ABSTRACT
BACKGROUND & AIMS: Increased intrahepatic resistance in cirrhosis is associated with reduced endothelial NO synthase (eNOS) activity and exacerbated by superimposed inflammation. NOSTRIN induces intracellular translocation of eNOS and reduces NO generation. Our aims were to quantify and compare hepatic expression of eNOS, NOSTRIN, NOSIP, and caveolin-1 in alcoholic cirrhosis with or without superimposed alcoholic hepatitis and in normal livers. METHODS: Biopsy specimens from 20 decompensated alcoholic cirrhotic patients with portal hypertension (10 with alcoholic hepatitis) and 6 normal livers were analyzed: real-time polymerase chain reaction for quantification of messenger RNA; Western blotting; and enzyme assays of eNOS in normal and diseased liver were performed. Localization and interaction of eNOS and NOSTRIN in liver was assessed by immunohistochemistry and co-immunoprecipitation. RESULTS: eNOS mRNA was significantly increased and eNOS activity decreased in alcoholic hepatitis patients, despite no differences in eNOS protein expression among the patients. Patients with alcoholic hepatitis had significantly higher hepatic levels of NOSTRIN and caveolin-1 mRNA compared with cirrhosis alone or normal biopsy specimens. A NOSTRIN splice variant, not present in normal tissue, was detected on mRNA and protein levels in all alcoholic patients. Coimmunoprecipitation demonstrated association among NOSTRIN, eNOS, and caveolin-1. CONCLUSIONS: An increase in mRNA and protein of NOSTRIN and its shortened variant in alcoholic hepatitis may partly account for the paradox of increased mRNA levels and normal protein expression but decreased enzymatic activity of eNOS in diseased liver. Such intracellular regulators of NO production may be important in the development of increased intrahepatic resistance in alcoholic hepatitis patients.
Subject(s)
Gene Expression , Genetic Variation , Hepatitis, Alcoholic/genetics , Hepatitis, Alcoholic/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Liver/metabolism , Adaptor Proteins, Signal Transducing , Animals , CHO Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caveolin 1/genetics , Caveolin 1/metabolism , Cell Line, Tumor , Cricetinae , Cricetulus , DNA-Binding Proteins , Female , Humans , Liver Cirrhosis/metabolism , Male , Middle Aged , Nitric Oxide Synthase Type III/genetics , Nitric Oxide Synthase Type III/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Tissue Distribution , Ubiquitin-Protein LigasesABSTRACT
Recently, we characterized a novel endothelial nitric-oxide synthase (eNOS)-interacting protein, NOSTRIN (for eNOS-trafficking inducer), which decreases eNOS activity upon overexpression and induces translocation of eNOS away from the plasma membrane. Here, we show that NOSTRIN directly binds to caveolin-1, a well-established inhibitor of eNOS. Because this interaction occurs between the N terminus of caveolin (positions 1-61) and the central domain of NOSTRIN (positions 323-434), it allows for independent binding of each of the two proteins to eNOS. Consistently, we were able to demonstrate the existence of a ternary complex of NOSTRIN, eNOS, and caveolin-1 in Chinese hamster ovary (CHO)-eNOS cells. In human umbilical vein endothelial cells (HUVECs), the ternary complex assembles at the plasma membrane upon confluence or thrombin stimulation. In CHO-eNOS cells, NOSTRIN-mediated translocation of eNOS involves caveolin in a process most likely representing caveolar trafficking. Accordingly, trafficking of NOSTRIN/eNOS/caveolin is affected by altering the state of actin filaments or cholesterol levels in the plasma membrane. During caveolar trafficking, NOSTRIN functions as an adaptor to recruit mediators such as dynamin-2 essential for membrane fission. We propose that a ternary complex between NOSTRIN, caveolin-1, and eNOS mediates translocation of eNOS, with important implications for the activity and availability of eNOS in the cell.
Subject(s)
Caveolin 1/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Multiprotein Complexes/metabolism , Nitric Oxide Synthase Type III/metabolism , Actins/metabolism , Adaptor Proteins, Signal Transducing , Animals , CHO Cells , Cell Membrane/metabolism , Cells, Cultured , Cholesterol/metabolism , Cricetinae , Cytoskeleton/metabolism , DNA-Binding Proteins , Dynamins/metabolism , Endothelial Cells/cytology , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Mice , NIH 3T3 Cells , Protein Binding , Protein TransportABSTRACT
NOSTRIN, an NO synthase binding protein, belongs to the PCH family of proteins, exposing a typical domain structure. While its SH3 domain and the C-terminal coiled-coil region cc2 have been studied earlier, the function of the N-terminal half comprising a Cdc15 domain with an FCH (Fes/CIP homology) region followed by a coiled-coil stretch cc1 is unknown. Here, we show that the FCH region is necessary and sufficient for membrane association of NOSTRIN, whereas the Cdc15 domain further specifies subcellular distribution of the protein. Thus, the FCH region and the Cdc15 domain fulfill complementary functions in subcellular targeting of NOSTRIN.
Subject(s)
Cell Cycle Proteins/metabolism , GTP-Binding Proteins/metabolism , Nitric Oxide Synthase/metabolism , Adaptor Proteins, Signal Transducing , Cell Cycle Proteins/genetics , DNA-Binding Proteins , GTP-Binding Proteins/genetics , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Nitric Oxide Synthase/genetics , Protein Transport/physiology , src Homology Domains/geneticsABSTRACT
Intracellular trafficking of endothelial nitric oxide synthase (eNOS) between different compartments is incompletely understood. Recently, we described a novel eNOS-interacting protein, NOSTRIN, which upon overexpression drives eNOS away from the plasma membrane towards intracellular compartments. Sequence similarity of NOSTRIN and pacsins/syndapins suggested a role for NOSTRIN in endocytosis. Accordingly, we show here that NOSTRIN interacts with the large GTPase dynamin and the actin nucleation promoting factor N-WASP by means of its SH3 domain, which also represents the docking site for eNOS. Via a coiled-coil region in the C-terminal portion of the protein, NOSTRIN oligomerizes, mainly forming trimers, which would allow simultaneous interaction with multiple binding partners of the SH3 domain. Consistent with this notion, expression of dynamin-2-GFP in CHO cells stably expressing eNOS (CHO-eNOS) results in recruitment of eNOS to dynamin-positive structures, only when NOSTRIN is present as well. Similarly, when N-WASP-GFP and NOSTRIN are co-expressed in CHO-eNOS cells, both proteins strongly co-localize with eNOS and are recruited to structures running along actin filaments. If, however, the actin cytoskeleton is depolymerized by cytochalasin D, NOSTRIN and eNOS are associated with extended structures in the cell periphery, possibly being unable to leave the plasma membrane. Together, these results indicate that NOSTRIN may facilitate endocytosis of eNOS by coordinating the function of dynamin and N-WASP.
Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/physiology , Endocytosis/physiology , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase/physiology , Actins/metabolism , Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport/metabolism , Animals , CHO Cells , COS Cells , Cell Cycle Proteins/chemistry , Chlorocebus aethiops , Cricetinae , DNA-Binding Proteins , Dynamins/metabolism , GTP-Binding Proteins/chemistry , Humans , Intracellular Signaling Peptides and Proteins , Multigene Family , Nitric Oxide Synthase/metabolism , Protein Binding/physiology , Protein Processing, Post-Translational , Protein Transport/physiology , Schizosaccharomyces pombe Proteins/chemistry , Sequence Homology, Amino Acid , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , src Homology Domains/physiologyABSTRACT
Mechanisms governing the activity of neuronal nitric oxide synthase (nNOS), the major source of nitric oxide (NO) in the nervous system, are not completely understood. We report here a protein-protein interaction between nNOS and NOSIP (nitric oxide synthase-interacting protein) in rat brain in vivo. NOSIP and nNOS are concentrated in neuronal synapses and demonstrate significant colocalization in various regions of the central and peripheral nervous systems. NOSIP produces a significant reduction in nNOS activity in a neuroepithelioma cell line stably expressing nNOS. Furthermore, overexpression of NOSIP in cultured primary neurons reduces the availability of nNOS in terminal dendrites. These results thus suggest that the interaction between NOSIP and nNOS is functionally involved in endogenous mechanisms regulating NO synthesis. Furthermore, we found that the subcellular distribution and expression levels of NOSIP are dynamically regulated by neuronal activity in vitro as well as in vivo, suggesting that NOSIP may contribute to a mechanism via which neuronal activity regulates the synaptic availability and activity of nNOS.
