Cis -acting regulation of splenic Art2 gene expression in inbred mouse strains

et al. 2009

Purinergic Signalling

ADP-ribosylation of cell surface proteins in mammalian cells is a post-translational modification by which ecto-ADP-ribosyltransferases (ARTs) transfer ADPribose from extracellular NAD to protein targets. The ART2 locus at murine chromosome 7 encompasses the tandem Art2a and Art2b genes that encode the distinct ART2.1 and ART2.2 proteins. Although both ecto-enzymes share 80% sequence identity, ART2.1 activity is uniquely regulated by an allosteric disulfide bond that is reducible in the presence of extracellular thiols, such as cysteine and glutathione, that accumulate in hypoxic and ischemic tissues. Previous studies have characterized the expression of ART2.1 and ART2.2 in murine T lymphocytes but not in other major classes of lymphoid and myeloid leukocytes. Here, we describe the expression of ART2.1 activity in a wide range of freshly isolated or tissue-cultured murine myeloid and lymphoid leukocytes. Spleen-derived macrophages, dendritic cells (DC), and B cells constitutively express ART2.1 as their predominant ART while spleen T cells express both ART2.1 and the thiol-independent ART2.2 isoform. Although bone-marrow-derived macrophages (BMDM) and dendritic cells (BMDC) constitutively express ART2.1 at low levels, it is markedly up-regulated when these cells are stimulated in vitro with IFNβ or IFNγ. ART2.1 expression and activity in splenic B cells is modestly up-regulated during incubation in vitro for 24 h, a condition that promotes B cell apoptosis. This increase in ART2.1 is attenuated by IL-4 (a B cell survival factor), but is not affected by IFNβ/γ, suggesting a possible induction of ART2.1 as an ancillary response to B cell apoptosis. In contrast, ART2.1 and ART2.2, which are highly expressed in freshly isolated splenic T cells, are markedly downregulated when purified T cells are incubated in vitro for 12-24 h. Studies with the BW5147 mouse thymocyte line verified basal expression of ART2.1 and ART2.2, as in primary spleen T cells, and demonstrated that both isoforms can be up-regulated when T cells are maintained in the presence of IFNs. Comparison of the surface proteins which are ADP-ribosylated by ART2.1 in the different leukocyte subtypes indicated both shared and cell-specific proteins as ART2.1 substrates. The LFA-1 integrin, a major target for ART2.2 in T cells, is also ADP-ribosylated by the ART2.1 expressed in macrophages. Thus, ART2.1, in contrast to ART2.2, is expressed in a broad range of myeloid and lymphoid leukocytes. The thiol redox-sensitive nature of this ecto-enzyme suggests an involvement in purinergic signaling that occurs in the combined context of inflammation and hypoxia/ischemia.

Section: Introductionsupporting

confidence: 51%

Basal and inducible expression of the thiol-sensitive ART2.1 ecto-ADP-ribosyltransferase in myeloid and lymphoid leukocytes

Hong

Brass²,

et al. 2009

Purinergic Signalling

“…3A). Note that BMDM yielded much stronger bands for ART2.1 than for ART2.2, whereas the inverse holds for BALB/c splenocytes that are known to express higher levels of ART2.2 than ART2.1 (28,29). We verified that the ART2.2 primers did not weakly cross-amplify ART2.1 cDNA by performing identical RT-PCR analyses using RNA from splenic lymphocytes of NZW mice that are natural art2b knockouts (32).…”

Section: Lps Selectively Induces Expression Of the Thiol-sensitive Armentioning

confidence: 50%

“…3B shows the schematic of this assay and Fig. 3C illustrates the robust etheno-ADP-ribosylation of multiple cell surface proteins observed in BALB/c splenic lymphocytes that are known to constitutively express ART2.2 (and to a lesser extent, also ART2.1) as a cell surface ectoenzyme (28,29).…”

Section: Lps Selectively Induces Expression Of the Thiol-sensitive Armentioning

confidence: 99%

“…T cells from ART2 knockout mice are resistant to NAD-induced apoptosis, whereas mice lacking ART2 exhibit increased survival in a model of NK T cell-mediated autoimmune hepatitis (25,26). Significantly, genetic background also affects expression of ART2.1 vs ART2.2 in different inbred mice strains (27)(28)(29). In BALB/c mice, both ART2.1 and ART2.2 are constitutively expressed in all subsets of naive T lymphocytes (30,31).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Lipopolysaccharide, IFN-γ, and IFN-β Induce Expression of the Thiol-Sensitive ART2.1 Ecto-ADP-Ribosyltransferase in Murine Macrophages

Hong

Brass²,

The Journal of Immunology

et al. 2007

Extracellular NAD modulates multiple immune and inflammatory responses via the activity of a family of ecto‐ADP‐ribosyltransferases (ARTs) that covalently modify target proteins at arginine or cysteine residues. Functional ARTs have been identified thus far in neutrophils and T cells but not macrophages. This study investigates the expression and function of ARTs by RT‐PCR and western blot analysis in primary bone‐marrow‐derived macrophages (BMDM) isolated from Balb/c mice. Although naive BMDM did not express any of the 6 murine ART genes at the mRNA or protein level, stimulation with lipopolysaccharide (LPS) or ligands for other Toll‐like receptors induced robust expression of ART2.1 as a GPI‐anchored cell surface ecto‐enzyme. ART2.1 expression was potentiated by pharmacological inhibition of ERK1/2 pathways but inhibited by pharmacological blockade of NFkappaB‐ or PI3K‐ signaling pathways. Significantly, catalytic enzyme activity of the inducible ART2.1 was strictly dependent on the presence of extracellular thiol reducing molecules such as cysteine or dithiolthreitol. These data indicate that modulation of immune function by NAD‐dependent ART2.1 activity in macrophages may be synergistically regulated by extracellular redox environments that occur during hypoxia or ishchemia. Support: NIH‐GM‐36387

“…C57BL/6 mice, for instance, express ART2.2 at very high level compared to other mouse strains although they all have rather similar T cell numbers. The origin of this variation is still unknown but might result from cis-acting regulatory elements in the promoters and their distribution in alternative splice variants [70,71]. Additionally, a polymorphism of ART2.1 and ART2.2 genes has been reported.…”

Section: Expression and Tissue Distribution Of Artsmentioning

confidence: 95%

Ecto-ADP-Ribosyltransferases (ARTs): Emerging Actors in Cell Communication and Signaling

Adriouch²,

Haag³

et al. 2004

CMC

105

Mammalian ecto ADP-ribosyltransferases (ARTs) constitute a family of structurally related proteins expressed on the cell surface or secreted in the extracellular compartment. Using NAD+ as substrate, they transfer ADP-ribose groups onto target proteins. In contrast to intracellular poly(ADP-ribosyl)transferases (PARPs), these enzymes transfer a single ADPR and are thus mono-ARTs. Five paralogs (ART1-5) have been cloned but only four of them are expressed in human due to a defective ART2 gene, and six in the mouse as the result of ART2 gene duplication. The recent determination of the crystal structure of rat ART2 reveals homologies with bacterial ART toxins and provides a molecular basis for understanding the specificity of ARTs for their targets. A combination of different technological approaches reveals that ecto-ARTs are expressed in different tissues with privileged sites such as heart and skeletal muscles for ART1, T lymphocytes for ART2 or testis for ART5. It also indicates that ART expression is highly regulated. ADP-ribosylation of target proteins on cell surfaces or circulating in body fluids leads to reversible post-translational modifications which can inhibit the targets, as known for bacterial ARTs, or activate them, as in the crosstalk between mouse ART2 and the cytolytic P2X7 receptor on T lymphocytes. ART activity in the extracellular compartment provides sophisticated regulatory mechanisms for cell communication. This designates ecto-ARTs as new candidates for drug targeting.