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.

Melatonin inhibits visfatin-induced inducible nitric oxide synthase expression and nitric oxide production in macrophages.

Aberrant expression of inducible nitric oxide synthase (iNOS) in macrophages, which has been reported to be suppressed by melatonin, has an important contribution in the development of pathological inflammation. Visfatin, an adipokine, regulates the expression of various inflammatory factors, leading to inflammation; however, the influence of visfatin on iNOS-driven processes in macrophages is unclear. Here, we report the assessment of the role of visfatin in the regulation of iNOS gene expression in macrophages. Our data show that the levels of iNOS protein in peritoneal macrophages as well as nitric oxide (NO) in blood plasma were significantly lower after lipopolysaccharide treatment in visfatin(+/-) mice than those in the WT mice. In addition, visfatin increases iNOS mRNA and protein levels in RAW 264.7 cells, along with increasing production of NO. The enhancement of iNOS expression was prevented by treating the cells with inhibitors of the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3), nuclear factor (NF)-κB, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase pathways. Our results also show that visfatin-induced iNOS expression and NO production were significantly inhibited by melatonin, an effect that was closely associated with a reduction in phosphorylated JAK2/STAT3 levels and with the inhibition of p65 translocation into nucleus. In conclusion, our data show, for the first time, that melatonin suppresses visfatin-induced iNOS upregulation in macrophages by inhibiting the STAT3 and NF-κB pathways. Moreover, our data suggest that melatonin could be therapeutically useful for attenuating the development of visfatin-iNOS axis-associated diseases.

Mutant p53-Notch1 Signaling Axis Is Involved in Curcumin-Induced Apoptosis of Breast Cancer Cells.

Notch1 has been reported to be highly expressed in triple-negative and other subtypes of breast cancer. Mutant p53 (R280K) is overexpressed in MDA-MB-231 triple-negative human breast cancer cells. The present study aimed to determine whether the mutant p53 can be a potent transcriptional activator of the Notch1 in MDA-MB-231 cells, and explore the role of this mutant p53-Notch1 axis in curcumin-induced apoptosis. We found that curcumin treatment resulted in an induction of apoptosis in MDA-MB-231 cells, together with downregulation of Notch1 and its downstream target, Hes1. This reduction in Notch1 expression was determined to be due to the decreased activity of endogenous mutant p53. We confirmed the suppressive effect of curcumin on Notch1 transcription by performing a Notch1 promoter-driven reporter assay and identified a putative p53-binding site in the Notch1 promoter by EMSA and chromatin immunoprecipitation analysis. Overexpression of mutant p53 increased Notch1 promoter activity, whereas knockdown of mutant p53 by small interfering RNA suppressed Notch1 expression, leading to the induction of cellular apoptosis. Moreover, curcumin-induced apoptosis was further enhanced by the knockdown of Notch1 or mutant p53, but it was decreased by the overexpression of active Notch1. Taken together, our results demonstrate, for the first time, that Notch1 is a transcriptional target of mutant p53 in breast cancer cells and suggest that the targeting of mutant p53 and/or Notch1 may be combined with a chemotherapeutic strategy to improve the response of breast cancer cells to curcumin.

The N-terminal ectodomain of Ninjurin1 liberated by MMP9 has chemotactic activity.

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.

Visfatin through STAT3 activation enhances IL-6 expression that promotes endothelial angiogenesis.

Signal transducer and activator of transcription 3 (STAT3) acts as a mediator and biomarker in endothelial activation. We have recently shown that a novel adipokine visfatin promotes endothelial angiogenesis. The present study was to determine whether visfatin affects STAT3 activity and to explore the potential target gene(s). Here, we found that visfatin induced the activation of STAT3, as characterized by increased tyrosine phosphorylation, nuclear translocation, and DNA-binding activity in human endothelial cells. In addition, visfatin significantly upregulated mRNA and protein levels of endothelial interleukin-6 (IL-6), which was blocked by a specific inhibitor of STAT3 signaling and by the transfection of siRNA specific for STAT3. Furthermore, visfatin-induced angiogenesis was reduced by the inhibition of STAT3 signaling or neutralization of IL-6 function, as measured by tube formation, rat aortic ring assay, and mouse Matrigel plug assay. Taken together, our results provide the first example of STAT3-dependent endothelial IL-6 induction by visfatin and of the role of IL-6 in mediating visfatin-induced angiogenesis.

The cleidocranial dysplasia-related R131G mutation in the Runt-related transcription factor RUNX2 disrupts binding to DNA but not CBF-beta.

Cleidocranial dysplasia (CCD) is caused by haploinsufficiency in RUNX2 function. We have previously identified a series of RUNX2 mutations in Korean CCD patients, including a novel R131G missense mutation in the Runt-homology domain. Here, we examine the functional consequences of the RUNX2(R131G) mutation, which could potentially affect DNA binding, nuclear localization signal, and/or heterodimerization with core-binding factor-beta (CBF-beta). Immunofluorescence microscopy and western blot analysis with subcellular fractions show that RUNX2(R131G) is localized in the nucleus. Immunoprecipitation analysis reveals that heterodimerization with CBF-beta is retained. However, precipitation assays with biotinylated oligonucleotides and reporter gene assays with RUNX2 responsive promoters together reveal that DNA-binding activity and consequently the transactivation of potential of RUNX2(R131G) is abrogated. We conclude that loss of DNA binding, but not nuclear localization or CBF-beta heterodimerization, causes RUNX2 haploinsufficiency in patients with the RUNX2(R131G) mutation. Retention of specific functions including nuclear localization and binding to CBF-beta of the RUNX2(R131G) mutation may render the mutant protein an effective competitor that interferes with wild-type function.

PEBP2-beta/CBF-beta-dependent phosphorylation of RUNX1 and p300 by HIPK2: implications for leukemogenesis.

The heterodimeric transcription factor RUNX1/PEBP2-beta (also known as AML1/CBF-beta) is essential for definitive hematopoiesis. Here, we show that interaction with PEBP2-beta leads to the phosphorylation of RUNX1, which in turn induces p300 phosphorylation. This is mediated by homeodomain interacting kinase 2 (HIPK2), targeting Ser(249), Ser(273), and Thr(276) in RUNX1, in a manner that is also dependent on the RUNX1 PY motif. Importantly, we observed the in vitro disruption of this phosphorylation cascade by multiple leukemogenic genetic defects targeting RUNX1/CBFB. In particular, the oncogenic protein PEBP2-beta-SMMHC prevents RUNX1/p300 phosphorylation by sequestering HIPK2 to mislocalized RUNX1/beta-SMMHC complexes. Therefore, phosphorylation of RUNX1 appears a critical step in its association with and phosphorylation of p300, and its disruption may be a common theme in RUNX1-associated leukemogenesis.

Upregulation of fibroblast growth factor-2 by visfatin that promotes endothelial angiogenesis.

Adipokines have been known to act as angiogenic regulators in the process of angiogenesis. Recently, we have demonstrated that visfatin, a novel adipokine, has angiogenic activity. However, little has been reported on the underlying mechanism of visfatin-induced angiogenesis. In this study, we report that visfatin-induced angiogenesis is mediated by endothelial fibroblast growth factor-2 (FGF-2). Visfatin increased the levels of FGF-2 mRNA and protein in human endothelial cells. The enhancement in FGF-2 expression was prevented by an inhibitor of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Furthermore, visfatin-induced angiogenesis was reduced by inhibition of FGF-2 receptor kinase or by neutralization of FGF-2 function. Taken together, our results indicate that visfatin-induced endothelial angiogenesis is composed largely of two sequential steps: the induction of Erk1/2-dependent FGF-2 gene expression by visfatin and the subsequent FGF-2-induced angiogenesis. These data further suggest an integral role for visfatin-FGF-2 signaling axis in modulating endothelial angiogenesis.