NINJ1 mediates plasma membrane rupture by cutting and releasing membrane disks.

The membrane protein NINJ1 mediates plasma membrane rupture in pyroptosis and other lytic cell death pathways. Here, we report the cryo-EM structure of a NINJ1 oligomer segmented from NINJ1 rings. Each NINJ1 subunit comprises amphipathic (⍺1, ⍺2) and transmembrane (TM) helices (⍺3, ⍺4) and forms a chain of subunits, mainly by the TM helices and ⍺1. ⍺3 and ⍺4 are kinked, and the Gly residues are important for function. The NINJ1 oligomer possesses a concave hydrophobic side that should face the membrane and a convex hydrophilic side formed by ⍺1 and ⍺2, presumably upon activation. This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. Live-cell and super-resolution imaging uncover ring-like structures on the plasma membrane that are released into the culture supernatant. Released NINJ1 encircles a membrane inside, as shown by lipid staining. Therefore, NINJ1-mediated membrane disk formation is different from gasdermin-mediated pore formation, resulting in membrane loss and plasma membrane rupture.

NINJ1 induces plasma membrane rupture and release of damage-associated molecular pattern molecules during ferroptosis.

Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent accumulation of oxidized phospholipids in cellular membranes, culminating in plasma membrane rupture (PMR) and cell lysis. PMR is also a hallmark of other types of programmed necrosis, such as pyroptosis and necroptosis, where it is initiated by dedicated pore-forming cell death-executing factors. However, whether ferroptosis-associated PMR is also actively executed by proteins or driven by osmotic pressure remains unknown. Here, we investigate a potential ferroptosis role of ninjurin-1 (NINJ1), a recently identified executor of pyroptosis-associated PMR. We report that NINJ1 oligomerizes during ferroptosis, and that Ninj1-deficiency protects macrophages and fibroblasts from ferroptosis-associated PMR. Mechanistically, we find that NINJ1 is dispensable for the initial steps of ferroptosis, such as lipid peroxidation, channel-mediated calcium influx, and cell swelling. In contrast, NINJ1 is required for early loss of plasma membrane integrity, which precedes complete PMR. Furthermore, NINJ1 mediates the release of cytosolic proteins and danger-associated molecular pattern (DAMP) molecules from ferroptotic cells, suggesting that targeting NINJ1 could be a therapeutic option to reduce ferroptosis-associated inflammation.

Deficiency of Ninjurin1 attenuates LPS/D-galactosamine-induced acute liver failure by reducing TNF-α-induced apoptosis in hepatocytes.

Nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a membrane protein that mediates cell adhesion. The role of Ninj1 during inflammatory response has been widely investigated in macrophages and endothelial cells. Ninj1 is expressed in various tissues, and the liver also expresses high levels of Ninj1. Although the hepatic upregulation of Ninj1 has been reported in human hepatocellular carcinoma and septic mice, little is known of its function during the pathogenesis of liver diseases. In the present study, the role of Ninj1 in liver inflammation was explored using lipopolysaccharide (LPS)/D-galactosamine (D-gal)-induced acute liver failure (ALF) model. When treated with LPS/D-gal, conventional Ninj1 knock-out (KO) mice exhibited a mild inflammatory phenotype as compared with wild-type (WT) mice. Unexpectedly, myeloid-specific Ninj1 KO mice showed no attenuation of LPS/D-gal-induced liver injury. Whereas, Ninj1 KO primary hepatocytes were relatively insensitive to TNF-α-induced caspase activation as compared with WT primary hepatocytes. Also, Ninj1 knock-down in L929 and AML12 cells and Ninj1 KO in HepG2 cells ameliorated TNF-α-mediated apoptosis. Consistent with in vitro results, hepatocyte-specific ablation of Ninj1 in mice alleviated LPS/D-gal-induced ALF. Summarizing, our in vivo and in vitro studies show that lack of Ninj1 in hepatocytes diminishes LPS/D-gal-induced ALF by alleviating TNF-α/TNFR1-induced cell death.

The Role of Ninjurin1 and Its Impact beyond the Nervous System.

Ninjurin1 (Ninj1) is a double-transmembrane cell surface protein that could promote nerve regeneration in the process of the peripheral nervous system injury and repairment. Nonetheless, the accurate function of Ninj1 in the central nervous system and outside the nervous system is not completely clear. According to the recent studies, we found that Ninj1 is also aberrantly expressed in various pathophysiological processes in vivo, including inflammation, tumorigenesis, and vascular, bone, and muscle homeostasis. These findings suggest that Ninj1 may play an influential role during these pathophysiological processes. Our review summarizes the diverse roles of Ninj1 in multiple pathophysiological processes inside and outside the nervous system. Ninj1 should be considered as an important and novel therapeutic target in certain diseases, such as inflammatory diseases and ischemic diseases. Our study provided a better understanding of Ninj1 in different pathophysiological processes and thereby provided the theoretical support for further research.

Ninjurin 1 has two opposing functions in tumorigenesis in a p53-dependent manner.

WT p53 is critical for tumor suppression, whereas mutant p53 promotes tumor progression. Nerve injury-induced protein 1 (Ninj1) is a target of p53 and forms a feedback loop with p53 by repressing p53 mRNA translation. Here, we show that loss of Ninj1 increased mutant p53 expression and, subsequently, enhanced cell growth and migration in cells carrying a mutant p53. In contrast, loss of Ninj1 inhibited cell growth and migration in cells carrying a WT p53. To explore the biological significance of Ninj1, we generated a cohort of Ninj1-deficient mice and found that Ninj1+/- mice were prone to systemic inflammation and insulitis, but not to spontaneous tumors. We also found that loss of Ninj1 altered the tumor susceptibility in both mutant p53 and p53-null background. Specifically, in a mutant p53(R270H) background, Ninj1 deficiency shortened the lifespan, altered the tumor spectrum, and increased tumor burden, likely via enhanced expression of mutant p53. In a p53-null background, Ninj1 deficiency significantly increased the incidence of T-lymphoblastic lymphoma. Taken together, our data suggest that depending on p53 genetic status, Ninj1 has two opposing functions in tumorigenesis and that the Ninj1-p53 loop may be targeted to manage inflammatory diseases and cancer.

Detrimental Role of Nerve Injury-Induced Protein 1 in Myeloid Cells under Intestinal Inflammatory Conditions.

Nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a cell-surface adhesion molecule that regulates cell migration and attachment. This study demonstrates the increase in Ninj1 protein expression during development of intestinal inflammation. Ninj1-deficient mice exhibited significantly attenuated bodyweight loss, shortening of colon length, intestinal inflammation, and lesser pathological lesions than wild-type mice. Although more severe inflammation and serious lesions are observed in wild-type mice than Ninj1-deficient mice, there were no changes in the numbers of infiltrating macrophages in the inflamed tissues obtained from WT and Ninj1-deficient mice. Ninj1 expression results in activation of macrophages, and these activated macrophages secrete more cytokines and chemokines than Ninj1-deficient macrophages. Moreover, mice with conditional deletion of Ninj1 in myeloid cells (Ninj1fl/fl; Lyz-Cre+) alleviated experimental colitis compared with wild-type mice. In summary, we propose that the Ninj1 in myeloid cells play a pivotal function in intestinal inflammatory conditions.

Ninjurin 1 mediates peripheral nerve regeneration through Schwann cell maturation of NG2-positive cells.

Ninjurin 1 (Ninj1) is identified as a peripheral nerve injury-induced protein. However, the role of Ninj1 in nerve regeneration is unclear. Schwann cells (SCs) and microvasculature are critical for peripheral nerve regeneration. SCs precursors and microvascular pericytes (PCs), which are nerve/glial antigen 2 (NG2)-positive cells are observed in peripheral nervous system. In this study, we investigated the role of Ninj1 in peripheral nerve regeneration using NG2+cell-specific inducible deletion of Ninj1 mouse model. The number of NG2+cells, which were associated with and without microvessels was increased after sciatic nerve crush injury. There was a significant increase in the expression of Ninj1 and EphA7 in the injured nerve tissue. This increase was mostly observed in NG2+cells. Genetic tracing of NG2+cells was performed using tamoxifen (Tam) treatment on NG2CreERT:R26R-tdTomato mice. The sciatic nerve was injured following the Tam-treatment, then tdTomato-expressing SCs were mostly observed in regenerated SCs at 21 days after nerve injury. Ninj1 gene knockout (Ninj1 KO) in NG2+cells was induced using NG2CreERT:Ninj1loxp mice. Tam-treated-NG2CreERT or Tam-nontreated NG2CreERT:Ninj1loxp mice were used as controls. Following Tam-treatment, the sciatic nerve in each group was injured. Ninj1KO significantly attenuated the expression of the myelin binding protein (MBP) as well as the number of myelinated axons. The expression of MBP in cultured SCs was significantly reduced by SiRNA-mediated Ninj1 knockdown (KD). Ninj1KD also attenuated the differentiation of SCs by isolated EphA7+multipotent PCs. The current data indicate that Ninj1 plays a vital role in peripheral nerve regeneration. This is observed particularly in the myelination process of NG2+cells including SCs precursors and multipotent PCs.

Ninjurin1 Assembles Into a Homomeric Protein Complex Maintained by N-linked Glycosylation.

Ninjurin1 (Ninj1) is a cell surface protein known as a homophilic adhesion molecule. Previous studies have shown a trans-interaction of Ninj1 between immune cells and endothelial cells; however, little is known about Ninj1 modification and structure in the cis-interaction. We showed that Ninj1 assembles into a homomeric complex via a cis-interaction mediated by the intracellular region and N-glycosylation at Asn60 . We identified cis-interaction between Ninj1 proteins using CFP- and YFP-tagged Ninj1 by Förster resonance energy transfer using a confocal microscope and fluorescence-activated cell sorter. We further observed the Ninj1 homomeric complexes composed of two to six monomeric Ninj1 molecules by a formaldehyde cross-linking assay. Co-immunoprecipitation assays with epitope-tagged truncated Ninj1 suggested that the intracellular region encompassing Leu101 -Ala110 participates in Ninj1 homomer assembly. Ninj1 N-glycosylation was characterized by treatment of tunicamycin and substitution of Asn to Gln or Ala. Fluorescence-activated cell sorting-based Förster resonance energy transfer assays further demonstrated that N-glycosylation is indispensable for the Ninj1 cis-interaction, and a formaldehyde cross-linking assay confirmed that interruption of N-glycosylation by Asn substitution disrupted Ninj1 homomeric complex formation. In silico analysis revealed that Ninj1 is highly conserved in vertebrates and that the conserved sequence contains an N-glycosylation motif and cis-interacting intracellular region, which participate in Ninj1 homomer assembly. Taken together, these data show that Ninj1 assembles into a homomeric protein complex and that N-glycosylation is a prerequisite for Ninj1 homomer assembly. J. Cell. Biochem. 118: 2219-2230, 2017. © 2017 Wiley Periodicals, Inc.

Ninjurin1 deficiency attenuates susceptibility of experimental autoimmune encephalomyelitis in mice.

Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, in vivo gene deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knock-out (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis. Ninjurin1 KO mice attenuated EAE susceptibility by reducing leukocyte recruitment into the injury regions of the spinal cord and showed less adhesion of leukocytes on inflamed retinal vessels in endotoxin-induced uveitis mice. Moreover, the administration of a custom-made antibody (Ab26-37) targeting the Ninjurin1 binding domain ameliorated the EAE symptoms, showing the contribution of its adhesion activity to leukocyte trafficking. In addition, we addressed the transendothelial migration (TEM) activity of bone marrow-derived macrophages and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO bone marrow-derived macrophages and siNinj1 Raw264.7 cells. Consistent with this, GFP-tagged mNinj1-overexpressing Raw264.7 cells increased their TEM activity. Taken together, we have clarified the contribution of Ninjurin1 to leukocyte trafficking in vivo and delineated its direct functions to TEM, emphasizing Ninjurin1 as a beneficial therapeutic target against inflammatory diseases such as multiple sclerosis.

Ninjurin1 enhances the basal motility and transendothelial migration of immune cells by inducing protrusive membrane dynamics.

Ninjurin1 is involved in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, by mediating leukocyte extravasation, a process that depends on homotypic binding. However, the precise regulatory mechanisms of Ninjurin1 during inflammation are largely undefined. We therefore examined the pro-migratory function of Ninjurin1 and its regulatory mechanisms in macrophages. Interestingly, Ninjurin1-deficient bone marrow-derived macrophages exhibited reduced membrane protrusion formation and dynamics, resulting in the impairment of cell motility. Furthermore, exogenous Ninjurin1 was distributed at the membrane of filopodial structures in Raw264.7 macrophage cells. In Raw264.7 cells, RNA interference of Ninjurin1 reduced the number of filopodial projections, whereas overexpression of Ninjurin1 facilitated their formation and thus promoted cell motility. Ninjurin1-induced filopodial protrusion formation required the activation of Rac1. In Raw264.7 cells penetrating an MBEC4 endothelial cell monolayer, Ninjurin1 was localized to the membrane of protrusions and promoted their formation, suggesting that Ninjurin1-induced protrusive activity contributed to transendothelial migration. Taking these data together, we conclude that Ninjurin1 enhances macrophage motility and consequent extravasation of immune cells through the regulation of protrusive membrane dynamics. We expect these findings to provide insight into the understanding of immune responses mediated by Ninjurin1.

Dynamics of 1α,25-dihydroxyvitamin D3-dependent chromatin accessibility of early vitamin D receptor target genes.

The signaling cascade of the transcription factor vitamin D receptor (VDR) is triggered by its specific ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study we demonstrate that in THP-1 human monocytic leukemia cells 87.4% of the 1034 most prominent genome-wide VDR binding sites co-localize with loci of open chromatin. At 165 of them 1α,25(OH)2D3 strongly increases chromatin accessibility and has at further 217 sites weaker effects. Interestingly, VDR binding sites in 1α,25(OH)2D3-responsive chromatin regions are far more often composed of direct repeats with 3 intervening nucleotides (DR3s) than those in ligand insensitive regions. DR3-containing VDR sites are enriched in the neighborhood of genes that are involved in controling cellular growth, while non-DR3 VDR binding is often found close to genes related to immunity. At the example of six early VDR target genes we show that the slope of their 1α,25(OH)2D3-induced transcription correlates with the basal chromatin accessibility of their major VDR binding regions. However, the chromatin loci controlling these genes are indistinguishable in their VDR association kinetics. Taken together, ligand responsive chromatin loci represent dynamically regulated contact points of VDR with the genome, from where it controls early 1α,25(OH)2D3 target genes.

Elevated Serum Ninjurin-1 Is Associated with a High Risk of Large Artery Atherosclerotic Acute Ischemic Stroke.

Ninjurin-1 is a novel adhesion molecule which is involved in many inflammatory diseases. Functional blockage of Ninjurin-1 has exerted an atheroprotective effect. The aim of the study is to explore the association between serum Ninjurin-1 and the risk of large artery atherosclerotic acute ischemic stroke. From August 2020 through December 2021, patients with large artery atherosclerotic acute ischemic stroke (LAA-AIS) admitted to the First Hospital Affiliated to Soochow University, and age- and sex-matched controls free of ischemic stroke were recruited. Serum Ninj1 was measured with an enzyme-linked immunosorbent assay. Multivariable logistic regression models were used to calculate the odds ratios and 95% confidence intervals of LAA-AIS associated with serum Ninj1 levels, and receiver operating characteristic (ROC) curves were performed to assess the improvement value of Ninj1 for the prediction of LAA-AIS after adding Ninj1 to established risk factors. Of the 110 patients and 110 age- and sex-matched controls free of ischemic stroke enrolled, serum Ninj1 levels in LAA-AIS patients were significantly higher than that in control group (142.70 ng/ml [IQR: 110.41-163.44] vs 101.62 ng/ml [IQR: 86.63-120.86], p < 0.001). In multivariable analysis, Ninj1 levels were expressed as continuous variable and ordinal variable (tertiles), and it turned out that Ninj1 levels were positively associated with increased risk of LAA-AIS, especially in tertile3 compared with tertile1 (adjusted OR = 12.567, 95%CI: 5.148-30.678, p < 0.001), and the adjusted odds OR per 10 ng/ml increment was 1.541, 95%CI: 1.348-1.763, p < 0.001. Furthermore, adding Ninj1 to a multivariate logistic model including conventional risk factors associated LAA-AIS improved the area under ROC curves from 0.787 to 0.874. Elevated circulating levels of Ninj1 were associated with increased risk of LAA-AIS, indicating that serum Ninj1 may act as a predictor independent of established conventional risk factors.

Oscillating Glucose Induces the Increase in Inflammatory Stress through Ninjurin-1 Up-Regulation and Stimulation of Transport Proteins in Human Endothelial Cells.

Clinical data implicate fluctuations of high levels of plasma glucose in cardiovascular diseases. Endothelial cells (EC) are the first cells of the vessel wall exposed to them. Our aim was to evaluate the effects of oscillating glucose (OG) on EC function and to decipher new molecular mechanisms involved. Cultured human ECs (EA.hy926 line and primary cells) were exposed to OG (5/25 mM alternatively at 3 h), constant HG (25 mM) or physiological concentration (5 mM, NG) for 72 h. Markers of inflammation (Ninj-1, MCP-1, RAGE, TNFR1, NF-kB, and p38 MAPK), oxidative stress (ROS, VPO1, and HO-1), and transendothelial transport proteins (SR-BI, caveolin-1, and VAMP-3) were assessed. Inhibitors of ROS (NAC), NF-kB (Bay 11-7085), and Ninj-1 silencing were used to identify the mechanisms of OG-induced EC dysfunction. The results revealed that OG determined an increased expression of Ninj-1, MCP-1, RAGE, TNFR1, SR-B1, and VAMP-3 andstimulated monocyte adhesion. All of these effects were induced bymechanisms involving ROS production or NF-kB activation. NINJ-1 silencing inhibited the upregulation of caveolin-1 and VAMP-3 induced by OG in EC. In conclusion, OG induces increased inflammatory stress, ROS production, and NF-kB activation and stimulates transendothelial transport. To this end, we propose a novel mechanism linking Ninj-1 up-regulation to increased expression of transendothelial transport proteins.

Preventive effects of cristacarpin on experimentally induced uveitis by targeting NF-κB.

Cristacarpin is a novel prenylated pterocarpan that reportedly exhibits broad anti-cancer activity by enhancing endoplasmic reticulum stress. However, whether and how cristacarpin affects in-flammatory processes remain largely unknown. In the present study, the anti-inflammatory effect of cristacarpin on lipopolysaccharide (LPS)-induced inflammation was investigated using zebrafish embryos, RAW 264.7 macrophages, and mouse uveitis models. In the non-toxic concentration range (from 20 to 100 µM), cristacarpin suppressed pro-inflammatory mediators such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, while stimulating anti-inflammatory mediators such as IL-4 and IL-10 in LPS-stimulated RAW 264.7 cells and uveitis mouse models. Cristacarpin decreased cell adhesion of macrophages through downregulation of the expression of Ninjurin1 and matrix metalloproteinases. Furthermore, cristacarpin reduced macrophage migration in zebrafish embryos in vivo. Cristacarpin also increased cytosolic levels of inhibitor of nuclear factor-κB and suppressed the nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells. Collectively, our results suggest that cristacarpin is a potential therapeutic candidate for developing ocular anti-inflammatory drugs.

Ninjurin1 drives lung tumor formation and progression by potentiating Wnt/ß-Catenin signaling through Frizzled2-LRP6 assembly.

BACKGROUND: Cancer stem-like cells (CSCs) play a pivotal role in lung tumor formation and progression. Nerve injury-induced protein 1 (Ninjurin1, Ninj1) has been implicated in lung cancer; however, the pathological role of Ninj1 in the context of lung tumorigenesis remains largely unknown. METHODS: The role of Ninj1 in the survival of non-small cell lung cancer (NSCLC) CSCs within microenvironments exhibiting hazardous conditions was assessed by utilizing patient tissues and transgenic mouse models where Ninj1 repression and oncogenic KrasG12D/+ or carcinogen-induced genetic changes were induced in putative pulmonary stem cells (SCs). Additionally, NSCLC cell lines and primary cultures of patient-derived tumors, particularly Ninj1high and Ninj1low subpopulations and those with gain- or loss-of-Ninj1 expression, and also publicly available data were all used to assess the role of Ninj1 in lung tumorigenesis. RESULTS: Ninj1 expression is elevated in various human NSCLC cell lines and tumors, and elevated expression of this protein can serve as a biomarker for poor prognosis in patients with NSCLC. Elevated Ninj1 expression in pulmonary SCs with oncogenic changes promotes lung tumor growth in mice. Ninj1high subpopulations within NSCLC cell lines, patient-derived tumors, and NSCLC cells with gain-of-Ninj1 expression exhibited CSC-associated phenotypes and significantly enhanced survival capacities in vitro and in vivo in the presence of various cell death inducers. Mechanistically, Ninj1 forms an assembly with lipoprotein receptor-related protein 6 (LRP6) through its extracellular N-terminal domain and recruits Frizzled2 (FZD2) and various downstream signaling mediators, ultimately resulting in transcriptional upregulation of target genes of the LRP6/ß-catenin signaling pathway. CONCLUSIONS: Ninj1 may act as a driver of lung tumor formation and progression by protecting NSCLC CSCs from hostile microenvironments through ligand-independent activation of LRP6/ß-catenin signaling.

Ninjurin1 Deletion in NG2-Positive Pericytes Prevents Microvessel Maturation and Delays Wound Healing.

The formation of mature vasculature through angiogenesis is essential for adequate wound healing, such that blood-borne cells, nutrients, and oxygen can be delivered to the remodeling skin area. Neovessel maturation is highly dependent on the coordinated functions of vascular endothelial cells and perivascular cells, namely pericytes (PCs). However, the underlying mechanism for vascular maturation has not been completely elucidated, and its role in wound healing remains unclear. In this study, we investigated the role of Ninjurin-1 (Ninj1), a new molecule mediating vascular maturation, in wound healing using an inducible PC-specific Ninj1 deletion mouse model. Ninj1 expression increased temporarily in NG2-positive PCs in response to skin injury. When tamoxifen treatment induced a decreased Ninj1 expression in PCs, the neovessels in the regenerating wound margins were structurally and functionally immature, but the total number of microvessels was unaltered. This phenotypic change is associated with a reduction in PC-associated microvessels. Wound healing was significantly delayed in the NG2-specific Ninj1 deletion mouse model. Finally, we showed that Ninj1 is a crucial molecule that mediates vascular maturation in injured skin tissue through the interaction of vascular endothelial cells and PCs, thereby inducing adequate and prompt wound healing.

Ninjurin-1: a biomarker for reflecting the process of neuroinflammation after spinal cord injury.

Previous studies have shown that Ninjurin-1 participates in cell trafficking and axonal growth following central and peripheral nervous system neuroinflammation. But its precise roles in these processes and involvement in spinal cord injury pathophysiology remain unclear. Western blot assay revealed that Ninjurin-1 levels in rats with spinal cord injury exhibited an upregulation until day 4 post-injury and slightly decreased thereafter compared with sham controls. Immunohistochemistry analysis revealed that Ninjurin-1 immunoreactivity in rats with spinal cord injury sharply increased on days 1 and 4 post-injury and slightly decreased on days 7 and 21 post-injury compared with sham controls. Ninjurin-1 immunostaining was weak in vascular endothelial cells, ependymal cells, and some glial cells in sham controls while it was relatively strong in macrophages, microglia, and reactive astrocytes. These findings suggest that a variety of cells, including vascular endothelial cells, macrophages, and microglia, secrete Ninjurin-1 and they participate in the pathophysiology of compression-induced spinal cord injury. All experimental procedures were approved by the Care and Use of Laboratory Animals of Jeju National University (approval No. 2018-0029) on July 6, 2018.

Ninjurin-1 upregulated by TNFα receptor 1 stimulates monocyte adhesion to human TNFα-activated endothelial cells; benefic effects of amlodipine.

AIMS: The objectives of the present study were to investigate the mechanisms of Ninj-1 regulation in TNFα-activated human endothelial cells (HEC), and to test if Amlodipine (AML) ameliorates the inflammatory stress by decreasing Ninj-1 expression. MAIN METHODS: TNFα-activated HEC with/without AML (0.1 µM and 1 µM) were used. TNFα-receptor 1 (TNFR1) was silenced and inhibitors for oxidative stress (N-acetyl cysteine), endoplasmic reticulum stress (salubrinal, 4-phenyl butyric acid), or NF-kB (Bay 11-7085) and p38 MAPK (SB203580) were used. Levels of Ninj-1, TNFR1, monocyte adhesion, endoplasmic reticulum stress (ERS) sensors, NADPH oxidase- and mitochondria-derived oxidative species were evaluated. KEY FINDINGS: The novel findings that we report here are: (i) silencing the endothelial TNFR1 leads to decreased Ninj-1 expression and diminished monocyte adhesion; (ii) increased oxidative stress, ERS and NF-kB activation enhance Ninj-1 expression and monocyte adhesion; (iii) up-regulation of endothelial Ninj-1 expression stimulates monocytes adhesion to TNFα - activated HEC; (iv) AML diminishes monocyte adhesion by reducing Ninj-1 expression through mechanisms involving the decrease of NADPH oxidase and mitochondria-dependent oxidative stress, ERS and NF-kB. In addition, AML alleviates apoptosis by reducing the pro-apoptotic CHOP expression and re-establishing the mitochondrial transmembrane potential. SIGNIFICANCE: The results of the present study suggest that Ninj-1 and the proteins involved in its regulation can be considered therapeutic targets for the alleviation of inflammation- dependent disorders. In addition, we demonstrate that some of the benefic effects of AML can be achieved through regulation of Ninj-1.

Radiation Potentiates Monocyte Infiltration into Tumors by Ninjurin1 Expression in Endothelial Cells.

Radiation is a widely used treatment for cancer patients, with over half the cancer patients receiving radiation therapy during their course of treatment. Considerable evidence from both preclinical and clinical studies show that tumor recurrence gets restored following radiotherapy, due to the influx of circulating cells consisting primarily of monocytes. The attachment of monocyte to endothelial cell is the first step of the extravasation process. However, the exact molecules that direct the transmigration of monocyte from the blood vessels to the tumors remain largely unknown. The nerve injury-induced protein 1 (Ninjurin1 or Ninj1) gene, which encodes a homophilic adhesion molecule and cell surface protein, was found to be upregulated in inflammatory lesions, particularly in macrophages/monocytes, neutrophils, and endothelial cells. More recently Ninj1 was reported to be regulated following p53 activation. Considering p53 has been known to be activated by radiation, we wondered whether Ninj1 could be increased in the endothelial cells by radiation and it might contribute to the recruiting of monocytes in the tumor. Here we demonstrate that radiation-mediated up-regulation of Ninj1 in endothelial cell lines such as human umbilical vein endothelial cells (HUVECs), EA.hy926, and immortalized HUVECs. Consistent with this, we found over-expressed Ninj1 in irradiated xenograft tumors, and increased monocyte infiltration into tumors. Radiation-induced Ninj1 was transcriptionally regulated by p53, as confirmed by transfection of p53 siRNA. In addition, Ninj1 over-expression in endothelial cells accelerated monocyte adhesion. Irradiation-induced endothelial cells and monocyte interaction was inhibited by knock-down of Ninj1. Furthermore, over-expressed Ninj1 stimulated MMP-2 and MMP-9 expression in monocyte cell lines, whereas the MMP-2 and MMP-9 expression were attenuated by Ninj1 knock-down in monocytes. Taken together, we provide evidence that Ninj1 is a key molecule that generates an interaction between endothelial cells and monocytes. This result suggests that radiation-mediated Ninj1 expression in endothelial cells could be involved in the post-radiotherapy recurrence mechanism.

Immunohistochemical localization of nerve injury-induced protein-1 in mouse tissues.

Nerve injury-induced protein (Ninjurin)-1 is a cell adhesion molecule that is upregulated in neurons and Schwann cells after transection injury in rats. In this study, we investigated the localization of Ninjurin-1 in various tissues, including the cerebrum, sciatic nerve, spleen, lung, stomach, ileum, colon, liver, pancreas, kidney, testis, and skin in C57BL/6 mice, using Western blotting and immunohistochemistry. Western blot analysis showed that Ninjurin-1 was differentially expressed among organs. Ninjurin-1 was abundant in skin and ileum, weakly expressed in cerebrum, and moderately expressed in the other organs studied. Immunohistochemical analysis largely confirmed the results of the western blot analysis with often localization of Ninjurin-1 in the regions with abundant connective tissues. In addition, Ninjurin-1 was differentially expressed in various cell types in the tissues under the investigation. These findings suggest that Ninjurin-1 may play organ-specific roles in development and homeostasis of many organs.