GBF1 and Arf1 interact with Miro and regulate mitochondrial positioning within cells.

The spatial organization of cells depends on coordination between cytoskeletal systems and intracellular organelles. The Arf1 small G protein and its activator GBF1 are important regulators of Golgi organization, maintaining its morphology and function. Here we show that GBF1 and its substrate Arf1 regulate the spatial organization of mitochondria in a microtubule-dependent manner. Miro is a mitochondrial membrane protein that interacts through adaptors with microtubule motor proteins such as cytoplasmic dynein, the major microtubule minus end directed motor. We demonstrate a physical interaction between GBF1 and Miro, and also between the active GTP-bound form of Arf1 and Miro. Inhibition of GBF1, inhibition of Arf1 activation, or overexpression of Miro, caused a collapse of the mitochondrial network towards the centrosome. The change in mitochondrial morphology upon GBF1 inhibition was due to a two-fold increase in the time engaged in retrograde movement compared to control conditions. Electron tomography revealed that GBF1 inhibition also resulted in larger mitochondria with more complex morphology. Miro silencing or drug inhibition of cytoplasmic dynein activity blocked the GBF1-dependent repositioning of mitochondria. Our results show that blocking GBF1 function promotes dynein- and Miro-dependent retrograde mitochondrial transport along microtubules towards the microtubule-organizing center, where they form an interconnected network.

Lipids and Their Trafficking: An Integral Part of Cellular Organization.

An evolutionarily conserved feature of cellular organelles is the distinct phospholipid composition of their bounding membranes, which is essential to their identity and function. Within eukaryotic cells, two major lipid territories can be discerned, one centered on the endoplasmic reticulum and characterized by membranes with lipid packing defects, the other comprising plasma-membrane-derived organelles and characterized by membrane charge. We discuss how this cellular lipid organization is maintained, how lipid flux is regulated, and how perturbations in cellular lipid homeostasis can lead to disease.

Fatty acid metabolism meets organelle dynamics.

Upon nutrient deprivation, cells metabolize fatty acids (FAs) in mitochondria to supply energy, but how FAs, stored as triacylglycerols in lipid droplets, reach mitochondria has been mysterious. Rambold et al. (2015) now show that FA mobilization depends on triacylglycerol lipolysis, whereas autophagy feeds the lipid droplet pool for continued fueling of mitochondria.

Emerging role of the scaffolding protein Dlg1 in vesicle trafficking.

Discs large 1 (Dlg1) is a modular scaffolding protein implicated in the control of cell polarity through assembly of specific multiprotein complexes, including receptors, ion channels and signaling proteins, at specialized zones of the plasma membrane. Recent data have shown that in addition to these well-known interaction partners, Dlg1 may also recruit components of the vesicle trafficking machinery either to the plasma membrane or to transport vesicles. Here, we discuss Dlg1 function in vesicle formation, targeting, tethering and fusion, in both the exocytotic and endocytotic pathways. These pathways contribute to cell functions as major and diverse as glutamatergic activity in the neurons, membrane homeostasis in Schwann cell myelination, insulin stimulation of glucose transport in adipocytes, or endothelial secretion of the hemostatic protein, von Willebrand factor (VWF).

Discs large 1 (Dlg1) scaffolding protein participates with clathrin and adaptator protein complex 1 (AP-1) in forming Weibel-Palade bodies of endothelial cells.

Weibel-Palade bodies (WPBs) are specific cigar-shaped granules that store von Willebrand factor (VWF) for its regulated secretion by endothelial cells. The first steps of the formation of these granules at the trans-Golgi network specifically require VWF aggregation and an external scaffolding complex that contains the adaptator protein complex 1 (AP-1) and clathrin. Discs large 1 (Dlg1) is generally considered to be a modular scaffolding protein implicated in the control of cell polarity in a large variety of cells by specific recruiting of receptors, channels, or signaling proteins to specialized zones of the plasma membrane. We propose here that in endothelial cells, Dlg1, in a complex with AP-1 and clathrin, participates in the biogenesis of WPBs. Supporting data show that Dlg1 colocalizes with microtubules, intermediate filaments, and Golgi markers. Tandem mass spectrometry experiments led to the identification of clathrin as an Dlg1-interacting partner. Interaction was confirmed by in situ proximity ligation assays. Furthermore, AP-1 and VWF immunoprecipitate and colocalize with Dlg1 in the juxtanuclear zone. Finally, Dlg1 depletion by siRNA duplexes disrupts trans-Golgi network morphology and WPB formation. Our results provide the first evidence for an unexpected role of Dlg1 in controlling the formation of specific secretory granules involved in VWF exocytosis in endothelial cells.

Identification of mitogen-activated protein/extracellular signal-responsive kinase kinase 2 as a novel partner of the scaffolding protein human homolog of disc-large.

Human disc-large homolog (hDlg), also known as synapse-associated protein 97, is a scaffold protein, a member of the membrane-associated guanylate kinase family, implicated in neuronal synapses and epithelial-epithelial cell junctions whose expression and function remains poorly characterized in most tissues, particularly in the vasculature. In human vascular tissues, hDlg is highly expressed in smooth muscle cells (VSMCs). Using the yeast two-hybrid system to screen a human aorta cDNA library, we identified mitogen-activated protein/extracellular signal-responsive kinase (ERK) kinase (MEK)2, a member of the ERK cascade, as an hDlg binding partner. Site-directed mutagenesis showed a major involvement of the PSD-95, disc-large, ZO-1 domain-2 of hDlg and the C-terminal sequence RTAV of MEK2 in this interaction. Coimmunoprecipitation assays in both human VSMCs and human embryonic kidney 293 cells, demonstrated that endogenous hDlg physically interacts with MEK2 but not with MEK1. Confocal microscopy suggested a colocalization of the two proteins at the inner layer of the plasma membrane of confluent human embryonic kidney 293 cells, and in a perinuclear area in human VSMCs. Additionally, hDlg also associates with the endoplasmic reticulum and microtubules in these latter cells. Taken together, these findings allow us to hypothesize that hDlg acts as a MEK2-specific scaffold protein for the ERK signaling pathway, and may improve our understanding of how scaffold proteins, such as hDlg, differentially tune MEK1/MEK2 signaling and cell responses.

Decreased number of circulating CD34+KDR+ cells in asymptomatic subjects with preclinical atherosclerosis.

OBJECTIVES: To assess whether circulating endothelial progenitor cells (CEPCs) can be considered as a cardiovascular risk marker before event has occurred, that is less firmly established than in clinically overt atherosclerosis. METHODS: Number of CD34+KDR+ cell number per ml blood was measured by flow cytometry in 84 untreated subjects without cardiovascular disease. Atherosclerotic plaque was detected by ultrasound in carotid, abdominal aortic and femoral sites and the number of sites affected by plaque among these three sites was counted as 0, 1, 2 or 3. Additionally, intima-media thickness (IMT) was measured by computerized ultrasound imaging of both common carotid segments. RESULTS: CD34+KDR+ cell number decreased by 48, 29 or 30% in the presence of carotid, aortic or femoral plaque (p<0.001, 0.05, 0.05, respectively) as compared to the absence of plaque and by 70% in the presence of three sites affected with plaque as compared with 0 site with plaque (p<0.01) but did not change with increasing IMT tertiles. Adjustment for Framingham risk score, that was also associated with decreased CD34+KDR+ cell number (p<0.001), made CD34+KDR+ cell number associations with plaque insignificant, except at the carotid site (p<0.01). CONCLUSIONS: Reduced CEPC number may participate to preclinical stage of atherosclerosis and provide additional information to traditional risk factors as regards global risk assessment.

Mitochondrial arginase II modulates nitric-oxide synthesis through nonfreely exchangeable L-arginine pools in human endothelial cells.

Reduced synthesis of nitric oxide (NO) contributes to the endothelial dysfunction and may be related to limited availability of L-arginine, the common substrate of constitutive nitric-oxide synthase (NOS) and cytosolic arginase I and mitochondrial arginase II. To determine whether arginases modulate the endothelial NO synthesis, we investigated the effects of the competitive arginase inhibitor N(omega)-hydroxy-nor-L-arginine (Nor-NOHA) on the activity of NOS, arginases, and L-arginine transporter and on NO release at surface of human umbilical vein endothelial cells (HUVECs). In unstimulated cells, Nor-NOHA dose-dependently reduced the arginase activity with maximal inhibition at 20 microM. When HUVECs were stimulated by thrombin without extracellular L-arginine, Nor-NOHA dose-dependently increased the NOS activity and the NO release with maximal effects at 20 microM. Extracellular L-arginine also dose-dependently increased NO release and arginase activity. When HUVECs were stimulated by thrombin in the presence of 100 microM L-arginine, NOS activity and NO release were similar in untreated and Nor-NOHA-treated cells. However, despite activation of L-arginine uptake, the inhibition of arginase activity by Nor-NOHA was still significant. The depletion of freely exchangeable L-arginine pools with extracellular L-lysine did not prevent Nor-NOHA from increasing the NO release. This indicates the presence of pools, which are accessible to NOS and arginase, but not exchangeable. Interestingly, the mitochondrial arginase II was constitutively expressed, whereas the cytosolic arginase I was barely detectable in HUVECs. These data suggest that endothelial NO synthesis depends on the activity of arginase II in mitochondria and l-arginine carriers in cell membrane.

Endothelial thrombomodulin induces Ca2+ signals and nitric oxide synthesis through epidermal growth factor receptor kinase and calmodulin kinase II.

Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca2+ signals with rapid phosphorylation of PLC(beta3) and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca2+ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase Cgamma1 and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca2+ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.

Cholinesterase activity in human pulmonary arteries and veins: correlation with mRNA levels.

Isolated intact human pulmonary arteries and veins were used to determine the acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) activities in the absence or presence of two selective cholinesterase (ChE) inhibitors, iso-OMPA or BW284c51, respectively. These results were compared with the mRNA levels for each enzyme in human pulmonary vessels. Total ChE activities measured in presence of acetylthiocholine (ACTI, 1 mM) in intact vascular preparations were 45+/-04 and 114+/-07 mU/g tissue in human pulmonary arteries (n=14) and veins (n=14), respectively. These activities were completely abolished in presence of 10 microM neostigmine. In both types of vessels AChE and BChE activities were observed. These activities were at least 2-fold higher in human pulmonary veins when compared with arteries and were correlated with the accumulation of the corresponding transcripts (n=8). In each type of vessel, similar total ChE activities were detected in homogenized and intact preparations, while in human bronchial preparations this activity was 5-fold higher in homogenates than in intact preparations. Together these results provide evidence that the ChE activities in human pulmonary vessels may be extracellular and that the higher activity measured in veins as compared to arteries was associated with the differential accumulation of the corresponding transcripts.

Prostacyclin release and receptor activation: differential control of human pulmonary venous and arterial tone.

1. In human pulmonary vascular preparations, precontracted arteries were more sensitive to the relaxant effect of acetylcholine (ACh) than veins (pD(2) values: 7.25+/-0.08 (n=23) and 5.92+/-0.09 (n=25), respectively). Therefore, the role of prostacyclin (PGI(2)) was explored to examine whether this mediator may be responsible for the difference in relaxation. 2. In the presence of the cyclooxygenase (COX) inhibitor, indomethacin (INDO), the ACh relaxations were reduced in arteries but not in veins. On the contrary, an inhibitor (l-NOARG) of the nitric oxide synthase blocked preferentially the relaxation in veins. 3. A greater release of 6-keto-PGF(1alpha), the stable metabolite of PGI(2), was observed in arterial preparations than in venous preparations when stimulated with either ACh or arachidonic acid (AA). 4. Exogenous PGI(2) produced a reduced relaxant effect in the precontracted vein when compared with the artery. In the presence of the EP(1)-receptor antagonist AH6809, the PGI(2) relaxation of veins was similar to arteries. 5. In veins, AA (0.1 mm) produced a biphasic response, namely, a contraction peak (0.4-0.5 g) followed by a relaxation. These contractions in venous preparations were abolished either in the absence of endothelium or in the presence of INDO or an EP(1)-receptor antagonist (AH6809, SC19220). In the arterial preparations AA induced only relaxations. 6. In both vascular preparations, COX-1 but not the COX-2 protein was detected in microsomal preparations derived from homogenized tissues or freshly isolated endothelial cells. 7. The differential vasorelaxations induced by ACh may be explained, in part, by a more pronounced production and release of PGI(2) in human pulmonary arteries than in the veins. In addition, while PGI(2) induced relaxation by activation of IP-receptors in both types of vessels, a PGI(2) constrictor effect was responsible for masking the relaxation in the veins by activation of the EP(1)-receptor.

Prostaglandin (PG) FP and EP1 receptors mediate PGF2alpha and PGE2 regulation of interleukin-1beta expression in Leydig cell progenitors.

Prostaglandins (PG) mediate IL-1beta regulation of several interleukin mRNAs in progenitor Leydig cells. PGE(2) and PGF(2alpha) potently reverse indomethacin (INDO; a cyclooxygenase inhibitor) inhibition of IL-1beta autoinduction. IL-1beta increases PGE(2) and PGF(2alpha) production. To determine the PG receptors involved in this regulation, this study established by RT-PCR and Western analyses which specific receptors for PGE(2) (EP receptors) and PGF(2alpha) (FP receptors) are expressed in progenitors. Pharmacological characterization of receptors involved in PGE(2) and PGF(2alpha) regulation of IL-1beta mRNA levels was ascertained using real-time PCR analyses. FP, EP(1), EP(2), and EP(4) receptor mRNAs and proteins, and an EP(3) receptor subtype were detected. IL-1beta treatment (24-h) significantly decreased EP(1) receptor levels; INDO abrogated this down-regulation. FP, EP(2), and EP(4) receptor levels increased after IL-1beta and IL-1beta + INDO. A selective FP agonist, cloprostenol (0.1 micro M), and PGF(2alpha) (10 micro M) had similar effects on IL-1beta mRNA levels in progenitors treated with IL-1beta + INDO. None of the EP(2)/EP(4) agonists [butaprost, misoprostol, or 11-deoxy PGE(1) (10 micro M)] affected IL-1beta mRNA levels. In contrast, EP(1)/EP(3) agonists (17-phenyl trinor PGE(2) and sulprostone) increased IL-1beta mRNAs in a dose-dependent manner. EP(1) receptor subtype-selective antagonist, SC-51322, blocked IL-1beta-induced and [IL-1beta + INDO + 17-phenyl trinor PGE(2)]-induced increases in IL-1beta mRNAs. Taken together, our data demonstrate that FP and EP(1) receptors mediate PGF(2alpha) and PGE(2) induction of progenitor IL-1beta expression.

Pharmacological evidence for a novel cysteinyl-leukotriene receptor subtype in human pulmonary artery smooth muscle.

1. To characterize the cysteinyl-leukotriene receptors (CysLT receptors) in isolated human pulmonary arteries, ring preparations were contracted with leukotriene C(4) (LTC(4)) and leukotriene D(4) (LTD(4)) in either the absence or presence of the selective CysLT(1) receptor antagonists, ICI 198615, MK 571 or the dual CysLT(1)/CysLT(2) receptor antagonist, BAY u9773. 2. Since the contractions induced by the cysteinyl-leukotrienes (cysLTs) in intact preparations failed to attain a plateau response over the concentration range studied, the endothelium was removed and the tissue treated continuously with indomethacin (Rubbed+INDO). In these latter preparations, the pEC(50) for LTC(4) and LTD(4) were not significantly different (7.61+/-0.07, n=20 and 7.96+/-0.09, n=22, respectively). However, the LTC(4) and LTD(4) contractions were markedly potentiated when compared with data from intact tissues. 3. Leukotriene E(4) (LTE(4)) did not contract human isolated pulmonary arterial preparations. In addition, treatment of preparations with LTE(4) (1 microM; 30 min) did not modify either the LTC(4) or LTD(4) contractions. 4. Treatment of preparations with the S-conjugated glutathione (S-hexyl-GSH; 100 microM, 30 min), an inhibitor of the metabolism of LTC(4) to LTD(4), did not modify LTC(4) contractions. 5. The pEC(50) values for LTC(4) were significantly reduced by treatment of the preparations with either ICI 198615, MK 571 or BAY u9773 and the pK(B) values were: 7.20, 7.02 and 6.26, respectively. In contrast, these antagonists did not modify the LTD(4) pEC(50) values. 6. These findings suggest the presence of two CysLT receptors on human pulmonary arterial vascular smooth muscle. A CysLT(1) receptor with a low affinity for CysLT(1) antagonists and a novel CysLT receptor subtype, both responsible for vasoconstriction. Activation of this latter receptor by LTC(4) and LTD(4) induced a contractile response which was resistant to the selective CysLT(1) antagonists (ICI 198615 and MK 571) as well as the non-selective (CysLT(1)/CysLT(2)) antagonist, BAY u9773.

Cyclooxygenase 2 pathway mediates IL-1beta regulation of IL-1alpha, -1beta, and IL-6 mRNA levels in Leydig cell progenitors.

Prostanoids are arachidonic acid (AA) metabolites derived from the cyclooxygenase (COX1 and COX2 isozymes) pathway and are involved in signal transduction pathways activated by distinct ILs. Although COX1 is the constitutive isoform of COX, IL-1beta is a potent inducer of COX2 expression in distinct cell types. This study was designed to determine whether cyclooxygenases could mediate endogenous cytokine regulation in rat progenitor Leydig cells. COX and IL (IL-1alpha, IL-1beta, and IL-6) mRNAs were measured by PCR and real-time PCR analyses, respectively. COX function was assessed using COX activity inhibitors: indomethacin (INDO; COX1 and COX2 inhibitor) and NS-398 (COX2 selective inhibitor). Our data indicate that endogenous progenitor COX1 mRNA levels are low and are not regulated by IL-1beta. In contrast, COX2 mRNA is induced by IL-1beta at 6, 9, and 24 h. IL-1beta induction of IL mRNAs was in part significantly impaired in the presence of INDO or NS-398. Among the prostanoids tested, prostaglandin E(2) (PGE(2)), PGF(2alpha), and carbaprostacyclin reversed the INDO inhibition of IL production. PGs alone have no (IL-1alpha and IL-1beta) or a modest (IL-6) effect on IL mRNA levels. PGE(2), PGF(2alpha), and PGI(2) measurements show that IL-1beta treatment significantly increases progenitor Leydig cell production of these PGs. Taken together, our data demonstrate that this COX2 cascade is a regulator of cytokines in Leydig progenitors.