ABSTRACT
Viral infection often trigger an ATM serine/threonine kinase (ATM)-dependent DNA damage response in host cells that suppresses viral replication. Viruses evolved different strategies to counteract this antiviral surveillance system. Here, we report that human herpesvirus 6B (HHV-6B) infection causes genomic instability by suppressing ATM signaling in host cells. Expression of immediate-early protein 1 (IE1) phenocopies this phenotype and blocks homology-directed double-strand break repair. Mechanistically, IE1 interacts with NBS1, and inhibits ATM signaling through two distinct domains. HHV-6B seems to efficiently inhibit ATM signaling as further depletion of either NBS1 or ATM do not significantly boost viral replication in infected cells. Interestingly, viral integration of HHV-6B into the host's telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair. Given that spontaneous IE1 expression has been detected in cells of subjects with inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases.
Subject(s)
Herpesvirus 6, Human , Immediate-Early Proteins , Roseolovirus Infections , Humans , Herpesvirus 6, Human/genetics , Herpesvirus 6, Human/metabolism , Roseolovirus Infections/genetics , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism , Virus Integration , Genomic Instability , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolismABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA-Binding Proteins/metabolism , Mad2 Proteins/metabolism , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , G2 Phase/genetics , HEK293 Cells , Humans , Mad2 Proteins/genetics , S Phase/genetics , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolismABSTRACT
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.
Subject(s)
DNA Breaks, Double-Stranded , Papillomaviridae/metabolism , Papillomavirus E7 Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Cell Line, Tumor , DNA Repair , Female , Genomic Instability , Homologous Recombination , Host-Pathogen Interactions , Humans , Papillomavirus E7 Proteins/genetics , Papillomavirus Infections/genetics , Papillomavirus Infections/virology , Signal Transduction , Tumor Suppressor p53-Binding Protein 1/metabolism , Ubiquitin/genetics , Ubiquitin-Protein Ligases/genetics , Uterine Cervical Neoplasms/virologyABSTRACT
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.
Subject(s)
Adaptor Proteins, Vesicular Transport/deficiency , Apoptosis , Bile Ducts/pathology , Cholestasis/complications , Hepatocytes/pathology , Liver Cirrhosis/pathology , Liver/injuries , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Cell Proliferation , Chemokines/metabolism , Cholestasis/pathology , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Hepatocytes/metabolism , Inflammation/pathology , Interleukin-6/metabolism , Ligation , Liver/metabolism , Liver/pathology , Liver Cirrhosis/complications , Liver Cirrhosis/metabolism , Mice, Inbred C57BL , Neutralization Tests , Phenotype , Sphingomyelin Phosphodiesterase/metabolismABSTRACT
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.
Subject(s)
5'-Nucleotidase/biosynthesis , Gene Expression Regulation/physiology , Liver Cirrhosis/metabolism , Myofibroblasts/metabolism , Transcription Factors/metabolism , Animals , DNA-Binding Proteins/metabolism , Male , Mice , Mice, Inbred C57BLABSTRACT
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.
Subject(s)
Adenosine/pharmacology , Bile Ducts/drug effects , Epithelial Cells/drug effects , Interleukin-6/genetics , Liver Cirrhosis, Biliary/genetics , Animals , Bile Ducts/metabolism , Bile Ducts/pathology , Cell Line , Epithelial Cells/metabolism , Gene Expression/drug effects , Humans , Interleukin-6/metabolism , Interleukin-6/pharmacology , Kaplan-Meier Estimate , Liver Cirrhosis, Biliary/metabolism , Mice, Inbred C57BL , Mice, Knockout , RNA Interference , Receptor, Adenosine A2B/genetics , Receptor, Adenosine A2B/metabolismABSTRACT
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.
Subject(s)
Brain Neoplasms/metabolism , Cell Proliferation , Chemokine CCL2/metabolism , Glioma/metabolism , Interleukin-8/metabolism , Receptors, Purinergic/physiology , Base Sequence , Brain Neoplasms/pathology , Cell Line, Tumor , DNA Primers , Glioma/pathology , Humans , Polymerase Chain Reaction , Receptors, Purinergic/genetics , Receptors, Purinergic/metabolism , Signal TransductionABSTRACT
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 αßMeATP, 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.
Subject(s)
Antigens, CD/genetics , Apyrase/genetics , Infertility, Male/genetics , Oligospermia/genetics , Receptors, Purinergic P2X1/genetics , Spermatozoa/physiology , Vas Deferens/enzymology , Adenosine Triphosphate/metabolism , Animals , Apyrase/deficiency , Ejaculation , Epididymis/enzymology , Epididymis/physiopathology , Female , Gene Expression Regulation , Infertility, Male/enzymology , Infertility, Male/physiopathology , Male , Mice , Mice, Knockout , Muscle Contraction , Muscle, Smooth/enzymology , Muscle, Smooth/physiopathology , Oligospermia/enzymology , Oligospermia/physiopathology , Oocytes/physiology , Receptors, Purinergic P2X1/metabolism , Sperm Capacitation , Vas Deferens/physiopathologyABSTRACT
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.
Subject(s)
Hepatic Stellate Cells/metabolism , Hepatocytes/metabolism , Purines/metabolism , Signal Transduction/physiology , Adenosine Triphosphatases/metabolism , Cell Line , Humans , Liver/cytology , Liver/metabolism , Receptors, Purinergic P1/metabolism , Receptors, Purinergic P2X/metabolism , Receptors, Purinergic P2Y/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.
Subject(s)
DNA Repair , Rad51 Recombinase , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Proteins/genetics , DNA/genetics , MitosisABSTRACT
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. Ca(2+) 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.
Subject(s)
Enteric Nervous System/metabolism , Esophagus/metabolism , Gastric Mucosa/metabolism , Pyrophosphatases/metabolism , Salivary Glands/metabolism , Analysis of Variance , Animals , Calcium/metabolism , Epithelial Cells/metabolism , Guinea Pigs , Immunohistochemistry , Male , Mice , Neurons/metabolism , Pyrophosphatases/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y(2) 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 P2Y(2) receptor (P2Y(2)R) expression are not known. We have identified a putative transcription start site in the P2Y(2)R gene and demonstrated acetylation of Lys(14) on histone H3 and Lys(8) on histone H4, thus suggesting that the chromatin associated with the P2Y(2) promoter is accessible to transcription factors. We also showed that the transcription factor NF-kappaB p65 regulates P2Y(2)R transcription under both proinflammatory and basal conditions. A NF-kappaB-responsive element was identified at -181 to -172 bp in the promoter region of P2Y(2). Hence, activation of P2Y(2)R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE(2) secretion by intestinal epithelial cells. These findings demonstrate that P2Y(2)R expression is regulated during intestinal inflammation through an NF-kappaB p65-dependent mechanism and could contribute not only to inflammatory bowel disease but also to other inflammatory diseases by regulating PG release.
Subject(s)
Cyclooxygenase 2/biosynthesis , Dinoprostone/metabolism , Intestinal Mucosa/metabolism , Receptors, Purinergic P2/genetics , Transcription Factor RelA/physiology , Transcription, Genetic/immunology , Up-Regulation/genetics , Up-Regulation/immunology , Animals , Caco-2 Cells , Cell Line , Cyclooxygenase 2/genetics , Humans , Inflammation Mediators/physiology , Intestinal Mucosa/cytology , Intestinal Mucosa/enzymology , Intestinal Mucosa/pathology , Promoter Regions, Genetic/immunology , Rats , Receptors, Purinergic P2/biosynthesis , Receptors, Purinergic P2/physiology , Receptors, Purinergic P2Y2ABSTRACT
Extracellular nucleotides and adenosine regulate endocrine pancreatic functions such as insulin secretion by Langerhans islet ß-cells via the activation of specific P2 and P1 receptors. Membrane-bound ectonucleotidases regulate the local concentration of these ligands and consequently control the activation of their receptors. The objective of this study was to identify and localize the major ectonucleotidases, namely NTPDases and ecto-5'-nucleotidase, present in the endocrine pancreas. In addition, the potential implication of ecto-ATPase activity on insulin secretion was investigated in the rat ß-cell line INS-1 (832/13). The localization of ectonucleotidase activity and protein was carried out in situ by enzyme histochemistry and immunolocalization in mouse, rat, and human pancreas sections. NTPDase1 was localized in all blood vessels and acini, and NTPDase2 was localized in capillaries of Langerhans islets and in peripheral conjunctive tissue, whereas NTPDase3 was detected in all Langerhans islet cell types. Interestingly, among the mammalian species tested, ecto-5'-nucleotidase was present only in rat Langerhans islet cells, where it was coexpressed with NTPDase3. Notably, the inhibition of NTPDase3 activity by BG0136 and NF279 facilitated insulin release from INS-1 (832/13) cells under conditions of low glycemia, probably by affecting P2 receptor activation. NTPDase3 activity also regulated the inhibitory effect of exogenous ATP in the presence of a high glucose concentration most likely by controlling adenosine production. In conclusion, all pancreatic endocrine cells express NTPDase3 that was shown to modulate insulin secretion in rat INS-1 (832/13) ß-cells. Ecto-5'-nucleotidase is expressed in rat Langerhans islet cells but absent in human and mouse endocrine cells.
Subject(s)
5'-Nucleotidase/physiology , Insulin/metabolism , Islets of Langerhans/enzymology , Islets of Langerhans/metabolism , Pyrophosphatases/physiology , Animals , Cell Line , Flow Cytometry , Humans , Immunohistochemistry , Insulin Secretion , Mice , Mice, Inbred C57BL , RatsABSTRACT
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.
Subject(s)
5'-Nucleotidase/metabolism , Gene Expression Regulation, Enzymologic , Genitalia, Male/enzymology , Animals , Blotting, Western , COS Cells , Chlorocebus aethiops , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Testis/enzymology , Vas Deferens/enzymologyABSTRACT
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.
Subject(s)
Actin Cytoskeleton/ultrastructure , Hepatic Stellate Cells/ultrastructure , Myofibroblasts/ultrastructure , Qb-SNARE Proteins/deficiency , Qc-SNARE Proteins/deficiency , Actin Cytoskeleton/chemistry , Actin Depolymerizing Factors/biosynthesis , Actins/analysis , Animals , Carbon Tetrachloride/toxicity , Cell Line , Cell Movement , Cell Separation , Gene Knockdown Techniques , Hepatic Stellate Cells/metabolism , Humans , Liver/cytology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/pathology , Mice , Qb-SNARE Proteins/antagonists & inhibitors , Qb-SNARE Proteins/genetics , Qb-SNARE Proteins/physiology , Qc-SNARE Proteins/antagonists & inhibitors , Qc-SNARE Proteins/genetics , Qc-SNARE Proteins/physiology , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Signal Transduction , Stress Fibers/chemistry , Stress Fibers/ultrastructure , Wound Healing , rho-Associated Kinases/physiologyABSTRACT
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's chronic response to radiation. In the current study, ten-month-old Sirt3-/- and Sirt3+/+ male mice received 24 Gy radiation targeted to liver. Six months after exposure, irradiated Sirt3-/- 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-1ß, TGF-ß) and fibrotic factors (Procollagen 1, α-SMA) were also measured in Sirt3-/- mice following 24 Gy IR. The alterations measured in enzymatic activities of catalase, glutathione peroxidase, and glutathione reductase in the livers of irradiated Sirt3-/- 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.
ABSTRACT
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 Müller 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 Müller 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.
Subject(s)
Adenosine Triphosphatases/metabolism , Antigens, CD/metabolism , Apyrase/metabolism , Neurons/enzymology , Retina/cytology , Animals , Male , Mice , Nerve Tissue Proteins/metabolism , Zebrafish , Zebrafish Proteins/metabolismABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/antagonists & inhibitors , Purinergic P2 Receptor Antagonists , Recombinant Proteins/antagonists & inhibitors , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/metabolism , Animals , Antigens, CD/genetics , Aorta, Thoracic/enzymology , Apyrase/deficiency , Apyrase/genetics , Benzenesulfonates/pharmacology , COS Cells , Cell Membrane , Chlorocebus aethiops , Humans , Hydrolysis , Mice , Mice, Knockout , Suramin/analogs & derivatives , Suramin/pharmacology , Transfection , Triazines/pharmacologyABSTRACT
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 with antisera directed against the glial glutamate/aspartate transporter (GLAST), the transduction pathway proteins phospholipase Cbeta2 (PLCbeta2) or the G-protein subunit alpha-gustducin, and serotonin (5HT) as markers of type I, II, and 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.