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Interstrand DNA crosslinks (ICLs) represent complex lesions that block essential biological processes, including DNA replication, recombination, and transcription. Several pathways have been involved in ICL repair, in particular nucleotide excision repair (NER), translesion DNA synthesis (TLS), Fanconi anemia (FA), and homologous recombination (HR). Still, the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genome-wide screening, we identified the poorly characterized C1orf112 (also known as Apolo1) as a novel sensitizer to the clinically relevant ICL-inducing agent mafosfamide. Consistently, we noted that low expression of C1orf112 correlates with increased sensitivity to a series of ICL agents and PARP inhibitors in a panel of cell lines. We showed that lack of C1orf112 does not impact the initial recruitment and ubiquitylation of FANCD2 at the ICL site but rather impairs the resolution of RAD51 from ICL-induced DSBs, thereby compromising homology-directed DNA repair pathways. Our proximal mapping of C1orf112 protein neighbours coupled to structure-function analysis revealed that C1orf112, through its WCF motif, forms a complex with the N-terminal domain of the AAA+ ATPase FIGNL1 and regulates the interaction of FIGNL1 with RAD51. Our work establishes the C1orf112-FIGNL1 complex as an integral part of the HR-mediated response to ICLs by regulating the unloading of RAD51 during ICL repair.

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell’s DNA replication and repair machineries to replicate their own genomes. How this host–pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins.

Hepatic stellate cells (HSC) are critical effector cells of liver fibrosis. In the injured liver, HSC differentiate into a myofibrobastic phenotype. A critical feature distinguishing myofibroblastic from quiescent HSC is cytoskeletal reorganization. Soluble NSF attachment receptor (SNARE) proteins are important in trafficking of newly synthesized proteins to the plasma membrane for release into the extracellular environment. The goals of this project were to determine the expression of specific SNARE proteins in myofibroblastic HSC and to test whether their alteration changed the HSC phenotype in vitro and progression of liver fibrosis in vivo. We found that HSC lack the t-SNARE protein, SNAP-25, but express a homologous protein, SNAP-23. Downregulation of SNAP-23 in HSC induced reduction in polymerization and disorganization of the actin cytoskeleton associated with loss of cell movement. In contrast, reduction in SNAP-23 in mice by monogenic deletion delayed but did not prevent progression of liver fibrosis to cirrhosis. Taken together, these findings suggest that SNAP-23 is an important regular of actin dynamics in myofibroblastic HSC, but that the role of SNAP-23 in the progression of liver fibrosis in vivo is unclear.

In patients with abdominal region cancers, ionizing radiation (IR)-induced long-term liver injury is a major limiting factor in the use of radiotherapy. Previously, the major mitochondrial deacetylase, sirtuin 3 (SIRT3), has been implicated to play an important role in the development of acute liver injury after total body irradiation but no studies to date have examined the role of SIRT3 in liver&rsquo, s chronic response to radiation. In the current study, ten-month-old Sirt3&minus, /&minus, and Sirt3+/+ male mice received 24 Gy radiation targeted to liver. Six months after exposure, irradiated Sirt3&minus, mice livers demonstrated histopathological elevations in inflammatory infiltration, the loss of mature bile ducts and higher DNA damage (TUNEL) as well as protein oxidation (3-nitrotyrosine). In addition, increased expression of inflammatory chemokines (IL-6, IL-1&beta, TGF-&beta, ) and fibrotic factors (Procollagen 1, &alpha, SMA) were also measured in Sirt3&minus, mice following 24 Gy IR. The alterations measured in enzymatic activities of catalase, glutathione peroxidase, and glutathione reductase in the livers of irradiated Sirt3&minus, mice also implied that hydrogen peroxide and hydroperoxide sensitive signaling cascades in the absence of SIRT3 might contribute to the IR-induced long-term liver injury.

Sortilin, a member of the vacuolar protein sorting 10 domain receptor family, traffics newly synthesized proteins from the trans-Golgi network to secretory pathways, endosomes, and cell surface. Sortilin-trafficked molecules, including IL-6 and acid sphingomyelinase (aSMase), mediate cholangiocyte proliferation and liver inflammation, hepatic stellate cell activation, hepatocyte apoptosis, and fibrosis. Based on these sortilin-regulated functions, we investigated its role in biliary damage leading to hepatocellular injury and fibrosis. Sortilin −/− mice displayed impaired inflammation and ductular reaction 3 days after bile duct ligation (BDL), as demonstrated by reduced cholangiocyte proliferation and activation and reduced serum IL-6. Interestingly, liver fibrosis was reduced in Sortilin −/− mice after both BDL and carbon tetrachloride treatment, in line with attenuated in vitro activation of Sortilin −/− hepatic stellate cells. Sortilin −/− hepatic aSMase activity was reduced in the BDL and carbon tetrachloride models and accompanied by reduced in vivo hepatocyte apoptosis. In addition, wild type (WT), but not Sortilin −/− hepatocytes, had increased aSMase-dependent susceptibility to bile acid–induced apoptosis in vitro . Mechanistically, short-term IL-6 neutralization in bile duct–ligated WT mice decreased hepatic inflammation and reactive cholangiocyte-derived cytokines and chemokines, without affecting fibrosis, whereas pharmacological inhibition of aSMase activity was not sufficient to attenuate hepatic fibrosis. Only combined IL-6 and aSMase inhibition significantly reduced fibrosis in bile duct–ligated WT mice. We conclude that sortilin regulates cholestatic liver damage and fibrosis via effects on both aSMase activity and serum IL-6.

DNA double‐strand breaks (DSBs) can be repaired by two major pathways: non‐homologous end‐joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)‐based approach, we identify 11 high‐confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ‐mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1‐, RIF1‐, and REV7‐dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR. Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision‐making process during DSB repair.

SUMMARYDNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of a previously undescribed factor, FAM35A/SHDL2, as a novel effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B-cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1- and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and another previously uncharacterized protein, C20orf196/SHDL1, which promotes NHEJ and limits HR. Together, these results establish FAM35A as a novel effector of REV7 in controlling the decision-making process during DSB repair.

Epithelial response to injury is critical to the pathogenesis of biliary cirrhosis, and IL-6 has been suggested as a mediator of this phenomenon. Several liver cell types can secrete IL-6 following activation by various signaling molecules including circulating adenosine. The aims of this study were to assess whether adenosine can induce IL-6 secretion by cholangiocytes via the A2b adenosine receptor (A2bAR) and to determine the effect of A2bAR-sensitive IL-6 release on injury response in biliary cirrhosis. Human normal cholangiocyte H69 cells were used for in vitro studies to determine the mechanism by which adenosine and the A2bAR induce release of IL-6. In vivo, control and A2bAR-deficient mice were used to determine the roles of A2bAR-sensitive IL-6 release in biliary cirrhosis induced by common bile duct ligation (BDL). Additionally, the response to exogenous IL-6 was assessed in C57BL/6 and A2bAR-deficient mice. Adenosine induced IL-6 mRNA expression and protein secretion via A2bAR activation. Although activation of A2bAR induced cAMP and intracellular Ca2+ signals, only the Ca2+ signals were linked to IL-6 upregulation. After BDL, A2bAR-deficient mice have impaired survival, which is further impaired by exogenous IL-6; however, decreased survival is not due to changes in fibrosis and no changes in inflammatory cells. Exogenous IL-6 is associated with the increased presence of bile infarcts. Extracellular adenosine induces cholangiocyte IL-6 release via the A2bAR. This signaling pathway is important in the pathogenesis of injury response in biliary cirrhosis but does not alter fibrosis. Adenosine upregulates IL-6 release by cholangiocytes via the A2bAR in a calcium-sensitive fashion. Mice deficient in A2bAR experience impaired survival after biliary cirrhosis induced by common bile duct ligation independent of changes in fibrosis.

Liver myofibroblasts are specialized effector cells that drive hepatic fibrosis, a hallmark process of chronic liver diseases, leading to progressive scar formation and organ failure. Liver myofibroblasts are increasingly recognized as heterogeneous with regards to their origin, phenotype, and functions. For instance, liver myofibroblasts express cell markers that are universally represented such as, ItgαV and Pdgfrβ, or restricted to a given subpopulation such as, Lrat exclusively expressed in hepatic stellate cells, and Gpm6a in mesothelial cells. To study liver myofibroblasts in vitro, we have previously generated and characterized a SV40-immortalized polyclonal rat activated portal fibroblast cell line called RGF-N2 expressing multiple mesothelin mRNA transcripts. Mesothelin, a cell-surface molecule expressed in normal mesothelial cells and overexpressed in several cancers such as, mesothelioma and cholangiocarcinoma, was recently identified as a key regulator of portal myofibroblast proliferation, and fibrosis progression in the setting of chronic cholestatic liver disease. Here, we identify novel mesothelin splice variants expressed in rat activated portal fibroblasts. RGF-N2 portal fibroblast cDNA was used as template for insertion of hemagglutinin tag consensus sequence into the complete open reading frame of rat mesothelin variant coding sequences by extension PCR. Purified amplicons were subsequently cloned into an expression vector for in vitro translation and transfection in monkey COS7 fibroblasts, before characterization of fusion proteins by immunoblot and immunofluorescence. We show that rat activated portal fibroblasts, hepatic stellate cells, and cholangiocarcinoma cells express wild-type mesothelin and additional splice variants, while mouse activated hepatic stellate cells appear to only express wild-type mesothelin. Notably, rat mesothelin splice variants differ from the wild-type isoform by their protein properties and cellular distribution in transfected COS7 fibroblasts. We conclude that mesothelin is a marker of activated murine liver myofibroblasts. Mesothelin gene expression and regulation may be critical in liver myofibroblasts functions and fibrosis progression.

International audience; In this work, we report that Entpd1(-/-) mice, deficient for the ectonucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), produce smaller litters (27% reduction) compared with wild-type C57BL6 animals. This deficit is linked to reduced in vivo oocyte fertilization by Entpd1(-/-) males (61 +/- 11% versus 88 +/- 7% for Entpd1(+/+)). Normal epididymal sperm count, spermatozoa morphology, capacitation, and motility and reduced ejaculated sperm number (2.4 +/- 0.5 versus 3.7 +/- 0.4 million for Entpd1(+/+)) pointed to vas deferens dysfunction. NTPDase1 was localized by immunofluorescence in the tunica muscularis of the vas deferens. Its absence resulted in a major ATP hydrolysis deficiency, as observed in situ by histochemistry and in primary smooth muscle cell cultures. In vitro, Entpd1(-/-) vas deferens displayed an exacerbated contraction to ATP, a diminished response to its non-hydrolysable analog alphabetaMeATP, and a reduced contraction to electrical field stimulation, suggesting altered P2X1 receptor function with a propensity to desensitize. This functional alteration was accompanied by a 3-fold decrease in P2X1 protein expression in Entpd1(-/-) vas deferens with no variation in mRNA levels. Accordingly, exogenous nucleotidase activity was required to fully preserve P2X1 receptor activation by ATP in vitro. Our study demonstrates that NTPDase1 is required to maintain normal P2X1 receptor functionality in the vas deferens and that its absence leads to impaired peristalsis, reduced spermatozoa concentration in the semen, and, eventually, reduced fertility. This suggests that alteration of NTPDase1 activity affects ejaculation efficacy and male fertility. This work may contribute to unveil a cause of infertility and open new therapeutic potentials.

Purinergic signaling regulates a diverse and biologically relevant group of processes in the liver. However, progress of research into functions regulated by purinergic signals in the liver has been hampered by the complexity of systems probed. Specifically, there are multiple liver cell subpopulations relevant to hepatic functions, and many of these have been effectively modeled in human cell lines. Furthermore, there are more than 20 genes relevant to purinergic signaling, each of which has distinct functions. Hence, we felt the need to categorize genes relevant to purinergic signaling in the best characterized human cell line models of liver cell subpopulations. Therefore, we investigated the expression of adenosine receptor, P2X receptor, P2Y receptor, and ecto-nucleotidase genes via RT-PCR in the following cell lines: LX-2, hTERT, FH11, HepG2, Huh7, H69, and MzChA-1. We believe that our findings will provide an excellent resource to investigators seeking to define functions of purinergic signals in liver physiology and liver disease pathogenesis.

Hepatic fibrosis represents a pathological wound healing and tissue repair process triggered in response to chronic liver injury. A heterogeneous population of activated non-parenchymal liver cells, known as liver myofibroblasts, functions as the effector cells in hepatic fibrosis. Upon activation, liver myofibroblasts become fibrogenic, acquiring contractile properties and increasing collagen production capacity, while developing enhanced sensitivity to endogenous molecules and factors released in the local microenvironment. Hepatic extracellular adenosine is a bioactive small molecule, increasingly recognized as an important regulator of liver myofibroblast functions, and an important mediator in the pathogenesis of liver fibrosis overall. Remarkably, ecto-5′-nucleotidase/Nt5e/Cd73 enzyme, which accounts for the dominant adenosine-generating activity in the extracellular medium, is expressed by activated liver myofibroblasts. However, the molecular signals regulating Nt5e gene expression in liver myofibroblasts remain poorly understood. Here, we show that activated mouse liver myofibroblasts express Nt5e gene products and characterize the putative Nt5e minimal promoter in the mouse species. We describe the existence of an enhancer sequence upstream of the mouse Nt5e minimal promoter and establish that the mouse Nt5e minimal promoter transcriptional activity is negatively regulated by an Elf2-like Ets-related transcription factor in activated mouse liver myofibroblasts.

Innate immune responses play a critical role in controlling acute infections due to Pseudomonas aeruginosa in both mice and in humans. In this review we focus on innate immune recognition and clearance mechanisms that are important for controlling P. aeruginosa in the mammalian lung, with particular attention to those that influence the outcome of in vivo infection in murine models.

Nucleotide-activated P2X channels and P2Y metabotropic receptors participate in nociceptive signaling. Agonist availability is regulated by nucleoside triphosphate diphosphohydrolase-1 (NTPDase) -1, -2, -3 and -8, a family of enzymes that hydrolyze extracellular ATP to generate adenosine diphosphate (ADP, a P2Y agonist) and monophosphate (AMP). They provide a major source of extracellular AMP, the substrate for adenosine production by ecto-5′-nucleotidase (NT5E), and thereby regulate adenosine (P1) receptor signaling. NTPDases vary in their efficiency of tri- and di-phosphate hydrolysis; therefore which family members are expressed impacts nucleotide availability and half-life. This study employed enzyme activity histochemistry to examine the distribution of ATPase activity and immunohistochemistry for NTPDase1, -2, -3 and -8 in dorsal root ganglion (DRG) and spinal cord. Nucleotidase activity was robust in spinal dorsal horn, confirming that nociceptive pathways are a major site of nucleotide transmission. In DRG, extensive staining revealed ATPase activity in a subset of neurons and in non-neuronal cells. mRNA for NTPDase1-3, but not NTPDase8, was detected in lumbar DRG and spinal cord. Immunoreactivity for NTPDase3 closely matched the distribution of ATPase activity, labeling DRG central projections in the dorsal root and superficial dorsal horn, as well as intrinsic spinal neurons concentrated in lamina II. In DRG, NTPDase3 co-localized with markers of nociceptors and with NT5E. In addition, labeling of a subset of larger-diameter neurons in DRG was consistent with intense staining of Meissner corpuscle afferents in glabrous skin. Merkel cells and terminal Schwann cells of hair follicle afferents were also labeled, but the axons themselves were negative. We propose that NTPDase3 is a key regulator of nociceptive signaling that also makes an unexpected contribution to innocuous tactile sensation.

Aim: Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, hormone synthesis and maintenance of fluid composition. Moreover, adenosine is a key molecule for sperm capacitation. Extracellular nucleotide and nucleoside levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family is the most abundant and effective to hydrolyse ATP and ADP to AMP. In the female reproductive tract three members of this family have been recently identified: NTPDase1, NTPDase2 and NTPDase3 (Histochem. Cell Biol.131, 2009, 615). The purpose of the present study was to characterize in this system the expression profile of ecto-5′-nucleotidase (CD73), the enzyme generating adenosine from AMP. Methods: Immunological techniques and in situ enzymatic assays were used to characterize the ecto-5′-nucleotidase expression in the mouse female reproductive tract along the four stages of the estrous cycle, that were determined by vaginal smear examination. Results: Ecto-5′-nucleotidase was abundantly detected in the corpora lutea of the ovaries, as well as in several epithelia, such as that of oviducts, uterus and endometrial glands. Marked changes in endometrial ecto-5′-nucleotidase expression and activity along the estrous cycle are described, these being maximum at estrus phase, coinciding with optimal female sexual receptivity. Conclusion: The adenosine generated thereby, besides other functions, might contribute to sperm capacitation, thus significantly influencing fertility.

Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y2 mRNA receptor expression in the human colon adenocarcinoma cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues of patient suffering from Crohn’s disease and ulcerative colitis. However, the transcriptional events regulating P2Y2 receptor (P2Y2R) expression are not known. We have identified a putative transcription start site in the P2Y2R gene and demonstrated acetylation of Lys14 on histone H3 and Lys8 on histone H4, thus suggesting that the chromatin associated with the P2Y2 promoter is accessible to transcription factors. We also showed that the transcription factor NF-κB p65 regulates P2Y2R transcription under both proinflammatory and basal conditions. A NF-κB-responsive element was identified at −181 to −172 bp in the promoter region of P2Y2. Hence, activation of P2Y2R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE2 secretion by intestinal epithelial cells. These findings demonstrate that P2Y2R expression is regulated during intestinal inflammation through an NF-κB p65-dependent mechanism and could contribute not only to inflammatory bowel disease but also to other inflammatory diseases by regulating PG release.

Bile ductular proliferation is markedly upregulated in biliary fibrosis and cirrhosis. However, the mechanisms regulating this upregulation in bile ductular proliferation have not been defined. Recently, we demonstrated that expression of the ectonucleotidase nucleoside triphosphate diphosphohydrolase-2 (NTPDase2/ Entpd2) by portal fibroblasts (PF) is a critical regulator of bile ductular proliferation. Since interleukin 6 (IL-6) is markedly upregulated in biliary cirrhosis, our aims were to determine the role and mechanism of IL-6 in the regulation of NTPDase2 by PF. We found that IL-6 downregulated NTPDase2 protein expression in a concentration-dependent and time-dependent fashion but did not alter PF α-smooth muscle actin expression. IL-6 markedly downregulated NTPDase2 mRNA expression. Expression of the IL-6 receptor gp130 but not the IL-6 receptor gp80 was detected in PF. Two transcription start sites were identified in rat Entpd2 by the method of RNA ligase-mediated rapid amplification of 5′ cDNA ends. The minimal promoter construct, but not shorter constructs, was downregulated by IL-6. Three putative IL-6 response elements were identified in silico and mutated. Mutation of all three response elements, but not fewer elements, completely abolished the IL-6 response. Thus IL-6 transcriptionally downregulates NTPDase2 expression by PF via actions at specific promoter elements independently of myofibroblastic differentiation. This effect may represent a novel signaling pathway by which bile ductular proliferation is dysregulated in biliary cirrhosis and thus provides a potential therapeutic approach for the regulation of bile ductular growth.

The plasma membrane bound nucleoside triphosphate diphosphohydrolase (NTPDase)-1, 2, 3 and 8 are major ectonucleotidases that modulate P2 receptor signaling by controlling nucleotides' concentrations at the cell surface. In this report, we systematically evaluated the effect of the commonly used P2 receptor antagonists reactive blue 2, suramin, NF279, NF449 and MRS2179, on recombinant human and mouse NTPDase1, 2, 3 and 8. Enzymatic reactions were performed in a Tris/calcium buffer, commonly used to evaluate NTPDase activity, and in a more physiological Ringer modified buffer. Although there were some minor variations, there were no major changes either in the enzymatic activity or in the profile of NTPDase inhibition between the two buffers. Except for MRS2179, all other antagonists significantly inhibited these ecto-ATPases; NTPDase3 being the most sensitive to inhibition and NTPDase8 the most resistant. Estimated IC(50) showed that human NTPDases were generally more sensitive to the P2 receptor antagonists tested than the corresponding mouse isoforms. NF279 and reactive blue 2 were the most potent inhibitors of NTPDases which almost completely abrogated their activity at the concentration of 100 microM. In conclusion, reactive blue 2, suramin, NF279 and NF449, at the concentrations commonly used to antagonize P2 receptors, inhibit the four major ecto-ATPases. This information may reveal useful for the interpretation of some pharmacological studies of P2 receptors. In addition, NF279 is a most potent non-selective NTPDase inhibitor. Although P2 receptor antagonists do not display a strict selectivity toward NTPDases, their IC(50) values may help to discriminate some of these enzymes.

Background and purpose: ARL 67156, 6-N,N-Diethyl-D-β-γ-dibromomethylene adenosine triphosphate, originally named FPL 67156, is the only commercially available inhibitor of ecto-ATPases. Since the first report on this molecule, various ectonucleotidases responsible for the hydrolysis of ATP at the cell surface have been cloned and characterized. In this work, we identified the ectonucleotidases inhibited by ARL 67156.

Evidence demonstrating that regular ingestion of coffee has salutary effects on patients with chronic liver disease is accumulating rapidly. Specifically, it appears that coffee ingestion can slow the progression of liver fibrosis, preventing cirrhosis and hepatocellular carcinoma (HCC). This should excite clinicians and scientists alike, since these observations, if true, would create effective, testable hypotheses that should lead to improved understanding on fibrosis pathogenesis and thus may generate novel pharmacologic treatments of patients with chronic liver disease.This review is designed to examine the relevant clinical and epidemiological data in critical fashion and to examine the putative pharmacological effects of coffee relevant to the pathogenesis of liver fibrosis and cirrhosis. We hope that this will inspire relevant critical analyses, especially among “coffee skeptics”. Of note, one major assumption made by this review is that the bulk of the effects of coffee consumption are mediated by caffeine, rather than by other chemical constituents of coffee. Our rationales for this assumption are threefold: first, caffeine’s effects on adenosinergic signaling provide testable hypotheses; second, although there are myriad chemical constituents of coffee, they are present in very low concentrations, and perhaps more importantly, vary greatly between coffee products and production methods (it is important to note that we do not dismiss the “botanical” hypothesis here; rather, we do not emphasize it at present due to the limitations of the studies examined); lastly, some (but not all) observational studies have examined both coffee and non-coffee caffeine consumption and found consistent effects, and when examined, no benefit to decaffeinated coffee has been observed. Further, in the interval since we examined this phenomenon last, further evidence has accumulated supporting caffeine as the effector molecule for coffee’s salutary effects.

The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5’-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis.

The presence of one or more calcium-dependent ecto-ATPases (enzymes that hydrolyze extracellular 5′-triphosphates) in mammalian taste buds was first shown histochemically. Recent studies have established that dominant ecto-ATPases consist of enzymes now called nucleoside triphosphate diphosphohydrolases (NTPDases). Massively parallel signature sequencing (MPSS) from murine taste epithelium provided molecular evidence suggesting that NTPDase2 is the most likely member present in mouse taste papillae. Immunocytochemical and enzyme histochemical staining verified the presence of NTPDase2 associated with plasma membranes in a large number of cells within all mouse taste buds. To determine which of the three taste cell types expresses this enzyme, double label assays were performed using antisera directed against the glial glutamate/aspartate transporter, (GLAST), the transduction pathway proteins phospholipaseC β2 (PLCβ2) or the G protein subunit α-gustducin, and serotonin (5HT) as markers of type I, II or III taste cells, respectively. Analysis of the double labeled sections indicates that NTPDase2 immunoreactivity is found on cell processes that often envelop other taste cells, reminiscent of type I cells. In agreement with this observation, NTPDase2 was located to the same membrane as GLAST, indicating that this enzyme is present in type I cells. The presence of ecto-ATPase in taste buds likely reflects the importance of ATP as an intercellular signaling molecule in this system.

The published epidemiological data demonstrating an inverse relationship between coffee (and potentially other caffeinated beverage) consumption and liver fibrosis and its downstream complications are weighty and rapidly accumulating. Several excellent recent reviews examine this evidence in great detail (1–3), and the overwhelming conclusion is that this inverse relationship is real – coffee drinking reduces liver fibrosis. Among the strongest studies to support this observation are the findings that, after adjustment for confounders, individuals in the highest quintile of caffeine consumption had less than one third the risk of ALT elevation of those in the lowest quintile (odds ratio (OR) 0.31, 95% CI 0.16–0.61) (4) and, perhaps more importantly, advanced liver fibrosis from chronic liver diseases of various etiologies is associated with reduced coffee and total caffeine consumption (5) with one study showing that the odds of having cirrhosis decreased with increasing daily consumption of coffee in a step-wise manner from an OR of 0.47 (95% CI 0.20–1.10) for patients consuming 1 cup of coffee per day to an OR of 0.16 (95% CI 0.05–0.50) for patients consuming 4 cups per day, compared to lifetime abstainers as the reference (OR 1.0) (6). Demonstrating the clinical significance of coffee consumption, Freedman and colleagues found that among patients with advanced fibrosis, those who consumed no coffee had a risk of hepatic decompensation or hepatocellular carcinoma (HCC) of 11.1 per 100 patient-years compared to just 6.3 per 100 patient-years in those consuming ≥ 3 cups of coffee per day, with no beneficial effect seen with tea or other sources of caffeine (7). Coffee consumption has also been shown to be associated with a lower risk of fatty liver disease (8), metabolic syndrome (9), and ultimately hepatocellular carcinoma (10). As a clinician or scientist interested in the pathogenesis of liver fibrosis, one may very well ask whether these findings are of great value.

Glioma cells release cytokines to stimulate inflammation that facilitates cell proliferation. Here, we show that Lipopolysaccharide (LPS) treatment could induce glioma cells to proliferate and this process was dependent on nucleotide receptor activation as well as interleukin-8 (IL-8/CXCL8) secretion. We observed that extracellular nucleotides controlled IL-8/CXCL8 and monocyte chemoattractant protein 1 (MCP-1/CCL2) release by U251MG and U87MG human glioma cell lines via P2X7 and P2Y6 receptor activation. The LPS-induced release of these cytokines was also modulated by purinergic receptor activation since IL-8 and MCP-1 release was decreased by the nucleotide scavenger apyrase as well as by the pharmacological P2Y6 receptor antagonists suramin and MRS2578. In agreement with these observations, the knockdown of P2Y6 expression decreased LPS-induced IL-8 release as well as the spontaneous release of IL-8 and MCP-1, suggesting an endogenous basal release of nucleotides. Moreover, high millimolar concentrations of ATP increased IL-8 and MCP-1 release by the glioma cells stimulated with suboptimal LPS concentration which were blocked by P2X7 and P2Y6 antagonists. Altogether, these data suggest that extracellular nucleotides control glioma growth via P2 receptor-dependent IL-8 and MCP-1 secretions.

Rationale Lack of an experimental model of portopulmonary hypertension (POPH) has been a major obstacle in understanding of pathophysiological mechanisms underlying the disease. Objective We investigated the effects of CCl4-mediated cirrhosis on the pulmonary vasculature, as an initial step towards an improved understanding of POPH. Methods And Results Male C57BL/6 mice received intraperitoneal injection of either sterile olive oil or CCl4 3 times/week for 12 weeks. Cirrhosis and portal hypertension were confirmed by evidence of bridging fibrosis and nodule formation in CCl4-treated liver determined by trichrome/picrosirius red staining and an increase in spleen weight/body weight ratio, respectively. Staining for the oxidative stress marker, 4-hydroxynonenal (4-HNE), was strong in the liver but was absent in the lung, suggesting that CCl4 did not directly induce oxidative injury in the lung. Pulmonary acceleration time (PAT) and the ratio of PAT/pulmonary ejection time (PET) measured by echocardiography were significantly decreased in cirrhotic mice. Increase in right ventricle (RV) weight/body weight as well as in the weight ratio of RV/(left ventricle + septum) further demonstrated the presence of pathological changes in the pulmonary circulation in these mice. Histological examination revealed that lungs of cirrhotic mice have excessive accumulation of perivascular collagen and thickening of the media of the pulmonary artery. Conclusion Collectively, our data demonstrate that chronic CCl4 treatment induces pathological changes in pulmonary circulation in cirrhotic mice. We propose that this murine cirrhotic model provides an exceptional tool for future studies of the molecular mechanisms mediating pulmonary vascular diseases associated with cirrhosis and for evaluation of novel therapeutic interventions.

Liver myofibroblasts derived from hepatic stellate cells (HSC) are critical mediators of liver fibrosis. Release of tissue inhibitor of metalloproteinase-1 (TIMP-1) advances liver fibrosis by blocking fibrinolysis. The mechanisms responsible for the posttranslational regulation of TIMP-1 by myofibroblastic HSC are unknown. Here, we demonstrate that TIMP-1 release by HSC is regulated in a posttranslational fashion via calcium-sensitive vesicular exocytosis. To our knowledge, this is the first article to directly examine vesicular trafficking in myofibroblastic HSC, potentially providing a new target to treat and or prevent liver fibrosis.

The most common cause of liver failure is cirrhosis, due to progressive liver fibrosis and other architectural changes in the liver. Fibrosis occurs after liver injury or stress and results directly from an imbalance between the processes of extracellular matrix synthesis (fibrogenesis) and degradation (fibrolysis). Although research studies have identified several promising targets at the molecular level, current therapies to prevent and treat hepatic fibrosis in patients have only shown limited success. It is well established that liver myofibroblasts are the primary effector cells responsible for the extensive extracellular matrix accumulation and scar formation observed during hepatic fibrosis, in both clinical and experimental settings. Thus, as the major fibrogenic cells implicated in wound healing and tissue repair response, liver myofibroblasts could represent excellent targets for antifibrotic therapies. Still, the exact natures and identities of liver myofibroblasts precursors have yet to be resolved, and their relative contribution to hepatic fibrosis to be determined. The goal of this review is to examine the relative importance of liver myofibroblast precursors in the pathogenesis of liver fibrosis.

Extracellular nucleotides and adenosine are biologically active molecules that bind members of the P2 and P1 receptor families, respectively. In the digestive system, these receptors modulate various functions, including salivary, gastric, and intestinal epithelial secretion and enteric neurotransmission. The availability of P1 and P2 ligands is modulated by ectonucleotidases, enzymes that hydrolyze extracellular nucleotides into nucleosides. Nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5′-nucleotidase are the dominant ectonucleotidases at physiological pH. While there is some information about the localization of ecto-5′-nucleotidase and NTPDase1 and -2, the distribution of NTPDase3 in the digestive system is unknown. We examined the localization of these ectonucleotidases, with a focus on NTPDase3, in the gastrointestinal tract and salivary glands. NTPDase1, -2, and -3 are responsible for ecto-ATPase activity in these tissues. Semiquantitative RT-PCR, immunohistochemistry, and in situ enzyme activity revealed the presence of NTPDase3 in some epithelial cells in serous acini of salivary glands and mucous acini and duct cells of sublingual salivary glands, in cells from the stratified esophageal and forestomach epithelia, and in some enteroendocrine cells of the gastric antrum. Interestingly, NTPDase2 and ecto-5′-nucleotidase are coexpressed with NTPDase3 in salivary gland cells and stratified epithelia. In the colon, neurons express NTPDase3 and glial cells express NTPDase2. Ca2+ imaging experiments demonstrate that NTPDases regulate P2 receptor ligand availability in the enteric nervous system. In summary, the specific localization of NTPDase3 in the digestive system suggests functional roles of the enzyme, in association with NTPDase2 and ecto-5′-nucleotidase, in epithelial functions such as secretion and in enteric neurotransmission.

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615-628, 2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main enzyme responsible for the generation of adenosine from AMP, namely the ecto-5'-nucleotidase (CD73). The enzyme was identified by immunological techniques and by in situ enzymatic assays, including inhibition experiments with alpha,beta-methylene-ADP, a specific CD73 inhibitor. High levels of ecto-5'-nucleotidase were detected in testes in association with both germinal and somatic cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5'-nucleotidase that, in concurrence with NTPDases, may impact male fertility.

Extracellular nucleotides might influence aspects of the biology of reproduction in that ATP affects smooth muscle contraction, participates in steroidogenesis and spermatogenesis, and also regulates transepithelial transport, as in oviducts. Activation of cellular nucleotide purinergic receptors is influenced by four plasma membrane-bound members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, namely NTPDase1, NTPDase2, NTPDase3, and NTPDase8 that differ in their ecto-enzymatic properties. The purpose of this study was to characterize the expression profile of the membrane-bound NTPDases in the murine female and male reproductive tracts by immunological techniques (immunolabelling, Western blotting) and by enzymatic assays, in situ and on tissue homogenates. Other than the expected expression on vascular endothelial and smooth muscle cells, NTPDase1 was also detected in Sertoli cells and interstitial macrophages in testes, in ovarian granulosa cells, and in apical cells from epididymal epithelium. NTPDase2 was largely expressed by cells in the connective tissue; NTPDase3 in secretory epithelia, and finally, NTPDase8 was not detected in any of the tissues studied here. In addition, NTPDase6 was putatively detected in Golgi-phase acrosome vesicles of round spermatids. This descriptive study suggests close regulation of extracellular nucleotide levels in the genital tract by NTPDases that may impact specific biological functions.

Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are a family of membrane-bound enzymes that hydrolyze extracellular di- and triphosphate nucleosides. E-NTPDases have been proposed to control extracellular nucleotide levels that mediate intercellular communication by binding to specific membrane receptors. Here we show a detailed immunocytochemical localization of two enzymes of the E-NTPDase family in the retinal layers of two vertebrate species, namely, the mouse and the zebrafish. In the mouse retina, NTPDase2 was chiefly localized in Muller glia and ganglion cell processes. NTPDase1 was located on neurons as well, since it was expressed by horizontal and ganglion cell processes, suggesting that nucleotides such as ATP and ADP can be hydrolyzed at the surface of these cells. NTPDase1 was also detected in intraretinal blood vessels of the mouse. Regarding zebrafish, NTPDases1 and 2 seem to be differentially localized in horizontal cell processes, photoreceptor segments, and ganglion cell dendrites and axons, but absent from Muller glia. Moreover, NTPDases1 and 2 appear to be expressed within the germinal margin of the zebrafish retina that contains proliferative and differentiating cells. Retinal homogenates from both species exhibited ecto-ATPase activity which might be attributed at least to NTPDases1 and 2, whose expression is described in this report. Our results suggest a compartmentalized regulation of extracellular nucleotide/nucleoside concentration in the retinal layers, supporting a relevant role for extracellular nucleotide mediated-signaling in vertebrate retinas.

Bile duct epithelia are the target of a number of "cholangiopathies" characterized by disordered bile ductular proliferation. Although mechanisms for bile ductular proliferation are unknown, recent evidence suggests that extracellular nucleotides regulate cell proliferation via activation of P2Y receptors. Portal fibroblasts may regulate bile duct epithelial P2Y receptors via expression of the ecto-nucleotidase NTPDase2. Thus, we tested the hypothesis that portal fibroblasts regulate bile duct epithelial proliferation via expression of NTPDase2. We generated a novel co-culture model of Mz-ChA-1 human cholangiocarcinoma cells and primary portal fibroblasts. Cell proliferation was measured by bromodeoxyuridine uptake. NTPDase2 expression was assessed by immunofluorescence and quantitative real-time reverse transcription PCR. NTPDase2 expression in portal fibroblasts was blocked using short interfering RNA. NTPDase2 overexpression in portal myofibroblasts isolated from bile duct-ligated rats was achieved by cDNA transfection. Co-culture of Mz-ChA-1 cells with portal fibroblasts decreased their proliferation to 26% of control. Similar decreases in Mz-ChA-1 proliferation were induced by the soluble ecto-nucleotidase apyrase and the P2 receptor inhibitor suramin. The proliferation of Mz-ChA-1 cells returned to baseline when NTPDase2 expression in portal fibroblasts was inhibited using NTPDase2-specific short interfering RNA. Untransfected portal myofibroblasts lacking NTPDase2 had no effect on Mz-ChA-1 proliferation, yet portal myofibroblasts transfected with NTPDase2 cDNA inhibited Mz-ChA-1 proliferation. We conclude that portal fibroblasts inhibit bile ductular proliferation via expression of NTPDase2 and blockade of P2Y activation. Loss of NTPDase2 may mediate the bile ductular proliferation typical of obstructive cholestasis. This novel cross-talk signaling pathway may mediate pathologic alterations in bile ductular proliferation in other cholangiopathic conditions.

Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with K (m) values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X(1-7) and P2Y(2,4,11) receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y(1,12,13) receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y(6) receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-8.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5'-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

We have cloned and characterized the nucleoside triphosphate diphosphohydrolase-3 (NTPDase3) from mouse spleen. Analysis of cDNA shows an open reading frame of 1587 base pairs encoding a protein of 529 amino acids with a predicted molecular mass of 58 953 Da and an estimated isoelectric point of 5.78. The translated amino acid sequence shows the presence of two transmembrane domains, eight potential N-glycosylation sites and the five apyrase conserved regions. The genomic sequence is located on chromosome 9F4 and is comprised of 11 exons. Intact COS-7 cells transfected with an expression vector containing the coding sequence for mouse NTPDase3 hydrolyzed P2 receptor agonists (ATP, UTP, ADP and UDP) but not AMP. NTPDase3 required divalent cations (Ca2+>Mg2+) for enzymatic activity. Interestingly, the enzyme had two optimum pHs for ATPase activity (pH 5.0 and 7.4) and one for ADPase activity (pH 8.0). Consequently, the ATP/ADP and UTP/UDP hydrolysis ratios were two to four folds higher at pH 5.0 than at pH 7.4, for both, intact cells and protein extracts. At pH 7.4 mouse NTPDase3 hydrolyzed ATP, UTP, ADP and UDP according to Michaelis–Menten kinetics with apparent Kms of 11, 10, 19 and 27 μM, respectively. In agreement with the Km values, the pattern of triphosphonucleoside hydrolysis showed a transient accumulation of the corresponding diphosphonucleoside and similar affinity for uracil and adenine nucleotides. NTPDase3 hydrolyzes nucleotides in a distinct manner than other plasma membrane bound NTPDases that may be relevant for the fine tuning of the concentration of P2 receptor agonists.

F. Kukulski, S.A. Levesque, E.G. Lavoie, J. Lecka, F. Bigonnesse, A.F. Knowles, S.C. Robson, T.L. Kirley & J. Sevigny (2005) Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8. Purinergic Signalling 1(2): 193–204. On page 194 of the above-mentioned article, in ‘Materials and methods’ under ‘Plasmids,’ second paragraph, the mouse NTPDase2 accession number AY37674 is incorrect and should read {"type":"entrez-nucleotide","attrs":{"text":"AY376711","term_id":"36312788","term_text":"AY376711"}}AY376711.