Jürgen Dedio

NOSIP, a novel modulator of endothelial nitric oxide synthase activity

Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+‐calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme;however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two‐hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl‐terminal region of the eNOS oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin‐1 scaffolding domain. NO production was significantly reduced in eNOS‐expressing CHO cells (CHO‐eNOS) that transiently overexpressed NOSIP. Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergentinsoluble to the detergent‐soluble fractions of CHO‐eNOS, and this translocation was completelyprevented by transient coexpression of NOSIP in CHO‐eNOS. Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO‐eNOS that was abolished in the presence of NOSIP. Subcellular fractionation studies identified eNOS in the caveolin‐rich membrane fractions of CHO‐eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments. We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.—Dedio, J., König, P., Wohlfart, P., Schroeder, C., Kummer, W., Muller‐Esterl, W. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J. 15, 79–89 (2001)

NOSTRIN: A protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase

Activity and localization of endothelial nitric oxide synthase (eNOS) is regulated in a remarkably complex fashion, yet the complex molecular machinery mastering stimulus-induced eNOS translocation and trafficking is poorly understood. In a search by the yeast two-hybrid system using the eNOS oxygenase domain as bait, we have identified a previously uncharacterized eNOS-interacting protein, dubbed NOSTRIN (for eNOS traffic inducer). NOSTRIN contains a single polypeptide chain of 506-aa residues of 58 kDa with an N-terminal cdc15 domain and a C-terminal SH3 domain. NOSTRIN mRNA is abundant in highly vascularized tissues such as placenta, kidney, lung, and heart, and NOSTRIN protein is expressed in vascular endothelial cells. Coimmunoprecipitation experiments demonstrated the eNOS–NOSTRIN interaction in vitro and in vivo, and NOSTRINs SH3 domain was essential and sufficient for eNOS binding. NOSTRIN colocalized extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells and in punctate cytosolic structures of CHO-eNOS cells. NOSTRIN overexpression induced a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release. We conclude that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS.

High molecular weight kininogen utilizes heparan sulfate proteoglycans for accumulation on endothelial cells.

Kininogens, the high molecular weight precursor of vasoactive kinins, bind to a wide variety of cells in a specific, reversible, and saturable manner. The cell docking sites have been mapped to domains D3 and D5H of kininogens; however, the corresponding cellular acceptor sites are not fully established. To characterize the major cell binding sites for kininogens exposed by the endothelial cell line EA.hy926, we digested intact cells with trypsin and other proteases and found a time- and concentration-dependent loss of 125I-labeled high molecular weight kininogen (H-kininogen) binding capacity (up to 82%), indicating that proteins are crucially involved in kininogen cell attachment. Cell surface digestion with heparinases similarly reduced kininogen binding capacity (up to 78%), and the combined action of heparinases and trypsin almost eliminated kininogen binding (up to 85%), suggesting that proteoglycans of the heparan sulfate type are intimately involved. Consistently, inhibitors such asp-nitrophenyl-β-d-xylopyranoside and chlorate interfering with heparan sulfate proteoglycan biosynthesis reduced the total number of kininogen binding sites in a time- and concentration-dependent manner (up to 67%). In vitro binding studies demonstrated that biotinylated H-kininogen binds to heparan sulfate glycosaminoglycans via domains D3 and D5H and that the presence of Zn2+ promotes this association. Cloning and over-expression of the major endothelial heparan sulfate-type proteoglycans syndecan-1, syndecan-2, syndecan-4, and glypican in HEK293t cells significantly increased total heparan sulfate at the cell surface and thus the number of kininogen binding sites (up to 3.3-fold). This gain in kininogen binding capacity was completely abolished by treating transfected cells with heparinases. We conclude that heparan sulfate proteoglycans on the surface of endothelial cells provide a platform for the local accumulation of kininogens on the vascular lining. This accumulation may allow the circumscribed release of short-lived kinins from their precursor molecules in close proximity to their sites of action.

Heparin-binding protein targeted to mitochondrial compartments protects endothelial cells from apoptosis

Neutrophil-borne heparin-binding protein (HBP) is a multifunctional protein involved in the progression of inflammation. HBP is stored in neutrophil granules and released upon stimulation of the cells in proximity to endothelial cells. HBP affects endothelial cells in multiple ways; however, the molecular and cellular mechanisms underlying the interaction of HBP with these cells are unknown. Affinity isolation and enzymatic degradation demonstrated that HBP released from human neutrophils binds to endothelial cell-surface proteoglycans, such as syndecans and glypican. Flow cytometry indicated that a significant fraction of proteoglycan-bound HBP is taken up by the endothelial cells, and we used radiolabeled HBP to determine the internalization rate of surface-bound HBP. Confocal and electron microscopy revealed that internalized HBP is targeted to perinuclear compartments of endothelial cells, where it colocalizes with mitochondria. Western blotting of isolated mitochondria from HBP-treated endothelial cells showed that HBP is present in 2 forms - 28 and 22 kDa. Internalized HBP markedly reduced growth factor deprivation-induced caspase-3 activation and protected endothelial cells from apoptosis, suggesting that uptake and intracellular routing of exogenous HBP to mitochondria contributes to the sustained viability of endothelial cells in the context of locally activated neutrophils.

Interleukin-1β stimulates acute phase response and C-reactive protein synthesis by inducing an NFκB- and C/EBPβ-dependent autocrine interleukin-6 loop

Cytokines interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) are involved in acute phase response (APR). C-reactive protein (CRP), the prototype acute phase protein, may represent an important component in the pathogenesis of arteriosclerosis and may also be a target for drug development. Inhibition of CRP synthesis is one potential strategy. Understanding CRP synthesis, however, is a prerequirement for the development of CRP-inhibitors. From studies in hepatoma cell lines, IL-1beta and IL-6 were considered as equal inductors of APR and CRP. We investigated IL-1beta- and IL-6-effects on primary human hepatocytes (PHH) and Hep3B-cells. Kupffer cell contamination in PHH preparations was <3%. In PHH, several APP like CRP, haptoglobin (HP), lipopolysaccharide-binding protein (LBP) or hepcidin (HAMP) were regulated similarly by IL-1beta and IL-6, though signal transduction pathways of these cytokines are different. In Hep3B-cells, APP were regulated exclusively by IL-6. IL-1beta induced IL-6-synthesis in PHH but not in Hep3B-cells. C/EBPbeta-overexpression in Hep3B-cells reconstituted IL-1beta-mediated IL-6/CRP inducibility. In PHH and in C/EBPbeta-overexpressing Hep3B-cells, neutralizing anti-IL-6-antibodies blocked IL-1beta-mediated APR. Inhibition of protein synthesis and NFkappaB-signalling blocked IL-1beta- but not IL-6-mediated CRP-expression in PHH, whereas Janus-Kinase-1-inhibition blocked IL-1beta- and IL-6-mediated APR. IL-1beta induces APR in PHH via an NFkappaB- and C/EBPbeta-dependent autocrine IL-6-loop. These findings partly reconcile the understanding of APR and may help to design a transcriptional suppressor of CRP for the treatment of cardiovascular disease.

Human homolog of mouse tescalcin associates with Na+/H+ exchanger type-1

A novel regulatory protein, tescalcin (TSC), recently isolated from mouse embryonic testes, has been implicated in gonadal differentiation. Employing the yeast two‐hybrid system with the Na+/H+ exchanger type‐1 (NHE1) carboxyterminal domain as a bait we have identified a novel NHE1‐associated protein of 214 amino acid residues representing the human homolog of mouse TSC (96.7% identity). Co‐precipitation experiments demonstrated the interaction of human TSC with NHE1 in vitro and in vivo, and 45Ca2+ overlay assay revealed that TSC binds Ca2+. Immunofluorescence studies indicated that TSC is prominent in cellular lamellipodia where it colocalizes with NHE1. Abundant expression of TSC mRNA in the heart suggests that TSC may play important role(s) in concert with NHE1 in cardiac tissues.

Hypoxia induces production of nitric oxide and reactive oxygen species in glomus cells of rat carotid body

The carotid body is an arterial chemoreceptor organ that senses arterial pO2 and pH. Previous studies have indicated that both reactive oxygen species (ROS) and nitric oxide (NO) are important potential mediators that may be involved in the response of the carotid body to hypoxia. However, whether their production by the chemosensitive elements of the carotid body is indeed oxygen-dependent is currently unclear. Thus, we have investigated their production under normoxic (20% O2) and hypoxic (1% O2) conditions in slice preparations of the rat carotid body by using fluorescent indicators and confocal microscopy. NO-synthesizing enzymes were identified by immunohistochemistry and histochemistry, and the subcellular localization of the NO-sensitive indicator diaminofluorescein was determined by a photoconversion technique and electron microscopy. Glomus cells of the carotid body responded to hypoxia by increases in both ROS and NO production. The hypoxia-induced increase in NO generation required (to a large extent, but not completely) extracellular calcium. Glomus cells were immunoreactive to endothelial NO synthase but not to the neuronal or inducible isoforms. Ultrastructurally, the NO-sensitive indicator was observed in mitochondrial membranes after exposure to hypoxia. The data show that glomus cells respond to exposure to hypoxia by the enhanced production of both ROS and NO. NO production by glomus cells is probably mediated by endothelial NO synthase, which is activated by calcium influx. The presence of NO indicator in mitochondria suggests the hypoxic regulation of mitochondrial function via NO in glomus cells.

Protein Kinase C Pathway Is Involved in Transcriptional Regulation of C-Reactive Protein Synthesis in Human Hepatocytes

Objective— C-Reactive protein (CRP) is the prototype acute phase protein and a cardiovascular risk factor. Interleukin-1&bgr; (IL-1&bgr;) and IL-6 stimulate CRP synthesis in hepatocytes. We searched for additional pathways regulating CRP expression. Methods and Results— Primary human hepatocytes (PHHs) were treated with IL-1&bgr;, IL-6, and protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). CRP was analyzed by quantitative RT-PCR and ELISA. PDBu significantly induced CRP transcription by 21.0±9.24-fold and protein release by 2.9±0.5-fold. Transcriptional regulation was studied in detail in hepatoma G2 (HepG2) cells stably transfected with the 1-kb CRP promoter (HepG2–ABEK14 cells). In these cells, PDBu significantly induced CRP transcription by 5.39±0.66-fold. Competetive inhibition with bisindolylmaleimide derivative LY333531 abolished PDBu-mediated promoter activation. Competetive inhibition with I&kgr;B kinase inhibitor I229 also inhibited PDBu effects. Importantly, IL-8 significantly induced CRP release in PHHs by 58.675±19.1-fold, which was blockable by LY333531. Conclusions— This study describes a novel PKC-dependent transcriptional regulation of CRP gene expression, which, in analogy to the classical IL-1&bgr; and IL-6 pathways, is operational in hepatocytes only. It also identifies IL-8 as a potential physiological PKC activator. HepG2–ABEK14 cells may be useful for high throughput screening to identify inhibitors of CRP synthesis for the prevention of cardiovascular disease.

NOSIP and its interacting protein, eNOS, in the rat trachea and lung.

Endothelial nitric oxide synthase (eNOS), the major nitric oxide (NO)-generating enzyme of the vasculature, is regulated through multiple interactions with proteins, including caveolin-1, Hsp90, Ca2+-calmodulin, and the recently discovered eNOS-interacting protein, NOSIP. Previous studies indicate that NOSIP may contribute to the intricate regulation of eNOS activity and availability. Because eNOS has been shown to be abundantly expressed in the airways, we determined the expression and cellular localization of NOSIP in rat trachea and lung by RT-PCR and immunohistochemistry and examined the interaction of NOSIP with eNOS in lung by coimmunoprecipitation. In tracheal epithelium and lung, NOSIP mRNA expression was prevalent, as shown by RT-PCR, and the corresponding protein interacted with eNOS, as demonstrated by coimmunoprecipitation. Using immunohistochemistry, we found both NOSIP and eNOS immunoreactivity in ciliated epithelial cells of trachea and bronchi, while Clara cells showed immunoreactivity for NOSIP only. NOSIP and eNOS were present in vascular and bronchial smooth muscle cells of large arteries and airways, whereas endothelial cells, as well as bronchiolar and arteriolar smooth muscle cells, exclusively stained for NOSIP. Our results point to functional role(s) of NOSIP in the control of airway and vascular diameter, mucosal secretion, NO synthesis in ciliated epithelium, and, therefore, of mucociliary and bronchial function.