Phosphorylation of tyrosine 801 of vascular endothelial growth factor receptor-2 is necessary for Akt-dependent endothelial nitric-oxide synthase activation and nitric oxide release from endothelial cells

Vascular endothelial growth factor (VEGF)-stimulated nitric oxide (NO) release from endothelial cells is mediated through the activation of VEGF receptor-2 (VEGFR-2). Herein, we have attempted to determine which autophosphorylated tyrosine residue on the VEGFR-2 is essential for VEGF-mediated endothelial nitric-oxide synthase (eNOS) activation and NO production from endothelial cells. Tyrosine residues 801, 1175, and 1214 of the VEGFR-2 were mutated to phenylalanine, and the mutated receptors were analyzed for their ability to stimulate NO production. We show, both in COS-7 cells cotransfected with the VEGFR-2 mutants and eNOS and in bovine aortic endothelial cells, that the Y801F-VEGFR-2 mutant is unable to stimulate NO synthesis and eNOS activation in contrast to the wild type, Y1175F-VEGFR-2, and Y1214F-VEGFR-2. However, the Y801F mutant retains the capacity to activate phospholipase C-gamma in contrast to the Y1175F-VEGFR-2. Interestingly, the Y801F-VEGFR-2, in contrast to the wild type receptor, does not fully activate phosphatidylinositol 3-kinase or recruit the p85 subunit upon receptor activation. This results in a complete incapacity of the Y801F-VEGFR-2 to stimulate Akt activation and eNOS phosphorylation on serine 1179 in endothelial cells. In addition, constitutive activation of Akt or a phosphomimetic mutant of eNOS (S1179D) fully rescues the inability of the Y801F-VEGFR-2 to induce NO release. Finally, we generated an antibody that specifically recognizes the phosphorylated form of tyrosine 801 of the VEGFR-2 and demonstrate that this residue is actively phosphorylated in response to VEGF stimulation of endothelial cells. We thus conclude that autophosphorylation of tyrosine residue 801 of the VEGFR-2 is essential for VEGF-stimulated NO production from endothelial cells, and this is primarily accomplished via the activation of phosphatidylinositol 3-kinase and Akt signaling to eNOS.     

Glucotoxicity and pancreatic proteomics

Chronic hyperglycaemia is one of the main characteristics of a diabetic state. This is also the first cause of diabetic complications. However, it is now generally accepted that glucotoxicity also participates in the worsening of type 2 diabetes, by affecting the secretion of β-cells. So far, different mechanisms have been proposed to explain the adverse effects of chronic hyperglycaemia. One of them suggests that the modulation of expression of several key proteins during a hyperglycaemia state, may explain the toxic effect of glucotoxicity. Therefore, proteomic analysis of biological samples represents an interesting method to study the effect of chronic hyperglycaemia on protein expression. The discovery of new proteins for which the expression could be modulated by chronic hyperglycaemia may probably help to better understand the mechanisms underlying glucotoxicity. In this review, we will first present an introduction of the different mechanisms known to be involved in the control of glucose homeostasis and in the development of glucotoxicity. In a second part, some proteomic data linked with the effect of glucotoxicity in pancreas, pancreatic islets and β-cells will be presented and discussed.     

The variant hERG/R148W associated with LQTS is a mutation that reduces current density on co-expression with the WT

Background

A variant of the ether-à-go-go related channel (hERG), p.Arg148Trp (R148W) was found at heterozygous state in two infants who died from sudden infant death syndrome (SIDS), one with documented prolonged QTc and Torsade de Pointes (TdP), and in an adult woman with QTc > 500 ms, atrioventricular block and TdP. This variant was previously reported in cases of severe ventricular arrhythmia but very rarely in control subjects. Its classification as mutation or polymorphism awaited electrophysiological characterization.

Methods

The properties of this N-terminal, proximal domain, hERG variant were explored in Xenopus oocytes injected with the same amount of RNA encoding for either hERG/WT or hERG/R148W or their equimolar mixture. The human ventricular cell (TNNP) model was used to test the effects of changes in hERG current.

Results

R148W alone produced a current similar to the WT (369 ± 76 nA (mean ± SEM), n = 13 versus 342 ± 55 nA in WT, n = 13), while the co-expression of 1/2 WT + 1/2 R148W lowered the current by 29% versus WT (243 ± 35 nA, n = 13, p < 0.05). The voltage dependencies of steady-state activation and inactivation were not changed in the variant alone or in co-expression with the WT. The time constants of fast recovery from inactivation and of fast and slow deactivation analyzed between − 120 and + 20 mV were not changed. The voltage-dependent distribution of the current amplitudes among fast-, slow- and non-deactivating fractions was unaltered. A 6.6% increase in APD90 from 323.5 ms to 345 ms was observed using the human cardiac ventricular myocyte model.

Conclusions

Such a decrease in hERG current as evidenced here when co-expressing the hERG/R148W variant with the WT may have predisposed to the observed long QT syndrome and associated TdP. Therefore, the heterozygous carriers of hERG/R148W may be at risk of cardiac sudden death.     

Role of RANKL (TNFSF11)-Dependent Osteopetrosis in the Dental Phenotype of Msx2 Null Mutant Mice

The MSX2 homeoprotein is implicated in all aspects of craniofacial skeletal development. During postnatal growth, MSX2 is expressed in all cells involved in mineralized tissue formation and plays a role in their differentiation and function. Msx2 null (Msx2 ^−/−) mice display complex craniofacial skeleton abnormalities with bone and tooth defects. A moderate form osteopetrotic phenotype is observed, along with decreased expression of RANKL (TNFSF11), the main osteoclast-differentiating factor. In order to elucidate the role of such an osteopetrosis in the Msx2 ^−/− mouse dental phenotype, a bone resorption rescue was performed by mating Msx2 ^−/− mice with a transgenic mouse line overexpressing Rank (Tnfrsf11a). Msx2 ^−/− Rank^Tg mice had significant improvement in the molar phenotype, while incisor epithelium defects were exacerbated in the enamel area, with formation of massive osteolytic tumors. Although compensation for RANKL loss of function could have potential as a therapy for osteopetrosis, but in Msx2 ^−/− mice, this approach via RANK overexpression in monocyte-derived lineages, amplified latent epithelial tumor development in the peculiar continuously growing incisor.