[Frontiers in Bioscience 13, 2588-2603, January 1, 2008]

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

Guido, Beldi 1, Keiichi, Enjyoji 2, Yan, Wu 3, Lindsay, Miller 4, Yara, Banz 5, Xiaofeng, Sun 6, Simon C. Robson 7

1 99 Brookline Avenue, Boston, MA 02215, 2 99 Brookline Avenue, Boston, MA 02215, 3 99 Brookline Avenue, Boston, MA 02215, 4 99 Brookline Avenue, Boston, MA 02215, 5 99 Brookline Avenue, Boston, MA 02215, 6 99 Brookline Avenue, Boston, MA 02215, 7 99 Brookline Avenue, Boston, MA 02215, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard University, Boston. MA

TABLE OF CONTENTS

1. Abstract 2. Introduction 3. Overview: principles and physiology 3.1. Hepatic purinergic signaling 3.2. Nucleotide release 3.3. Purinergic receptors 3.4. Ectonucleotidases 3.4.1. Ecto- nucleoside triphosphate diphosphohydrolases (NTPDases of the CD39 family) 3.4.2. Nucleotide pyrophosphatase/phosphodiesterases (NPP) 3.4.3. Alkaline phosphatases (ALP) 3.4.4. Ecto-5’-nucleotidase / CD73 3.5. Distribution and functions (hepatocytes, cholangiocytes / bile ducts, vasculature, stellate cells / portal fibroblasts, Kupffer cells, liver associated lymphocytes) 3.5.1. Hepatocytes 3.5.1.1. Purinergic receptors 3.5.1.2. Ectonucleotidases 3.5.2. Cholangiocytes / bile ducts 3.5.2.1. Purinergic receptors 3.5.2.2. Ectonucleotidases 3.5.3. Vasculature 3.5.3.1.Purinergic receptors 3.5.3.2. Ectonucleotidases 3.5.4. Stellate cells / portal fibroblasts 3.5.4.1. Purinergic receptors 3.5.4.2. Ectonucleotidases 3.5.5. Kupffer cells 3.5.5.1.Purinergic receptors 3.5.5.2. Ectonucleotidases 3.5.6. Liver associated lymphocytes 3.5.6.1.Purinergic receptors 3.5.6.2. Ectonucleotidases 4. Impact of purinergic signaling 4.1. Hepatic homeostasis in health (bile flow, nucleotide synthesis and salvage, metabolism) 4.1.1. Bile flow 4.1.2. Nucleotide synthesis and salvage 4.1.3. Metabolism (glucose/fatty acids) 4.2. Liver disease (vascular injury, rejection, regeneration, tumors, fibrogenesis and cirrhosis, cachexia) 4.2.1. Vascular injury 4.2.2. Rejection 4.2.3. Regeneration 4.2.4. Tumors 4.2.5. Fibrogenesis and cirrhosis 4.2.6. Cachexia 5. Conclusions and perspective 6. Acknowledgments 7. References

1. ABSTRACT platelets within the injured vasculature and bind specific cell-surface type-2 purinergic (P2) receptors. This process Extracellular nucleotides (e.g. ATP, UTP, ADP) drives vascular inflammation and thrombosis within grafted are released by activated endothelium, leukocytes and organs. Importantly, there are also vascular

2588 Purinergic signaling in liver transplantation ectonucleotidases i.e. ectoenzymes that hydrolyze and drives differentiation to promote cellular immune extracellular nucleotides in the blood to generate responses (8). Depending upon the receptor subtype, the nucleosides (viz. adenosine). Endothelial cell cell types and signaling pathway involved, these receptors NTPDase1/CD39 has been shown to critically modulate trigger and mediate short-term (acute) processes that affect levels of circulating nucleotides. This process tends to limit metabolism, adhesion, activation or migration. Activation the activation of platelet and leukocyte expressed P2 of select receptors in a repetitive manner also has a receptors and also generates adenosine to reverse profound impact upon long-term reactions, including cell inflammatory events. This vascular protective CD39 proliferation, differentiation and apoptosis, as seen in activity is rapidly inhibited by oxidative reactions, such as several chronic inflammatory states. is observed with liver ischemia reperfusion injury. In this review, we chiefly address the impact of these signaling The third, and final, component of purinergic cascades following liver transplantation. Interestingly, the signaling systems comprises ectonucleotidases (9). These hepatic vasculature, hepatocytes and all non-parenchymal ectoenzymes hydrolyze extracellular nucleotides to cell types express several components co-ordinating the generate nucleosides that in turn activate adenosine purinergic signaling response. With hepatic and vascular receptors, often with opposing effects as compared to those dysfunction, we note heightened P2- expression and mediated by P2-receptors. Within the past decade, alterations in ectonucleotidase expression and function that ectonucleotidases belonging to several enzyme families may predispose to progression of disease. In addition to have been discovered, cloned and further characterized by documented impacts upon the vasculature during pharmacological means. Modulated, distinct engraftment, extracellular nucleotides also have direct ectonucleotidase expression may regulate nucleotide- influences upon liver function and bile flow (both under mediated signaling in the vasculature in both temporal and physiological and pathological states). We have recently spatial manners. Vascular endothelial and accessory cell shown that alterations in purinergic signaling mediated by activation with associated proliferative responses within altered CD39 expression have major impacts upon hepatic grafts may have important consequences for platelet metabolism, repair mechanisms, regeneration and activation, thrombogenesis, angiogenesis, vascular associated immune responses. Future clinical applications remodeling and the metabolic milieu of the vasculature, in in transplantation might involve new therapeutic modalities response to inflammatory stress and/or immune reactions using soluble recombinant forms of CD39, altering (9). expression of this ectonucleotidase by drugs and/or using small molecules to inhibit deleterious P2-mediated This review considers important P2-receptors and signaling while augmenting beneficial adenosine-mediated major ectonucleotidases of the vascular endothelium and effects within the transplanted liver. liver, and relates the expression and functions of these to vascular injury, thromboregulatory issues, rejection 2. INTRODUCTION patterns, metabolic disturbances and disordered regeneration seen in certain hepatic grafts. Purinergic signaling comprises release of extracellular nucleotides, stimulation of purinergic 3. OVERVIEW: PRINCIPLE AND PHYSIOLOGY receptors and the modulation of nucleotide levels by ectoenzymes. Over the past decade, many advances have 3.1. Hepatic purinergic signaling been made in the study of purinergic receptors and the States of biological stress may lead to alteration regulation of extracellular nucleotide concentrations (1). It of release or uptake of nucleotides or may alternatively is now generally accepted that extracellular nucleotides decrease enzymatic function of ectonucleotidases (9). (e.g. ATP, UTP, ADP), and the derivative nucleosides (e.g. Besides being implicated in thrombosis, inflammation and adenosine from ATP), are released in a regulated manner vascular injury, purinergic signaling regulates important by cells to provide the primary components for purinergic hepatic processes such as bile secretion, glycogen responses (2). Specific nucleotide/nucleoside mediators metabolism and responses to insulin. Hence, the liver is of bind defined purinergic receptors that comprise the second major importance for purine homeostasis and for the requirement for this intricate signaling network. Almost all regulation of nucleotide synthesis. This highly cells express cell-surface type-2 purinergic (P2) receptors metabolically active organ provides the bulk of all purine for nucleotides and adenosine or type-1 purinergic (P1) precursor substrates for the peripheral tissues (10, 11). receptors. There are seven ionotropic (P2X), at least eight Salvage of nucleotides in bile was recognized over 40 years metabotropic (P2Y) and four adenosine receptor subtypes ago. This process is associated with both systemic that have been identified and characterized to date (3-7). nucleotide homeostasis and the potential for local Hepatic parenchymal and non-parenchymal cells express intercellular signaling within the canalicular networks and multiple P2X and P2Y receptor subtypes (see later). These biliary systems (12). receptors operate in both auto- and paracrine loops and are thought to play a complex, important role in the regulation Within the liver, ATPase activity has been shown of vascular and immune cell-mediated responses. For to be associated with the vasculature (NTPDase1), example ATP stimulation of endothelium and lymphocytes periportal fibroblasts (NTPDase2) and bile canaliculus largely induces pro-inflammatory responses, such as the (NTPDase8). Other ATPases such as NTPDase3 and release of interleukin-1 and interleukin-6. Exposure of NTPDase5 have been found in hepatic tissue, but their dendritic cells (DC) to extracellular ATP induces migration functional relevance is still unclear.

2589 Purinergic signaling in liver transplantation

Table 1. Expression and function of P2-receptors and ectonucleotidases by cellular components of the liver Cellular component Expression Function References Hepatocytes P2Y1, P2Y2, P2Y4, P2Y6, P2Y13, NTPDase8 (apical Glycogen metabolism 108-111 membrane) Insulin resistance Cholangiocytes P2Y1, P2Y2, P2Y4, P2Y6, P2X2, P2X3, P2X4, P2X6 Bile (anion) secretion 115, 117 Nucleotide salvage Canalicular contraction Interaction with hepatocytes Adenosine resorption from bile Endothelial cells P2Y1, P2Y2, P2Y6, P2X4, P2X7 NO release 96-102 NTPDase1 Secretion of prostaglandins E2 Vascular smooth muscle cells P2Y1, P2Y2, P2Y6, P2X4, P2X7 Portal vein contraction 48, 50, 164, 174 Hepatic stellate cells P2Y2, P2Y4, P2Y6, NTPDase2 Secretion of Prostaglandin F2 and D2 60, 83-85 Cell contraction Portal fibroblasts NTPDase2 Hepatic fibrosis 48, 92, 93, 95 Kupffer cell/macrophages P2Y1, P2Y2, P2Y4, P2Y6, P2X1, P2X4, P2X7, Killing of intracellular pathogens 120-134 NTPDase1 Secretion of prostaglandin E2 Interleukin 6 Liver associated lymphocytes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y14 Modulate Concanavalin A mediated hepatitis 135-145 NK, NKT, T cells, B cells P2X1, P2X2, P2X4, P2X7, NTPDase1 Neutrophils P2Y2 Chemotaxis 174

After hepatic ischemia and reperfusion injury, adenosine from the bile (25). Salvage of nucleotides in bile vascular NTPDase activity is lost and deletion of may be crucial in the maintenance of appropriate NTPDase1 in mice leads to significantly increased injury nucleotide/nucleoside concentrations within hepatocytes or and decreased survival. Furthermore, post-transplant within the entire organism (11). biochemical activity of NTPDase1 is lost initially postoperatively and is reestablished within days to weeks 3.3. Purinergic receptors later in the surviving, potentially accommodated grafts Over fifteen P2-receptors of different specificities (13). transmit signals from extracellular nucleotides, triggering and modulating vascular and immune cell activation In the following sections, we focus on the processes, metabolism, nitric oxide (NO) release, adhesion, expression and function of purinergic P2 receptors and migration, proliferation and apoptosis (26, 27). Since the ectonucleotidases within hepatic tissue (Table 1) (14). We original cloning of the P2Y2R subtype, at least eight P2YR describe the known implications of purinergic signaling in and seven P2XR subtypes have been cloned and processes such as vascular injury, modulation of bile and functionally identified. metabolism e.g. of blood glucose and tissue nucleotide levels. We also briefly allude to diseases that are impacted There are two main families of nucleotide upon by perturbations in nucleotide flux e.g. liver fibrosis receptors: P2X are "rapid" ligand-gated ion channels and tumor formation. permeable for Na+, K+ and also Ca2+ (subtypes P2X1-7) (28) and P2Y are the "slow" metabotropic receptors (P2Y1, 3.2. Nucleotide release 2, 4, 6, 11-14). P2Y receptors are 7-transmembrane Gq- or Extracellular nucleotides and nucleosides are Gi-protein linked and initiate signal transduction coupled to released in a regulated manner from cells by a variety of activation of phospholipase C, or to inhibition of adenylate mechanisms. Such pathways include exocytosis of cyclase, respectively (29). ATP/UTP-containing vesicles, facilitated diffusion via connexin-43 hemichannels, by putative ABC transporters There are also four known P1 cell surface or potentially by poorly understood electrodiffusional receptors for nucleosides, chiefly adenosine (A1, A2a, A2b movements through ATP/nucleotide channels (2, 15-21). and A3) that are classified according to their affinities for Under pathophysiological conditions, the release of adenosine and variant coupling to adenylate cyclase (30, nucleotides and the expression of purinergic receptors is 31). increased markedly in injured or stressed cells (17). Depending upon the repertoire of receptors and Extracellular ATP entering the liver by the portal signaling components, P2R influence cellular activation, vein is rapidly metabolized after a single passage through proliferation and the induction of apoptosis. For example, the liver (22). Therefore, metabolic stimulation by in the vascular system, extracellular nucleotides and extracellular nucleotides might expected to be more nucleosides can influence platelet activation, thrombosis, pronounced in the periportal region. In addition, ATP can inflammatory processes, cardiac function and vasomotor be released by hepatocytes into different extracellular responses, (32-35). ATP and ADP appear to regulate compartments: via basolateral, sinusoidal or apical exocrine hemostasis through the activation of platelet P2 receptors. routes. Secretion of ATP into the bile is mediated by an ADP is a major platelet recruiting factor originating from increase in cholangiocyte cell volume, which stimulates platelet dense granules, released upon activation whereas nucleotide release by vesicular exocytosis (23, 24). ATP derived from the same sources is considered a Canalicular nucleotide and nucleoside levels are further competitive antagonist of ADP for platelet P2Y1 receptors regulated and controlled by the presence of a canalicular (17, 36, 37). Platelet P2Y12 is perhaps the best known Na+-dependent nucleoside transporter that removes purinergic receptor and several drugs (thienopyridine

2590 Purinergic signaling in liver transplantation

desensitizing receptors and/or channels (P2Y1, P2Y2, P2X1 and P2X3) contrast with slow desensitizing receptors such as the P2Y6, P2X2 and P2X7 receptors (the last has proven to be very important in inflammatory reactions); P2X4 is intermediate in this regard (57).

3.4. Ectonucleotidases Ectonucleotidases consist of families of nucleotide metabolizing enzymes that are expressed on the plasma membrane and have externally orientated active sites (Figure 1). These ectoenzymes operate in concert or consecutively and metabolize nucleotides to the respective nucleoside analogs. They have the potential to decrease extracellular concentrations of nucleotides and to generate nucleosides. The relative contribution of the distinct

enzymes to the modulation of purinergic signaling may Figure 1. Principles of purinergic signaling in the hepatic depend on the availability and preference of substrates and microenvironment. Nucleotides are released from on cell and tissue distribution. parenchymal cells, non-parenchymal cells and platelets. These nucleotides then activate P2 receptors or are Ectonucleotidases modulate P2-receptor- hydrolyzed by NTPDases and 5’nucleotidase to adenosine, mediated signaling. Alterations in extracellular which activates P1 receptors. nucleotide levels can increase or decrease P2 activity or lead to P2 receptor desensitization (58). Desensitization agents e.g. clopidogrel) are used in clinical practice to of P2 receptors is dose dependent. In vitro the target ADP-mediated cardiovascular thrombosis (38). desensitization of P2-mediated anion secretion responses requires 10-min preincubations with the P2Y2 ATP (and UTP) also stimulate endothelial P2Y receptor agonist UTP. Recovery from UTP or ATP- receptors to release prostacyclin (PGI2) and nitric oxide induced desensitization can either be rapid (<10 min) at (NO); two vasodilators and inhibitors of platelet preincubation concentrations of approximately 2 µM or aggregation (39-43). This latter protective action of ATP requires progressively longer time periods at higher may limit the extent of intravascular platelet aggregation concentrations. Furthermore, generation of extracellular and to help localize thrombus formation to areas of adenosine not only abrogates nucleotide-mediated vascular damage (41, 44, 45). The receptors P2Y1 and effects but also activates adenosine receptors, often with P2Y2 on vascular endothelial cells, smooth muscle cells opposing (patho-) physiological effects. and monocytes are also important receptors in the mediation of vascular inflammation (46-48). Ectonucleotidases also produce the key molecules for purine salvage and consequent replenishment In addition, ATP also stimulates P2X receptors of ATP stores within multiple cell types (59). Indeed, to cause plasma membrane permeabilization, induction although nucleotides do not appear to be taken up by cells, of apoptosis, organic anion transport, and stimulation of dephosphorylated nucleoside derivatives interact with Ca2+ mobilization (33, 49). The active form of ATP that several specific transporters to enable intracellular uptake causes membrane permeabilization is a fully ionized via membrane passage (11, 60). tetrabasic ion (or ATP4) that interacts with the unique multimeric P2X7 receptors expressed on endothelium In the liver functional ATPases were shown as and monocyte-macrophages (50). This nucleotide is a early as 1960 to be associated with canalicular plasma relative inhibitor of NTPDases, as these are divalent membranes in association with alkaline phosphatases (61). cation dependent enzymes (51). Such P2X7 receptors This canalicular ecto-ATPase activity was subsequently shown to be modulated by bile duct ligation and to be are also stimulated by the nonhydrolyzable ATP analog + + ATP-γ-S but are unresponsive to UTP (35, 52). The distinct from the classical Na K -ATPase-pumps that are major effect of P2X7 receptors is the induction of preferentially expressed on the basolateral membranes (11). cellular activation and apoptosis (52, 53). The initially cloned putative ecto-ATPase was not a true ATPase but rather shared significant sequence homology Pathways of nucleotide-mediated signaling are with several canalicular glycoproteins such as C-Cam105 further complicated by P2-receptor desensitization (11). phenomena. Thus, adenine nucleotides may also directly limit ADP-mediated reactivity of platelets (33, 36, 54, 55). There are four major families of Like other members of the G protein coupled receptor ectonucleotidases in the liver microenvironment namely family, some P2Y receptors readily undergo agonist- CD39/NTPDases (ecto-nucleoside triphosphate induced desensitization (56). This may involve diphosphohydrolases), nucleotide phosphorylation of the receptor by multiple protein kinases pyrophosphatase/phosphodiesterase (NPP)-type ecto- (27). For unclear reasons, P2X- and P2Y-type receptors phosphodiesterases, alkaline phosphatases and ecto-5’- differ widely with respect to desensitization rates: rapidly nucleotidases/CD73 (62).

2591 Purinergic signaling in liver transplantation

3.4.1. Ecto- nucleoside triphosphate in response to ATP or UTP, or in response to ADP after diphosphohydrolases (NTPDases of the CD39 family) conversion to ATP (76-79). On the basis of the It is now known that there are three pharmacological profile of the nucleotide-induced Ca2+- ectonucleotidases largely responsible for the bulk of responses in human hepatocytes, it is believed that ATPase activity in the liver: NTPDase1, which is present extracellular ATP and UTP increase [Ca2+]i by activating on Kupffer and vascular endothelial cells, NTPDase2, P2Y2 and possibly P2Y4 receptors coupled to the Ca2+- which is expressed by portal fibroblasts and activated phosphatidylinositol signaling cascade (80). The major hepatic stellate cells and NTPDase8 that seems to be the metabolic effects of ATP include modulation of glycogen major ATPase of the hepatic canaliculus. Other hepatic metabolism by glycogenolysis and inactivation of glycogen NTPDases such as NTPDase3 on stellate cells and synthesis (81). For example, it has been demonstrated that NTPDase5 have been described on liver cells, however, activation of P2Y1 receptors in rat hepatocytes functional data is still lacking. substantially stimulates glycogen phosphorylase (82).

3.4.2. Nucleotide pyrophosphatase/phosphodiesterases 3.5.1.2. Ectonucleotidases (NPP) The major ectonucleotidases activity of NPP hydrolyze pyrophosphate or phosphodiester hepatocytes lies within the bile canalicular domain and is bonds of nucleotides, metabolizing ATP to AMP and comparable to what is described for the cholangiocyte, as diphosphates. The family of NPPs consists of seven members discussed below. (NPP1-7). NPP1 is expressed in hepatocytes and localizes basolateral membrane (63-65). The expression of NPP1 is 3.5.2. Cholangiocytes / bile ducts associated by hepatocellular growth. It is absent during the 3.5.2.1. Purinergic receptors fetal period and decreased during liver regeneration following Cholangiocytes express mRNA of distinct P2Y 70% partial hepatectomy (66). This observation underscores receptor family members P2Y1, P2Y2, P2Y4 and P2Y6 the relevance of ATP for hepatocellular proliferation (67). (83). Microperfusion of intrahepatic bile ducts by apyrase NPP3 is the major NPP isoenzyme at the apical membrane of and the P2 receptor inhibitor suramin also reveals the both hepatocytes and cholangiocytes (64, 65). presence of functional apical P2 receptors (60). Similar experiments using the perfusion of ATP-γ-S show net 3.4.3. Alkaline phosphatases (ALP) alkalization of bile ducts (83). Alkaline phosphatases are hydrolases responsible for removing phosphate groups in the 5- and 3- positions The apical purinergic receptor agonist preference from many types of molecules, including nucleotides, is consistent with the P2Y2 subtype and this receptor has proteins, and alkaloids. As the name suggests, alkaline been shown to be a marker of the apical cilia (60, 84). On phosphatases are most effective in an alkaline environment. the basolateral membrane the function of P2Y receptors Biliary and canalicular expression of ALP may provide a appears to be more complex. The kinetics of ATP defense mechanism modulating endotoxin toxicity, not degradation in apical and basolateral compartments are addressed here in detail (68)) distinct, suggesting that there are domain-specific signaling pathways that contribute to purinergic responses in 3.4.4. Ecto-5’-nucleotidase / CD73 polarized cholangiocytes (60, 83). Reverse transcriptase Ecto-5’-nucleotidase (CD73; EC 3.1.3.5) PCR from cultured normal rat cholangiocytes shows terminates the dephosphorylation cascade of nucleotides to transcripts for P2X receptors P2X2, P2X3, P2X4, and adenosine (69). This enzyme has been detected in the P2X6. In lysates of cholangiocytes, P2X4 protein can be canalicular plasma membrane, in the connective tissue of also detected, and immunohistochemical staining of intact the portal triads and central veins, and HSC (11, 70). Ecto- rat liver reveals P2X4 protein within intrahepatic bile ducts. 5’-nucleotidase is co-expressed with NTPDase8 on the Of P2X receptors, P2X4 is chiefly involved in regulation of canalicular membrane, thereby regulating biliary nucleotide bile secretion in vitro (85). Hepatocytes and bile ductular and nucleoside levels (71). cells have been shown to interact and communicate via ATP release in a paracrine manner and nucleotides may be 3.5. Distribution and functions involved in the regulation of canalicular contraction and bile 3.5.1. Hepatocytes secretion. ATP/UTP within the cholangiole stimulates anion 3.5.1.1. Purinergic receptors and fluid secretion. The binding of nucleotides to apical Select P2X and P2Y purinergic receptors are P2Y2-receptors may facilitate the coordination of bile expressed by hepatocytes (Figure 1) (72, 73). Of the P2Y formation as a consequence of the paracrine hepatobiliary receptor family, mRNA species of P2Y1, P2Y2, P2Y4, coupling. (85) P2Y6 and P2Y13 have been observed. Of these, only the P2Y1, P2Y2 and P2Y13 receptors appear to be of 3.5.2.2. Ectonucleotidases functional relevance for the hepatocyte (74, 75). P2X Functional ATPases were previously shown to be receptors have been noted in association with the apical associated with bile canalicular plasma membranes by membrane as shown below. histochemical techniques (12). The corresponding enzyme was originally identified as c-CAM105, but this turned out Extracellular nucleotides have several effects on to be incorrect (86-88). Recent studies have revealed that isolated hepatocytes such as repetitive changes of transients the canalicular ecto-ATPase corresponds to NTPDase8 in cytosolic free calcium concentration ([Ca (2+)]i) directly (89).

2592 Purinergic signaling in liver transplantation

nucleotides, sinusoidal endothelial cells secrete Prostaglandin E2 (PGE2) in a P2Y dependent manner (95).

On vascular smooth muscle cells, P2Y1, P2Y2, P2Y6, P2X1, P2X3, P2X4, P2X5 and P2X7 have been described (96-101). Vasoconstriction is the major effect on arterial smooth muscle cells in response to ATP and UDP and is mediated by P2X and P2Y6 receptors (96, 97). In the portal vein, vascular responses seem to be mediated by P2X and P2Y2 receptors (98, 99, 102).

3.5.3.2. Ectonucleotidases In the liver NTPDase1 can be detected immunohistochemically on endothelial cells of muscularized vessels and Kupffer cells (Figure 3) (103). Figure 2. P2 receptor expression by hepatic sinusoidal The NTPDase/CD39 family includes genes that share endothelial cells. High levels of mRNA for P2Y1, P2Y2, considerable sequence homology inclusive of five P2X4 and P2X7 are noted. The expression of P2Y6 is weak conserved regions (102). CD39 was originally and no signals for P2Y4, P2X1 and P2X2 are noted. characterized as a cell activation marker, identified on B cells, subsets of activated NK-cells and T-lymphocytes (104-106). We and others have since demonstrated that CD39 is an NTPDase (EC 3.6.1.5) that hydrolyzes both extracellular ATP and ADP to AMP (50, 105, 107). Members of this family of ectoenzymes are all membrane- attached ab initio, with external-oriented active sites. Select members such as NTPDase5 and NTPDase6 are solubilized after proteolytic degradation. Quiescent sinusoidal endothelial cells do not express CD39. However, under specific conditions of activation e.g. proliferation after partial hepatectomy, expression of CD39 on sinusoidal endothelial cells is notably upregulated and is of substantial functional relevance (Beldi et al, manuscript submitted). In all human and murine specimens examined to date, neither canalicular nor biliary expression of NTPDase1 has been observed, whereas strong expression on Kupffer cells and

endothelial cells of muscularized vessels is found (Figure Figure 3. Immunohistochemical patterns of expression of 3) (108). CD39 in donor human liver. Endothelial cells of the portal vein (pv), hepatic artery (ha) and Kupffer cells strongly Interactions between hepatic NTPDases and P2 express CD39. Quiescent sinusoidal endothelial cells and may exist, as NTPDases may modulate hepatocyte or bile bile duct (bd) epithelia do not stain for CD39. duct epithelial P2-receptor activity. However, there is no current data to support this hypothesis. Other evidence Human and rat NTPDase8 cDNA has been suggests that certain P2-receptors expressed in the cloned and the genes are located on chromosome loci 9q34 gastrointestinal tract can undergo desensitization in the and 3p13, respectively (71, 89). NTPDase8 resembles the Cd39-null mouse (58). chicken ecto-ATPase cloned from oviduct and liver (90, 91). Major ATPase and ADPase activity was detected in 3.5.4. Stellate cells / portal fibroblasts the bile canaliculi by immunohistochemistry after 3.5.4.1. Purinergic receptors incubation with nucleotides. It has been proposed that Quiescent hepatic stellate cells (HSC) can NTPDase8 is the canalicular ecto-ATPase and that it is function as pericytes in the liver and express the P2Y subtypes P2Y2 and P2Y4 that are activated by ATP and responsible for a major proportion of canalicular NTPDase activity (71). UTP. Activated HSC in turn express the P2Y subtype P2Y6 (108). Early reports have shown secretion of prostaglandins 3.5.3. Vasculature F2 and D2 after stimulation with ATP (109). Recent studies 3.5.3.1.Purinergic receptors revealed that P2Y receptors link extracellular ATP to Functional expression profiles of P2Y1 and P2Y2 inositol triphosphate-mediated cytosolic calcium signals receptors have been described for the hepatic artery and resulting in localized calcium signaling and cell contraction portal vein (92, 93). These endothelial cells of the hepatic (110, 111). artery and portal vein mediate nitric oxide (NO) release by P2Y mediated mechanisms (92-94)., We have detected Portal fibroblasts show functional P2Y activity mRNA for P2Y1, P2Y2, P2Y6, P2X4 and P2X7 receptors and express NTPDase2. NTPDase2 (or CD39L1/Cd39L1) in sinusoidal endothelial cells (Figure 2). In response to was originally cloned from rat brain and chicken smooth

2593 Purinergic signaling in liver transplantation muscle and was characterized later from human/mouse 3.5.6. Liver associated lymphocytes sources (112-114). This protein shares 40% sequence 3.5.6.1.Purinergic receptors homology with rat NTPDase1 but has a 32-fold preference Natural killer (NK) and natural killer T (NKT) for hydrolysis of ATP over ADP (leading to sustained cells as well as B cells express a variety of P2 receptors. accumulation of ADP). Functional and molecular Lymphocytes express P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, expression of NTPDase2 has been shown in portal P2Y12 and P2Y14 as well as P2X1, P2X2, P2X4 and P2X7 fibroblasts, near the basolateral membranes of bile duct (135-143). In the liver activation of P2X7 by released epithelia (115). This distribution may represent a nucleotides has been shown to mediate, at least in part, the previously unrecognized mechanism for regulation of injury noted in Concanavalin A dependent autoimmune nucleotide signaling in bile ducts and other epithelia. Under hepatitis (144). basal conditions portal fibroblasts hydrolyze ATP to AMP and thereby inhibit activation of basolateral P2Y receptor 3.5.6.2. Ectonucleotidases expression by bile duct epithelia. After injury, such as NTPDase1/CD39 is expressed on subsets of liver following bile duct ligation, portal fibroblasts transform lymphocytes such as NK, NKT and B cells. We have into portal myofibroblasts. Loss of NTPDase2 might recently shown, that NTPDase1 is expressed on regulatory predispose to stimulation of proliferation of bile duct T cells (145). epithelia (116). Decreased levels of NTPDase2 expression in human biliary cirrhosis, as well as in models of bile duct 4. IMPACT OF PURINERGIC SIGNALING ligation in the rat, have been observed. Expression of NTPDase 2 also shifts from the portal area to the areas with 4.1.Hepatic homeostasis in health bridging fibrous bands in cirrhosis with hepatitis C (117). 4.1.1. Bile flow Bile acid secretion is disturbed after liver 3.5.4.2. Ectonucleotidases transplantation, thereby possibly exacerbating hepatic Hepatic stellate cells express CD39 mRNA both ischemia and reperfusion injury (146, 147). Extracellular under quiescent and activated conditions (108). Upon nucleotides are released into the canaliculus and modulate activation HSC also express NTPDase2 (108, 115). The bile secretion. The biliary epithelium and hepatocytes functional relevance of NTPDase2 on HSC remains constitutively release ATP into the bile (11). In biliary unclear. NTPDase 2 is also expressed on portal fibroblasts epithelium ATP is stored in vesicles and is released in as discussed below. In vivo, NTPDase2 is not expressed on response to cell swelling (23); concentrations of canalicular hepatocytes (118). However, certain wild-type mouse adenine nucleotides in bile samples are estimated to be hepatoma cells also contain both constitutive and inducible 5±0.9µM (148, 149). Extracellular nucleotides entering the ecto-ATPase activities with unclear cellular localization bile ducts are potent stimuli for secretion of bile fluid (118). NTPDase2 was also found to be expressed on mouse including anions, and canalicular contraction, which is in hepatocytes in vitro after dioxin (2,3,7,8- part explained by interaction and communication between tetrachlorodibenzo-p-dioxin) treatment, but this remains hepatocytes and bile duct cells via local ATP release (25, controversial (119). 149-151). These effects are mediated and coordinated by apical P2Y2 receptors and NTPDase8 (71, 83, 89). 3.5.5. Kupffer cells 3.5.5.1.Purinergic receptors 4.1.2. Nucleotide synthesis and salvage There are limited data concerning the expression Canalicular localization of NTPDase8 in tandem and function of P2 receptors on Kupffer cells. Peritoneal with ecto-5’-ectonucleotidase suggests involvement in the macrophages typically express mRNA for P2Y1, P2Y2, regulation of nucleoside salvage and consequently P2Y6 and possibly P2Y4 as well as P2X1, P2X4 and P2X7 nucleotide homeostasis and purine salvage (71). Ultimately, receptors (120-122). P2X7 activation is associated with the generation of extracellular adenosine from Interleukin-1 secretion and killing of Mycobacteria, dephosphorylated ATP not only activates adenosine Chlamydia and fungi in monocyte-macrophages (52, 123- receptors but also produces the key molecule for purine 128). Polymorphisms of P2X7 have been associated with salvage and consequent replenishment of ATP within many decreased killing of Mycobacteria (129-131). Furthermore cell types (11, 59). In the canaliculus Na+ dependent secretion of interleukin-6 possibly by stimulation of P2X4 nucleoside transporters might further aid in concentrating receptors activates innate immunity (132). In one report, nucleotides within the bile (152). Nucleoside transporters liver injury by ATP was attenuated by administration of are of major importance to organs and cells incapable of de gadolinium chloride, which depletes Kupffer cells; Suramin novo nucleotide synthesis such as the brain, muscle, a P2 receptor antagonist has comparable effect. (133). intestinal mucosa and bone marrow (10, 25). Prostaglandin E2 is released after P2Y receptor activation in non-parenchymal cells (95). Erythrocytes also have these nucleoside transporters and may facilitate the transport of purines 3.5.5.1. Ectonucleotidases between hepatocytes and dependent tissues (153). As the NTPDase1/CD39 is expressed on Kupffer cells, liver appears to be a major source of purines for these however its function remains unclear. In theory, NTPDase1 tissues, curtailment of nucleotide loss into the bile may be would modulate P2 receptor function on Kupffer cells and important to maintain appropriate nucleotide/nucleoside mimic effects on macrophage function with respect to concentrations within hepatocytes (11). Thus, cytokine production (132, 134). dephosphorylation of nucleotides by ectonucleotidases may

2594 Purinergic signaling in liver transplantation be critical for appropriate systemic purine homeostasis production of prostacyclin (PGI2) and NO; two (25). vasodilators and inhibitors of platelet aggregation (39, 40).

4.1.3. Metabolism (glucose/fatty acids) Administration of soluble NTPDases in the blood Extracellular nucleotides are involved in the stream or up regulation of CD39 post-adenoviral infection regulation of glycogen, fatty acid metabolism and insulin has been shown to prolong transplanted graft survival and resistance. Extracellular nucleotides activate glycogen abrogate platelet sequestration within the vasculature. storage and are glycogenolytic (81). Overall glycogen Finally, mutant mice deficient in Cd39 have evidence for a phosphorylase and the generation of inositol triphosphate vascular-based thrombotic state, paradoxically associated (IP3) are stimulated. Glycogen synthase is inhibited, with marked prolongation of experimental bleeding times adenosine 3': 5’-cyclic monophosphate levels are lowered and acquired platelet hypofunction (164). and activation of phospholipase D is decreased (81, 154- 156). In addition, administered ATP appears to facilitate 4.2.2. Rejection up-regulation of the gluconeogenic enzyme, Purinergic receptors and ectonucleotidases are phosphoenolpyruvate carboxykinase, and down-regulation expressed on a variety of immune cells. However, data of the glycolytic enzyme, pyruvate kinase; hence promoting concerning hepatic graft survival are lacking. NTPDase1 glucose output by the liver (157, 158). Of the P2Y deficiency appears to have two opposing impacts on receptors that mainly control metabolism and proliferation, nucleotide-mediated signaling, that is, inhibition via P2Y2 seems to primarily control both glycogen metabolism desensitization of some but not all P2 receptors and and proliferation-associated responses such as increased augmentation of certain other responses via impaired [Ca (2+)](c) and modulation of mitogen-activated protein hydrolysis of their ligands (134). Consequently, in Cd39 kinase cascades (82). null mice, defects in dendrititc cell function, antigen presentation, T cell responses to haptens (type IV Effects of extracellular ATP on fatty acid hypersensitivity reactions) and delayed cellular rejection metabolism indicate inhibition of acetyl-CoA carboxylase responses are observed (8, 131, 134). We have noted activity and fatty acid synthesis with a concomitant membrane expression of CD39 by classic T-regulatory decrease of intracellular malonyl-CoA concentrations (Treg) cells that appears to be of crucial importance in the (159). immunomodulation of skin allograft rejection (145). These T cell subsets have recently been shown to directly Purinergic signaling also impacts upon insulin influence chronic vascular injury (165). signaling. Disordered regulation of extracellular nucleotides induces insulin resistance. Cd39 knockout mice 4.2.3. Regeneration show impaired glucose tolerance and sensitivity to insulin. In the area of split liver and living donor related Furthermore, plasma insulin levels are significantly higher liver transplantation liver regeneration has gained when compared to wild-type mice. Hyperinsulinemic additional interest in order to maintain and increase euglycemic clamp studies have revealed altered glucose regenerative capacities of remaining and transplanted liver metabolism in the livers of mice null for CD39/NTPDase1. lobes. ATP activates proliferation of hepatocytes in vitro The impaired sensitivity was accompanied by increased and in vivo and modulates growth factor responses possibly susceptibility to activation of c-jun N-terminal kinase /SAP via P2Y2 receptor activation (67). The response of by extracellular ATP in CD39 null mice liver, (Enjyoji at hepatocytes is comparable to early signaling events al, submitted). associated with liver regeneration such as transient activation of c-jun N-terminal kinase and immediate early 4.2. Liver disease genes. VEGF receptor 2 signaling, which is of importance 4.2.1. Vascular injury during liver regeneration, is modulated by the P2Y2 Many processes of vascular injury result in the receptor (48). In mice null for CD39 liver regeneration is release of adenine nucleotides that exert a variety of significantly decreased. Stimulation of cultured liver inflammatory effects on endothelial cells, platelets and sinusoidal endothelial cells (LSEC) with UTP and VEGF leukocytes (reviewed in (9, 33, 160-162)). Perturbation of leads to enhanced VEGF receptor 2 phosphorylation that is nucleotide signaling in the vasculature can influence abrogated in cultures with Cd39 -/- LSEC (Beldi G, Wu Y, vasomotor responses, platelet activation, inflammatory Sun X Imai M, Enjoji K, Zimmermann H, Candinas D, processes and also cardiac function (32). Robson SC, The vascular ectonucleotidase CD39 is permissive for sinusoidal endothelial cell proliferation In the setting of organ transplantation NTPDase1 during liver regeneration Hepatology 44 (4): 190A-191A 8 activity is lost with reperfusion or rejection and up- Suppl. 1 2006). regulation is seen with graft survival (13). In a model of total hepatic ischemia and reperfusion injury we have 4.2.4. Tumors shown a significantly decreased survival associated with Recurrence of hepatocellular carcinoma is an increased biochemical and structural liver injury in Cd39- important problem post-transplantation, especially in null animals as compared to wild-type controls (163). patients with larger tumors exceeding standard selection Furthermore, ADP is a major platelet recruiting factor criteria for liver transplantation (166, 167). As shown originating from platelet dense granules during activation above, deletion of Cd39 and perturbations in extracellular (17, 36, 37). ATP also stimulates endothelial cell nucleotides influence hepatocellular proliferation.

2595 Purinergic signaling in liver transplantation

Curiously, mice null for Cd39 also exhibit an increased modulation by NTPDases of the CD39 family in the incidence of spontaneous and diethyl nitrosamine induced regulation of hepatic blood flow, bile production, hepatocellular carcinoma (Wu Y, Sun X, Imai M, Sultan B, homeostasis and metabolism. These pathways impact upon Csizmadia E, Enjyoji K, Jackson S, Usheva A, Robson SC. vascular and immune responses to transplantation. In Modulation of RAS/ERK signaling by CD39/ENTPD1 addition to graft injury, disordered bile flow and ongoing during liver regeneration. Hepatology 2005 42, 4, Suppl.1, graft ischemia secondary to vascular rejection may impact Abst.1138, page 643A). Although the mechanism is upon the outcome of organ transplantation. unclear, mitogenic effects of extracellular nucleotides may predispose to the development of malignancy in these Additionally, purinergic receptors and CD39 are livers. There are also documented associations between not only expressed on endothelial cells and hepatic CD39 and RanBPM, a membrane scaffolding protein that sinusoidal cells but also at high levels on Treg (145, 171, may be responsible for heightened levels of hepatocyte Ras 172) and NKT cell populations (Beldi et al, submitted). It is activation observed following genetic deletion of CD39. particularly important that hepatic metabolic changes can This, in turn, may predispose to hepatocarcinogenesis be caused by extracellular nucleotides thought to serve (168). primarily as inflammatory mediators. These new data provide evidence for integration of nucleotide-mediated 4.2.5. Fibrogenesis and cirrhosis vascular and immune responses and associated metabolic Liver fibrosis and cirrhosis due to an array of disturbances e.g. insulin resistance that are so crucial to the underlying etiologies are the leading cause world wide of clinical outcomes following hepatic allografting. end stage liver disease. In patients post liver transplantation with hepatitis B and C, fibrosis has additionally been seen Our expectation is that therapeutic avenues will in conditions such as fibrosing cholestatic syndrome (169, develop using soluble CD39/NTPDase1 and/or small 170). The main cellular components that cause hepatic molecules to enhance NTPDase ecto-enzymatic functions fibrosis, namely portal fibroblasts and HSC, contain a that may be used together with selective adenosine receptor variety of P2 receptors and express NTPDase2. In this agonists. Other approaches to inhibit NTPDase activity system, closely regulated extracellular nucleotides can may be considered to slow progression of fibrosis e.g. in activate HSC leading to transcription of procollagen-1 chronic pancreatitis. It is of interest that certain drugs (108). The proliferation of bile duct epithelium which is already in clinical practice influence CD39 function and important for fibrogenesis is inhibited by NTPDase2 on expression. Commonly prescribed lipid lowering therapies portal fibroblasts leading to blockade of P2Y activation that have salutary effects on the vasculature (e.g. HMG- (116). CoA reductase inhibitors) and membrane phospholipids have in parallel important positive effects on endothelial 4.2.6. Cachexia cell NTPDase activity (173). Individuals with liver disease may develop metabolic imbalances with cholestasis, consequent bone Studies are planned to address the efficacy of marrow suppression, wasting or severe neurological CD39-based therapeutics in the setting of hepatic manifestations (including cerebral edema) that may reperfusion injury and in the appropriate immune progress to coma and death over days to weeks. modulation of hepatic allograft rejection. Hypothetically, uncompensated failure of hepatic nucleotide synthetic pathways and disordered salvage may 6. ACKNOWLEDGEMENTS contribute, at least in part, to these complications of acute or chronic liver failure. SCR acknowledges grant support from NIH HL57307, HL63972 and HL076540. GB thanks the Swiss The cachexia seen in untreated AIDS patients National Research Foundation for grant support. (PASMA- also has a component that may be associated with purine 115700, PBBEB-112764). 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Rosenberg: Targeted pyrophosphatase/phosphodiesterase, HSC: hepatic stellate disruption of cd39/ATP diphosphohydrolase results in cell, NK: natural killer cell, NKT: natural killer T cell,

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Treg: T regulatory cells, VEGF: Vascular endothelial growth factor, LSEC liver sinusoidal endothelial cell

Key Words: Transplantation, Liver, Purinergic Receptors, Bile, CD39, ecto-ATPase, Endothelium, Immunology, Metabolism, NTPDase, Platelet, Vasculature, Review

Send correspondence to: Simon C. Robson, MD, Liver and Transplantation Centers, Room 301, Research North, Beth Israel Deaconess Medical Center, 99 Brookline Ave, Boston MA 02215, Tel: 617-632-0831, Fax: 617 632 1861, E-mail: [email protected] http://www.bioscience.org/current/vol13.htm

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