The degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. NaV1.5 (also known as SCN5A) Na(+) channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that NaV1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that NaV1.5 colocalises with Na(+)/H(+) exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of NaV1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4-7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, NaV1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.
Abstract Sterile inflammation is a key determinant of myocardial reperfusion injuries. It participates in infarct size determination in acute myocardial infarction and graft rejection following heart transplantation. We previously showed that P2Y11 exerted an immunosuppressive role in human dendritic cells, modulated cardiofibroblasts’ response to ischemia/reperfusion in vitro and delayed graft rejection in an allogeneic heterotopic heart transplantation model. We sought to investigate a possible role of P2Y11 in the cellular response of cardiomyocytes to ischemia/reperfusion. We subjected human AC16 cardiomyocytes to 5 h hypoxia/1 h reoxygenation (H/R). P2Y11R (P2Y11 receptor) selective agonist NF546 and/or antagonist NF340 were added at the onset of reoxygenation. Cellular damages were assessed by LDH release, MTT assay and intracellular ATP level; intracellular signaling pathways were explored. The role of P2Y11R in mitochondria-derived ROS production and mitochondrial respiration was investigated. In vitro H/R injuries were significantly reduced by P2Y11R stimulation at reoxygenation. This protection was suppressed with P2Y11R antagonism. P2Y11R stimulation following H 2 O 2 -induced oxidative stress reduced mitochondria-derived ROS production and damages through PKCε signaling pathway activation. Our results suggest a novel protective role of P2Y11 in cardiomyocytes against reperfusion injuries. Pharmacological post-conditioning targeting P2Y11R could therefore contribute to improve myocardial ischemia/reperfusion outcomes in acute myocardial infarction and cardiac transplantation.
Vascular dysfunction in cardiovascular diseases includes vasomotor response impairments, endothelial cells (ECs) activation, and smooth muscle cells (SMCs) proliferation and migration to the intima. This results in intimal hyperplasia and vessel failure. We previously reported that activation of the P2Y11 receptor (P2Y11R) in human dendritic cells, cardiofibroblasts and cardiomyocytes was protective against hypoxia/reoxygenation (HR) lesions. In this study, we investigated the role of P2Y11R signaling in vascular dysfunction. P2Y11R activity was modulated using its pharmacological agonist NF546 and antagonist NF340. Rat aortic rings were exposed to angiotensin II (AngII) and evaluated for their vasomotor response. The P2Y11R agonist NF546 reduced AngII-induced vascular dysfunction by promoting EC-dependent vasorelaxation, through an increased nitric oxide (NO) bioavailability and reduced AngII-induced H2O2 release; these effects were prevented by the use of the P2Y11R antagonist NF340. Human vascular SMCs and ECs were subjected to AngII or H/R simulation in vitro. P2Y11R agonist modulated vasoactive factors in human ECs, that is, endothelial nitric oxide synthase (eNOS) and endothelin-1, reduced SMC proliferation and prevented the switch towards a synthetic phenotype. H/R and AngII increased ECs secretome-induced SMC proliferation, an effect prevented by P2Y11R activation. Thus, our data suggest that P2Y11R activation may protect blood vessels from HR-/AngII-induced injury and reduce vascular dysfunctions. These results open the way for new vasculoprotective interventions.
Objective: High salt diet is related to the development of hypertension, increases skin dendritic cell (DC) density as well as interstitial sodium concentration (up to 180 mol/L). Recent in vitro studies indicated that high sodium concentrations polarize the T-cell immune response toward TH17. DC play a major role in innate and adaptive immune system. Interestingly, hypertension was recently shown to be transferable to normotensive animals through infusion of DC from hypertensive animals. However, the effects of high sodium concentration on DC function remain unknown. Design and method: In this in vitro study, we analyzed how high NaCl concentration interferes with LPS-induced DC maturation. DC were derived from human blood monocytes that were differentiated for 6 days into DCs in RPMI 10% Bovine calf serum supplemented with GMCSF (66 ng/ml) and IL4 (25 ng/ml) then stimulated during 48 h with LPS 2.5 ng/ml at increasing concentrations of NaCl [140 160 180 200 Mm]. We analyzed cell viability by Annexin V and Propidium iodide staining and cell expression of CCR7, CD25, CD86 and CD83 by flow cytometry. We also studied secreted cytokines such as IL-12p70 by ELISA method. Results: DC viability was maintained until a concentration of [200 mM] NaCl (above, viability was markedly reduced to 5%). High NaCl concentration reduced the expression of maturation markers (CD25+ 91.3 ± 2.8 vs 79.6 ± 5.9%, CD86+: 92.5 ± 4.4 vs 77.2 ± 3.0%, CD83+: 91.8 ± 4.4 vs 77.2 ± 5.6% with significates differences p < 0.01) for 140 mM and 200 mM NaCl, respectively. Of note, the expression of CCR7 diminished from (47.4 ± 12.5 to 19.4 ± 12.2% with significates differences p < 0.01) for 140 mM and 200 mM. We could reveal by ELISA method a significant IL12-p70 cytokine decrease from (225 ± 94.5 to 65.5 ± 25.7 pg/ml with significates differences p < 0.05) for 140 mM and 200 mM NaCl, respectively. Conclusions: High NaCl concentration reduced maturation and migration capacity of human DC, decreases IL12 production thus altering the polarization pathway towards Th1 immune cells which may directly affect our immune response against viral infection. Whether DC submitted to high NaCl concentrations may implicate specific cellular signaling pathways remains unknown.
Ischemia reperfusion injuries are involved in the physiopathology of heart transplantation where they will increase graft rejection. Ischemia generates cellular stress leading to ATP release in the extracellular medium that may activate purinergic receptors. These receptors may play important regulatory roles. We already showed that the purinergic receptor P2Y11 (P2Y11R) exhibited an immunosuppressive role in human dendritic cells in vitro (Chadet et al., 2015). We sought here for a possible role of P2Y11R in the cellular response of cardiomyocytes to ischemia/reperfusion. We used a human cell line of adult ventricular cardiomyocytes (AC16) and a rat cardiomyoblast cell line (H9c2). Both were subjected to 5h of hypoxia (1% O2, PBS) and 1h of reoxygenation (21% O2, DMEM) (H/R). Viability/mortality was assessed by MTT Assay, ATPi level and LDH release. We then confirmed our in vitro data in an in vivo model of allogeneic heterotopic heart transplantation in mice. Hearts from BalbC mice were intra-abdominall...
Abstract High concentrations of extracellular ATP (eATP) resulting from cell damage may be found during an ischemia/reperfusion (I/R) episode at the site of injury. eATP activates purinergic receptors in dendritic cells (DCs) and may inhibit inflammation. This immunosuppressive activity could be of interest in the field of I/R, which is an inflammatory condition involved in myocardial infarction, stroke, and solid organ transplantation. However, the specific purinergic receptor responsible for this effect remains to be identified. In this study, we report that eATP induced maturation of human monocyte-derived DCs. Additionally, eATP inhibited IL-12 production whereas IL-10 levels remained unchanged in activated DCs. These effects were prevented by the P2Y11R antagonist NF340. Interestingly, a 5-h hypoxia prevented the effects of eATP on cytokine production whereas a 1-h hypoxia did not affect the eATP-mediated decrease of IL-12 and IL-6. We showed a time-dependent downregulation of P2Y11R at both mRNA and protein levels that was prevented by knocking down hypoxia-inducible factor-1α. In this study, we showed an immunosuppressive role of P2Y11R in human DCs. Additionally, we demonstrated that the time-dependent downregulation of P2Y11R by hypoxia orientates DCs toward a proinflammatory phenotype that may be involved in post-I/R injuries as observed after organ transplantation.
