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.
Synopsis
Induction of ferroptosis by lipid peroxidation can lead to plasma membrane rupture, but the mediators of this rupture are unknown. This study identifies Ninjurin-1 (NINJ1) as the executor of plasma membrane permeabilization in ferroptotic macrophages and fibroblasts.
Ferroptosis induction leads to NINJ1 activation and oligomerization in macrophages and fibroblasts.
NINJ1 activation occurs downstream of lipid peroxidation and calcium influx, the first steps of ferroptosis.
NINJ1 activation triggers the last steps of ferroptosis, i.e., loss of membrane integrity and rupture.
NINJ1 controls the release of cytosolic proteins and danger-associated molecular pattern molecules from ferroptotic cells.
Ninjurin-1 (NINJ1) oligomerizes and mediates loss of plasma membrane integrity as well as plasma membrane rupture in ferroptotic cells.
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.
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.
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.
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.
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.
•Ninj-1 upregulation stimulates monocyte adhesion.•TNFR1 silencing reduce Ninj-1 expression and monocyte adhesion.•Oxidative and/or endoplasmic reticulum stress stimulate Ninj-1 expression.•NF-kB inhibition determines the down-regulation of Ninj-1 expression.•AML reduces Ninj-1 by inhibiting oxidative and endoplasmic reticulum stress.
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•GEx diminishes MCP-1, VCAM-1 and monocyte adhesion in TNFα-exposed HEC.•GEx anti-inflammatory action was correlated with the decrease of Ninj-1 and TNFR1.•GEx decreases intracellular ...RAGE expression and increases sRAGE levels.•GEx decrease ROS by reducing p22phox and NOX4 and activating Nrf2/HO-1 axis.
Dysfunction of endothelial cells (EC) is important for atherosclerosis progression. The aim of this study was to evaluate the potential of ginger extract (GEx), 6-gingerol (6-G) and 6-shogaol (6-Sh) to reverse EC dysfunction and to investigate its mechanism of action, using cultured human EC incubated with TNFα. The results showed that GEx decreases monocyte chemoattractant protein-1, vascular cell adhesion molecule-1 and monocyte adhesion. This decrease was associated with the: (1) decrease of ninjurin-1 expression; (2) reduction of TNFα receptor1 and of the receptor for advanced glycation end-products expression (RAGE), in parallel with the increase of soluble RAGE; (3) decrease of NADPH oxidase subunits expression; (4) activation of antioxidant Nrf2 and heme oxygenase-1, and (5) inhibition of NF-kB. The benefic effects of 6-G and 6-Sh were weaker than those of GEx (GEx > 6-Sh > 6-G). In conclusion, GEx might be a promising alternative to ameliorate disorders in which oxidative stress and inflammation are important.
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.
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.
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.
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.
Objective: To evaluate and compare the gene expression profiles of Ninjurin-1 and Ninjurin-2 with the expressions of L1 family of cell adhesion molecules (NCAM-L1), neuralcell adhesion molecules ...(NCAM), and N-cadherin during the course of neural differentiationof mouse neural stem cells (NSCs).Materials and Methods: Briefly, for neural stem cell isolation, the frontal part of an adultmouse brain was minced in phosphate buffered saline (PBS) and digested by an enzymesolution which contained hyaluronidase and trypsin. Isolated cells were culturedin medium supplemented by epidermal growth factor (EGF) and basic fibroblast growthfactor (bFGF). After seven days, primary neurospheres appeared in culture medium. Aftertransfer to poly-L-ornithine coated dishes that contained culture medium supplementedwith 1% fetal bovine serum (FBS), the primary neurospheres differentiated into neural-likeand neuroglial-like cells. Differentiated cells were examined by morphological, immunocytochemical,and molecular evaluations.Results: Our results showed that isolated cells from the preventricular area of mouseadult brain proliferated in medium which contained EGF and bFGF, and expansion of thecells continued until passage 14 without losing morphological and neurogenesis capacity.Multiple passaging confirmed the stemness nature of the isolated cells. The isolatedNSCs were able to differentiate into neural-like and neuroglial-like cells after transfer topoly-L-ornithine coated dishes that contained culture medium supplemented with 1%FBS. Molecular studies for NCAM, NCAM-L1, and N-Cadherin genes, as well as immunocytochemicalanalysis for NCAM-L1 and NCAM proved the differentiation. Our dataalso revealed, for the first time, gene expression profiling of Ninurin-1 and Ninjurin-2, twonovel cell adhesion molecules (CAMs), during the course of differentiation of neural stemcells.Conclusion: The induction of neural differentiation in mouse NSCs initiates the expressionof NCAM-L1, NCAM, and N-cadherin which can be the proof of complete neural differentiation.In addition, our results indicate that the expression of Ninjurin-1 increases afterneural induction much like the expressions of NCAM-L1, NCAM, and N-cadherin. Thisdata suggests the probable importance of Ninjurin-1 in both the morphology and functionof neural cells. The data in this study also reveals that the expression of Ninjurin-2, in contrastwith Ninjurin-1, initiates and continues until the differentiation termination point. Thissuggests that although Ninjurin-1 and Ninjurin-2 share conserved hydrophobic regions fortheir transmembrane domains, their roles in nerve cells are probably different.
To investigate the regulatory effects of ninjurin-1 on adhesion of myeloid cells in the retina at the early stage of diabetic rats.
Experimental study. The rat diabetic model was induced by ...intraperitoneal injection of streptozotocin. After 2 months of diabetes induction, 27 diabetic rats were randomly chosen and assigned to 3 groups, including diabetes and phosphate buffered saline (PBS) injection group (group B), diabetes and anti-Ninj-1 injection group (group C) and diabetes and anti-IgG injection group (group D), with 9 rats in each group. Nine age matched health rats were chosen as control group (group A). Retinal leukostasis was quantified with acridine orange leukocyte fluorography. Retinal myeloid cell infiltration activity was measured by enzyme linked immunosorbent assay of myeloperoxidase (MPO). The differences of the mean values among the four groups were analyzed by one-factor analysis of variance. The multiple comparisons of the mean values among the four groups were analyzed by LSD-t analysis.