The role of Nogo and the mitochondria-endoplasmic reticulum unit in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is caused by excessive proliferation of vascular cells, which occlude the lumen of pulmonary arteries (PAs) and lead to right ventricular failure. The cause of the vascular remodeling in PAH remains unknown, and the prognosis of PAH remains poor. Abnormal mitochondria in PAH PA smooth muscle cells (SMCs) suppress mitochondria-dependent apoptosis and contribute to the vascular remodeling. We hypothesized that early endoplasmic reticulum (ER) stress, which is associated with clinical triggers of PAH including hypoxia, bone morphogenetic protein receptor II mutations, and HIV/herpes simplex virus infections, explains the mitochondrial abnormalities and has a causal role in PAH. We showed in SMCs from mice that Nogo-B, a regulator of ER structure, was induced by hypoxia in SMCs of the PAs but not the systemic vasculature through activation of the ER stress-sensitive transcription factor ATF6. Nogo-B induction increased the distance between the ER and mitochondria and decreased ER-to-mitochondria phospholipid transfer and intramitochondrial calcium. In addition, we noted inhibition of calcium-sensitive mitochondrial enzymes, increased mitochondrial membrane potential, decreased mitochondrial reactive oxygen species, and decreased mitochondria-dependent apoptosis. Lack of Nogo-B in PASMCs from Nogo-A/B-/- mice prevented these hypoxia-induced changes in vitro and in vivo, resulting in complete resistance to PAH. Nogo-B in the serum and PAs of PAH patients was also increased. Therefore, triggers of PAH may induce Nogo-B, which disrupts the ER-mitochondria unit and suppresses apoptosis. This could rescue PASMCs from death during ER stress but enable the development of PAH through overproliferation. The disruption of the ER-mitochondria unit may be relevant to other diseases in which Nogo is implicated, such as cancer or neurodegeneration.

Endothelial reticulon-4B (Nogo-B) regulates ICAM-1-mediated leukocyte transmigration and acute inflammation.

The reticulon (Rtn) family of proteins are localized primarily to the endoplasmic reticulum (ER) of most cells. The Rtn-4 family, (aka Nogo) consists of 3 splice variants of a common gene called Rtn-4A, Rtn-4B, and Rtn-4C. Recently, we identified the Rtn-4B (Nogo-B) protein in endothelial and smooth muscle cells of the vessel wall, and showed that Nogo-B is a regulator of cell migration in vitro and vascular remodeling and angiogenesis in vivo. However, the role of Nogo-B in inflammation is still largely unknown. In the present study, we use 2 models of inflammation to show that endothelial Nogo-B regulates leukocyte transmigration and intercellular adhesion molecule-1 (ICAM-1)-dependent signaling. Mice lacking Nogo-A/B have a marked reduction in neutrophil and monocyte recruitment to sites of inflammation, while Nogo-A/B(-/-) mice engrafted with wild-type (WT) bone marrow still exhibit impaired inflammation compared with WT mice engrafted with Nogo-A/B(-/-) bone marrow, arguing for a critical role of host Nogo in this response. Using human leukocytes and endothelial cells, we show mechanistically that the silencing of Nogo-B with small interfering RNA (siRNA) impairs the transmigration of neutrophils and reduces ICAM-1-stimulated phosphorylation of vascular endothelial-cell cadherin (VE-cadherin). Our results reveal a novel role of endothelial Nogo-B in basic immune functions and provide a key link in the molecular network governing endothelial-cell regulation of diapedesis.

Epithelial reticulon 4B (Nogo-B) is an endogenous regulator of Th2-driven lung inflammation.

Nogo-B is a member of the reticulon family of proteins (RTN-4B) that is highly expressed in lung tissue; however, its function remains unknown. We show that mice with Th2-driven lung inflammation results in a loss of Nogo expression in airway epithelium and smooth muscle compared with nonallergic mice, a finding which is replicated in severe human asthma. Mice lacking Nogo-A/B (Nogo-KO) display an exaggerated asthma-like phenotype, and epithelial reconstitution of Nogo-B in transgenic mice blunts Th2-mediated lung inflammation. Microarray analysis of lungs from Nogo-KO mice reveals a marked reduction in palate lung and nasal clone (PLUNC) gene expression, and the levels of PLUNC are enhanced in epithelial Nogo-B transgenic mice. Finally, transgenic expression of PLUNC into Nogo-KO mice rescues the enhanced asthmatic-like responsiveness in these KO mice. These data identify Nogo-B as a novel protective gene expressed in lung epithelia, and its expression regulates the levels of the antibacterial antiinflammatory protein PLUNC.

Identification and regulation of reticulon 4B (Nogo-B) in renal tubular epithelial cells.

Nogo-B is a member of the reticulon family of proteins that has been implicated in diverse forms of vascular injury. Although Nogo-B is expressed in renal tissues, its localization and function in the kidney have not been examined. Here, we report that Nogo-B is expressed specifically in the epithelial cells of the distal nephron segments in the murine kidney. After unilateral ureteral obstruction (UUO) and ischemia/reperfusion, Nogo-B gene and protein levels increased dramatically in the kidney. This increase was driven in part by injury-induced de novo expression in proximal tubules. Examination of Nogo-B immunostaining in human biopsy specimens from patients with acute tubular necrosis showed similar increases in Nogo-B in cortical tubules. Mice genetically deficient in Nogo-A/B were indistinguishable from wild-type (WT) mice based on histological appearance and serum analyses. After UUO, there was a significant delay in recruitment of macrophages to the kidney in the Nogo-A/B-deficient mice. However, measurements of fibrosis, inflammatory gene expression, and histological damage were not significantly different from WT mice. Thus, Nogo-B is highly expressed in murine kidneys in response to experimental injuries and may serve as a marker of diverse forms of renal injury in tissues from mice and humans. Furthermore, Nogo-B may regulate macrophage recruitment after UUO, although it does not greatly affect the degree of tissue injury or fibrosis in this model.

Reticulon 4B (Nogo-B) is necessary for macrophage infiltration and tissue repair.

Blood vessel formation during ischemia and wound healing requires coordination of the inflammatory response with genes that regulate blood vessel assembly. Here we show that the reticulon family member 4B, aka Nogo-B, is upregulated in response to ischemia and is necessary for blood flow recovery secondary to ischemia and wound healing. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. Bone marrow-derived macrophages isolated from Nogo knock-out mice have reduced spreading and chemotaxis due to impaired Rac activation. Bone marrow reconstitution experiments show that Nogo in myeloid cells is necessary to promote macrophage homing and functional recovery after limb ischemia. Thus, endogenous Nogo coordinates macrophage-mediated inflammation with arteriogenesis, wound healing, and blood flow control.

In vivo modulation of Nogo-B attenuates neointima formation.

Nogo-B was recently identified as a novel vascular marker; the normally high vascular expression of Nogo-B is rapidly lost following vascular injury. Here we assess the potential therapeutic effects of Ad-Nogo-B delivery to injured vessels in vivo. Nogo-B overexpression following Ad-Ng-B infection of vascular smooth muscle cells (VSMCs) was shown to block proliferation and migration in a dose-dependent manner in vitro. We next assessed the effects of Ad-Ng-B treatment on neointima formation in two in vivo models of acute vascular injury. Adventitial delivery of Ad-Ng-B to wire-injured murine femoral arteries led to a significant decrease in the intimal area [0.014 mm(2) versus 0.030 mm(2) (P = 0.049)] and the intima:media ratio [0.78 versus 1.67 (P = 0.038)] as compared to the effects of Ad-beta-Gal control virus at 21 days after injury. Similarly, lumenal delivery of Ad-Ng-B to porcine saphenous veins prior to carotid artery grafting significantly reduced the intimal area [2.87 mm(2) versus 7.44 mm(2) (P = 0.0007)] and the intima:media ratio [0.32 versus 0.55 (P = 0.0044)] as compared to the effects following the delivery of Ad- beta-Gal, at 28 days after grafting. Intimal VSMC proliferation was significantly reduced in both the murine and porcine disease models. Gene delivery of Nogo-B exerts a positive effect on vascular injury-induced remodeling and reduces neointimal development in two arterial and venous models of vascular injury.

Endothelial cells promote human immunodeficiency virus replication in nondividing memory T cells via Nef-, Vpr-, and T-cell receptor-dependent activation of NFAT.

Human endothelial cells (ECs) enhance human immunodeficiency virus (HIV) replication within CD4(+) memory T cells by 50,000-fold in a Nef-dependent manner. Here, we report that EC-mediated HIV type 1 replication is also dependent on an intact vpr gene. Moreover, we demonstrate that despite a requirement for engaging major histocompatibility complex (MHC) class II molecules and costimulators, EC-stimulated virus-producing cells (p24(high) T cells) do not proliferate, nor are they arrested in the cell cycle. Rather, they are minimally activated, sometimes expressing CD69 but not CD25, HLA-DR, VLA-1, or effector cytokines. Blocking antibodies to interleukin 2 (IL-2), IL-6, IL-7, or tumor necrosis factor do not inhibit viral replication. Cyclosporine effectively inhibits viral replication, as does disruption of the NFAT binding site in the viral long terminal repeat. Furthermore, in the presence of ECs, suboptimal T-cell receptor (TCR) stimulation with phytohemagglutinin L supports efficient viral replication, and suboptimal stimulation with toxic shock syndrome toxin 1 leads to viral replication selectively in the TCR-stimulated, Vbeta2-expressing T cells. Collectively, these data indicate that ECs provide signals that promote Nef- and Vpr-dependent HIV replication in memory T cells that have been minimally activated through their TCRs. Our studies suggest a mechanism for HIV replication in vivo within the reservoir of circulating memory CD4(+) T cells that persist despite antiretroviral therapy and further suggest that maintenance of immunological memory by MHC class II-expressing ECs via TCR signaling may contribute to HIV rebound following cessation of antiretroviral therapy.

Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC).

Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC-expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP-1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino-terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax.

Sulfation of tyrosine 174 in the human C3a receptor is essential for binding of C3a anaphylatoxin.

The complement anaphylatoxin C3a and its cellular seven-transmembrane segment receptor, C3aR, are implicated in a variety of pathological inflammatory processes. C3aR is a G-protein-coupled receptor with an exceptionally large second extracellular loop of 172 amino acids. Previously reported deletion studies have shown that at least part of this region plays a critical role in binding C3a. Our data now demonstrate that five tyrosines in the second extracellular loop of the C3aR are posttranslationally modified by the addition of sulfate. Blocking sulfation by mutation of tyrosine to phenylalanine at positions 184, 188, 317, and/or 318 does not affect ligand binding or signal transduction. However, when tyrosine 174 is mutated to phenylalanine, binding of native C3a is completely blocked. This variant efficiently mobilizes calcium in response to synthetic C3a agonist peptides, but not to native C3a. This finding is consistent with a two-site model of ligand association typical of many peptide ligand-receptor interactions and identifies sulfotyrosine 174 as the critical C3a docking site. Tyrosine sulfation in the amino-terminal extracellular domain has been shown to be important in several other seven-transmembrane segment receptors. Our data now demonstrate that tyrosine sulfate in other extracellular domains can function for ligand interactions as well.

Tyrosine sulfation of human antibodies contributes to recognition of the CCR5 binding region of HIV-1 gp120.

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 infection. Here, we show that a number of human antibodies directed against gp120 are tyrosine sulfated at their antigen binding sites. Like that of CCR5, antibody association with gp120 is dependent on sulfate moieties, enhanced by CD4, and inhibited by sulfated CCR5-derived peptides. Most of these antibodies preferentially associate with gp120 molecules of CCR5-utilizing (R5) isolates and neutralize primary R5 isolates more efficiently than laboratory-adapted isolates. These studies identify a distinct subset of CD4-induced HIV-1 neutralizing antibodies that closely emulate CCR5 and demonstrate that tyrosine sulfation can contribute to the potency and diversity of the human humoral response.

Tyrosine-sulfated peptides functionally reconstitute a CCR5 variant lacking a critical amino-terminal region.

Entry of most primary human immunodeficiency virus, type 1 (HIV-1) isolates into their target cells requires the cellular receptor CD4 and the G protein-coupled chemokine coreceptor CCR5. An acidic, tyrosine-rich, and tyrosine-sulfated domain of the CCR5 amino terminus plays a critical role in the ability of CCR5 to serve as an HIV-1 coreceptor, and tyrosine-sulfated peptides based on this region physically associate with the HIV-1 envelope glycoprotein gp120 and slow HIV-1 entry into CCR5-expressing cells. Here we show that the same tyrosine-sulfated peptides, but not their unsulfated analogs, can restore the HIV-1 coreceptor activity of a CCR5 variant lacking residues 2-17 of its amino terminus. Additionally, these sulfated peptides restored the ability of this CCR5 variant to mobilize calcium in response to the chemokines macrophage inflammatory factors 1alpha and 1beta. These observations show that a tyrosine-sulfated region of the CCR5 amino terminus can function independently to mediate association of chemokines and the HIV-1 envelope glycoprotein with the remaining domains of CCR5.

The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 alpha association and HIV-1 entry.

The chemokine receptor CXCR4 plays critical roles in development, immune function, and human immunodeficiency virus type 1 (HIV-1) entry. Here we demonstrate that, like the CC-chemokine receptors CCR5 and CCR2b, CXCR4 is posttranslationally modified by sulfation of its amino-terminal tyrosines. The sulfate group at tyrosine 21 contributes substantially to the ability of CXCR4 to bind its ligand, stromal derived factor 1 alpha. Tyrosine sulfation plays a less significant role in CXCR4-dependent HIV-1 entry than in CCR5-dependent HIV-1 entry. In some cell lines, CXCR4 is efficiently modified by a chondroitin sulfate chain at serine 18, but neither HIV-1 entry nor stromal derived factor 1 alpha binding was affected by loss of this glycosaminoglycan. These data demonstrate a functional role for tyrosine sulfate in the CXC-chemokine receptor family and underscore a general difference in HIV-1 utilization of CCR5 and CXCR4.