Keratin filaments mediate the expansion of extra-embryonic membranes in the post-gastrulation mouse embryo.

Mesoderm arises at gastrulation and contributes to both the mouse embryo proper and its extra-embryonic membranes. Two-photon live imaging of embryos bearing a keratin reporter allowed recording filament nucleation and elongation in the extra-embryonic region. Upon separation of amniotic and exocoelomic cavities, keratin 8 formed apical cables co-aligned across multiple cells in the amnion, allantois, and blood islands. An influence of substrate rigidity and composition on cell behavior and keratin content was observed in mesoderm explants. Embryos lacking all keratin filaments displayed a deflated extra-embryonic cavity, a narrow thick amnion, and a short allantois. Single-cell RNA sequencing of sorted mesoderm cells and micro-dissected amnion, chorion, and allantois, provided an atlas of transcriptomes with germ layer and regional information. It defined the cytoskeleton and adhesion expression profile of mesoderm-derived keratin 8-enriched cells lining the exocoelomic cavity. Those findings indicate a novel role for keratin filaments in the expansion of extra-embryonic structures and suggest mechanisms of mesoderm adaptation to the environment.

A structural model of the iRhom-ADAM17 sheddase complex reveals functional insights into its trafficking and activity.

Several membrane-anchored signal mediators such as cytokines (e.g. TNFα) and growth factors are proteolytically shed from the cell surface by the metalloproteinase ADAM17, which, thus, has an essential role in inflammatory and developmental processes. The membrane proteins iRhom1 and iRhom2 are instrumental for the transport of ADAM17 to the cell surface and its regulation. However, the structure-function determinants of the iRhom-ADAM17 complex are poorly understood. We used AI-based modelling to gain insights into the structure-function relationship of this complex. We identified different regions in the iRhom homology domain (IRHD) that are differentially responsible for iRhom functions. We have supported the validity of the predicted structure-function determinants with several in vitro, ex vivo and in vivo approaches and demonstrated the regulatory role of the IRHD for iRhom-ADAM17 complex cohesion and forward trafficking. Overall, we provide mechanistic insights into the iRhom-ADAM17-mediated shedding event, which is at the centre of several important cytokine and growth factor pathways.

The keratin-desmosome scaffold: pivotal role of desmosomes for keratin network morphogenesis.

Desmosome-anchored keratin intermediate filaments (KFs) are essential for epithelial coherence. Yet, desmosomal KF attachment and network organization are still unexplored in vivo. We, therefore, monitored KF network morphogenesis in fluorescent keratin 8 knock-in murine embryos revealing keratin enrichment at newly formed desmosomes followed by KF formation, KF elongation and KF fusion. To examine details of this process and its coupling to desmosome formation, we studied fluorescent keratin and desmosomal protein reporter dynamics in the periphery of expanding HaCaT keratinocyte colonies. Less than 3 min after the start of desmosomal proteins clustering non-filamentous keratin enriched at these sites followed by KF formation and elongation. Subsequently, desmosome-anchored KFs merged into stable bundles generating a rim-and-spokes system consisting of subcortical KFs connecting desmosomes to each other and radial KFs connecting desmosomes to the cytoplasmic KF network. We conclude that desmosomes are organizing centers for the KF cytoskeleton with a hitherto unknown nucleation capacity.

A rim-and-spoke hypothesis to explain the biomechanical roles for cytoplasmic intermediate filament networks.

Textbook images of keratin intermediate filament (IF) networks in epithelial cells and the functional compromization of the epidermis by keratin mutations promulgate a mechanical role for this important cytoskeletal component. In stratified epithelia, keratin filaments form prominent radial spokes that are focused onto cell-cell contact sites, i.e. the desmosomes. In this Hypothesis, we draw attention to a subset of keratin filaments that are apposed to the plasma membrane. They form a rim of filaments interconnecting the desmosomes in a circumferential network. We hypothesize that they are part of a rim-and-spoke arrangement of IFs in epithelia. From our review of the literature, we extend this functional role for the subplasmalemmal rim of IFs to any cell, in which plasma membrane support is required, provided these filaments connect directly or indirectly to the plasma membrane. Furthermore, cytoplasmic IF networks physically link the outer nuclear and plasma membranes, but their participation in mechanotransduction processes remain largely unconsidered. Therefore, we also discuss the potential biomechanical and mechanosensory role(s) of the cytoplasmic IF network in terms of such a rim (i.e. subplasmalemmal)-and-spoke arrangement for cytoplasmic IF networks.

Central role of the TIR-domain-containing adaptor-inducing interferon-ß (TRIF) adaptor protein in murine sterile liver injury.

Multiple pathways drive the sterile injury response in the liver; however, it is unclear how the type of cells injured or the mechanism of injury activates these pathways. Here, we use a model of selective hepatocyte death to investigate sterile liver injury. In this model, the TIR-domain-containing adaptor-inducing interferon-ß (TRIF) was a central mediator of the resulting intrahepatic inflammatory response that was independent of both upstream Toll-like receptor (TLR) 4 signaling and downstream type I interferon (IFN) signaling. TRIF was required for induction of interleukin (IL)-10, IL-6, and IL-1ß cytokines. Conversely, although induction of C-C motif chemokine ligand (CCL) 2 and C-X-C motif chemokine ligand (CXCL) 1 chemokines and up-regulation of chemokine (Ccl2, Ccl7, Cxcl1, Cxcl2, and Cxcl10) and cell-adhesion (intracellular adhesion molecule 1 and vascular cell adhesion molecule 1) genes involved in myeloid cell recruitment was reduced in a majority of TRIF-/- mice, a subset of TRIF-/- mice showed breakthrough inflammation and the ability to induce these genes and proteins, indicating that redundant pathways exist to respond to hepatocyte death. Furthermore, we found that hepatocytes themselves were the main responders to hepatocyte death, increasing transcription of genes involved in myeloid cell recruitment more than either liver sinusoidal endothelial cells or Kupffer cells. CONCLUSION: Our studies define a TRIF-dependent, TLR4- and type I IFN-independent pathway of sterile liver injury in which hepatocytes are both the targets of damage and the principal responding cell type. (Hepatology 2017;65:1336-1351).

Nucleotide-Induced Membrane-Proximal Proteolysis Controls the Substrate Specificity of T Cell Ecto-ADP-Ribosyltransferase ARTC2.2.

ARTC2.2 is a toxin-related, GPI-anchored ADP-ribosyltransferase expressed by murine T cells. In response to NAD(+) released from damaged cells during inflammation, ARTC2.2 ADP-ribosylates and thereby gates the P2X7 ion channel. This induces ectodomain shedding of metalloprotease-sensitive cell surface proteins. In this study, we show that ARTC2.2 itself is a target for P2X7-triggered ectodomain shedding. We identify the metalloprotease cleavage site 3 aa upstream of the predicted GPI anchor attachment site of ARTC2.2. Intravenous injection of NAD(+) increased the level of enzymatically active ARTC2.2 in serum, indicating that this mechanism is operative also under inflammatory conditions in vivo. Radio-ADP-ribosylation assays reveal that shedding refocuses the target specificity of ARTC2.2 from membrane proteins to secretory proteins. Our results uncover nucleotide-induced membrane-proximal proteolysis as a regulatory mechanism to control the substrate specificity of ARTC2.2.

Leukocytes require ADAM10 but not ADAM17 for their migration and inflammatory recruitment into the alveolar space.

Inflammation is a key process in various diseases, characterized by leukocyte recruitment to the inflammatory site. This study investigates the role of a disintegrin and a metalloproteinase (ADAM) 10 and ADAM17 for leukocyte migration in vitro and in a murine model of acute pulmonary inflammation. Inhibition experiments or RNA knockdown indicated that monocytic THP-1 cells and primary human neutrophils require ADAM10 but not ADAM17 for efficient chemokine-induced cell migration. Signaling and adhesion events that are linked to cell migration such as p38 and ρ GTPase-family activation, F-actin polymerization, adhesion to fibronectin, and up-regulation of α5 integrin were also dependent on ADAM10 but not ADAM17. This was confirmed with leukocytes isolated from mice lacking either ADAM10 or ADAM17 in all hematopoietic cells (vav 1 guanine nucleotide exchange factor [Vav]-Adam10(-/-) or Vav-Adam17(-/-) mice). In lipopolysaccharide-induced acute pulmonary inflammation, alveolar recruitment of neutrophils and monocytes was transiently increased in Vav-Adam17(-/-) but steadily reduced in Vav-Adam10(-/-) mice. This deficit in alveolar leukocyte recruitment was also observed in LysM-Adam10(-/-) mice lacking ADAM10 in myeloid cells and correlated with protection against edema formation. Thus, with regard to leukocyte migration, leukocyte-expressed ADAM10 but not ADAM17 displays proinflammatory activities and may therefore serve as a target to limit inflammatory cell recruitment.

A transmembrane C-terminal fragment of syndecan-1 is generated by the metalloproteinase ADAM17 and promotes lung epithelial tumor cell migration and lung metastasis formation.

Syndecan-1 is a heparan sulfate proteoglycan expressed by endothelial and epithelial cells and involved in wound healing and tumor growth. Surface-expressed syndecan-1 undergoes proteolytic shedding leading to the release of the soluble N-terminal ectodomain from a transmembrane C-terminal fragment (tCTF). We show that the disintegrin and metalloproteinase (ADAM) 17 generates a syndecan-1 tCTF, which can then undergo further intra-membrane proteolysis by γ-secretase. Scratch-induced wound closure of cultured lung epithelial A549 tumor cells associates with increased syndecan-1 cleavage as evidenced by the release of shed syndecan-1 ectodomain and enhanced generation of the tCTF. Both wound closure and the associated syndecan-1 shedding can be suppressed by inhibition of ADAM family proteases. Cell proliferation, migration and invasion into matrigel as well as several signaling pathways implicated in these responses are suppressed by silencing of syndecan-1. These defects of syndecan-1 deficient cells can be overcome by overexpression of syndecan-1 tCTF or a corresponding tCTF of syndecan-4 but not by overexpression of a tCTF lacking the transmembrane domain. Finally, lung metastasis formation of A549 cells in SCID mice was found to be dependent on syndecan-1, and the presence of syndecan-1 tCTF was sufficient for this activity. Thus, the syndecan-1 tCTF by itself is capable of mediating critical syndecan-1-dependent functions in cell proliferation, migration, invasion and metastasis formation and therefore can replace full length syndecan-1 in the situation of increased syndecan-1 shedding during cell migration and tumor formation.

Keratins as the main component for the mechanical integrity of keratinocytes.

Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.

Plasticity of cytoplasmic intermediate filament architecture determines cellular functions.

Cytoplasmic intermediate filaments endow cells with mechanical stability. They are subject to changes in morphology and composition if needed. This remodeling encompasses entire cells but can also be restricted to specific intracellular regions. Intermediate filaments thereby support spatially and temporally defined cell type-specific functions. This review focuses on recent advances in our understanding of how intermediate filament dynamics affect the underlying regulatory pathways. We will elaborate on the role of intermediate filaments for the formation and maintenance of surface specializations, cell migration, contractility, organelle positioning, nucleus protection, stress responses and axonal conduction velocity. Together, the selected examples highlight the modulatory role of intermediate filament plasticity for multiple cellular functions.

Bioprinting-associated pulsatile hydrostatic pressure elicits a mild proinflammatory response in epi- and endothelial cells.

During nozzle-based bioprinting, like inkjet and microextrusion, cells are subjected to hydrostatic pressure for up to several minutes. The modality of the bioprinting-related hydrostatic pressure is either constant or pulsatile depending on the technique. We hypothesized that the difference in the modality of hydrostatic pressure affects the biological response of the processed cells differently. To test this, we used a custom-made setup to apply either controlled constant or pulsatile hydrostatic pressure on endothelial and epithelial cells. Neither bioprinting procedure visibly altered the distribution of selected cytoskeletal filaments, cell-substrate adhesions, and cell-cell contacts in either cell type. In addition, pulsatile hydrostatic pressure led to an immediate increase of intracellular ATP in both cell types. However, the bioprinting-associated hydrostatic pressure triggered a pro-inflammatory response in only the endothelial cells, with an increase of interleukin 8 (IL-8) and a decrease of thrombomodulin (THBD) transcripts. These findings demonstrate that the settings adopted during nozzle-based bioprinting cause hydrostatic pressure that can trigger a pro-inflammatory response in different barrier-forming cell types. This response is cell-type and pressure-modality dependent. The immediate interaction of the printed cells with native tissue and the immune system in vivo might potentially trigger a cascade of events. Our findings, therefore, are of major relevance in particular for novel intra-operative, multicellular bioprinting approaches.

Regionally specific levels and patterns of keratin 8 expression in the mouse embryo visceral endoderm emerge upon anterior-posterior axis determination.

The mechanical properties of the different germ layers of the early mammalian embryo are likely to be critical for morphogenesis. Cytoskeleton components (actin and myosin, microtubules, intermediate filaments) are major determinants of epithelial plasticity and resilience to stress. Here, we take advantage of a mouse reporter for Keratin 8 to record the pattern of the keratin intermediate filaments network in the first epithelia of the developing mouse embryo. At the blastocyst stage, Keratin 8 is strongly expressed in the trophectoderm, and undetectable in the inner cell mass and its derivatives, the epiblast and primitive endoderm. Visceral endoderm cells that differentiate from the primitive endoderm at the egg cylinder stage display apical Keratin 8 filaments. Upon migration of the Anterior Visceral Endoderm and determination of the anterior-posterior axis, Keratin 8 becomes regionally distributed, with a stronger expression in embryonic, compared to extra-embryonic, visceral endoderm. This pattern emerges concomitantly to a modification of the distribution of Filamentous (F)-actin, from a cortical ring to a dense apical shroud, in extra-embryonic visceral endoderm only. Those regional characteristics are maintained across gastrulation. Interestingly, for each stage and region of the embryo, adjacent germ layers display contrasted levels of keratin filaments, which may play a role in their adaptation to growth and morphological changes.

Quantitative mapping of keratin networks in 3D.

Mechanobiology requires precise quantitative information on processes taking place in specific 3D microenvironments. Connecting the abundance of microscopical, molecular, biochemical, and cell mechanical data with defined topologies has turned out to be extremely difficult. Establishing such structural and functional 3D maps needed for biophysical modeling is a particular challenge for the cytoskeleton, which consists of long and interwoven filamentous polymers coordinating subcellular processes and interactions of cells with their environment. To date, useful tools are available for the segmentation and modeling of actin filaments and microtubules but comprehensive tools for the mapping of intermediate filament organization are still lacking. In this work, we describe a workflow to model and examine the complete 3D arrangement of the keratin intermediate filament cytoskeleton in canine, murine, and human epithelial cells both, in vitro and in vivo. Numerical models are derived from confocal airyscan high-resolution 3D imaging of fluorescence-tagged keratin filaments. They are interrogated and annotated at different length scales using different modes of visualization including immersive virtual reality. In this way, information is provided on network organization at the subcellular level including mesh arrangement, density and isotropic configuration as well as details on filament morphology such as bundling, curvature, and orientation. We show that the comparison of these parameters helps to identify, in quantitative terms, similarities and differences of keratin network organization in epithelial cell types defining subcellular domains, notably basal, apical, lateral, and perinuclear systems. The described approach and the presented data are pivotal for generating mechanobiological models that can be experimentally tested.

Desmoplakin Maintains Transcellular Keratin Scaffolding and Protects From Intestinal Injury.

BACKGROUND & AIMS: Desmosomes are intercellular junctions connecting keratin intermediate filaments of neighboring cells. The cadherins desmoglein 2 (Dsg2) and desmocollin 2 mediate cell-cell adhesion, whereas desmoplakin (Dsp) provides the attachment of desmosomes to keratins. Although the importance of the desmosome-keratin network is well established in mechanically challenged tissues, we aimed to assess the currently understudied function of desmosomal proteins in intestinal epithelia. METHODS: We analyzed the intestine-specific villin-Cre DSP (DSPΔIEC) and the combined intestine-specific DSG2/DSPΔIEC (ΔDsg2/Dsp) knockout mice. Cross-breeding with keratin 8-yellow fluorescent protein knock-in mice and generation of organoids was performed to visualize the keratin network. A Dsp-deficient colorectal carcinoma HT29-derived cell line was generated and the role of Dsp in adhesion and mechanical stress was studied in dispase assays, after exposure to uniaxial cell stretching and during scratch assay. RESULTS: The intestine of DSPΔIEC mice was histopathologically inconspicuous. Intestinal epithelial cells, however, showed an accelerated migration along the crypt and an enhanced shedding into the lumen. Increased intestinal permeability and altered levels of desmosomal proteins were detected. An inconspicuous phenotype also was seen in ΔDsg2/Dsp mice. After dextran sodium sulfate treatment, DSPΔIEC mice developed more pronounced colitis. A retracted keratin network was seen in the intestinal epithelium of DSPΔIEC/keratin 8-yellow fluorescent protein mice and organoids derived from these mice presented a collapsed keratin network. The level, phosphorylation status, and solubility of keratins were not affected. Dsp-deficient HT29 cells had an impaired cell adhesion and suffered from increased cellular damage after stretch. CONCLUSIONS: Our results show that Dsp is required for proper keratin network architecture in intestinal epithelia, mechanical resilience, and adhesion, thereby protecting from injury.

Effective targeting of microglial P2X7 following intracerebroventricular delivery of nanobodies and nanobody-encoding AAVs.

The P2X7 ion channel is a key sensor for extracellular ATP and a key trigger of sterile inflammation. Intravenous injection of nanobodies that block P2X7 has shown to be beneficial in mouse models of systemic inflammation. P2X7 has also emerged as an attractive therapeutic target for inflammatory brain diseases. However, little is known about the ability of nanobodies to cross the BBB. Here we evaluated the ability of P2X7-specific nanobodies to reach and to block P2X7 on microglia following intravenous or intracerebral administration. For this study, we reformatted and sequence-optimized P2X7 nanobodies for higher stability and elevated isoelectric point. Following injection of nanobodies or nanobody-encoding adeno-associated viral vectors (AAV), we monitored the occupancy and blockade of microglial P2X7 in vivo using ex vivo flow cytometry. Our results show that P2X7 on microglia was within minutes completely occupied and blocked by intracerebroventricularly injected nanobodies, even at low doses. In contrast, very high doses were required to achieve similar effects when injected intravenously. The endogenous production of P2X7-antagonistic nanobodies following intracerebral or intramuscular injection of nanobody-encoding AAVs resulted in a long-term occupancy and blockade of P2X7 on microglia. Our results provide new insights into the conditions for the delivery of nanobodies to microglial P2X7 and point to AAV-mediated delivery of P2X7 nanobodies as a promising strategy for the treatment of sterile brain inflammation.

A disintegrin and metalloproteinase 17 (ADAM17) mediates inflammation-induced shedding of syndecan-1 and -4 by lung epithelial cells.

Syndecans are cell surface proteoglycans that bind and modulate various proinflammatory mediators and can be proteolytically shed from the cell surface. Within the lung, syndecan-1 and -4 are expressed as transmembrane proteins on epithelial cells and released in the bronchoalveolar fluid during inflammation. We here characterize the mechanism leading to the generation of soluble syndecan-1 and -4 in cultured epithelial cells and murine lung tissue. We show that the bladder carcinoma epithelial cell line ECV304, the lung epithelial cell line A459 and primary alveolar epithelial cells express and constitutively release syndecan-1 and -4. This release involves the activity of the disintegrin-like metalloproteinase ADAM17 as demonstrated by use of specific inhibitors and lentivirally transduced shRNA. Stimulation of epithelial cells with PMA, thrombin, or proinflammatory cytokines (TNFalpha/IFNgamma) led to the down-regulation of surface-expressed syndecan-1 and -4, which was associated with a significant increase of soluble syndecans and cell-associated cleavage fragments. The enhanced syndecan release was not related to gene induction of syndecans or ADAM17, but rather due to increased ADAM17 activity. Soluble syndecan-1 and -4 were also released into the bronchoalveolar fluid of mice. Treatment with TNFalpha/IFNgamma increased ADAM17 activity and syndecan release in murine lungs. Both constitutive and induced syndecan shedding was prevented by the ADAM17 inhibitor. ADAM17 may therefore be an important regulator of syndecan functions on inflamed lung epithelium.

Regulated release and functional modulation of junctional adhesion molecule A by disintegrin metalloproteinases.

Junctional adhesion molecule A (JAM-A) is a transmembrane adhesive glycoprotein that participates in the organization of endothelial tight junctions and contributes to leukocyte transendothelial migration. We demonstrate here that cultured endothelial cells not only express a cellular 43-kDa variant of JAM-A but also release considerable amounts of a 33-kDa soluble JAM-A variant. This release is enhanced by treatment with proinflammatory cytokines and is associated with the down-regulation of surface JAM-A. Inhibition experiments, loss/gain-of-function experiments, and cleavage experiments with recombinant proteases indicated that cleavage of JAM-A is mediated predominantly by the disintegrin and metalloproteinase (ADAM) 17 and, to a lesser extent, by ADAM10. Cytokine treatment of mice increased JAM-A serum level and in excised murine aortas increased ADAM10/17 activity correlated with enhanced JAM-A release. Functionally, soluble JAM-A blocked migration of cultured endothelial cells, reduced transendothelial migration of isolated neutrophils in vitro, and decreased neutrophil infiltration in a murine air pouch model by LFA-1- and JAM-A-dependent mechanisms. Therefore, shedding of JAM-A by inflamed vascular endothelium via ADAM17 and ADAM10 may not only generate a biomarker for vascular inflammation but could also be instrumental in controlling JAM-A functions in the molecular zipper guiding transendothelial diapedesis of leukocytes.

NAD+ and ATP released from injured cells induce P2X7-dependent shedding of CD62L and externalization of phosphatidylserine by murine T cells.

Extracellular NAD(+) and ATP trigger the shedding of CD62L and the externalization of phosphatidylserine on murine T cells. These events depend on the P2X(7) ion channel. Although ATP acts as a soluble ligand to activate P2X(7), gating of P2X(7) by NAD(+) requires ecto-ADP-ribosyltransferase ART2.2-catalyzed transfer of the ADP-ribose moiety from NAD(+) onto Arg125 of P2X(7). Steady-state concentrations of NAD(+) and ATP in extracellular compartments are highly regulated and usually are well below the threshold required for activating P2X(7). The goal of this study was to identify possible endogenous sources of these nucleotides. We show that lysis of erythrocytes releases sufficient levels of NAD(+) and ATP to induce activation of P2X(7). Dilution of erythrocyte lysates or incubation of lysates at 37 degrees C revealed that signaling by ATP fades more rapidly than that by NAD(+). We further show that the routine preparation of primary lymph node and spleen cells induces the release of NAD(+) in sufficient concentrations for ART2.2 to ADP-ribosylate P2X(7), even at 4 degrees C. Gating of P2X(7) occurs when T cells are returned to 37 degrees C, rapidly inducing CD62L-shedding and PS-externalization by a substantial fraction of the cells. The "spontaneous" activation of P2X(7) during preparation of primary T cells could be prevented by i.v. injection of either the surrogate ART substrate etheno-NAD or ART2.2-inhibitory single domain Abs 10 min before sacrificing mice.

Differential regulation of P2X7 receptor activation by extracellular nicotinamide adenine dinucleotide and ecto-ADP-ribosyltransferases in murine macrophages and T cells.

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X(7) purinergic receptor (P2X(7)R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X(7)R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X(7)R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X(7)R function in murine macrophages. Coexpression of the cloned murine P2X(7)R with ART2.1 or ART2.2 in HEK293 cells verified that P2X(7)R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X(7)R. Rather, NAD potentiated ATP-dependent P2X(7)R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X(7)R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X(7)R channel opening in T cells, this P2X(7)R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X(7)R signaling in murine macrophages and also suggest that the cellular context in which P2X(7)R signaling occurs differs between myeloid and lymphoid leukocytes.