Guillaume Arguin

P2Y6 receptor contributes to neutrophil recruitment to inflamed intestinal mucosa by increasing CXC chemokine ligand 8 expression in an AP‐1‐dependent manner in epithelial cells

Background: Inflammatory bowel diseases are characterized by the presence of CXCL8 at the site of lesions resulting in neutrophil recruitment and loss of tissue functions. We report that P2Y6 receptor activation stimulates CXCL8 expression and release by intestinal epithelial cells (IECs). In this context, we investigated if uridine 5′‐diphosphate (UDP) enemas stimulate neutrophil recruitment to the mucosa of mice suffering from colitis‐like disease and we characterized the signaling events linking P2Y6 to CXCL8 expression in IEC. Methods: Neutrophil recruitment was monitored by immunofluorescence and FACS analysis. Expression of Cxcl1, a mouse functional homolog of CXCL8, was determined by quantitative real‐time polymerase chain reaction (qPCR). Pharmacological inhibitors and interfering RNAs were used to characterize the signaling pathway. The outcomes of these treatments on protein phosphorylation and on CXCL8 expression were characterized by western blots, qPCR, luciferase, and chromatin immunoprecipitation (ChIP) assays. Results: Mutation of the AP‐1 site in the CXCL8 core promoter abolished the UDP‐stimulating effect. The c‐fos/c‐jun dimer was identified as the AP‐1 complex regulating CXCL8 in response to UDP stimulation. Regulation of CXCL8 expression by P2Y6 required PKC&dgr; activation upstream of the signaling pathway composed of MEK1/2‐ERK1/2 and c‐fos. UDP administration to mice suffering from colitis‐like disease increased the number of neutrophil infiltrating the mucosa, correlating with Cxcl1 increased expression in IEC and the severity of inflammation. Conclusions: This study not only describes the P2Y6 signaling mechanism regulating CXCL8 expression in IEC, but it also illustrates the potential of targeting P2Y6 to reduce intestinal inflammation. (Inflamm Bowel Dis 2012)

Protein kinase C phosphorylates the inositol 1,4,5-trisphosphate receptor type 2 and decreases the mobilization of Ca2+in pancreatoma AR4-2J cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor channel, which plays a major (IP(3)R) is an intracellular Ca(2+) role in Ca(2+) signalling. Three isoforms of IP(3)R have been identified (IP(3)R-1, IP(3)R-2 and IP(3)R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP(3)R are poorly understood. AR4-2J cells, which express almost exclusively (~86%) the IP(3)R-2, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) influences IP(3)R-2-mediated Ca(2+) release. Using an immunoprecipitation approach, we confirmed that AR4-2J cells express almost exclusively the IP(3)R-2 isoform. Using an in vitro phosphorylation assay, we showed that the immunopurified IP(3)R-2 was efficiently phosphorylated by exogenous PKC. In intact AR4-2J cells metabolically labelled with (32)Pi, we showed that phorbol-12-myristate-13-acetate (PMA) and Ca(2+) mobilizing agonists cause the phosphorylation of IP(3)R-2. In saponin-permeabilized AR4-2J cells, IP(3)-induced Ca(2+) release was reduced after a pre-treatment with PMA or with exogenous PKC. PMA also reduced the Ca(2+) response of intact AR4-2J cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. These results demonstrate that PKC decreases the Ca(2+)mobilizing activity of IP(3)R-2 and thus exerts a negative feedback on the agonists-induced Ca(2+) response of AR4-2J cells.

Angiotensin IV interacts with a juxtamembrane site on AT4/IRAP suggesting an allosteric mechanism of enzyme modulation

Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II, binds to a specific receptor (AT(4)) that has recently been identified as the transmembrane aminopeptidase insulin-regulated aminopeptidase (IRAP) based on the fact that the two proteins share several pharmacological and biochemical properties. Our binding studies indicated that bovine heart expresses relatively large amounts (1.2 pmol/mg protein) of high-affinity binding sites for Ang IV (K(d)=1.8 nM). A photoaffinity-labeling approach combined with mild trypsin digestion revealed that the AT(4) receptor of bovine heart is a single transmembrane domain protein (153 kDa) with a large extracellular fragment (143 kDa). After alkaline denaturation of the AT(4) receptor, trypsin digestion produced two small membrane-associated fragments (16.9 and 6.6 kDa). These results suggest that Ang IV interacts with a juxtamembrane domain of AT(4) receptor. The location of the juxtamembrane site of contact was different from that of the active site of IRAP, suggesting that Ang IV uses an allosteric mechanism to modulate the activity of the AT(4)/IRAP.

cAMP-dependent protein kinase enhances inositol 1,4,5-trisphosphate-induced Ca2+ release in AR4-2J cells.

In non‐excitable cells, the inositol 1,4,5‐trisphosphate receptor (IP3R), a ligand‐gated Ca2+ channel, plays an important role in the control of intracellular Ca2+. There are three subtypes of IP3R that are differentially distributed among cell types. AR4‐2J cells express almost exclusively the IP3R‐2 subtype. The purpose of this study was to investigate the effect of cAMP‐dependent protein kinase (PKA) on the activity of IP3R‐2 in AR4‐2J cells. We showed that immunoprecipitated IP3R‐2 is a good substrate for PKA. Using a back‐phosphorylation approach, we showed that endogenous PKA phosphorylates IP3R‐2 in intact AR4‐2J cells. Pretreatment with PKA enhanced IP3‐induced Ca2+ release in permeabilized AR4‐2J cells. Pretreatment with the cAMP generating agents forskolin and vasoactive intestinal peptide (VIP) enhanced carbachol (Cch)‐induced and epidermal growth factor (EGF)‐induced Ca2+ responses in intact AR4‐2J cells. Our results are consistent with an enhancing effect of PKA on IP3R‐2 activity. This conclusion supports the emerging concept of crosstalk between Ca2+ signaling and cAMP pathways and thus provides another way by which Ca2+ signals are finely encoded within non‐excitable cells. J. Cell. Biochem. 101: 609–618, 2007.

UDP made a highly promising stable, potent, and selective P2Y6-receptor agonist upon introduction of a boranophosphate moiety

P2Y(6) nucleotide receptor (P2Y(6)-R) plays important physiological roles, such as insulin secretion and reduction of intraocular pressure. However, this receptor is still lacking potent and selective agonists to be used as potential drugs. Here, we synthesized uracil nucleotides and dinucleotides, substituted at the C5 and/or P(α) position with methoxy and/or borano groups, 18-22. Compound 18A, R(p) isomer of 5-OMe-UDP(α-B), is the most potent and P2Y(6)-R selective agonist currently known (EC(50) 0.008 μM) being 19-fold more potent than UDP and showing no activity at uridine nucleotide receptors, P2Y(2)- and P2Y(4)-R. Analogue 18A was highly chemically stable under conditions mimicking gastric juice acidity (t(1/2) = 16.9 h). It was more stable to hydrolysis by nucleotide pyrophosphatases (NPP1,3) than UDP (15% and 28% hydrolysis by NPP1 and NPP3, respectively, vs 50% and 51% hydrolysis of UDP) and metabolically stable in blood serum (t(1/2) = 17 vs 2.4, 11.9, and 21 h for UDP, 5-OMe-UDP, and UDP(α-B), respectively). This newly discovered highly potent and physiologically stable P2Y(6)-R agonist may be of future therapeutic potential.

P2Y2 receptor promotes intestinal microtubule stabilization and mucosal re-epithelization in experimental colitis†

P2Y2 receptor expression is increased in intestinal epithelial cells (IECs) during inflammatory bowel diseases (IBDs). In this context, P2Y2 stimulates PGE2 release by IECs, suggesting a role in wound healing. For this study, we have used the non‐cancerous IEC‐6 cell line. IEC‐6 cell migration was determined using Boyden chambers and the single‐edged razor blade model of wounding. The receptor was activated using ATP, UTP, or 2‐thioUTP. Pharmacological inhibitors, a blocking peptide, a neutralizing antibody and interfering RNAs were used to characterize the signaling events. Focal adhesions and microtubule (MT) dynamics were determined by immunofluorescence using anti‐vinculin and anti‐acetylated‐α‐tubulin antibodies, respectively. In vivo, the dextran sodium sulfate mouse model of colitis was used to characterize the effects of P2Y2 agonist 2‐thioUTP on remission. We showed that P2Y2 increased cell migration and wound closure by recruiting Go protein with the cooperation of integrin αv. Following P2Y2 activation, we demonstrated that GSK3β activity was inhibited in response to Akt activation. This leads to MT stabilization and increased number of focal adhesions. In vivo, P2Y2 activation stimulates remission, as illustrated by a reduction in the disease activity index values and histological scores as compared to control mice. These findings highlight a novel function for this receptor in IECs. They also illustrate that P2Y receptors could be targeted for the development of innovative therapies for the treatment of IBDs. J. Cell. Physiol. 228: 99–109, 2013.

Glucose transporter 2 expression is down regulated following P2X7 activation in enterocytes

With the diabetes epidemic affecting the world population, there is an increasing demand for means to regulate glycemia. Dietary glucose is first absorbed by the intestine before entering the blood stream. Thus, the regulation of glucose absorption by intestinal epithelial cells (IECs) could represent a way to regulate glycemia. Among the molecules involved in glycemia homeostasis, extracellular ATP, a paracrine signaling molecule, was reported to induce insulin secretion from pancreatic β cells by activating P2Y and P2X receptors. In rats jejunum, P2X7 expression was previously immunolocalized to the apex of villi, where it has been suspected to play a role in apoptosis. However, using an antibody recognizing the receptor extracellular domain and thus most of the P2X7 isoforms, we showed that expression of this receptor is apparent in the top two‐thirds of villi. These data suggest a different role for this receptor in IECs. Using the non‐cancerous IEC‐6 cells and differentiated Caco‐2 cells, glucose transport was reduced by more than 30% following P2X7 stimulation. This effect on glucose transport was not due to P2X7‐induced cell apoptosis, but rather was the consequence of glucose transporter 2 (Glut2)s internalization. The signaling pathway leading to P2X7‐dependent Glut2 internalization involved the calcium‐independent activation of phospholipase Cγ1 (PLCγ1), PKCδ, and PKD1. Although the complete mechanism regulating Glut2 internalization following P2X7 activation is not fully understood, modulation of P2X7 receptor activation could represent an interesting approach to regulate intestinal glucose absorption. J. Cell. Physiol. 228: 120–129, 2013.

Multidrug Resistance-Associated Protein 2 Expression Is Upregulated by Adenosine 5’-Triphosphate in Colorectal Cancer Cells and Enhances Their Survival to Chemotherapeutic Drugs

Extracellular adenosine 5’-triphosphate (ATP) is a signaling molecule that induces a plethora of effects ranging from the regulation of cell proliferation to modulation of cancerous cell behavior. In colorectal cancer, ATP was reported to stimulate epithelial cell proliferation and possibly promote resistance to anti-cancer treatments. However, the exact role of this danger-signaling molecule on cancerous intestinal epithelial cells (IECs) in response to chemotherapeutic agents remains unknown. To address how ATP may influence the response of cancerous IECs to chemotherapeutic agents, we used Caco-2 cells, which display enterocyte-like features, to determine the effect of ATP on the expression of multidrug resistance-associated protein 2 (MRP2). Gene and protein expression were determined by quantitative real-time PCR (qRT-PCR) and Western blotting. Resistance to etoposide, cisplatin and doxorubicin was determined by MTT assays in response to ATP stimulation of Caco-2 cells and in cells for which MRP2 expression was down-regulated by shRNA. ATP increased the expression of MRP2 at both the mRNA and protein levels. MRP2 expression involved an ATP-dependent stimulation of the MEK/ERK signaling pathway that was associated with an increase in relative resistance of Caco-2 cells to etoposide. Abolition of MRP2 expression using shRNA significantly reduced the protective effect of MRP2 toward etoposide as well as to cisplatin and doxorubicin. This study describes the mechanism by which ATP may contribute to the chemoresistance of cancerous IECs in colorectal cancer. Given the heterogeneity of colorectal adenocarcinoma responses to anti-cancer drugs, these findings call for further study to understand the role of P2 receptors in cancer drug therapy and to develop novel therapies aimed at regulating P2 receptor activity.

The Transcription Factors NFAT and CREB Have Different Susceptibilities to the Reduced Ca2+ Responses Caused by the Knock Down of Inositol Trisphosphate Receptor in HEK 293A Cells

Background/Aims: The inositol 1,4,5-trisphosphate receptor (IP₃R), a ligand-gated Ca²⁺ channel, plays an important role in the control of intracellular Ca²⁺. Three isoforms of IP₃R have been identified and most cell types express different proportions of these isoforms. The purpose of this study was to investigate how IP₃R signalling is involved in the activation of the Ca²⁺-sensitive transcription factors NFAT and CREB. Methods: Each IP₃R isoform expressed in HEK 293A cells was knocked down using selective siRNA. Free intracellular Ca²⁺ was monitored spectrofluometrically. NFAT and CREB activities were measured with luciferase reporter constructs. Results: IP₃R-2-knocked down HEK 293A cells showed a deficient CCh-induced Ca²⁺ response that could be rescued by co-stimulation with VIP, a cAMP increasing agonist. NFAT transcriptional activity, but not CREB transcriptional activity, was significantly reduced in IP₃R-2-knocked down HEK 293A cells. Overexpression of IP₃R-1 could fully compensate for IP₃R-2 knock down to mobilize Ca²⁺ and to activate NFAT. Conclusion: Our results show that the knock down of IP₃R-2 significantly reduced the intracellular Ca²⁺ response of HEK 293 cells. This reduced Ca²⁺ response did not affect the activation of CREB but significantly decreased the activation of NFAT, suggesting that the Ca²⁺ signals required for the activation of NFAT are stronger than those required for the activation of CREB.

The loss of P2X7 receptor expression leads to increase intestinal glucose transit and hepatic steatosis

In intestinal epithelial cells (IEC), it was reported that the activation of the P2X7 receptor leads to the internalization of the glucose transporter GLUT2, which is accompanied by a reduction of IEC capacity to transport glucose. In this study, we used P2rx7−/− mice to decipher P2X7 functions in intestinal glucose transport and to evaluate the impacts on metabolism. Immunohistochemistry analyses revealed the presence of GLUT2 at the apical domain of P2rx7−/− jejunum enterocytes. Positron emission tomography and biodistribution studies demonstrated that glucose was more efficiently delivered to the circulation of knockout animals. These findings correlated with increase blood glucose, insulin, triglycerides and cholesterol levels. In fact, P2rx7−/− mice had increased serum triglyceride and cholesterol levels and displayed glucose intolerance and resistance to insulin. Finally, P2rx7−/− mice developed a hepatic steatosis characterized by a reduction of Acaca, Acacb, Fasn and Acox1 mRNA expression, as well as for ACC and FAS protein expression. Our study suggests that P2X7 could play a central role in metabolic diseases.