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LIVER AND BILIARY TRACT
1Liver and Transplantation Centers, Beth Israel Deaconess Medical Center, Harvard University, Boston, Massachusetts; 2Department of General Surgery, University of Heidelberg, Heidelberg, Germany; and 3Institute of Immunology, University of Heidelberg, Heidelberg, Germany
Submitted 13 June 2006 ; accepted in final form 14 August 2006
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ABSTRACT |
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type-2 purinergic receptors; chronic pancreatitis; pancreatic cancer; pancreas
Extracellular nucleotides regulate cell-cell communication via purinergic/pyrimidinergic P2 receptors (P2Rs). There are two main families of extracellular nucleotide receptors: P2X are ligand-gated ion channels, permeable for Na+, K+, and also Ca2+ (subtypes P2X1–7); and P2Y are G protein-coupled receptors that initiate signal transduction-coupled activation of phospholipase C, protein kinase C, and adenylate cyclases (5, 6, 13). Importantly, select P2Y receptors induce downstream effects acting through the inositol trisphosphate pathway, mediated by intracellular Ca2+ (10).
CD39 is an ectoenzyme, now referred to as nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), which hydrolyzes extracellular nucleotides (12). Vascular CD39 is the dominant endothelial ectonucleotidase and hydrolyzes both ATP and ADP in the plasma to AMP, ultimately to adenosine (12). Another NTPDase associated with the vasculature is the cell-associated ecto-ATPase (CD39L1 or NTPDase2) (9).
NTPDases functionally interact with P2Y receptors (12). For example, combinations of NTPDases have the capacity to terminate P2R signaling, modulate receptor desensitization, alter specificities of the response, or even generate signaling molecules (ADP) from precursors (ATP) (25). Extracellular ATP, UTP, and UDP are also known to be potent growth factors for vascular smooth muscle cells through the activation of P2Y receptors (9, 15). ATP, UDP, and others, acting at P2X7 receptor and presumably also P2Y receptor, trigger IL-1 and TNF release from activated macrophages and endothelium (8).
NTPDases are not only expressed on smooth muscle and endothelial cells. Dranoff et al. (9) have described expression of NTPDase2/CD39L1 on portal fibroblasts where this specific localization may modulate novel functional interactions of these stromal cells.
Functions of CD39 may not be limited to vascular mechanisms, such as platelet aggregation (16), thromboregulatory functions (26), and other cell-cell interactions (18) crucial for the maintenance of the vascular integrity. Recently, infiltrating immune cells that dedifferentiate to fibrocytes promoting scar formation have been described in lung, liver, and pancreatic lesions (4). Any proposed expression of CD39 by these cells and other vascular and stromal elements in scar tissues could play a significant role in the modulation of fibrogenesis within diseased pancreatic tissues and indeed elsewhere.
In this study we have explored patterns of expression of CD39 and P2R in human normal pancreas (NP), CP, and PaCa. In addition, we have correlated the altered patterns of clinical data with levels of expression of ectonucleotidases and P2R with the clinicopathological state.
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MATERIALS AND METHODS |
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The studies were approved by the Human Subject Committee of the University of Heidelberg (Heidelberg, Germany).
Real-time RT-PCR detection. Tissue samples were collected in RNAlater (Ambion, Austin, TX) and stored at –20°C until analysis. Tissue was disrupted by one run with the RiboLyser (ThermoHYBAID, Heidelberg, Germany) in lysing matrix-D tubes (Q-BIOgen, Heidelberg, Germany) containing 400 µl of lysis buffer from the MagnaPure mRNA Isolation Kit II (Roche Diagnostics, Mannheim, Germany). The RiboLyser tubes were centrifuged at 4°C for 1 min at 13,000 rpm. The lysate (300 µl) was then collected and mixed with 600 µl capture buffer containing oligo(dT). After centrifugation at 13,000 rpm for 5 min, 880 µl of this mix were transferred into a MagnaPure sample cartridge, and mRNA was isolated with the MagnaPure-LC device using the mRNA-II standard protocol. The elution volume was set to 50 µl. An aliquot of 8.2 µl RNA was reverse transcribed using avian myeloblastosis virus-RT and oligo(dT) as primer (First Strand cDNA synthesis kit, Roche) according to the manufacturer's protocol in a thermocycler. After termination of the cDNA synthesis, the reaction mix was diluted to a final volume of 500 µl and stored at –20°C until PCR analysis.
Specific primer sets for CD39, CD39L1, P2X7, P2Y1, P2Y2, and P2Y6 optimized for the LightCycler (RAS, Mannheim, Germany) were developed and purchased from SEARCH-LC (Heidelberg, Germany). The PCR was performed with the LightCycler FastStart DNA Sybr Green I kit (RAS) according to the protocol provided in the parameter specific kits. To control for specificity of the amplification products, a melting curve analysis was performed. No amplification of unspecific products was observed. The copy number was calculated from a standard curve, obtained by plotting known input concentrations of four different plasmids at log dilutions to the PCR-cycle number (CP) at which the detected fluorescence intensity reaches a fixed value. This approach dramatically decreased variations due to handling errors over several logarithmic dilution steps.
To correct for differences in the content of total RNA, calculated copy numbers were normalized according to the average expression of two housekeeping genes, cyclophilin B and hypoxanthine-guanine phosphoribosyltransferase. Values were thus given as input adjusted copy number per microliter of cDNA.
Cell lysate preparation and Western blot analysis. Cell lysates were prepared by using a homogenate buffer containing 20 mM Tris, 100 mM NaCl, 0.1 mM PMSF, 10 µg/ml aprotinin, and 1% NP-40. After sonication and centrifugation (12,000 rpm for 15 min at 4°C), protein concentrations were measured with the Bradford method (Bio-Rad, Hercules, CA). SDS-polyacrylamide gel electrophoresis techniques were employed to separate proteins (40 µg/lane) on 4% to 15% linear gradient gel, both under reducing and nonreducing (only CD39) conditions. Proteins were then transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA) by semidry electroblotting and probed with either CD39 (Ancell), CD39L1 rabbit polyclonal (BZ3–4F), P2X7 (Alomone, APR-004), P2Y2 (Alomone, APR-010), or for control GAPDH (Amblion) primary antibodies. P2Y1 and P2Y6 antibodies from Santa Cruz Biotechnology were found to be nonspecific on testing and were not used further. Bands were visualized by using horseradish peroxidase-conjugated secondary antibodies donkey anti-rabbit (CD39L1, P2X7, and P2Y2), goat anti-mouse (CD39 and GAPDH), and mouse anti-goat (P2Y1) (1:85,000; Pierce, Rockford, IL). Equal gel loading was either confirmed using Ponceau red staining (not shown) of the blotted membrane or by stripping the blots and reprobing with GAPDH antibody.
To validate P2R expression (P2X7 and P2Y2), primary antibodies against P2X7 and P2Y2 were blocked with the specific antigen provided by the manufacturer (Alomone). Internal controls show complete negative blots (not shown) that confirmed that detected signals were specific.
For the semiquantitative estimates of protein concentration by image densitometric analysis, the IMAGE Station 2000MM from Kodak was used. Kodak 1D software was used to digitalize data, according to the manufacturer's protocols.
Immunohistochemistry. Surgical specimens of NP, CP, and PaCa were embedded in Triangle Biomedical Sciences tissue-freezing medium (American Master Tech Scientific, Lodi, CA) and immediately snap frozen in isopentane, cooled on liquid nitrogen, and stored at –80°C. Five-micrometer serial cryostat sections were fixed in ice-cold acetone (Sigma) for 10 min and rinsed in PBS. IgG binding sites were blocked with appropriate control serum diluted 1:5 in solution 9 [PBS, pH 7.4, supplemented with 0.1% bovine serum albumin, 150 mM tranexamic acid, 20 µg/ml aprotinin (3–7 trypsin inhibitory U/mg), 1.8 mM ethylenediaminetetraacetic acid, and 2 mM iodoacetic acid] and further supplemented with 2% 3-omega fatty acid (Sigma) for 1 h at room temperature. Sections were then incubated overnight with primary antibody for CD39, CD39L1, P2X7, and P2Y2 at 4°C, rinsed with PBS, and then incubated with 3% H2O2 in methanol for 5 min to deplete endogenous peroxidase. After incubation with the appropriate biotinylated IgG for F(ab')2 fragment of IgG for 30 min, staining was performed with the Vectastatin ABC elite kit (Vector) with diaminobenzidine as the peroxidase substrate. Sections were counterstained with Mayer's hematoxylin, dehydrated, cleared in xylene, and mounted in Permount. All antibodies were diluted in solution 9, unless otherwise indicated. Optimal antibody concentrations were determined by serial dilution.
Statistical analysis. Results are expressed as median and range and/or means (SD). For statistical analysis, the Mann-Whitney test was used. Survival curves were computed by the Kaplan-Meier method as analyzed by the log-rank test. Significance was defined as P < 0.05. For survival analysis, the samples were segregated according to the fold increase in mRNA expression levels compared with normal controls in positive and negative tumors. The cutoff levels (number of mRNA copies) were set at 410 for CD39 and 33 for P2Y2.
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RESULTS |
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Overexpression of CD39 and purinergic receptors.
Low levels of CD39, CD39L1, P2X7, P2Y2, and P2Y6 expression were noted in normal representative pancreatic biopsies (Fig. 2A). CD39 and especially P2X7 were upregulated and highly expressed in CP. CD39L1 and P2Y2 were moderately expressed in CP where levels were comparable with findings in NP. PaCa samples exhibited heightened levels of CD39 and P2Y2, validating mRNA findings of overexpression. High levels of both intact CD39 (78 kDa) and truncated proteolytic fragments (50 kDa) were observed. P2X7, expressed as a double band at 
79 kDa, was overexpressed in CP. In contrast to elevated P2X7 in CP, P2Y2 (47 kDa) was only specifically overexpressed in PaCa (Fig. 2A). Measurements of protein expression in relation to GAPDH expression revealed a trend toward increased expression by 2.75-fold (P = NS) in the instance of CD39 levels in CP. In PaCa, CD39 expression was, however, significantly increased by 2.75-fold (P = 0.0070) (Fig. 2B). Expression levels of P2X7 show a 3.0-fold increase in CP and a 2.2-fold increase of protein expression in PaCa compared with those if P2X7 protein expression (in NP). Although, at the mRNA level, P2X7 was overexpressed in CP compared with NP, and, at the protein level, differences in P2X7 expression between CP and NP did not achieve statistical significance (P = 0.11). P2Y2 protein expression levels were comparable in both NP and CP (1.7-time increase, P = NS), but there was a 3.1-fold increase in PaCa compared with NP (P = 0.0281).
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In PaCa, CD39 was highly expressed around malignant cells in stromal elements. However, cancer cells, as well as normal acinar cells, do not express CD39 to any extent. CD39L1 localization to nerves can be clearly shown in Fig. 3F, where strong CD39L1 signals are detected. Interseptal fibroblasts also stain moderately for CD39L1. Moderate to weak signals are encountered in the cytosol of cancerous ductal cells and acini. P2X7 was almost undetectable in PaCa, other than clear positive staining in immunocytes. P2Y2 expression retained specific expression in interseptal fibroblasts but not ductal and acini cells. The signal intensity is high and consistent with Western blot analysis and mRNA data. P2Y2 were faintly expressed by the common fibroblast-like cells of the abundant present desmoplastic reaction (Fig. 3, C, F, I, and L).
High CD39 expression in PaCa correlates with better clinical outcomes. To evaluate the clinical significance of mRNA expression in PaCa samples, RT-PCR results were correlated with survival data and histopathological parameters after tumor resection (Table 2). Patients whose tumors exhibited overall elevated high levels of CD39 mRNA had significantly longer median postoperative survival periods [CD39: median, 33 mo; and mean, 29.7 mo (SD 20.94)] than patients whose tumors exhibited low to moderate CD39 mRNA levels [CD39: median, 14 mo; and mean, 20.1 mo (SD 16.56)]. These differences were statistically significant when analyzed by the log-rank test (CD39: P = 0.0466) (Fig. 4). There were no significant relationships noted for CD39L1, P2Y2, and P2Y6 mRNA expression; postoperative patient survival; and tumor stage (Table 2).
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DISCUSSION |
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The data obtained from control biopsies might explain observed differences in the sensitivity of NP to purinergic agonists. Interestingly, acinar cells do not express P2X7 receptors, whereas pancreatic ductal cells do (24, 28). In general, pancreatic ducts show little desensitization of purinergic receptors, but only very few pancreatic acini show functional P2Rs (24, 28). Pancreatic acini release ATP after cholinergic stimulation, and this may lead to selective signaling, since acini lacking P2 expression could avoid being stimulated by the released ATP.
Inflammation in pancreatic disease is a complex pathophysiological process associated with leukocyte infiltration, tissue injury, cell death, and release of nucleotides and cytokines. These actions are relevant stimuli for altered NTPDase activity and increased levels of hydrolysis of ATP and ADP that could modify P2R-mediated responses under inflammatory conditions. For example, P2X7 receptor activation of caspase-1 by macrophages is dependent on prestimulation of these cells with LPS or other Toll-like receptors ligands (17). In addition, P2Y receptors induce downstream effects via inositol trisphosphate-mediated pathways that are also modulated by several other signals (10).
Possible links between fibrosis and the impact of P2R in modulating hepatic fibrogenesis have been proposed by Dranoff et al. (11). Hepatic stellate cells (HSC) expressing P2Y subtypes differ in their relative activation states with high expression of P2Y2 and P2Y4 under basal or quiescent states, whereas HSC, activated by ATP and UTP, express high levels of P2Y6. Activation of HSC also leads to heightened expression of CD39L1/NTPDase2. Linked to this mechanism, extracellular UDP can be shown to triple mRNA levels of procollagen-1, a major constituent of extracellular matrix (11). P2Y receptors (P2Y2 and P2Y6) are highly expressed in fibroblast-like cells and may regulate procollagen-1 transcription in pancreatic disease. These data indicate interesting future targets for pharmacological interventions in fibrogenic diseases.
Comparable factors might be associated with the observed upregulation of NTPDases and P2Y receptors in PaCa. Furthermore, our data suggest, at least in part, that there are correlations between possible CD39 and P2R interactions/regulation (e.g., P2Y1 and P2Y2) within tumor matrix in human PaCa.
Further considerations of the impact of NTPDases on fibrosis and specific localizations have been described (9). CD39L1 expression has been shown on the plasma membrane of fibroblast-like cells adjacent to the basolateral aspect of bile duct epithelia (the area where portal fibroblast are located) (9). Our findings show similar patterns for pancreatic fibroblast-like cells around pancreatic ducts in NP. In PaCa, but not CP, CD39L1 expression is highly upregulated. These alterations in NTPDase expression might alter extracellular nucleotide signaling in pancreatic periductular fibroblasts in the setting of malignancy and desmoplasia.
Recent studies in mouse pancreas show that CD39 is mainly expressed in endothelium but not in ductal cells (20). By electron microscopy, granules in mouse pancreatic acini are shown to express CD39 and CD39L1. Additionally, Sorensen et al. (28) have described ATP as a potential paracrine regulator between acini and ducts. Furthermore, CD39 can be released with a suitable stimulus into the lumen (28). Differences in CD39 localization and distribution of P2 and adenosine receptors might be responsible for functional alterations in the acinar-ductal system of injured rat pancreas.
In comparison to the upregulation of CD39 in desmoplastic area of PaCa, we have demonstrated upregulation of CD39 expression in CP, a novel finding not yet described. Kittel et al. (19) have demonstrated CD39 expression, albeit low, in cancerous ducts with only weak staining of connective tissues around the duct. We also show significant correlations between expression of CD39 and postoperative survival of patients with PaCa. These data might infer that samples derived from patients with low CD39 expression are linked to later-stage malignancy. This would support our hypothesis that high CD39 expression in the tumor matrix might provide protective effects for patients with PaCa.
These findings potentially implicate distinct roles of CD39 and purinergic signaling in tissue remodeling, fibrogenesis, and probably tumor progression. P2Rs might become important mediators of downstream effects of nucleotide processing and also show modulation in inflammatory and neoplastic conditions. Mechanisms of downregulation of CD39 expression during tumor progression are not completely understood.
It is possible that the induction of CD39 is an initial trigger of P2R upregulation/desensitization in inflammation and desmoplasia. The specific localization of CD39 in pathological pancreas tissues, mainly on fibroblasts and endothelium as well as P2Y2 specific on fibroblasts, might link pancreatic inflammation and desmoplasia to purinergic signaling.
P2X7 is known to have proinflammatory properties and is connected to cytokine release (e.g., IL-1
and IL-18), nitric oxide generation and cytotoxicity, and other relevant processes in inflammation (3, 7, 14). Their various P2X and P2Y receptors are expressed in immunocytes. P2X7 has been demonstrated to be a functional receptor in the majority of these immune cells (6). We have shown that P2X7 is highly upregulated in CP but not in PaCa and might therefore be linked with pancreatic inflammation. P2X7 may serve as an immunomodulator in this disease via modulated actions of tissue macrophages and other immune and inflammatory cells (Fig. 3H).
Expression of P2X7 receptors in diseased pancreatic acini may, however, be pathogenetic, since acinar cells are packed with nucleotide granules that might be activated and cause tissue injury. The ATP release from acini may initiate its action downstream of the acini level in ductules and larger pancreatic ducts (28).
The fact that P2Y2 is highly upregulated at the mRNA level in PaCa and is localized to fibroblasts in close vicinity to invading cancerous ducts may contribute to distinct roles in cancer progression/inhibition by facilitating angiogenesis or fibrogenesis. These processes of tissue remodeling are known to be highly dysregulated in PaCa. Acini contain low numbers of functional P2Rs (24). This may indicate that changes in the pancreatic tissue P2 repertoire of receptors results in disturbed purinergic signaling.
Progressive tissue changes result in pancreatic parenchymal atrophy, fibrosis, and long-term pancreatic insufficiency. The roles of the inflammatory actions, as well as the desmoplastic process in chronic pancreatitis and PaCa, are still poorly understood. However, this present study provides insights into possible mechanistic involvement of purinergic signaling and NTPDases in diseases of the pancreas. Overexpression of CD39 and correlation to long-term survival after tumor resection for PaCa also suggest a potential, highly novel role for purinergic signaling in PaCa.
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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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410) and P2Y2 (B) (cutoff