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CD73 or CD39 Deletion Reveals Different Mechanisms of Formation for Spontaneous and Mechanically Stimulated Adenosine and Sex Specific Compensations in ATP Degradation.

ABSTRACT: Adenosine is important for local neuromodulation, and rapid adenosine signaling can occur spontaneously or after mechanical stimulation, but little is known about how adenosine is formed in the extracellular space for those stimulations. Here, we studied mechanically stimulated and spontaneous adenosine to determine if rapid adenosine is formed by extracellular breakdown of adenosine triphosphate (ATP) using mice globally deficient in extracellular breakdown enzymes, either CD39 (nucleoside triphosphate diphosphohydrolase 1, NTPDase1) or CD73 (ecto-5'-nucleotidase). CD39 knockout (KO) mice have a lower frequency of spontaneous adenosine events than wild-type (WT, C57BL/6). Surprisingly, CD73KO mice demonstrate sex differences in spontaneous adenosine; males maintain similar event frequencies as WT, but females have significantly fewer events and lower concentrations. Examining the mRNA expression of other enzymes that metabolize ATP revealed tissue nonspecific alkaline phosphatase (TNAP) was upregulated in male CD73KO mice, but not secreted prostatic acid phosphatase (PAP) or transmembrane PAP. Thus, TNAP upregulation compensates for CD73 loss in males but not in females. These sex differences highlight that spontaneous adenosine is formed by metabolism of extracellular ATP by many enzymes. For mechanically stimulated adenosine, CD39KO or CD73KO did not change stimulation frequency, concentration, or t_1/2. Thus, the mechanism of formation for mechanically stimulated adenosine is likely direct release of adenosine, different than spontaneous adenosine. Understanding these different mechanisms of rapid adenosine formation will help to develop pharmacological treatments that differentially target modes of rapid adenosine signaling, and all treatments should be studied in both sexes, given possible differences in extracellular ATP degradation.

SUBMITTER: Wang Y

PROVIDER: S-EPMC7335217 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Synapsed cells can communicate using exocytosed nucleotides like adenosine triphosphate (ATP). Ectonucleotidases localized to synaptic junctions degrade nucleotides into metabolites like adenosine monophosphate (AMP) or adenosine. Oftentimes nucleotide degradation occurs in a sequential manner, of which ATP degradation by CD39 and CD73 is a representative example. Here, CD39 first converts ATP and adenosine diphosphate (ADP) into AMP, after which AMP is dephosphorylated into adenosine by CD73. Hence, the concerted activity of CD39 and CD73 can help shape cellular responses to extracellular ATP. In a previous study, we demonstrated that coupled CD39 and CD73 activity within synapse-like junctions is strongly controlled by the enzymes' co-localization, their surface charge densities, and the electrostatic potential of the surrounding cell membranes. In this study, we demonstrate that crowders within synaptic junctions, which can include globular proteins like cytokines and membrane-bound proteins, impact coupled CD39 and CD73 ectonucleotidase activity and, in turn, the availability of intrasynapse ATP. Specifically, we developed a spatially explicit, reaction-diffusion model for the coupled conversion of ATP → AMP and AMP → adenosine in a model synaptic junction with crowders that is solved via the finite element method. Our modeling results suggest that the association rate for ATP to CD39 is strongly influenced by the density of intrasynaptic protein crowders, as increasing crowder density generally suppressed ATP association kinetics. Much of this suppression can be rationalized based on a loss of configurational entropy. The surface charges of crowders can further influence the association rate, with the surprising result that favorable crowder-nucleotide electrostatic interactions can yield CD39 association rates that are faster than crowder-free configurations. However, attractive crowder-nucleotide interactions decrease the rate and efficiency of adenosine production, which in turn increases the availability of ATP and AMP within the synapse relative to crowder-free configurations. These findings highlight how CD39 and CD73 ectonucleotidase activity, electrostatics, and crowding within synapses influence the availability of nucleotides for intercellular communication.

Project description:BackgroundOvarian cancer (OvCA) tissues show abundant expression of the ectonucleotidases CD39 and CD73 which generate immunomodulatory adenosine, thereby inhibiting cytotoxic lymphocytes. Little, however, is known about the effect of adenosine on myeloid cells. Considering that tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) constitute up to 20 % of OvCA tissue, we investigated the effect of adenosine on myeloid cells and explored a possible contribution of myeloid cells to adenosine generation in vitro and ex vivo.MethodsMonocytes were used as human blood-derived myeloid cells. After co-incubation with SK-OV-3 or OAW-42 OvCA cells, monocyte migration was determined in transwell assays. For conversion into M2-polarized "TAM-like" macrophages, monocytes were co-incubated with OAW-42 cells. Ex vivo TAMs were obtained from OvCA ascites. Macrophage phenotypes were investigated by intracellular staining for IL-10 and IL-12. CD39 and CD73 expression were assessed by FACS analysis both on in vitro-induced TAM-like macrophages and on ascites-derived ex situ-TAMs. Myeloid cells in solid tumor tissue were analyzed by immunohistochemistry. Generation of biologically active adenosine by TAM-like macrophages was measured in luciferase-based reporter assays. Functional effects of adenosine were investigated in proliferation-experiments with CD4(+) T cells and specific inhibitors.ResultsWhen CD39 or CD73 activity on OvCA cells were blocked, the migration of monocytes towards OvCA cells was significantly decreased. In vivo, myeloid cells in solid ovarian cancer tissue were found to express CD39 whereas CD73 was mainly detected on stromal fibroblasts. Ex situ-TAMs and in vitro differentiated TAM-like cells, however, upregulated the expression of CD39 and CD73 compared to monocytes or M1 macrophages. Expression of ectonucleotidases also translated into increased levels of biologically active adenosine. Accordingly, co-incubation with these TAMs suppressed CD4(+) T cell proliferation which could be rescued via blockade of CD39 or CD73.ConclusionAdenosine generated by OvCA cells likely contributes to the recruitment of TAMs which further amplify adenosine-dependent immunosuppression via additional ectonucleotidase activity. In solid ovarian cancer tissue, TAMs express CD39 while CD73 is found on stromal fibroblasts. Accordingly, small molecule inhibitors of CD39 or CD73 could improve immune responses in ovarian cancer.

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CD73 or CD39 Deletion Reveals Different Mechanisms of Formation for Spontaneous and Mechanically Stimulated Adenosine and Sex Specific Compensations in ATP Degradation.

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