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1.
Exp Mol Med ; 50(4): 1-13, 2018 04 27.
Article in English | MEDLINE | ID: mdl-29700280

ABSTRACT

Liver fibrosis can be reversed by removing its causative injuries; however, the molecular mechanisms mediating the resolution of liver fibrogenesis are poorly understood. We investigate the role of a scaffold protein, A-Kinase Anchoring Protein 12 (AKAP12), during liver fibrosis onset, and resolution. Biliary fibrogenesis and fibrosis resolution was induced in wild-type (WT) or AKAP12-deficient C57BL/6 mice through different feeding regimens with 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing chow. AKAP12 expression in portal fibroblasts (PFs) and liver sinusoidal endothelial cells (LSECs) gradually decreased as fibrosis progressed but was restored after cessation of the fibrotic challenge. Histological analysis of human liver specimens with varying degrees of fibrosis of different etiologies revealed that AKAP12 expression diminishes in hepatic fibrosis from its early stages onward. AKAP12 KO mice displayed reduced fibrosis resolution in a DDC-induced biliary fibrosis model, which was accompanied by impaired normalization of myofibroblasts and capillarized sinusoids. RNA sequencing of the liver transcriptome revealed that genes related to ECM accumulation and vascular remodeling were mostly elevated in AKAP12 KO samples. Gene ontology (GO) and bioinformatic pathway analyses identified that the differentially expressed genes were significantly enriched in GO categories and pathways, such as the adenosine 3',5'-cyclic monophosphate (cAMP) pathway. Knockdown of the AKAP12 gene in cultured primary PFs revealed that AKAP12 inhibited PF activation in association with the adenosine 3',5'-cyclic monophosphate (cAMP) pathway. Moreover, AKAP12 knockdown in LSECs led to enhanced angiogenesis, endothelin-1 expression and alterations in laminin composition. Collectively, this study demonstrates that AKAP12-mediated regulation of PFs and LSECs has a central role in resolving hepatic fibrosis.


Subject(s)
A Kinase Anchor Proteins/genetics , Cell Cycle Proteins/genetics , Fibroblasts/pathology , Gene Expression Regulation , Liver Cirrhosis/genetics , Liver/pathology , A Kinase Anchor Proteins/analysis , Animals , Cell Cycle Proteins/analysis , Cell Line , Fibroblasts/metabolism , Humans , Liver/cytology , Liver/metabolism , Liver Cirrhosis/pathology , Male , Mice, Inbred C57BL , Mice, Knockout , Transcriptome
2.
Oncotarget ; 8(40): 68517-68529, 2017 Sep 15.
Article in English | MEDLINE | ID: mdl-28978134

ABSTRACT

SAM domain and HD domain containing protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase (dNTPase) that inhibits retroviruses by depleting intracellular deoxynucleotide triphosphates (dNTPs) in non-cycling myeloid cells. Although SAMHD1 is expressed ubiquitously throughout the human body, the molecular mechanisms regulating its enzymatic activity and function in non-immune cells are relatively unexplored. Here, we demonstrate that the dNTPase activity of SAMHD1 is regulated by acetylation, which promotes cell cycle progression in cancer cells. SAMHD1 is acetylated at residue lysine 405 (K405) in vitro and in vivo by an acetylatransferase, arrest defective protein 1 (ARD1). Acetylated SAMHD1 wildtype proteins have enhanced dNTPase activity in vitro, whereas non-acetylated arginine substituted mutants (K405R) do not. K405R mutant expressing cancer cells have reduced G1/S transition and slower proliferation compared to wildtype. SAMHD1 acetylation levels are strongest during the G1 phase, indicating a role during G1 phase. Collectively, these findings suggest that SAMHD1 acetylation enhances its dNTPase activity and promotes cancer cell proliferation. Therefore, SAMHD1 acetylation may be a potent therapeutic target for cancer treatment.

3.
Oncotarget ; 8(34): 57216-57230, 2017 Aug 22.
Article in English | MEDLINE | ID: mdl-28915666

ABSTRACT

Aurora kinase A (AuA) is a prerequisite for centrosome maturation, separation, and mitotic spindle assembly, thus, it is essential for cell cycle regulation. Overexpression of AuA is implicated in poor prognosis of many types of cancer. However, the regulatory mechanisms underlying the functions of AuA are still not fully understood. Here, we report that AuA colocalizes with arrest defective protein 1 (ARD1) acetyltransferase during cell division and cell migration. Additionally, AuA is acetylated by ARD1 at lysine residues at positions 75 and 125. The double mutations at K75/K125 abolished the kinase activity of AuA. Moreover, the double mutant AuA exhibited diminished ability to promote cell proliferation and cell migration. Mechanistic studies revealed that AuA acetylation at K75/K125 promoted cell proliferation via activation of cyclin E/CDK2 and cyclin B1. In addition, AuA acetylation stimulated cell migration by activating the p38/AKT/MMP-2 pathway. Our findings indicate that ARD1-mediated acetylation of AuA enhances cell proliferation and migration, and probably contributes to cancer development.

4.
J Cell Biochem ; 118(8): 2219-2230, 2017 08.
Article in English | MEDLINE | ID: mdl-28067406

ABSTRACT

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.


Subject(s)
Cell Adhesion Molecules, Neuronal/chemistry , Cell Adhesion Molecules, Neuronal/metabolism , Nerve Growth Factors/chemistry , Nerve Growth Factors/metabolism , Animals , Bone Marrow Cells/metabolism , Cell Adhesion Molecules, Neuronal/genetics , Cells, Cultured , Flow Cytometry , Fluorescence Resonance Energy Transfer , Glycosylation/drug effects , HEK293 Cells , Humans , Immunoblotting , Immunoprecipitation , Male , Mice , Mice, Knockout , Microscopy, Confocal , NIH 3T3 Cells , Nerve Growth Factors/genetics , Protein Multimerization/drug effects , Protein Multimerization/genetics , Tunicamycin/pharmacology
5.
Mol Neurobiol ; 54(9): 7353-7368, 2017 11.
Article in English | MEDLINE | ID: mdl-27815839

ABSTRACT

Over the last few decades, molecular neurobiology has uncovered many genes whose deficiency in mice results in behavioral traits associated with human neuropsychiatric disorders such as autism, obsessive-compulsive disorder (OCD), and schizophrenia. However, the etiology of these common diseases remains enigmatic with the potential involvement of a battery of genes. Here, we report abnormal behavioral phenotypes of mice deficient in a cell adhesion molecule Ninjurin 1 (Ninj1), which are relevant to repetitive and anxiety behaviors of neuropsychiatric disorders. Ninj1 knockout (KO) mice exhibit compulsive grooming-induced hair loss and self-made lesions as well as increased anxiety-like behaviors. Histological analysis reveals that Ninj1 is predominantly expressed in cortico-thalamic circuits, and neuron-specific Ninj1 conditional KO mice manifest aberrant phenotypes similar to the global Ninj1 KO mice. Notably, the brains of Ninj1 KO mice display altered synaptic transmission in thalamic neurons as well as a reduced number of functional synapses. Moreover, the disruption of Ninj1 leads to glutamatergic abnormalities, including increased ionotropic glutamate receptors but reduced glutamate levels. Furthermore, chronic treatment with fluoxetine, a drug reportedly ameliorates compulsive behaviors in mice, prevents progression of hair loss and alleviates the compulsive grooming and anxiety-like behavior of Ninj1 KO mice. Collectively, our results suggest that Ninj1 could be involved in neuropsychiatric disorders associated with impairments of repetitive and anxiety behaviors.


Subject(s)
Anxiety/genetics , Anxiety/metabolism , Cell Adhesion Molecules, Neuronal/deficiency , Cell Adhesion Molecules, Neuronal/genetics , Compulsive Behavior/genetics , Compulsive Behavior/metabolism , Nerve Growth Factors/deficiency , Nerve Growth Factors/genetics , Animals , Anxiety/psychology , Cells, Cultured , Compulsive Behavior/psychology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout
6.
Nat Commun ; 7: 12882, 2016 10 06.
Article in English | MEDLINE | ID: mdl-27708256

ABSTRACT

Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.


Subject(s)
HSP70 Heat-Shock Proteins/metabolism , N-Terminal Acetyltransferase A/metabolism , N-Terminal Acetyltransferase E/metabolism , Protein Refolding , Acetylation , Animals , Apoptosis , Caspases/metabolism , Cell Survival , Green Fluorescent Proteins/chemistry , HEK293 Cells , Humans , Molecular Chaperones/chemistry , Mutation , Protein Binding , Protein Domains , Protein Processing, Post-Translational , RNA, Small Interfering/metabolism , Stress, Physiological , Zebrafish
7.
Int J Oncol ; 49(4): 1407-14, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27498767

ABSTRACT

Cirrhosis, the end-stage of hepatic fibrosis, is not only life-threatening by itself, but also a causative factor of liver cancer. Despite efforts to develop treatment for liver fibrosis, there are no approved agents as anti-fibrotic drugs to date. In the present study, we aimed to investigate the anti-fibrotic effect of the AMP-activated protein kinase (AMPK) activator, HL156A. A mouse model of thioacetamide (TAA)-induced liver fibrosis was used to examine the effect of HL156A in vivo. Mice received either TAA alone or a combination of TAA and HL156A intraperitoneally for a total duration of 6 weeks. Including HL156A during exposure to TAA significantly reduced extracellular matrix (ECM) deposition and production of the hepatic transforming growth factor-ß1 (TGF-ß1). Immunohistochemical analysis revealed that the activation of hepatic stellate cells and the capillarization of liver sinusoids were also diminished significantly by HL156A co-treatment. The anti-fibrotic effect of HL156A was further studied in vitro by using a rat hepatic stellate cell line, HSC-T6 cells. The induction of α-smooth muscle actin (α-SMA) by TGF-ß1 treatment was reversed by HL156A, which was likely via the activation of AMPK. Moreover, HL156A showed anti-inflammatory effects on macrophages. Treatment with HL156A diminished LPS-induced activation of both Raw264.7 macrophage cells and primary cultured mouse macrophages. Taken together, these results imply that the AMPK activator HL156A inhibits hepatic fibrosis via multiple mechanisms and could be a potentially effective agent for fibrosis treatment.


Subject(s)
Guanidines/administration & dosage , Hepatic Stellate Cells/drug effects , Liver Cirrhosis/drug therapy , Macrophages/drug effects , Pyrrolidines/administration & dosage , Thioacetamide/adverse effects , Actins/metabolism , Animals , Cell Line , Disease Models, Animal , Extracellular Matrix/drug effects , Gene Expression Regulation/drug effects , Guanidines/pharmacology , Hepatic Stellate Cells/metabolism , Humans , Injections, Intraperitoneal , Liver Cirrhosis/chemically induced , Liver Cirrhosis/metabolism , Macrophages/metabolism , Mice , Pyrrolidines/pharmacology , RAW 264.7 Cells , Rats , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta1/pharmacology
8.
Int J Oncol ; 48(2): 821-8, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26677008

ABSTRACT

Ninjurin1 is a transmembrane protein involved in macrophage migration and adhesion during inflammation. It was recently reported that repression of Ninjurin1 attenuated the lipopolysaccharide (LPS)-induced inflammatory response in macrophages; however, the precise mechanism by which Ninjurin1 modulates LPS-induced inflammation remains poorly understood. In the present study, we found that the interaction between Ninjurin1 and LPS contributed to the LPS-induced inflammatory response. Notably, pull-down assays using lysates from HEK293T cells transfected with human or mouse Ninjurin1 and biotinylated LPS (LPS-biotin) showed that LPS directly bound Ninjurin1. Subsequently, LPS binding assays with various truncated forms of Ninjurin1 protein revealed that amino acids (aa) 81-100 of Ninjurin1 were required for LPS binding. In addition, knockdown experiments using Ninj1 siRNA resulted in decreased nitric oxide (NO) and tumor necrosis factor-α (TNFα) secretion upon LPS treatment in Raw264.7 cells. Collectively, our results suggest that Ninjurin1 regulates the LPS-induced inflammatory response through its direct binding to LPS, thus, identifying Ninjurin1 as a putative target for the treatment of inflammatory diseases, such as sepsis and inflammation-associated carcinogenesis.


Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Inflammation/chemically induced , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Nerve Growth Factors/metabolism , Amino Acids/metabolism , Animals , Cell Adhesion/drug effects , Cell Line , Cell Movement/physiology , HEK293 Cells , Humans , Macrophages/metabolism , Mice , Nitric Oxide/metabolism , Protein Binding/physiology , Tumor Necrosis Factor-alpha/metabolism
9.
Int J Oncol ; 46(1): 99-106, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25338643

ABSTRACT

ARD1 is an acetyltransferase with several variants derived from alternative splicing. Among ARD1 variants, mouse ARD1(225) (mARD1(225)), mouse ARD1(235) (mARD1(235)), and human ARD1(235) (hARD1(235)) have been the most extensively characterized and are known to have different biological functions. In the present study, we demonstrated that mARD1(225), mARD1(235), and hARD1(235) 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 mARD1(225), mARD1(235), and hARD1(235) undergo similar autoacetylation with the target site conserved at the Lys136 residue. Moreover, functional investigations revealed that the role of mARD1(225) autoacetylation is completely distinguishable from that of mARD1(235) and hARD1(235). Under hypoxic conditions, mARD1(225) autoacetylation inhibited tumor angiogenesis by decreasing the stability of hypoxia-inducible factor-1α (HIF-1α). Autoacetylation stimulated the catalytic activity of mARD1(225) to acetylate Lys532 of the oxygen-dependent degradation (ODD) domain of HIF-1α, leading to the proteosomal degradation of HIF-1α. In contrast, autoacetylation of mARD1(235) and hARD1(235) 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.


Subject(s)
Carcinogenesis/genetics , N-Terminal Acetyltransferase A/genetics , N-Terminal Acetyltransferase A/metabolism , N-Terminal Acetyltransferase E/genetics , N-Terminal Acetyltransferase E/metabolism , Polymorphism, Single Nucleotide , Acetylation , Amino Acid Sequence , Carcinogenesis/metabolism , Cell Proliferation/genetics , Cells, Cultured , Feedback, Physiological , HeLa Cells , Human Umbilical Vein Endothelial Cells , Humans , Hypoxia/genetics , Hypoxia/metabolism , Hypoxia/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Molecular Sequence Data , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Protein Isoforms/genetics , Protein Stability
10.
Nat Commun ; 5: 4952, 2014 Sep 17.
Article in English | MEDLINE | ID: mdl-25229625

ABSTRACT

The meninges forms a critical epithelial barrier, which protects the central nervous system (CNS), and therefore its prompt reconstruction after CNS injury is essential for reducing neuronal damage. Meningeal cells migrate into the lesion site after undergoing an epithelial-mesenchymal transition (EMT) and repair the impaired meninges. However, the molecular mechanisms of meningeal EMT remain largely undefined. Here we show that TGF-ß1 and retinoic acid (RA) released from the meninges, together with oxygen tension, could constitute the mechanism for rapid meningeal reconstruction. AKAP12 is an effector of this mechanism, and its expression in meningeal cells is regulated by integrated upstream signals composed of TGF-ß1, RA and oxygen tension. Functionally, AKAP12 modulates meningeal EMT by regulating the TGF-ß1-non-Smad-SNAI1 signalling pathway. Collectively, TGF-ß1, RA and oxygen tension can modulate the dynamic change in AKAP12 expression, causing prompt meningeal reconstruction after CNS injury by regulating the transition between the epithelial and mesenchymal states of meningeal cells.


Subject(s)
A Kinase Anchor Proteins/metabolism , Cell Cycle Proteins/metabolism , Central Nervous System/injuries , Gene Expression Regulation , Meninges/metabolism , Oxygen/chemistry , Animals , Arachnoid/metabolism , Brain/metabolism , Cell Line, Tumor , Epithelial Cells/cytology , Epithelial-Mesenchymal Transition , Humans , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Receptors, Retinoic Acid/metabolism , Retinal Pigment Epithelium/cytology , Signal Transduction , Snail Family Transcription Factors , Transcription Factors/metabolism , Transforming Growth Factor beta1/metabolism , Tretinoin/metabolism
11.
PLoS One ; 9(8): e105185, 2014.
Article in English | MEDLINE | ID: mdl-25133627

ABSTRACT

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 (hARD1ΔN), which can no longer access the nucleus, resulted in cell morphology changes and cellular growth impairment. Notably, hARD1ΔN-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.


Subject(s)
Cell Nucleus/metabolism , N-Terminal Acetyltransferase A/metabolism , N-Terminal Acetyltransferase E/metabolism , Nuclear Localization Signals/metabolism , Cell Cycle/genetics , Cell Cycle/physiology , Cell Line , HEK293 Cells , HeLa Cells , Humans , N-Terminal Acetyltransferase A/genetics , N-Terminal Acetyltransferase E/genetics , Nuclear Localization Signals/genetics , Protein Transport/genetics , Protein Transport/physiology , S Phase/genetics , S Phase/physiology
12.
Oncotarget ; 5(13): 5087-99, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24970818

ABSTRACT

Overexpression of Notch1 has been associated with breast cancer. We recently showed that visfatin stimulates breast cancer cell proliferation and invasion. The present study was undertaken to determine whether Notch1 signaling is affected by visfatin and to characterize the functional role of the visfatin-Notch1 axis in breast cancer. Visfatin and Notch1 were expressed at higher levels in breast tumors than in matched control tissues. Visfatin induced Notch1 expression in MDA-MB-231 breast cancer cell line and in nontransformed MCF10A mammary epithelial cells, whereas visfatin depletion reduced Notch1 mRNA and protein levels. Depletion of Notch1 in MDA-MB-231 cells attenuated cell growth in vitro and in vivo; visfatin depletion produced similar effects, but was less potent. Additionally, Notch1 depletion inhibited cell proliferation induced by visfatin. Analysis of the signaling pathways underlying visfatin-mediated Notch1 upregulation revealed that visfatin activated NF-κB p65. Blockade of NF-κB signaling suppressed the effects of visfatin on Notch1 upregulation and breast cancer cell proliferation. Breast tumors expressing high levels of NF-κB p65 exhibited increased expression of Notch1. Our results demonstrate that the visfatin-Notch1 axis contributes to breast tumor growth through the activation of the NF-κB pathway. Study of the visfatin-Notch1 axis may offer new therapeutic directions for breast cancer.


Subject(s)
Breast Neoplasms/genetics , Cell Proliferation , Nicotinamide Phosphoribosyltransferase/genetics , Receptor, Notch1/genetics , Animals , Apoptosis/genetics , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , MCF-7 Cells , Mice, Nude , Nicotinamide Phosphoribosyltransferase/metabolism , RNA Interference , Receptor, Notch1/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factor RelA/metabolism , Tumor Burden/genetics , Up-Regulation , Xenograft Model Antitumor Assays/methods
13.
J Biol Chem ; 289(32): 21926-36, 2014 Aug 08.
Article in English | MEDLINE | ID: mdl-24917672

ABSTRACT

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.


Subject(s)
Cell Adhesion Molecules, Neuronal/physiology , Cell Movement/physiology , Macrophages/physiology , Nerve Growth Factors/physiology , Animals , Cell Adhesion/physiology , Cell Adhesion Molecules, Neuronal/deficiency , Cell Adhesion Molecules, Neuronal/genetics , Cell Line , Cell Membrane/physiology , Cells, Cultured , Endothelial Cells/physiology , Gene Knockdown Techniques , Inflammation/etiology , Inflammation/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Nerve Growth Factors/deficiency , Nerve Growth Factors/genetics , Neuropeptides/metabolism , Pseudopodia/physiology , RNA Interference , rac1 GTP-Binding Protein/metabolism
14.
PLoS One ; 9(4): e94695, 2014.
Article in English | MEDLINE | ID: mdl-24760034

ABSTRACT

The repair process after CNS injury shows a well-organized cascade of three distinct stages: inflammation, new tissue formation, and remodeling. In the new tissue formation stage, various cells migrate and form the fibrotic scar surrounding the lesion site. The fibrotic scar is known as an obstacle for axonal regeneration in the remodeling stage. However, the role of the fibrotic scar in the new tissue formation stage remains largely unknown. We found that the number of A-kinase anchoring protein 12 (AKAP12)-positive cells in the fibrotic scar was increased over time, and the cells formed a structure which traps various immune cells. Furthermore, the AKAP12-positive cells strongly express junction proteins which enable the structure to function as a physical barrier. In in vivo validation, AKAP12 knock-out (KO) mice showed leakage from a lesion, resulting from an impaired structure with the loss of the junction complex. Consistently, focal brain injury in the AKAP12 KO mice led to extended inflammation and more severe tissue damage compared to the wild type (WT) mice. Accordingly, our results suggest that AKAP12-positive cells in the fibrotic scar may restrict excessive inflammation, demonstrating certain mechanisms that could underlie the beneficial actions of the fibrotic scar in the new tissue formation stage during the CNS repair process.


Subject(s)
A Kinase Anchor Proteins/metabolism , Cell Cycle Proteins/metabolism , Central Nervous System/metabolism , Fibrosis/metabolism , A Kinase Anchor Proteins/genetics , Animals , Blotting, Western , Cell Cycle Proteins/genetics , Fibrosis/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Rats, Wistar , Wound Healing/genetics , Wound Healing/physiology
15.
J Biol Chem ; 289(6): 3328-38, 2014 Feb 07.
Article in English | MEDLINE | ID: mdl-24347169

ABSTRACT

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.


Subject(s)
Bone Marrow Cells/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Cell Movement , Encephalomyelitis, Autoimmune, Experimental/metabolism , Macrophages/metabolism , Nerve Growth Factors/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Bone Marrow Cells/pathology , Cell Adhesion Molecules, Neuronal/antagonists & inhibitors , Cell Adhesion Molecules, Neuronal/genetics , Cell Line , Disease Susceptibility , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/therapy , Macrophages/pathology , Mice , Mice, Knockout , Multiple Sclerosis/genetics , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Multiple Sclerosis/therapy , Nerve Growth Factors/antagonists & inhibitors , Nerve Growth Factors/genetics
16.
Korean J Physiol Pharmacol ; 17(4): 291-7, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23946688

ABSTRACT

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.

17.
J Pineal Res ; 55(3): 294-303, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23869429

ABSTRACT

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.


Subject(s)
Antioxidants/pharmacology , Cytokines/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Macrophages, Peritoneal/enzymology , Melatonin/pharmacology , Nicotinamide Phosphoribosyltransferase/metabolism , Nitric Oxide Synthase Type II/biosynthesis , Nitric Oxide/biosynthesis , Animals , Cell Line , Cytokines/genetics , Gene Expression Regulation, Enzymologic/genetics , Janus Kinase 2/genetics , Janus Kinase 2/immunology , Janus Kinase 2/metabolism , Janus Kinase 3/genetics , Janus Kinase 3/metabolism , Mice , Mice, Mutant Strains , Mitogen-Activated Protein Kinase 8/genetics , Mitogen-Activated Protein Kinase 8/metabolism , Mitogen-Activated Protein Kinase 9/genetics , Mitogen-Activated Protein Kinase 9/metabolism , Nicotinamide Phosphoribosyltransferase/genetics , Nitric Oxide/genetics , Nitric Oxide Synthase Type II/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Up-Regulation/drug effects , Up-Regulation/genetics
18.
Biochem Biophys Res Commun ; 428(4): 438-44, 2012 Nov 30.
Article in English | MEDLINE | ID: mdl-23142597

ABSTRACT

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.


Subject(s)
Cell Adhesion Molecules, Neuronal/chemistry , Chemotactic Factors/chemistry , Matrix Metalloproteinase 9/chemistry , Nerve Growth Factors/chemistry , Amino Acid Sequence , Animals , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Chemokines/chemistry , Chemokines/genetics , Chemokines/metabolism , Chemotactic Factors/genetics , Chemotactic Factors/metabolism , HEK293 Cells , Humans , Kidney/metabolism , Leucine/chemistry , Liver/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary
19.
Endocrinology ; 153(2): 554-63, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22186408

ABSTRACT

Obesity is frequently associated with breast cancer. Such associations are possibly mediated by adipokines. Visfatin, an adipokine, has recently been shown to be related to the development and progression of breast cancer. Therefore, the down-regulation of visfatin may be a novel strategy for breast cancer therapy. Curcumin has anticancer activities by modulating multiple signaling pathways and genes. The purpose of this study was to investigate whether visfatin gene expression is affected by curcumin in human breast cancer cells and to characterize the functional role of visfatin in breast cancer. We found that the mRNA and protein levels of visfatin were down-regulated by curcumin in MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cells, along with decreased activity of constitutive nuclear factor (NF)-κB. We confirmed the repressive effect of curcumin on visfatin transcription by performing a visfatin promoter-driven reporter assay and identified two putative NF-κB-binding sites on visfatin promoter that are important for this effect. EMSA and chromatin immunoprecipitation analysis indicated the binding of p65 to the visfatin promoter, which was effectively blocked by curcumin. Enforced expression of p65 protein increased visfatin promoter activity, whereas blocking NF-κB signaling suppressed visfatin gene expression. Visfatin could enhance the invasion of MDA-MB-231 cells and also attenuate curcumin-induced inhibition of cell invasion; on the other hand, visfatin knockdown by small interfering RNA led to the reduction of cell invasion. Our data demonstrate, for the first time, that curcumin down-regulates visfatin gene expression in human breast cancer cells by a mechanism that is, at least in part, NF-κB dependent and suggest that visfatin may contribute to breast cancer cell invasion and link obesity to breast cancer development and progression.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Curcumin/pharmacology , Cytokines/metabolism , Down-Regulation/drug effects , Nicotinamide Phosphoribosyltransferase/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cytokines/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Invasiveness , Nicotinamide Phosphoribosyltransferase/genetics , Obesity/metabolism , Promoter Regions, Genetic , Response Elements
20.
Cardiovasc Res ; 89(2): 436-45, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-20817637

ABSTRACT

AIMS: Our aims were to determine the role of Notch1 in mediating visfatin-induced angiogenesis and to explore potential target genes involved. METHODS AND RESULTS: Inhibition of Notch signalling attenuated visfatin-induced angiogenesis in vitro, ex vivo, and in vivo. Visfatin increased γ-secretase activity, Notch1 cleavage and activation, and Hes1 gene induction. Visfatin also stimulated fibroblast growth factor-2 (FGF-2) gene expression in a Notch1-dependent manner. Enforced expression of active Notch1 intracellular domain increased FGF-2 protein levels and stimulated endothelial tube formation, whereas blocking Notch1 signalling or knockdown of Notch1 by small interfering RNA suppressed visfatin-induced FGF-2 up-regulation and angiogenesis. Reporter analysis of FGF-2 promoter revealed the presence of CSL (CBF-1, suppressor of hairless, LAG-1)-binding site, and chromatin immunoprecipitation analysis demonstrated the binding of Notch1-CSL complex to this site in response to visfatin. CONCLUSION: Our data provide the first example of Notch1-dependent endothelial FGF-2 induction by visfatin and of Notch1 activation in visfatin-stimulated endothelial angiogenesis, suggesting that the signalling axis of visfatin/Notch1/angiogenic factors like FGF-2 might be a valuable target for pathological angiogenesis.


Subject(s)
Cytokines/metabolism , Endothelial Cells/enzymology , Fibroblast Growth Factor 2/metabolism , Neovascularization, Physiologic , Nicotinamide Phosphoribosyltransferase/metabolism , Receptor, Notch1/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Binding Sites , Cells, Cultured , Chick Embryo , Chromatin Immunoprecipitation , Dipeptides/pharmacology , Endothelial Cells/drug effects , Enzyme Inhibitors/pharmacology , Fibroblast Growth Factor 2/genetics , Genes, Reporter , Homeodomain Proteins/genetics , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Male , Mice , Mice, Inbred C57BL , Neovascularization, Physiologic/drug effects , Promoter Regions, Genetic , RNA Interference , Rats , Rats, Sprague-Dawley , Receptor, Notch1/genetics , Recombinant Proteins/metabolism , Signal Transduction , Transcription Factor HES-1 , Transfection , Up-Regulation
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