61) leads to the generation of diacylglycerol and inositol trisphosphate, which signal through PKC and the elevation of cytosolic Ca2+ levels, respectively (25). Direct activation of PKC using the phorbol ester PMA, or elevation of intracellular Ca2+ with the Ca2+ ionophore ionomycin, did not however, mimic the adenosine effect, but in contrast caused an elevation of cellular DPPIV (Fig. 4A). In addition, neither of two agents that are inhibitory for PKC, GF-109203X (75), or calphostin C (39) interfered with adenosine-evoked suppression of DPPIV (Fig. 4B). These observations exclude the involvement of the phosphoinositide cycle and PKC from the DPPIV downregulatory response caused by adenosine.
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We next explored whether adenosine might be activating protein tyrosine kinase (PTK) pathways. We used two broad-spectrum PTK inhibitors to assess the role of PTKs in DPPIV downregulation in response to adenosine. Genistein is a potent inhibitor of most cellular PTKs (1). At concentrations of 20 µM (Fig. 4D) or 50 µM (data not shown) genistein failed to block the adenosine downregulation of DPPIV on HT-29 cells. We also used herbimycin A, a PTK inhibitor that is relatively selective for src-like PTK (37). Herbimycin A at 1 µM also did not block the adenosine effect (Fig. 4D).
Adenosine downregulation of DPPIV is dependent on increased protein tyrosine phosphatase activity.
Certain effects of adenosine have been shown to involve serine/threonine protein phosphatases (41, 52); while we and others have recently found that adenosine action may also be linked to the activation of protein tyrosine phosphatase (PTP) activity (30, 50, 83). We therefore examined whether the downregulatory response of DPPIV that we see here might occur through the adenosine activation of phosphatase(s). We first tested two commercially available broadly inhibitory cocktails of agents against serine/threonine and tyrosine protein phosphatases. Inhibition of serine/threonine phosphatases did not abrogate the adenosine effect at up to 0.2% vol/vol cocktail (Fig. 5A), or at a higher concentration (0.5% vol/vol, data not shown) that caused substantial (>60%) loss of cell viability. However, the tyrosine phosphatase inhibitor cocktail caused a progressive dampening of the adenosine response up to 0.2% vol/vol cocktail (Fig. 5B) and complete abrogation of the adenosine-induced downregulation of DPPIV at 0.5% vol/vol (data not shown), although that high concentration again led to a substantial (50%) loss of cell viability.
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1 µM, reaching complete cell death at 5 µM) over the 48-h treatment period. However, no inhibition of the adenosine response was evident in experiments with phenylarsine oxide at 0.5 µM or 1 µM (data not shown). Taken together, these data demonstrate the involvement of a PTP(s) in downregulation of cell-surface DPPIV on HT-29 cells exposed to adenosine. The selectivity to certain PTP inhibitors and not others should provide clues as to the particular PTP that is activated by adenosine.
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Inhibition of p38 with SB-203580 (12, 36) did not block or enhance the adenosine effect (Fig. 7A). Similarly, the JNK inhibitor SP-600125 (7, 27) failed to block or enhance the adenosine downregulation of DPPIV (Fig. 7B). Although inhibition of JNK led to a dose-dependent increase in baseline DPPIV expression (mean increase, 70.5% at 20 µM SP-600125), there was no change in the extent of the adenosine depression. However, the effect of PD-98059, a MAPK kinase (MEK1) inhibitor (2), was more informative (Fig. 7C). First, treatment of HT-29 cells with PD-98059 by itself caused a downregulation of DPPIV cell-surface protein comparable to adenosine. This is consistent with the notion that a decrease in the active tyrosine-phosphorylated form of ERK, caused either through adenosine-stimulated PTP activity, or through PD-98059-mediated inhibition of MAPKK (MEK), is linked to a decline in DPPIV. In addition, PD-98059 plus adenosine caused a greater but not additive decrease in DPPIV, consistent with these two interventions acting through the same process (Fig. 7C). These findings provide indirect evidence that the adenosine-mediated downregulation of DPPIV involves the ERK1/2 pathway and is associated with a decrease in ERK1/2 activation.
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100 µM (Fig. 8B). This dose-response relationship exactly parallels the concentration dependence of the adenosine downregulation of DPPIV following a single adenosine dose (72). A reduction in overall tyrosine protein phosphorylation in the 40–50 kDa range of proteins from adenosine-treated HT-29 cells was also observed after Western blot analysis of total cellular lysates with anti-phosphotyrosine mAb (data not shown), consistent with a reduction in ERK1/2 phosphorylation. These findings, Figs. 6, A and B, and 7C, strongly argue that the adenosine reduction, together with those of cell-surface DPPIV in HT-29 cells occurs in close association with negative regulation of the ERK1/2 signaling pathway.
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45% increase in ERK2 phosphorylation at the same concentration (5 nM) that led to a 40% increase in the amount of DPPIV protein at the cell surface (Fig. 4A).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We confirmed here that the downregulation of DPPIV by adenosine involved regulation of mRNA levels, and was not purely a perturbation of cellular trafficking of mature DPPIV protein to or from the cell surface, although such changes in trafficking may occur. The decline in mRNA expression (50%) is at first sight somewhat small compared with such changes in many other model systems. However, the decrease was maintained over a period of some 36 h and was similar in magnitude to the decrease in DPPIV protein. It is therefore consistent with the ensuing protein response. The 50% decrease in the abundance of cell-surface DPPIV is likely important because its impact is to alter enzyme activities (dipeptidase and deaminase) and cell-extracellular matrix interactions that collectively provide a multiple and magnified response. This is shown by observations that adenosine-treated cells, due to downregulated DPPIV, migrate at less than half the rate of untreated cells on cellular fibronectin (72) and that the decline in ecto-ADA bound to DPPIV leads to an increase in the half-life of adenosine in HT-29 cell cultures from 2 h (48) to >24 h (60). Given that adenosine is immunosuppressive, mitogenic for tumor cells, and also angiogenic (4, 33, 34, 43, 44, 47, 48) the latter event would have major consequences in the context of an intact tumor.
Because we had excluded any possible action of adenosine on intracellular targets such as the adenylyl cyclase "P" site (72), our expectation was that one of the existing well-characterized adenosine receptor subtypes (A1, A2A, A2B, or A3) would be involved in triggering this process. However, numerous experiments using eight different established antagonists (and with particular attention paid to the A2B and A3 receptors that are most robustly expressed on the HT-29 cells; M. Mujoomdar and J. Blay, unpublished observations) failed to block the adenosine response. Most convincingly, when a combination of antagonists against all four subtypes (at the highest feasible concentrations) was used, there was absolutely no reduction in the adenosine-evoked downregulation of DPPIV. Furthermore, the broadly selective agonists NECA (A1, A2, A3) and R-PIA (A1, A2) failed to mimic the action of adenosine even at concentrations as high as 30 µM. Finally, the failure of the PKA inhibitor Rp-cAMPs to alter the adenosine-mediated downregulation of DPPIV shows that the major signaling route employed by all four of the adenosine receptor subtypes, the cAMP signaling pathway, is not involved. We are therefore compelled toward the conclusion that the adenosine downregulation of DPPIV does not occur through the conventional adenosine receptors so far known.
Other researchers (21, 23, 28, 81) have also found evidence for the existence of atypical, or nonclassic, adenosine receptor subtypes. We have already established that high levels of adenosine are sensed at the cell surface, rather than evoking a change in DPPIV after entry into the cell through nucleoside transporters (72). The downregulation is not simply a cell-surface perturbation triggered by extracellular adenosine that leads to intracellular sequestration of DPPIV (as for example occurs with Caco-2 cells treated with forskolin; Ref. 5). In addition to the alteration in mRNA, the time course is relatively slow (maximum reached at 48 h; Ref. 72), and we show here that there is clearly an intermediary intracellular signaling pathway that leads to the decline in DPPIV protein.
Our extensive studies using inhibitors of signaling pathways lead us to the conclusion that the adenosine effect on DPPIV is exerted through intermediate steps that involve 1) activation of a protein tyrosine phosphatase that is sensitive to inhibition by orthovanadate and bpV(phen), and 2) reduction in the activation of ERK1/2 MAPK. Our conclusion that ERK1/2 is involved in regulation of DPPIV levels is further supported by our demonstration that PMA, which in contrast to adenosine, increases the expression of cell-surface DPPIV, also acts oppositely to increase the activation of ERK1/2 over the same timeframe.
Regulation of ERK1/2 (p42/p44) MAPK in response to adenosine has been reported in other cellular systems. Human A1, A2A, A2B, and A3 adenosine receptors expressed in Chinese hamster ovary cells [and A2A receptors expressed in human embryonic kidney-293 (HEK-293) cells] all couple to the ERK1/2 MAPK pathway, but in each case adenosine or an appropriate synthetic agonist increase the phosphorylation of ERK1/2 (16, 64, 67, 76). Such expression models may risk revealing pathways that normally do not play a major role (26, 32). However, stimulation of ERK1/2 through endogenous adenosine receptors (mainly A2) has also been observed, in primary human endothelial cells (A2A; Ref. 68), untransfected HEK-293 human embryonic kidney cells (A2B; Ref. 26), HMC-1 human mast cells (A2B; Ref. 24), BR canine mastocytoma cells (A2B; Ref. 26), PC12 rat pheochromocytoma cells (A2A; Ref. 3) and XS-106 mouse dendritic cells (A2B and A3; Ref. 17). There are data showing that adenosine may inhibit the stimulation of ERK1/2 phosphorylation by other factors, including thrombin (32) and NGF (3). However, this report is the first observation, to our knowledge, of adenosine itself causing negative regulation of the ERK1/2 signaling pathway. It is notable that most studies of ERK1/2 activation have used stable ligands such as NECA or agents selective for the appropriate receptor subtype, whereas we would argue, based on earlier observations (48), that adenosine itself may elicit different cellular responses to its analogs.
The dose-response relationship for adenosine inhibition of ERK1/2 phosphorylation in HT-29 cells exactly parallels that of the reduction in cell-surface protein, indicating a single affinity of ligand interaction for the two adenosine effects, which is consistent with linkage to a common receptor that initiates the response. However, our results differ from other studies of ERK1/2 activation not only in that we see an inhibition, rather than a stimulation, of activity by adenosine (or analogs), but that we fail to attribute the sensing of the initial signal to any of the four well-characterized receptor subtypes. This is not an artifact of inappropriate adenosine concentration, as our data show sensitivity to adenosine down to the 10–4 M range without inhibiting its metabolism; and we routinely treat with 10–30 µM adenosine in the presence of 2.5 µM coformycin or 20 µM EHNA to inhibit ADA-mediated breakdown. This contrasts, for example, with the millimolar dosing used by Harrington and colleagues (30) in their studies of adenosine p38
modulation, which leads to apoptosis.
The lack of evidence for the involvement of cAMP pathways in adenosine-induced DPPIV downregulation is consistent with data showing that adenosine modulates ERK1/2 signaling irrespective of whether increases (A2A, A2B) or decreases (A1, A3) in cAMP would be expected to occur (16, 17, 64, 67, 76); and that stable cAMP analogs do not produce changes in ERK1/2 (67, 68). Furthermore, the dose-response relationships for ERK1/2 phosphorylation vs. cAMP accumulation in sensitive cells differ by two orders of magnitude (64). The situation differs in adenosine receptor-transfected Chinese hamster ovary and HEK-293 cell models, in which cAMP seems to play a major role (see Refs. 26, 32).
In contrast, we have found striking evidence for adenosine stimulation of a protein phosphatase activity that is able to produce the dephosphorylation of ERK1/2 that we have observed. The individual PTP inhibitors orthovanadate and bpV(phen) completely abrogated both the adenosine suppression of ERK1/2 activation and the downregulation of cell-surface DPPIV. This observation parallels our findings in a recent exploration of adenosine inhibition of IL-2 signaling in T cells (83). In that work, we found that adenosine activates Src homology PTP 2 (SHP-2) to reduce the tyrosine phosphorylation of STAT5a/b, and that this is blocked by orthovanadate and bpV(phen).
The PTP target that is the focus of adenosine action and which is inhibited by orthovanadate and bpV(phen) is currently under investigation. Our preliminary studies have excluded the involvement of SHP-2, which in any case is usually coupled to an increase in ERK activation (13, 69), as well as SHP-1 and PTP1B: sodium stibogluconate, at a concentration of 100 µM (105 µg/ml), which is sufficient to inhibit these three PTPs (54), produced no change in the decrease in DPPIV levels due to adenosine (data not shown). We are currently using other approaches to identify which of the many other potential ERK-specific phosphatases (56, 59, 85) is/are activated by adenosine to reduce ERK1/2 phosphorylation and lead to DPPIV downregulation.
In summary, we have identified a pathway leading from the encounter of the cancer cell with adenosine at concentrations that exist in the tumor extracellular fluid, to the downregulation of DPPIV and its associated functions. This pathway involves the activation of a PTP by adenosine through a route that is independent of the existing adenosine receptor subtypes, and does not require participation of the PKA, PLC, PKC, PI3K, or PTK pathways. Activation of the PTP by adenosine is associated with the reduced tyrosine phosphorylation and activity of ERK1/2 MAPK, which is required for the downregulation of DPPIV to occur. The steps that are involved in initial sensing of the adenosine signal, and the precise phosphatase(s) involved in ERK1/2 suppression, remain to be elucidated. Protein-tyrosine phosphatases play a substantial role in regulation of solid cancers, particularly in colorectal carcinoma (53, 79). Understanding this particular pathway should allow us to be able to block the downregulation of DPPIV due to adenosine and its adverse consequences, leading to new ways of interfering with tumor expansion and spread. The independence of this DPPIV-downregulatory pathway from known adenosine receptors is an advantage, as the conventional signaling pathways for adenosine are involved in numerous aspects of physiologic regulation (such as blood flow), which we would prefer to spare in any pharmacological intervention focusing on adenosine's tumor-enhancing actions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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) or N6-(L-2-phenylisopropyl)adenosine (R-PIA; 
) at the concentrations indicated. Forty-eight hours later, surface expression of DPPIV was measured by radioantibody binding assay. The data are means ± SE (n = 4). **P < 0.01, significant reduction by adenosine.
