CD73 Regulation Research Articles

Downregulation of hepatic ecto‐5′‐nucleotidase (CD73) in a mouse model of alcoholic liver injury and in patients with alcoholic steatohepatitis

Chronic liver diseases are a significant and growing medical problem in need of therapeutic advances. Strategies with the greatest potential to contribute broad impacts are those that can block the disease process early, and independently of the underlying etiology. The enzyme ecto‐5′‐nucleotidase (CD73), which metabolizes extracellular adenosine monophosphate (AMP) to adenosine, represents a promising novel liver disease target. Previous studies with CD73−/− mice suggested that CD73 significantly contributes to various forms of liver injury, including alcoholic steatosis. However, the functional and regulatory mechanisms of CD73 during liver injury are poorly understood. The objective of this study was to determine the dynamics of CD73 regulation in response to ethanol in vivo and ex vivo, and to compare these changes to human ASH patients.MethodsEthanol administration was performed using the Lieber DeCarli method in female C57BL/6J mice (14–16 weeks of age), with 1 or 2 week duration of ethanol diet, and a final concentration of 3.3% or 5% vol/vol ethanol (n=4–8 mice per group, 3 independent studies). Freshly isolated sandwich‐cultured hepatocytes from female C57BL/6J mice were treated with ethanol (50 or100mM for 3h or 24h), followed by RNA isolation, quantitative real‐time PCR and Western blot. CD73 gene expression analysis was performed on biopsy specimens from human ASH patients. Biochemical and histological assessment of CD73 expression and enzymatic activity was performed on liver lysates, cryosections, and isolated mouse hepatocytes using commercially available assays, western blotting, and enzyme histochemistry procedures.ResultsCD73 protein expression in the livers of mice receiving high dose of ethanol were significantly downregulated, by 32% (p<0.05) after one week, and 51% (p<0.01) after two weeks, with concomitant decrease in cell surface AMPase activity. This effect was milder in the group receiving the lower ethanol dose, where no changes were observed after 1 week, and 20% reduction in CD73 expression was seen after 2 weeks. Expression levels of CD73 mRNA in biopsy specimens from patients with ASH (n=10) were downregulated to 58+11.1% compared to control human livers (n=2). In contrast, ethanol treatment of isolated mouse hepatocytes caused a transcriptional CD73 upregulation and a significant redistribution of cell surface AMPase activity, demonstrating that CD73 is a target of the ethanol effects ex vivo.ConclusionCD73 expression and enzymatic activity are downregulated in response to ethanol in mouse ethanol‐treated livers and in patients with alcoholic liver disease. Since CD73 downregulation in response to ethanol in the mouse model occurs early (i.e. prior to the development of steatosis), our findings point to possible non‐enzymatic pathways for CD73‐dependent signaling during chronic alcohol‐induced liver injury. Additional mechanistic studies are needed to reconcile the ex vivo and in vivo effects of CD73 to better understand its regulation and signaling function during hepatocyte injury and liver disease.Support or Funding InformationSupported by NIH grants DK093776 and DK102450

Abstract P5-04-13: Toll like receptor-9 and CD73 may act on the same pathway to induce immunosuppression in triple negative breast cancer cells

Abstract Toll like-receptor-9 (TLR9) is an innate immune system DNA-receptor which is also widely expressed in breast cancer cell lines and in clinical breast cancer specimens. Although TLR9 ligands (such as bacterial DNA and synthetic, CpG-sequence containing oligonucleotides) induce TLR9-mediated invasion in breast cancer cells in vitro, the contribution of this protein to breast cancer pathophysiology remains unclear. We showed previously that tumor TLR9 expression is a highly significant prognostic factor in breast cancer, but only in tumors that are triple negative (TNBC). Specifically, low tumor TLR9 expression in TNBC is associated with a significantly shortened disease-specific survival. Our published, preclinical results further suggest that tumor TLR9 expression may be an important determinant of tumor immunophenotype and that patients with low-TLR9 TNBC may not gain the immunogenic benefit from chemotherapy. A possible mechanistic explanation for the change in the immunophenotype of low TLR9-TNBC tumors involves CD73, which is a 5’ectonucleotidase that converts extracellular adenosine monophosphate (AMP) into the highly immunosuppressive adenosine. CD73 is also expressed in breast cancer cells and in clinical specimens. High CD73 expression has been associated with poor prognosis, but similar with TLR9 expression, the prognostic significance was specific only for TNBC. There are no previous reports on TLR9 regulation of CD73 in any cancer. Interestingly, however, TLR9 deficiency was shown to promote CD73 expression of T-cells in a mouse model of diabetes. To begin to test the hypothesis that TLR9 affects CD73 expression in TNBC, we studied CD73 mRNA and protein expression in control and TLR9 siRNA TNBC cells in vitro. We discovered that TLR9 siRNA TNBC cells express significantly higher CD73 mRNA concentrations than control siRNA cells specifically in hypoxia. Similar findings were observed with immunofluorescence. Our results suggest that the lack or TLR9 in TNBC may allow high CD73 expression and through this mechanism result in an immunosuppressive phenotype of the tumors. The immunosuppression then would explain the poor prognosis associated with low TLR9-TNBC tumors. We are currently studying this with clinical TNBC specimens. Citation Format: Katri S Selander, Johanna M Tuomela, Mikko Mella, Joonas Kauppila, Jouko Sandholm, Gennady Yegutkin, Peeter Karihtala, Arja Jukkola-Vuorinen, Kirsi-Maria Haapasaari, Katri S Vuopala, Kevin W Harris. Toll like receptor-9 and CD73 may act on the same pathway to induce immunosuppression in triple negative breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-13.

Alternative splicing of human NT5E in cirrhosis and hepatocellular carcinoma produces a negative regulator of ecto-5'-nucleotidase (CD73).

Ecto-5'-nucleotidase (CD73), encoded by NT5E, is the major enzymatic source of extracellular adenosine. CD73 controls numerous pathophysiological responses and is a potential disease target, but its regulation is poorly understood. We examined NT5E regulation by alternative splicing. Genomic database analysis of human transcripts led us to identify NT5E-2, a novel splice variant that was expressed at low abundance in normal human tissues but was significantly up-regulated in cirrhosis and hepatocellular carcinoma (HCC). NT5E-2 encodes a shorter CD73 isoform we named CD73S. The presence of CD73S protein, which lacks 50 amino acids, was detected in HCC using an isoform-specific antibody. A noncanonical mouse mRNA, similar to human CD73S, was observed, but the corresponding protein was undetectable. The two human isoforms exhibited functional differences, such that ectopic expression of canonical CD73 (CD73L) in human HepG2 cells was associated with decreased expression of the proliferation marker Ki67, whereas CD73S expression did not have an effect on Ki67 expression. CD73S was glycosylated, catalytically inactive, unable to dimerize, and complexed intracellularly with the endoplasmic reticulum chaperone calnexin. Furthermore, CD73S complexed with CD73L and promoted proteasome-dependent CD73L degradation. The findings reveal species-specific CD73 regulation, with potential significance to cancer, fibrosis, and other diseases characterized by changes in CD73 expression and function.

Reply

We thank Dr. French for his comments and questions about our study on the role of the ecto-nucleotidase CD73 in mouse liver Mallory-Denk body (MDB) formation.1 CD73 is expressed in multiple tissues, including the lung, heart, brain, kidney, liver, and the immune system.2 Although formation of extracellular adenosine is considered its primary function, CD73 also may act in a number of other ways independent of its enzymatic role.3 As Dr. French pointed out, our findings that CD73−/− mice are resistant to MDB formation is somewhat counterintuitive, given our initial observation that the MDB-susceptible mouse strain (C57BL/6J) is characterized by having lower CD73 mRNA and protein levels under basal conditions compared to the MDB-resistant C3H/He strain. However, we also showed that after 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treatment there was a near complete loss of cell surface CD73 (AMPase) activity, but only a 60% decrease in overall CD73 activity (measured biochemically after tissue lysis). This observation, together with our findings of DDC-induced loss of plasma membrane CD73 in isolated hepatocytes, illustrates the complex nature of CD73 regulation in this model of liver injury. Therefore, the subcellular distribution (organellar versus plasma membrane) and regulation (such as glycosylation changes and enzymatic activity) of CD73 during hepatocellular injury are likely a more important determinant of hepatocyte injury than the overall CD73 expression levels. Furthermore, the MDB resistance in CD73−/− mice also may be immune-related, given the known major role of CD73 in regulating inflammatory and immune responses.4 For example, in absence of CD73 to catalyze the final and irreversible step in the conversion of extracellular adenosine triphosphate (ATP) to adenosine, the ATP levels in the vicinity of damaged and dying cells may significantly increase. This can accelerate the clearance of damaged cells by promoting phagocytosis by way of a number of mechanisms.5 Altered purinergic signaling on other immune cell types, such as regulatory T cells,6 also may alter the course of liver injury progression in CD73−/− mice. In summary, resistance to MDB formation in CD73−/− mice likely is related to a number of hepatocyte-intrinsic and -extrinsic mechanisms. Generation of mice with targeted deletion of CD73 from specific cell types should help resolve many outstanding questions pertaining to the functions of this enzyme in the liver. Natasha T. Snider, Ph.D. M. Bishr Omary, M.D., Ph.D. Department of Molecular & Integrative Physiology University of Michigan Medical School Ann Arbor, MI

Activated hepatic stellate cells upregulate transcription of ecto-5′-nucleotidase/CD73 via specific SP1 and SMAD promoter elements

Adenosine is a potent modulator of liver fibrosis and inflammation. Adenosine has been shown to regulate such diverse activities as chemotaxis, contraction, and matrix production in hepatic stellate cells (HSC). Ecto-5'-nucleotidase/CD73 [EC 3.1.3.5] is the rate-limiting enzyme in adenosine production. Cd73-deficient mice are resistant to experimental liver fibrosis and have impaired adenosine generation. However, cell-specific expression and regulation of CD73 within the fibrotic liver have not been defined. In particular, prior evidence demonstrating that liver myofibroblasts, the cells believed to be responsible for matrix formation in the liver, express CD73 is lacking. Thus we tested the hypothesis that HSC and portal fibroblasts (PF), cells that undergo differentiation into liver myofibroblasts, express CD73 in a regulated fashion. We found that CD73 is weakly expressed in quiescent HSC and PF but is markedly upregulated at the transcriptional level in myofibroblastic HSC and PF. We furthermore found that CD73 protein and its functional activity are strongly increased in fibrous septa in rats subjected to experimental fibrosis. To determine the mechanism for the upregulation of Cd73 gene, we cloned the rat Cd73 promoter and then used serial truncation and site-directed mutagenesis to identify key regulatory elements. We identified two consensus SP1 motifs and one SMAD binding site, each of which was necessary for Cd73 gene upregulation. In conclusion, activated HSC upregulate Cd73 gene expression, via specific SP1 and SMAD promoter elements, after myofibroblastic differentiation. The ecto-5'-nucleotidase/CD73 enzyme is a novel cellular marker of activated liver myofibroblasts in vivo and in vitro and thus represents a promising molecular target for antifibrotic therapies in liver diseases.