Arrest defective 1 (ARD1) is an acetyltransferase that is highly conserved across organisms, from yeasts to humans. The high homology and widespread expression of ARD1 across multiple species and tissues signify that it serves a fundamental role in cells. Human ARD1 (hARD1) has been suggested to be involved in diverse biological processes, and its role in cell proliferation and cancer development has been recently drawing attention. However, the subcellular localization of ARD1 and its relevance to cellular function remain largely unknown. Here, we have demonstrated that hARD1 is imported to the nuclei of proliferating cells, especially during S phase. Nuclear localization signal (NLS)-deleted hARD1 (hARD1?N), which can no longer access the nucleus, resulted in cell morphology changes and cellular growth impairment. Notably, hARD1?N-expressing cells showed alterations in the cell cycle and the expression levels of cell cycle regulators compared to hARD1 wild-type cells. Furthermore, these effects were rescued when the nuclear import of hARD1 was restored by exogenous NLS. Our results show that hARD1 nuclear translocation mediated by NLS is required for cell cycle progression, thereby contributing to proper cell proliferation.
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.
ARD1 is an acetyltransferase with several variants derived from alternative splicing. Among ARD1 variants, mouse ARD1225 (mARD1225), mouse ARD1235 (mARD1235), and human ARD1235 (hARD1235) have been the most extensively characterized and are known to have different biological functions. In the present study, we demonstrated that mARD1225, mARD1235, and hARD1235 have conserved autoacetylation activities, and that they selectively regulate distinct roles of ARD1 variants in tumorigenesis. Using purified recombinants for ARD1 variants, we found that mARD1225, mARD1235, and hARD1235 undergo similar autoacetylation with the target site conserved at the Lys136 residue. Moreover, functional investigations revealed that the role of mARD1225 autoacetylation is completely distinguishable from that of mARD1235 and hARD1235. Under hypoxic conditions, mARD1225 autoacetylation inhibited tumor angiogenesis by decreasing the stability of hypoxia-inducible factor-1? (HIF-1?). Autoacetylation stimulated the catalytic activity of mARD1225 to acetylate Lys532 of the oxygen-dependent degradation (ODD) domain of HIF-1?, leading to the proteosomal degradation of HIF-1?. In contrast, autoacetylation of mARD1235 and hARD1235 contributed to cellular growth under normoxic conditions by increasing the expression of cyclin D1. Taken together, these data suggest that autoacetylation of ARD1 variants differentially regulates angiogenesis and cell proliferation in an isoform-specific manner.
Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple sclerosis (MS). However, in vivo gene-deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knockout (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis (EIU). 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 EIU 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 (BMDMs) and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO BMDMs 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 MS.
Nerve injury induced protein 1, Ninj1 (Ninjurin1) is a cell surface protein that is induced by nerve injury and promotes axonal growth in the peripheral nervous system. However, the function of Ninj1 in the vascular system and central nervous system (CNS) is incompletely understood. Here we review recent studies that have shed further light on the role and regulation of Ninj1 in vascular remodeling and inflammation. Increasing evidence suggests that Ninj1 mediates cell communication and enhances the entry, migration, and activity of leukocytes such as monocytes and macrophages in developmental processes and inflammatory responses. Moreover, our recent studies show that Ninj1 regulates close interaction between leukocytes and vascular endothelial cells in vascular remodeling and inflamed CNS. Additionally, Ninj1 enhances the apoptosis-inducing activity of leukocytes and is cleaved by MMPs, resulting in loss of adhesion during tissue remodeling. The collective data described here show that Ninj1 is required for the entry, adhesion, activation, and movement of leukocytes during tissue remodeling and might be a potential therapeutic target to regulate the adhesion and trafficking of leukocytes in inflammation and leukocyte-mediated diseases such as multiple sclerosis, diabetic retinopathy, and neuropathy.
Ninjurin1 (nerve injury-induced protein, Ninj1) is an adhesion molecule that is essential for cell-to-cell interactions. However, little is known about the function of Ninj1 in the central nervous system (CNS). To address its role in the CNS, we analyzed the expression pattern of Ninj1 in normal rats and in an experimental autoimmune encephalomyelitis (EAE) model. Ninj1 was expressed in three major compartments of brains, meninges, the choroid plexus, and parenchymal perivascular spaces. In the EAE brains, Ninj1 was strongly expressed in myeloid cells (macrophages/monocytes and neutrophils) and partially expressed in endothelial cells (ECs). Furthermore, Ninj1 enhanced adhesion between BV2 cells (murine monocyte lineage microglia) and HBMECs (human brain microvascular endothelial cells). Collectively, our findings suggest that Ninj1 may mediate the entry of myeloid cells into the CNS in normal and EAE brains, and it is a potential therapeutic target for regulating myeloid cell trafficking across the blood-brain barrier (BBB) in CNS immune processes.
Ninjurin1 is known as an adhesion molecule promoting leukocyte trafficking under inflammatory conditions. However, the posttranslational modifications of Ninjurin1 are poorly understood. Herein, we defined the proteolytic cleavage of Ninjurin1 and its functions. HEK293T cells overexpressing the C- or N-terminus tagging mouse Ninjurin1 plasmid produced additional cleaved forms of Ninjurin1 in the lysates or conditioned media (CM). Two custom-made anti-Ninjurin1 antibodies, Ab(1-15) or Ab(139-152), specific to the N- or C-terminal regions of Ninjurin1 revealed the presence of its shedding fragments in the mouse liver and kidney lysates. Furthermore, Matrix Metalloproteinase (MMP) 9 was responsible for Ninjurin1 cleavage between Leu(56) and Leu(57). Interestingly, the soluble N-terminal Ninjurin1 fragment has structural similarity with well-known chemokines. Indeed, the CM from HEK293T cells overexpressing the GFP-mNinj1 plasmid was able to attract Raw264.7 cells in trans-well assay. Collectively, we suggest that the N-terminal ectodomain of mouse Ninjurin1, which may act as a chemoattractant, is cleaved by MMP9.
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