Nathalie Suarez Gonzalez

Involvement of multiple P2Y receptors and signaling pathways in the action of adenine nucleotides diphosphates on human monocyte‐derived dendritic cells

Adenosine 5′‐triphosphate (ATP), which is released from necrotic cells, induces a semimaturation state of dendritic cells (DC), characterized by the up‐regulation of costimulatory molecules and the inhibition of proinflammatory cytokines. This action is mediated by cyclic adenosine monophosphate (cAMP) and involves the P2Y11 receptor. As DC express the ecto‐enzyme CD39, which converts ATP into adenosine 5′‐diphosphate (ADP), the effects of adenine nucleotides diphosphates on molecular signaling [intracellular calcium ([Ca2+]i), cAMP, extracellular signal‐regulated kinase 1 (ERK1)], costimulatory molecule expression (CD83), and cytokine production [interleukin (IL)‐12, tumor necrosis factor α (TNF‐α), IL‐10] were investigated in human monocyte‐derived DC. ADP, 2‐methylthio‐ADP, and ADPβS had no effect on cAMP, increased [Ca2+]i, and stimulated the phosphorylation of ERK1. The effect on ERK1 was inhibited by AR‐C69931MX, a P2Y12 and P2Y13 antagonist. On the contrary the effect on [Ca2+]i was neither inhibited by AR‐C69931MX or by the P2Y1 antagonist MRS‐2179. Both effects were inhibited by pertussis toxin. ADPβS alone was less potent for up‐regulation of CD83 than ATPγS and did not increase the CD83 expression by DC stimulated with lipopolysaccharide (LPS). Similar to ATPγS, ADPβS inhibited the release of IL‐12p40, IL‐12p70, and TNF‐α stimulated by LPS (1–100 ng/ml). The inhibitory effect of ADPβS on IL‐12 release was neither reversed by AR‐C69931MX or by MRS‐2179. The two nucleotides had opposite effects on IL‐10 production: inhibition by ADPβS and potentiation by ATPγS. In conclusion, ATP can modulate the function of DC, directly via a cAMP increase mediated by the P2Y11 receptor and indirectly via its degradation into ADP, which acts via Gi‐coupled receptors coupled to ERK activation and calcium mobilization. These distinct mechanisms converge on the inhibition of inflammatory cytokine production, particularly IL‐12, but have a differential effect on IL‐10.

Extracellular Adenine Nucleotides Inhibit the Activation of Human CD4+ T Lymphocytes

ATP has been reported to inhibit or stimulate lymphoid cell proliferation, depending on the origin of the cells. Agents that increase cAMP, such as PGE2, inhibit human CD4+ T cell activation. We demonstrate that several ATP derivatives increase cAMP in both freshly purified and activated human peripheral blood CD4+ T cells. The rank order of potency of the various nucleotides was: adenosine 5′-O-(3-thiotriphosphate) (ATPγS) ≈ 2′- and 3′-O-(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP ≫ dATP, 2-propylthio-β,γ-dichloromethylene-d-ATP, UDP, UTP. This effect did not involve the activation of A2Rs by adenosine or the synthesis of prostaglandins. ATPγS had no effect on cytosolic calcium, whereas BzATP induced an influx of extracellular calcium. ATPγS and BzATP inhibited secretion of IL-2, IL-5, IL-10, and IFN-γ; expression of CD25; and proliferation after activation of CD4+ T cells by immobilized anti-CD3 and soluble anti-CD28 Abs, without increasing cell death. Taken together, our results suggest that extracellular adenine nucleotides inhibit CD4+ T cell activation via an increase in cAMP mediated by an unidentified P2YR, which might thus constitute a new therapeutic target in immunosuppressive treatments.

The fate of P2Y-related orphan receptors: GPR80/99 and GPR91 are receptors of dicarboxylic acids.

Several orphan G protein-coupled receptors are structurally close to the family of P2Y nucleotide receptors: GPR80/99 and GPR91 are close to P2Y1/2/4/6/11 receptors, whereas GPR87, H963 and GPR34 are close to P2Y12/13/14. Over the years, several laboratories have attempted without success to identify the ligands of those receptors. In early 2004, two papers have been published: One claiming that GPR80/99 is an AMP receptor, called P2Y15, and the other one showing that GPR80/99 is a receptor for α-ketoglutarate, while GPR91 is a succinate receptor. The accompanying paper by Qi et al. entirely supports that GPR80/99 is an α-ketoglutarate receptor and not an AMP receptor. The closeness of dicarboxylic acid and P2Y nucleotide receptors might be linked to the negative charges of both types of ligands and the involvement of conserved Arg residues in their neutralization.

HDGF is dephosphorylated during the early steps of endothelial cell apoptosis in a caspase-dependent way.

We were looking by a proteomic approach for new phospho‐proteins involved during the early steps of the TNF + cycloheximide (CHX)‐induced apoptosis—preceding mitochondrial membrane permeabilization—of endothelial cells (BAEC). In the present study, we observed on the autoradiography from 2D gel of 32P‐labeled samples a string of proteins undergoing a complete dephosphorylation after 1 h of stimulation with TNF + CHX—while mitochondrial membrane permeabilization was observed after 3 h—identified the different spots by mass spectrometry as one and only protein, HDGF, and confirmed the identity by western blot. The intensity of the 2D phosphorylation pattern of HDGF was correlated with the amount of apoptosis induced by TNF + CHX and TNF or CHX alone and this event was inhibited by the Caspase specific inhibitor zVADfmk. Moreover the TNF + CHX‐treatment did not affect the nuclear localization of GFP‐HDGF. Taken together, our data suggest an involvement of HDGF during the initiation phase of the apoptotic process downstream from an initiator Caspase and a regulation of this protein by phosphorylation in the nucleus. J. Cell. Biochem. 104: 1161–1171, 2008.