RESUMO
The final control of renal water reabsorption occurs in the collecting duct (CD) and relies on regulated expression of aquaporin-2 (AQP2) in principal CD cells. AQP2 transcription is primarily induced by type 2 vasopressin receptor (V2R)-cAMP-protein kinase A (PKA) signaling but also by other factors, including TonEBP and NF-κB. NAPDH oxidase 4 (NOX4) represents a major source of reactive oxygen species (ROS) in the kidney. Because NOX-derived ROS may alter PKA, TonEBP and NF-κB activity, we examined the effects of NOX4 depletion on AQP2 expression. Depleted NOX4 expression by siRNA (siNOX4) in mpkCCDcl4 cells attenuated increased AQP2 mRNA expression by arginine vasopressin (AVP) but not by hypertonicity, which induces both TonEBP and NF-κB activity. AVP-induced AQP2 expression was similarly decreased by the flavoprotein inhibitor diphenyleneiodonium. siNOX4 altered neither TonEBP nor NF-κB activity but attenuated AVP-inducible cellular cAMP concentration, PKA activity and CREB phosphorylation as well as AQP2 mRNA expression induced by forskolin, a potent activator of adenylate cyclase. The repressive effect of siNOX4 on AVP-induced AQP2 mRNA expression was abolished by the non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and was significantly decreased by selective PDE antagonists cilostamide and rolipram, but not vinpocetine, which respectively target PDE3, PDE4 and PDE1. Thus, by inhibiting PDE3 and PDE4 activity NOX4-derived ROS may contribute to V2R-cAMP-PKA signaling and enhance AQP2 transcription.
Assuntos
Aquaporina 2/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , NADPH Oxidases/deficiência , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , 1-Metil-3-Isobutilxantina , Animais , Aquaporina 2/genética , Arginina Vasopressina/metabolismo , Western Blotting , AMP Cíclico/metabolismo , Túbulos Renais Coletores/citologia , Camundongos , NADPH Oxidase 4 , Quinolonas , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Rolipram , Transdução de Sinais/fisiologiaRESUMO
Recently, we showed that connexin37 (Cx37) protects against early atherosclerotic lesion development by regulating monocyte adhesion. The expression of this gap junction protein is altered in mouse and human atherosclerotic lesions; it is increased in macrophages newly recruited to the lesions and disappears from the endothelium of advanced plaques. To obtain more insight into the molecular role of Cx37 in advanced atherosclerosis, we used micro-array analysis for gene expression profiling in aortas of ApoE(-/-) and Cx37(-/-)ApoE(-/-) mice before and after 18 weeks of cholesterol-rich diet. Out of >15,000 genes, 106 genes were significantly differentially expressed in young mice before diet (P-value of <0.05, fold change of >0.7 or <-0.7, and intensity value >2.2 times background). Ingenuity pathway analysis (IPA) revealed differences in genes involved in cell-to-cell signaling and interaction, cellular compromise and nutritional disease. In addition, we identified 100 genes that were significantly perturbed after the cholesterol-rich diet. Similar to the analysis on 10-week-old mice, IPA revealed differences in genes involved in cell-to-cell signaling and interaction as well as to immuno-inflammatory disease. Furthermore, we found important changes in genes involved in vascular calcification and matrix degradation, some of which were confirmed at protein level by (immuno-)histochemistry. In conclusion, we suggest that Cx37 deficiency alters the global differential gene expression profiles in young mice towards a pro-inflammatory phenotype, which are then further influenced in advanced atherosclerosis. The results provide new insights into the significance of Cx37 in plaque calcification.
Assuntos
Aterosclerose/patologia , Conexinas/fisiologia , Animais , Apolipoproteínas E/deficiência , Aterosclerose/genética , Aterosclerose/metabolismo , Calcinose/patologia , Colesterol na Dieta/administração & dosagem , Perfilação da Expressão Gênica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteína alfa-4 de Junções ComunicantesRESUMO
A polymorphism in the human Cx37 gene (C1019T), resulting in a non-conservative amino acid change in the regulatory C-terminus of the Cx37 protein (P319S), has been proposed as a prognostic marker for atherosclerosis. We have recently demonstrated that Cx37 hemichannels control the initiation of atherosclerotic plaque development by regulating ATP-dependent monocyte adhesion in atherosclerosis-susceptible apolipoprotein E-deficient mice. In this study, we have measured the electrical properties of Cx37 hemichannels (HCs) and gap junction channels (GJCs) with voltage-clamp methods. To this end, we have transfected hCx37-P319, hCx37-S319 or empty pIRES-eGFP vector cDNA into communication-deficient HeLa cells. In clones expressing similar levels of Cx37, exposure of single cells to low-Ca(2+) solution induced a voltage-sensitive HC current. The analysis yielded a bell-shaped function g(hc)=f(V(m)) (g(hc): normalized conductance at steady state; V(m): membrane potential) with a maximum around V(m)=-30 mV. The peak g(hc) of Cx37-P319 was 3-fold larger than that of Cx37-S319 HCs. Experiments on cell pairs revealed that Cx37-P319 GJCs exhibited a 1.5-fold larger unitary conductance than Cx37-S319 GJCs. Hence, the larger peak g(hc) of the former may reflect a larger conductance of their HCs. Using the same clones, we found that Cx37-P319 cells released more ATP and were less adhesive than Cx37-S319 cells. The reduction in adhesiveness of Cx37-expressing cells was prevented by extracellular apyrase. We conclude that the differences in biophysical properties between polymorphic HCs may be responsible for inequality in ATP release between Cx37-P319 and Cx37-S319 cells, which results in differential cell adhesion.
Assuntos
Conexinas/genética , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Polimorfismo Genético , Trifosfato de Adenosina/metabolismo , Adesão Celular , Fenômenos Eletrofisiológicos , Células HeLa , Humanos , Transfecção , Proteína alfa-4 de Junções ComunicantesRESUMO
We previously reported that reducing the expression of the gap junction protein connexin (Cx)43 in mice restricts intimal thickening formation after acute vascular injury by limiting the inflammatory response and the proliferation and migration of smooth muscle cells (SMCs) toward the damaged site. SMC populations isolated from porcine coronary artery exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). S-SMCs are predominant in the normal media, whereas R-SMCs are recovered in higher proportion from stent-induced intimal thickening, suggesting that they participate in the restenotic process. Here, we further investigate the relationship between connexin expression and SMC phenotypes using porcine coronary artery SMCs. Cx40 was highly expressed in normal media of porcine coronary artery in vivo, whereas Cx43 was barely detectable. In contrast, Cx40 was downregulated and Cx43 was markedly upregulated in stent-induced intimal thickening. In vitro, S-SMCs expressed Cx40 and Cx43. In R-SMCs, Cx43 expression was increased and Cx40 was absent. We confirmed that S-SMCs treated with platelet-derived growth factor-BB acquire an R phenotype. This was accompanied by an upregulation of Cx43 and a loss of Cx40. Importantly, platelet-derived growth factor-BB-induced S-to-R phenotypic change was prevented by a reduction of Cx43 expression with antisense, ie, S-SMCs retained their typical elongated appearance and the expression of alpha-smooth muscle actin, a well-known SMC differentiation marker, whereas the expression of S100A4, a typical marker of R-SMCs, was prevented. In conclusion, limiting Cx43 expression in S-SMCs prevents platelet-derived growth factor-BB-induced S-to-R modulation. This suggests that Cx43 may be an additional target for local delivery strategies aimed at reducing restenosis.
Assuntos
Conexina 43/metabolismo , Estenose Coronária/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Actinas/metabolismo , Animais , Becaplermina , Diferenciação Celular , Movimento Celular , Forma Celular , Células Cultivadas , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Conexinas/metabolismo , Estenose Coronária/etiologia , Estenose Coronária/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Feminino , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Peptídeos/farmacologia , Fenótipo , Proteínas Proto-Oncogênicas c-sis , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas S100/metabolismo , Transdução de Sinais/efeitos dos fármacos , Stents/efeitos adversos , Sus scrofa , Fatores de Tempo , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Proteína alfa-5 de Junções ComunicantesRESUMO
A genetic polymorphism in the human gene encoding connexin37 (CX37, encoded by GJA4, also known as CX37) has been reported as a potential prognostic marker for atherosclerosis. The expression of this gap-junction protein is altered in mouse and human atherosclerotic lesions: it disappears from the endothelium of advanced plaques but is detected in macrophages recruited to the lesions. The role of CX37 in atherogenesis, however, remains unknown. Here we have investigated the effect of deleting the mouse connexin37 (Cx37) gene (Gja4, also known as Cx37) on atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice, an animal model of this disease. We find that Gja4(-/-)Apoe(-/-) mice develop more aortic lesions than Gja4(+/+)Apoe(-/-) mice that express Cx37. Using in vivo adoptive transfer, we show that monocyte and macrophage recruitment is enhanced by eliminating expression of Cx37 in these leukocytes but not by eliminating its expression in the endothelium. We further show that Cx37 hemichannel activity in primary monocytes, macrophages and a macrophage cell line (H36.12j) inhibits leukocyte adhesion. This antiadhesive effect is mediated by release of ATP into the extracellular space. Thus, Cx37 hemichannels may control initiation of the development of atherosclerotic plaques by regulating monocyte adhesion. H36.12j macrophages expressing either of the two CX37 proteins encoded by a polymorphism in the human GJA4 gene show differential ATP-dependent adhesion. These results provide a potential mechanism by which a polymorphism in CX37 protects against atherosclerosis.