NT-proANP and NT-proBNP circulating levels as predictors of cardiovascular outcome following coronary stent implantation.

BACKGROUND: Natriuretic peptides are diagnostic/prognostic biomarkers in major cardiovascular diseases. We aimed at assessing the predictive role of N-terminal pro-A-type (NT-proANP) and pro-B-type (NT-proBNP) natriuretic peptides levels toward cardiovascular outcome in both stable and unstable coronary artery disease (CAD) patients after percutaneous coronary intervention (PCI) in a non-primary PCI setting. METHODS: A total of 395 patients undergoing PCI with stent implantation for either stable angina (SA) or non ST-elevation acute coronary syndrome (NSTE-ACS) were enrolled. Pre-procedural NT-proANP and NT-proBNP levels were measured. Occurrence of major adverse cardiac events (MACEs), composite of cardiac death, non-fatal myocardial infarction, and clinically driven target lesion revascularization (c-TLR), was the endpoint of the study. Follow up mean time was 48.53±14.69months. RESULTS: MACEs occurred in forty-four patients (11%) during follow up. Both NT-proANP levels [3170 (2210-4630) vs 2283 (1314-3913) fmol/mL, p=0.004] and NT-proBNP levels [729 (356-1353) vs 511 (267-1006) fmol/mL, p=0.04] were significantly higher in patients with MACEs compared to patients without MACEs. Similar results were found when considering hard MACEs (myocardial infarction and cardiac death). NT-proANP levels were significantly higher in patients with c-TLR compared with patients without c-TLR [3705 (2766-5184) vs 2343 (1340-3960) fmol/mL, p=0.021]. At multivariate analysis, NT-proANP levels were a significant predictor of MACEs (HR 1.09, 95% CI 1.03-1.18, p=0.04). Kaplan-Meyer curves revealed that patients with elevated NT-proANP levels (>2.100fmol/mL) had a lower MACE free survival (p=0.003). CONCLUSIONS: Both NT-proANP and NT-proBNP levels were higher in CAD patients experiencing MACEs following PCI in a non-primary setting. Notably, only NT-proANP levels significantly affected prognosis after PCI.

Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease.

BACKGROUND: The genetic basis of stroke susceptibility remains to be elucidated. STR1 quantitative trait locus (STR1/QTL) was identified on rat chromosome 1 of stroke-prone spontaneously hypertensive rat (SHRSP) upon Japanese-style stroke-permissive diet (JD), and it contributes to 20% of the stroke phenotype variance. METHODS AND RESULTS: Nine hundred eighty-six probe sets mapping on STR1 were selected from the Rat RAE230A array and screened through a microarray differential expression analysis in brains of SHRSP and stroke-resistant SHR (SHRSR) fed with either regular diet or JD. The gene encoding Ndufc2 (NADH dehydrogenase [ubiquinone] 1 subunit), mapping 8 Mb apart from STR1/QTL Lod score peak, was found significantly down-regulated under JD in SHRSP compared to SHRSR. Ndufc2 disruption altered complex I assembly and activity, reduced mitochondrial membrane potential and ATP levels, and increased reactive oxygen species production and inflammation both in vitro and in vivo. SHRSR carrying heterozygous Ndufc2 deletion showed renal abnormalities and stroke occurrence under JD, similarly to SHRSP. In humans, T allele variant at NDUFC2/rs11237379 was associated with significant reduction in gene expression and with increased occurrence of early-onset ischemic stroke by recessive mode of transmission (odds ratio [OR], 1.39; CI, 1.07-1.80; P=0.012). Subjects carrying TT/rs11237379 and A allele variant at NDUFC2/rs641836 had further increased risk of stroke (OR=1.56; CI, 1.14-2.13; P=0.006). CONCLUSIONS: A significant reduction of Ndufc2 expression causes complex I dysfunction and contributes to stroke susceptibility in SHRSP. Moreover, our current evidence may suggest that Ndufc2 can contribute to an increased occurrence of early-onset ischemic stroke in humans.

T2238C ANP gene variant and risk of recurrent acute coronary syndromes in an Italian cohort of ischemic heart disease patients.

BACKGROUND: The role of C2238/atrial natriuretic peptide (ANP) minor allele, at the T2238C ANP gene variant, as a predisposing risk factor for acute cardiovascular events, has been previously reported. We aimed at evaluating, by a retrospective approach, the long-term impact of C2238/ANP-minor allele carrier status toward the risk of recurrent acute coronary syndromes (re-ACS) in an Italian cohort of ischemic heart disease patients. METHODS: A total of 379 patients (males = 80.5%; mean age = 62.5 ±â€Š9.2 years) presenting with ACS were retrospectively analyzed. Mean follow-up was 5.1 ±â€Š3.5 years (range 1-26 years). Occurrence of new episodes of unstable angina, non-ST-segment elevation myocardial infarction and STE myocardial infarction over the years was recorded and compared between subjects not carrying and carrying C2238/ANP-minor allele. RESULTS: At univariate analysis, C2238/ANP-minor allele carrier status and treatment with beta-blocker, aspirin and statin were associated with risk of re-ACS. Multivariate analysis confirmed that hypercholesterolemia (P < 0.0001) and C2238/ANP-minor allele carrier status (P < 0.05) were both significantly and independently associated with increased risk of re-ACS. Both treatments with beta-blocker and with statin were significantly associated with reduced risk of re-ACS (P = 0.01 and P < 0.01, respectively). Age above 55 years was associated with recurrence of ACS in C2238/ANP-minor allele carriers (hazard ratio 1.427, 95% confidence interval 1.066-1.911, P = 0.017). Kaplan-Meier curves confirmed highest risk of new events occurrence in C2238/ANP-minor allele carriers (P = 0.035). CONCLUSIONS: The present results demonstrate that C2238/ANP-minor allele carrier status is an independent risk factor for ACS recurrence in an Italian cohort of ischemic heart disease patients over the long term, and they support the role of C2238/ANP-minor allele as a negative prognostic factor in coronary artery disease patients.

Differential modulation of AMPK/PPARα/UCP2 axis in relation to hypertension and aging in the brain, kidneys and heart of two closely related spontaneously hypertensive rat strains.

OBJECTIVES: We examined expression protein of AMPK/SIRT1/PGC1α/PhoxO3a/PPARα/UCP2 pathway in brain, kidneys and heart of stroke-prone spontaneously hypertensive rat (SHRSP) vs stroke-resistant SHR (SHRSR) at different weeks of age, up to one year, in order to test the hypothesis that abnormalities within this pathway could associate with higher susceptibility of SHRSP to develop hypertension-related vascular damage. BACKGROUND: SHRSP develops severe hypertension and related target organ damage. Marked reduction of uncoupling protein 2 (UCP2) expression upon high salt-low potassium diet associates with increased renal injury in SHRSP. UCP2 may represent a key mitochondrial protein involved in cardiovascular damage. RESULTS: At 2 months of age a significant down-regulation of UCP2 expression at both mRNA and protein levels was found, along with reduced protein expression of all components of UCP2 regulatory pathway, in tissues of SHRSP but not of SHRSR, that progressed with hypertension development and aging. A significant increase of both oxidative stress and inflammation was detected in tissues of SHRSP as a function of age. SBP levels were significantly higher in SHRSP than SHRSR at 3 months of age and thereafter. At one year of age, higher degree of renal damage, with proteinuria and severe glomerular and tubulo-interstitial fibrosis, of cerebral damage, with significant vessel extravasation and stroke occurrence, and of myocardial damage was detected in SHRSP than SHRSR.  CONCLUSIONS: The early significant reduced expression of the antioxidant AMPK/PPARα/UCP2 pathway that progressed throughout lifetime may contribute to explain higher predisposition of SHRSP to oxidative stress dependent target organ damage in the context of severe hypertension.

Protective effects of Brassica oleracea sprouts extract toward renal damage in high-salt-fed SHRSP: role of AMPK/PPARα/UCP2 axis.

OBJECTIVES: Renal damage precedes occurrence of stroke in high-sodium/low-potassium-fed stroke-prone spontaneously hypertensive rat (SHRSP). We previously reported a marked suppression of uncoupling protein-2 (UCP2) upon high-salt Japanese-style diet in SHRSP kidneys. Vegetable compounds are known to exert protective effects in cardiovascular diseases. We aimed at evaluating the impact of Brassica oleracea sprouts juice toward renal damage in Japanese diet-fed SHRSP and exploring the role of 5'-adenosine monophosphate-activated protein kinase (AMPK)/NAD-dependent deacetylase sirtuin-1 (SIRT1)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α)/peroxisome proliferator-activated receptor-α (PPARα)/UCP2 axis. METHODS: SHRSP received Japanese diet for 4 weeks. A group of SHRSP received Japanese diet and B. oleracea. A third group received Japanese diet, B. oleracea, and PPARα inhibitor (GW6471). A group of SHRSP fed with regular diet served as control. RESULTS: Japanese diet induced marked increases of oxidative stress, inflammation, and proteinuria, along with glomerular and tubular damage, as compared with regular diet. A significant suppression of AMPK/UCP2 pathway was observed. Despite Japanese diet feeding, concomitant administration of B. oleracea prevented oxidative stress accumulation, inflammation, renal damage, and proteinuria. All components of the UCP2 regulatory pathway were significantly increased by B. oleracea. Superoxide dismutase 2 and phosphoendothelial nitric oxide synthase were also stimulated. Addition of PPARα inhibitor to B. oleracea and Japanese diet significantly reduced the B. oleracea beneficial effects. SBP levels were comparable among the different groups of rats.In vitro, UCP2 inhibition by genipin offset the antioxidant effect of B. oleracea in renal mesangial and proximal tubular cells. CONCLUSION: B. oleracea administration prevented renal damage in salt-loaded SHRSP, independently from SBP, with parallel stimulation of AMPK/SIRT1/PGC1α/PPARα/UCP2 axis. Stimulation of the latter mechanism may provide relevant renal protective effect and play a therapeutic role in target organ damage progression in hypertension.

The C2238/αANP variant is a negative modulator of both viability and function of coronary artery smooth muscle cells.

BACKGROUND: Abnormalities of vascular smooth muscle cells (VSMCs) contribute to development of vascular disease. Atrial natriuretic peptide (ANP) exerts important effects on VSMCs. A common ANP molecular variant (T2238C/αANP) has recently emerged as a novel vascular risk factor. OBJECTIVES: We aimed at identifying effects of CC2238/αANP on viability, migration and motility in coronary artery SMCs, and the underlying signaling pathways. METHODS AND RESULTS: Cells were exposed to either TT2238/αANP or CC2238/αANP. At the end of treatment, cell viability, migration and motility were evaluated, along with changes in oxidative stress pathway (ROS levels, NADPH and eNOS expression), on Akt phosphorylation and miR21 expression levels. CC2238/αANP reduced cell vitality, increased apoptosis and necrosis, increased oxidative stress levels, suppressed miR21 expression along with consistent changes of its molecular targets (PDCD4, PTEN, Bcl2) and of phosphorylated Akt levels. As a result of increased oxidative stress, CC2238/αANP markedly stimulated cell migration and increased cell contraction. NPR-C gene silencing with specific siRNAs restored cell viability, miR21 expression, and reduced oxidative stress induced by CC2238/αANP. The cAMP/PKA/CREB pathway, driven by NPR-C activation, significantly contributed to both miR21 and phosphoAkt reduction upon CC2238/αANP. miR21 overexpression by mimic-hsa-miR21 rescued the cellular damage dependent on CC2238/αANP. CONCLUSIONS: CC2238/αANP negatively modulates viability through NPR-C/cAMP/PKA/CREB/miR21 signaling pathway, and it augments oxidative stress leading to increased migratory and vasoconstrictor effects in coronary artery SMCs. These novel findings further support a damaging role of this common αANP variant on vessel wall and its potential contribution to acute coronary events.

C2238 atrial natriuretic peptide molecular variant is associated with endothelial damage and dysfunction through natriuretic peptide receptor C signaling.

RATIONALE: C2238 atrial natriuretic peptide (ANP) minor allele (substitution of thymidine with cytosine in position 2238) associates with increased risk of cardiovascular events. OBJECTIVE: We investigated the mechanisms underlying the vascular effects of C2238-αANP. METHODS AND RESULTS: In vitro, human umbilical vein endothelial cell were exposed to either wild-type (T2238)- or mutant (C2238)-αANP. Cell survival and apoptosis were tested by Trypan blue, annexin V, and cleaved caspase-3 assays. C2238-αANP significantly reduced human umbilical vein endothelial cell survival and increased apoptosis. In addition, C2238-αANP reduced endothelial tube formation, as assessed by matrigel. C2238-αANP did not differentially modulate natriuretic peptide receptor (NPR)-A/B activity with respect to T2238-αANP, as evaluated by intracellular cGMP levels. In contrast, C2238-αANP, but not T2238-αANP, markedly reduced intracellular cAMP levels in an NPR-C-dependent manner. Accordingly, C2238-αANP showed higher affinity binding to NPR-C, than T2238-αANP. Either NPR-C inhibition by antisense oligonucleotide or NPR-C gene silencing by small interfering RNA rescued survival and tube formation of human umbilical vein endothelial cell exposed to C2238-αANP. Similar data were obtained in human aortic endothelial cell with NPR-C knockdown. NPR-C activation by C2238-αANP inhibited the protein kinase A/Akt1 pathway and increased reactive oxygen species. Adenovirus-mediated Akt1 reactivation rescued the detrimental effects of C2238-αANP. Overall, these data indicate that C2238-αANP affects endothelial cell integrity through NPR-C-dependent inhibition of the cAMP/protein kinase A/Akt1 pathway and increased reactive oxygen species production. Accordingly, C2238-αANP caused impairment of acetylcholine-dependent vasorelaxation ex vivo, which was rescued by NPR-C pharmacological inhibition. Finally, subjects carrying C2238 minor allele showed early endothelial dysfunction, which highlights the clinical relevance of our results. CONCLUSIONS: C2238-αANP reduces endothelial cell survival and impairs endothelial function through NPR-C signaling. NPR-C targeting represents a potential strategy to reduce cardiovascular risk in C2238 minor-allele carriers.

Differential modulation of uncoupling protein 2 in kidneys of stroke-prone spontaneously hypertensive rats under high-salt/low-potassium diet.

The stroke-prone spontaneously hypertensive rat (SHRsp) represents an animal model of increased susceptibility to high-salt diet-induced cerebral and renal vascular injuries. High blood pressure and genetic factors are viewed as major contributing factors. In high-salt-loaded SHRsp and stroke-resistant SHR animals, we determined blood pressure levels, degree of kidney lesions, renal uncoupling protein 2 (UCP2) gene and protein expression levels along with rattus norvegicus (rno)-microRNA (miR) 24 and 34a gene expression, nuclear factor-κB protein levels, and oxidative stress. In vitro, UCP2 gene silencing was performed in renal mesangial cells. We found more severe degree of renal damage in SHRsp at the end of 4-week high-salt dietary treatment as compared with stroke-resistant SHR, despite comparable blood pressure levels, along with increased rate of inflammation and oxidative stress. Kidney UCP2 gene and protein expression levels were significantly downregulated under high-salt diet in SHRsp, but not in stroke-resistant SHR. Differential UCP2 regulation was paralleled by differential expression of kidney rno-miR 24 and 34a, known to target UCP2 gene, in the 2 strains. UCP2 gene silencing in renal mesangial cells led to increased rate of reactive oxygen species generation, increased inflammation and apoptosis, reduced cell vitality, and increased necrosis. In conclusion, high-salt diet downregulates the antioxidant UCP2-dependent mechanism in kidneys of SHRsp, but not of stroke-resistant SHR. A parallel differential kidney miR regulation under high-salt diet in the 2 strains may contribute to the differential UCP2 modulation. UCP2 is a critical protein to prevent oxidative stress damage in renal mesangial cells in vitro.

Association of a single nucleotide polymorphism of the NPR3 gene promoter with early onset ischemic stroke in an Italian cohort.

BACKGROUND: NPR3, located on human chromosome 5 (5p14-p13), encodes the natriuretic peptide receptor type C (NPR-C) that is mainly known as the natriuretic peptide clearance receptor. Involvement of NPR3 in susceptibility to cardiovascular diseases, i.e. hypertension, has been previously shown. With regard to stroke predisposition, evidence for a potential role of genetic variation within or nearby NPR3 has been suggested by a previous genome wide association study. METHODS: We investigated the contribution to early-onset ischemic stroke susceptibility of the NPR3 -55 C>A transition by genotyping this variant in an Italian cohort of 368 cases and 335 controls. RESULTS: In a multivariable logistic regression analysis adjusting for age, gender, hypertension, hypercholesterolemia, smoking habit and diabetes, a significant association of the -55 AA genotype with stroke was observed (OR=3.2, 95% CI 1.2-8.3, p=0.016). Remarkably, the polymorphism remained associated with stroke after adjusting for hypertensive status. CONCLUSION: Our observation obtained in an Italian cohort of early onset ischemic strokes suggests that a NPR3 promoter gene variant could have a role on cerebrovascular disease susceptibility.

Influence of rs5065 atrial natriuretic peptide gene variant on coronary artery disease.

OBJECTIVES: The aim of this study was to investigate the impact of rs5065 atrial natriuretic peptide (ANP) gene variant on coronary artery disease (CAD) and its outcomes and to gain potential mechanistic insights on the association with CAD. BACKGROUND: Either modified ANP plasma levels or peptide structural alterations have been involved in development of cardiovascular events. METHODS: Three hundred ninety-three control subjects and 1,004 patients undergoing coronary angiography for suspected CAD (432 stable angina [SA], 572 acute coronary syndrome [ACS]) were genotyped for rs5065 ANP gene variant. Data in SA and ACS groups were replicated in an independent population of 482 stable angina patients (rSA) and of 675 ACS patients, respectively. Clinical follow-up was available for both SA and rSA patients. Plasma N-terminal-proANP, myeloperoxidase, lipoprotein-associated phospholipase A2, and oxidized low-density lipoprotein were assessed in a subgroup of rSA patients. RESULTS: rs5065 minor allele (MA) was an independent predictor of ACS (odds ratio: 1.90; 95% confidence interval: 1.40 to 2.58, p < 0.001). At follow-up, rs5065 MA was independently associated with a significantly higher rate of major adverse cardiovascular events in the SA group, p < 0.001. Data were replicated in the rSA group at follow-up (p = 0.008). Cox proportional hazard analysis tested by 4 models confirmed higher major adverse cardiovascular events risk in rs5065 MA carriers in both SA and rSA cohorts. Significantly higher myeloperoxidase levels were detected in rs5065 MA carriers (n = 597 [345 to 832 µg/l] vs. n = 488 [353 to 612 µg/l], p = 0.038). No association of rs5065 was observed with N-terminal-proANP levels. CONCLUSIONS: The MA of rs5065 ANP gene variant associates with increased susceptibility to ACS and has unfavorable prognostic value in CAD.

Identification of a novel MYBPC3 gene variant in a patient with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder characterized by cardiac hypertrophy caused by mutations in sarcomere protein genes. MYBPC3 mutations are reported as a frequent cause of HCM. We aimed to identify the gene mutation underlying HCM in an Italian patient and his family composed of 13 relatives. Mutation screening of 658 known mutations was performed using a rapid and efficient mutation detection system based on semiautomated MALDI-TOF mass spectrometry using the Sequenom MassArray System and iPLEX Gold genotyping chemistry. Subsequently, direct sequencing of the coding exons and flanking intronic regions was performed for the most suitable HCM genes (MYBPC3, MYH7, TNNT2, TNNI3, and TPM1) in the index patient. We found a novel MYBPC3 gene mutation: G13999T (Gln689His). No other sarcomere gene mutation was found in this family. This genetic variant, which changes the last amino acid of MYBPC3 exon 21, affects a highly conserved residue. Furthermore, the Gln689His does not appear in public databases and has never been described as a polymorphism. The potential pathogenic role of this novel mutation was underlined by its absence in a sample of healthy subjects (n = 122) from the general Italian population. In summary, a novel MYBPC3 gene mutation has been identified in a patient affected by HCM, whereas it was absent in 244 reference alleles.

Determinants of N-terminal proatrial natriuretic peptide plasma levels in a survey of adult male population from Southern Italy.

OBJECTIVES: Natriuretic peptides control cardiovascular functions through diuretic, natriuretic, and vasodilatory properties. Several anthropometric, cardiac and renal variables were found to be independently correlated to their levels. Few studies, however, systematically investigated the independent determinants of natriuretic peptide levels in large populations. DESIGN: The present analysis was carried out in a large unselected sample of adult male population in Southern Italy (The Olivetti Heart Study, n = 806 men, mean age = 59.5, range 35-82 years). We examined the relationship of plasma natriuretic peptide-proatrial natriuretic peptide (NT-proANP) levels with relevant anthropometric, clinical and biochemical variables; the impact of age; and the association of NT-proANP levels with cardiovascular risk. RESULTS: NT-proANP was directly associated to age, pulse pressure (PP), renal sodium fractional excretion (FENa) (P < 0.005), and inversely to diastolic blood pressure (DBP), heart rate (HR), creatinine clearance, body mass index (BMI), arm and leg circumferences (P < 0.005). After adjustment for age, DBP, creatinine clearance, FENa and HR remained independent determinants of NT-proANP levels (all P < 0.01, cumulative R = 0.186). Upon stratification of our population by tertile of age, NT-proANP was significantly associated (P

Reactive oxygen species-mediated effects on vascular remodeling induced by human atrial natriuretic peptide T2238C molecular variant in endothelial cells in vitro.

OBJECTIVES: T2238C ANP (atrial natriuretic peptide) gene variant has been associated with increased cardiovascular risk in humans and with a significant pharmacogenomic effect on cardiovascular disease outcome in hypertensive patients. We investigated the impact of T2238C ANP gene variant on oxidative stress production, cell proliferation and migration, angiogenesis and vascular remodeling in human umbilical vein endothelial cells in vitro. METHODS: Differentially expressed genes in human umbilical vein endothelial cells exposed to either wild-type (TT2238) or mutant (CC2238) alpha-ANP were characterized by real time-PCR-macroarray analysis using human oxidative stress, angiogenesis and matrix arrays. Reactive oxygen species (ROS) production was determined by dihydroethidium and by evaluation of dichlorofluorescein content. NADPH oxidase gp91phox subunit was investigated by western blotting. Endothelial cell proliferation, migration and tube formation were characterized both in the presence and in the absence of NADPH oxidase inhibition. RESULTS: Compared with TT2238, CC2238 alpha-ANP altered the redox state balance of the cells in a more significant manner, favoring ROS production and reducing antioxidative stress response. Gene expression of molecules involved in atherogenesis and vascular remodeling was enhanced. In contrast to TT2238 peptide, CC2238 was unable to stimulate cell proliferation and it markedly inhibited endothelial cell tube formation. NADPH oxidase inhibition restored the cell proliferative properties under CC2238 peptide exposure. CONCLUSION: CC2238 alpha-ANP led to ROS accumulation and increased expression of genes related to atherosclerosis and vascular remodeling in human umbilical vein endothelial cells. As a consequence of NADPH-derived ROS, blunted endothelial cell proliferation and impaired endothelial cell tube formation were observed. These in-vitro effects may link the T2238C alpha-ANP variant to enhanced susceptibility to vascular damage in vivo.

Phosphodiesterase 4D and 5-lipoxygenase activating protein genes and risk of ischemic stroke in Sardinians.

Genetic factors contribute to the risk of ischemic stroke (IS). The phosphodiesterase-4D (PDE4D) and the 5-lipoxygenase activating protein (ALOX5AP) genes were identified as contributors to stroke in an Icelandic population. In an attempt to better define the contributory role of PDE4D and ALOX5AP genes to the risk of IS in humans, we carried out the present association study in a well-characterized, earlier published, genetically homogenous population from the island of Sardinia, Italy. In this cohort, including 294 cases and 235 controls, age, hypertension, hypercholesterolemia, and atrial fibrillation represent risk factors for IS. The PDE4D gene was evaluated by four single nucleotide polymorphisms (SNP32, SNP45, SNP83, SNP87) and by the microsatellite AC008818-1; the ALOX5AP gene was characterized by three SNPs (SG13S32, SG13S89, ALO2A). The results of our study provide no evidence of association between any single PDE4D and ALOX5AP gene variant with the risk of IS in the Sardinian cohort. Haplotype analysis, including that constructed with allele 0 of microsatellite AC008818-1 and SNP45 of the PDE4D gene, was also negative. In conclusion, we found no evidence of association between PDE4D and ALOX5AP genes and the risk of IS in a genetically homogenous population from Sardinia.

Endogenously activated mGlu5 metabotropic glutamate receptors sustain the increase in c-Myc expression induced by leukaemia inhibitory factor in cultured mouse embryonic stem cells.

We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.