RESUMO
Synapses are fundamental units of communication in the brain. The prototypical synapse-organizing complex neurexin-neuroligin mediates synapse development and function and is central to a shared genetic risk pathway in autism and schizophrenia. Neurexin's role in synapse development is thought to be mediated purely by its protein domains, but we reveal a requirement for a rare glycan modification. Mice lacking heparan sulfate (HS) on neurexin-1 show reduced survival, as well as structural and functional deficits at central synapses. HS directly binds postsynaptic partners neuroligins and LRRTMs, revealing a dual binding mode involving intrinsic glycan and protein domains for canonical synapse-organizing complexes. Neurexin HS chains also bind novel ligands, potentially expanding the neurexin interactome to hundreds of HS-binding proteins. Because HS structure is heterogeneous, our findings indicate an additional dimension to neurexin diversity, provide a molecular basis for fine-tuning synaptic function, and open therapeutic directions targeting glycan-binding motifs critical for brain development.
Assuntos
Heparitina Sulfato/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Drosophila , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Glicopeptídeos/análise , Heparitina Sulfato/química , Humanos , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso , Moléculas de Adesão de Célula Nervosa/antagonistas & inibidores , Moléculas de Adesão de Célula Nervosa/genética , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Alinhamento de SequênciaRESUMO
Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.
Assuntos
Anticorpos Monoclonais , Membrana Celular , Inflamação , Fígado , Fatores de Crescimento Neural , Traumatismo por Reperfusão , Animais , Camundongos , Alanina Transaminase , Alarminas , Anticorpos Monoclonais/imunologia , Aspartato Aminotransferases , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/imunologia , Moléculas de Adesão Celular Neuronais/ultraestrutura , Morte Celular , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Concanavalina A , Galactosamina , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Inflamação/patologia , Lactato Desidrogenases , Fígado/patologia , Microscopia Eletrônica , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/ultraestrutura , Infiltração de Neutrófilos , Traumatismo por Reperfusão/patologiaRESUMO
[Figure: see text].
Assuntos
Anticorpos Neutralizantes/farmacologia , Aterosclerose/prevenção & controle , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Proteínas da Matriz Extracelular/antagonistas & inibidores , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacologia , Animais , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Camundongos Transgênicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Placa Aterosclerótica , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteína Reelina , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Transdução de Sinais , Células U937Assuntos
Autoanticorpos/sangue , Doenças Autoimunes/diagnóstico , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Imunoterapia , Doenças Neurodegenerativas/etiologia , Idoso , Doenças Autoimunes/terapia , Diagnóstico Diferencial , Transtornos Neurológicos da Marcha/etiologia , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Masculino , Doença dos Neurônios Motores/etiologia , Transtornos do Sono-Vigília/etiologiaRESUMO
It has been shown that overexpression of activated leukocyte cell adhesion molecule (ALCAM) is involved in development of resistance to tamoxifen therapy and promotion of cell invasion, migration and metastasis in ER+ breast cancer cells. Thus, we hypothesized that blockade of ALCAM interconnections with antibodies could be an effective approach for reversing mentioned negative events associated with ALCAM overexpression in breast cancer cells. Here, an anti-ALCAM scFv was recombinantly expressed and used throughout study for examination of the putative anticancer effects of ALCAM blockade. The anti-ALCAM scFv coding sequence was obtained from GenBank database and after addition of a 6× His-tag moiety, signal peptide and flanking sequences, the whole construct was expressed in Escherichia coli. Tamoxifen resistant MCF7 cells were then pretreat for 24 hours with purified recombinant anti-ALCAM scFv prior to administration of tamoxifen. In parallel, the cytotoxicity profile of anti-ALCAM scFv and tamoxifen co-treatments against tamoxifen resistant and sensitive MCF7 cell lines was also evaluated using CompuSyn software. The invasion/migration inhibitory effects of anti-ALCAM scFv on MDA-MB-231 cells were also evaluated. Pretreatment with anti-ALCAM scFv could successfully enhance anti-proliferative effects of tamoxifen against resistant MCF-7 cell lines. Furthermore, the combination of 19.2:1 of tamoxifen to anti-ALCAM scFv demonstrated synergistic cell inhibitory effect against tamoxifen resistant MCF7 cell lines. Also, incubating MDA-MB-231 cell lines with anti-ALCAM scFv resulted in a 30% and 25% reduction in number of invaded and migrated cells respectively. Overall, application of anti-ALCAM scFv could significantly suppress cancer cells metastasis in vitro and modulate tamoxifen resistant ER+ MCF7 cell line's sensitivity to tamoxifen. SIGNIFICANCE OF THE STUDY: Acquisition of resistance to tamoxifen therapy is one of the major challenges associated with cancer chemotherapy, gradually turning a responsive tumour into a refractory more invasive one which ultimately ends in disease progression and relapse. Here, we reported expression of an anti-ALCAM scFv, capable of increasing the sensitivity of tamoxifen resistant ER+ MCF-7 cells to tamoxifen therapy following a 24-hour pretreatment period. In addition, we demonstrated that the anti-ALCAM scFv monotherapy was also capable of suppressing invasion and migration of MDA-MB-231 cells in Boyden chamber assays.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Proteínas Fetais/antagonistas & inibidores , Tamoxifeno/farmacologia , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Allergic asthma causes morbidity in many subjects, and novel precision-directed treatments would be valuable. OBJECTIVE: We sought to examine the role of a novel innate molecule, repulsive guidance molecule b (RGMb), in murine models of allergic asthma. METHODS: In models of allergic asthma using ovalbumin or cockroach allergen, mice were treated with anti-RGMb or control mAb and examined for airway inflammation and airway hyperreactivity (AHR), a cardinal feature of asthma. The mechanisms by which RGMb causes airways disease were also examined. RESULTS: We found that blockade of RGMb by treatment with anti-RGMb mAb effectively blocked the development of airway inflammation and AHR. Importantly, blockade of RGMb completely blocked the development of airway inflammation and AHR, even if treatment occurred only during the challenge (effector) phase. IL-25 played an important role in these models of asthma because IL-25 receptor-deficient mice did not develop disease after sensitization and challenge with allergen. RGMb was expressed primarily by innate cells in the lungs, including bronchial epithelial cells (known producers of IL-25), activated eosinophils, and interstitial macrophages, which in the inflamed lung expressed the IL-25 receptor and produced IL-5 and IL-13. We also found that neogenin, the canonical receptor for RGMb, was expressed by interstitial macrophages and bronchial epithelial cells in the inflamed lung, suggesting that an innate RGMb-neogenin axis might modulate allergic asthma. CONCLUSIONS: These results demonstrate an important role for a novel innate pathway in regulating type 2 inflammation in patients with allergic asthma involving RGMb and RGMb-expressing cells, such as interstitial macrophages and bronchial epithelial cells. Moreover, targeting this previously unappreciated innate pathway might provide an important treatment option for allergic asthma.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/tratamento farmacológico , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Alérgenos/imunologia , Animais , Asma/imunologia , Hiper-Reatividade Brônquica/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Moléculas de Adesão Celular Neuronais/imunologia , Baratas/imunologia , Feminino , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Macrófagos/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/genética , Receptores de Interleucina/genética , Receptores de Interleucina/imunologiaRESUMO
The Stabilin receptors are systemic clearance receptors for some classes of chemically modified nucleic acid therapeutics. In this study, the recombinant human secreted ecto-domain of the small isoform of Stabilin-2 (s190) was purified from cell culture and evaluated for direct binding with a multitude of antisense oligonucleotides (ASOs) using a fluorescence polarization-based assay. The tested ASOs varied in their backbone composition, modification of the ribose 2' position, overall length of the oligo, and sequence of the nucleotide bases. A fully phosphorothioate (PS) ASO with a 5-10-5 pattern of flanking 2'- O-methoxyethyl modifications was then used to test the effects of pH and salt concentration on receptor binding. These tests concluded that the PS backbone was the primary determinant for ASO binding and that decreasing pH and increasing salt generally increased the rate of ligand dissociation and fit within the biological parameters expected of a constitutive recycling receptor. These results will be useful in the rational design of therapeutic oligonucleotides for enhancing their affinity or avoidance of the Stabilin receptors.
Assuntos
Moléculas de Adesão Celular Neuronais/química , Polarização de Fluorescência , Oligodesoxirribonucleotídeos Antissenso/química , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Humanos , Concentração de Íons de Hidrogênio , Domínios Proteicos , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
Ongoing efforts to remove pathological inflammatory stimuli are crucial for the protection of endothelial cells in diabetes. Nerve injury-induced protein 1 (Ninj1) is an adhesion molecule that not only contributes to inflammation but also regulates the apoptosis of endothelial cells. In the present study, Ninj1 was found highly expressed in endothelial cells in Type 2 diabetic mice and increased in high-glucose (HG) cultured HUVECs. Furthermore, we found that Ninj1 levels are up-regulated in endothelial cells in clinical specimens of diabetic patients when compared with nondiabetic tissues, indicating a biological correlation between Ninj1 and endothelial pathophysiology in diabetic condition. Functional blocking of Ninj1 promoted endothelial tube formation and eNOS phosphorylation in the HG condition. Additionally, blocking Ninj1 inhibited the activation of caspase-3 and increased the Bcl-2/Bax ratio, thus inhibiting HUVECs apoptosis induced by HG. HG-induced ROS overproduction, p38 MAPK and NF-κB activation, and the overexpression of VCAM-1, ICAM-1, MCP-1, and IL-6 genes were ameliorated after Ninj1 was blocked. Using the signaling pathway inhibitor LY294002, we found that Bcl-2 expression and eNOS phosphorylation after Ninj1 blockade were regulated via PI3K/Akt signaling pathway. The in vivo endothelial contents, α-SMA+PECAM-1+ vascular numbers, and blood perfusion in the hindlimb were markedly up-regulated after Ninj1 was blocked. According to our findings, functional blocking of Ninj1 shows protective effects on diabetic endothelial cells both in vitro and in vivo Thus, we consider Ninj1 to be a potential therapeutic target for preventing endothelial dysfunction in diabetes mellitus.
Assuntos
Anticorpos Bloqueadores/farmacologia , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais/efeitos dos fármacos , Fatores de Crescimento Neural/antagonistas & inibidores , Animais , Moléculas de Adesão Celular Neuronais/imunologia , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacosRESUMO
The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas α1 has a more conventional role of maintaining ion homeostasis. The ß subunit differentially regulates maturation, trafficking, and activity of α-ß heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three ß isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with ß2 in T-tubules of cardiac myocytes, forming α2ß2 heterodimers. We have expressed human α1ß1, α2ß1, α2ß2, and α2ß3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2ß2 and α2ß3 differ significantly from both α2ß1 and α1ß1 in having a higher K0.5K+ and lower K0.5Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2ß2 and α2ß3 over α1ß1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2ß2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2ß2 for cardiac muscle contractility. The high sensitivity of α2ß2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2ß2-selective digoxin derivatives for reducing cardiotoxicity.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Inibidores Enzimáticos/química , Miocárdio/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Animais , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/química , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/química , Dimerização , Inibidores Enzimáticos/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Camundongos , Miocárdio/química , Potássio/química , Potássio/metabolismo , Sódio/química , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Penile erection is a neurovascular phenomenon, and erectile dysfunction (ED) is caused mainly by vascular risk factors or diseases, neurologic abnormalities, and hormonal disturbances. Men with diabetic ED often have severe endothelial dysfunction and peripheral nerve damage, which result in poor response to oral phosphodiesterase-5 inhibitors. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. Here, we demonstrate in streptozotocin-induced diabetic mice that inhibition of the Ninj1 pathway by administering Ninj1-neutralizing antibody (Ninj1-Ab) or by using Ninj1-knockout mice successfully restored erectile function through enhanced penile angiogenesis and neural regeneration. Angiopoietin-1 (Ang1) expression was down-regulated and angiopoietin-2 expression was up-regulated in the diabetic penis compared with that in controls, and these changes were reversed by treatment with Ninj1-Ab. Ninj1 blockade-mediated penile angiogenesis and neural regeneration as well as recovery of erectile function were abolished by inhibition of Ang1-Tie2 (tyrosine kinase with Ig and epidermal growth factor homology domain-2) signaling with soluble Tie2 antibody or Ang1 siRNA. The present results suggest that inhibition of the Ninj1 pathway will be a novel therapeutic strategy for treating ED.
Assuntos
Anticorpos Neutralizantes/farmacologia , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Complicações do Diabetes/tratamento farmacológico , Disfunção Erétil/tratamento farmacológico , Neovascularização Fisiológica/fisiologia , Fatores de Crescimento Neural/antagonistas & inibidores , Regeneração Nervosa/fisiologia , Ereção Peniana/fisiologia , Análise de Variância , Angiopoietina-1/metabolismo , Animais , Western Blotting , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/imunologia , Primers do DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/imunologia , Regeneração Nervosa/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Ereção Peniana/efeitos dos fármacos , Receptor TIE-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Reelin is an extracellular matrix glycoprotein involved in the modulation of synaptic plasticity and essential for the proper radial migration of cortical neurons during development and for the integration and positioning of dentate granular cell progenitors; its expression is down-regulated as brain maturation is completed. Trimethyltin (TMT) is a potent neurotoxicant which causes selective neuronal death mainly localised in the CA1-CA3/hilus hippocampal regions. In the present study we analysed the expression of reelin and the modulation of endogenous neurogenesis in the postnatal rat hippocampus during TMT-induced neurodegeneration (TMT 6 mg/kg). Our results show that TMT administration induces changes in the physiological postnatal decrease of reelin expression in the hippocampus of developing rats. In particular, quantitative analysis of reelin-positive cells evidenced, in TMT-treated animals, a persistent reelin expression in the stratum lacunosum moleculare of Cornu Ammonis and in the molecular layer of Dentate Gyrus. In addition, a significant decrease in the number of bromodeoxyuridine (BrdU)-labeled newly-generated cells was also detectable in the subgranular zone of P21 TMT-treated rats compared with P21 control animals; no differences between P28 TMT-treated rats and age-matched control group were observed. In addition the neuronal commitment of BrdU-positive cells appeared reduced in P21 TMT-treated rats compared with P28 TMT-treated animals. Thus TMT treatment, administrated during development, induces an early reduction of endogenous neurogenesis and influences the hippocampal pattern of reelin expression in a temporally and regionally specific manner, altering the physiological decrease of this protein.
Assuntos
Moléculas de Adesão Celular Neuronais/biossíntese , Proteínas da Matriz Extracelular/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Neurogênese/fisiologia , Serina Endopeptidases/biossíntese , Compostos de Trimetilestanho/farmacologia , Animais , Animais Recém-Nascidos , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Neurogênese/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Ratos , Ratos Wistar , Proteína Reelina , Serina Endopeptidases/genéticaRESUMO
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.
Assuntos
Células da Medula Óssea/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular , Encefalomielite Autoimune Experimental/metabolismo , Macrófagos/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Células da Medula Óssea/patologia , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/terapia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/genéticaRESUMO
The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Rede Nervosa/crescimento & desenvolvimento , Rede Nervosa/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Moléculas de Adesão de Célula Nervosa/antagonistas & inibidores , Moléculas de Adesão de Célula Nervosa/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canais de Potássio Shab/genética , Canais de Potássio Shab/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND AND PURPOSE: MicroRNA (miR)-200c increases rapidly in the brain after transient cerebral ischemia but its role in poststroke brain injury is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-200c. We hypothesized that miR-200c contributes to injury from transient cerebral ischemia by targeting reelin. METHODS: Brain infarct volume, neurological score and levels of miR-200c, reelin mRNA, and reelin protein were assessed in mice subjected to 1 hour of middle cerebral artery occlusion with or without intracerebroventricular infusion of miR-200c antagomir, mimic, or mismatch control. Direct targeting of reelin by miR-200c was assessed in vitro by dual luciferase assay and immunoblot. RESULTS: Pretreatment with miR-200c antagomir decreased post-middle cerebral artery occlusion brain levels of miR-200c, resulting in a significant reduction in infarct volume and neurological deficit. Changes in brain levels of miR-200c inversely correlated with reelin protein expression. Direct targeting of the Reln 3' untranslated region by miR-200c was verified with dual luciferase assay. Inhibition of miR-200c resulted in an increase in cell survival subsequent to in vitro oxidative injury. This effect was blocked by knockdown of reelin mRNA, whereas application of reelin protein afforded protection. CONCLUSIONS: These findings suggest that the poststroke increase in miR-200c contributes to brain cell death by inhibiting reelin expression, and that reducing poststroke miR-200c is a potential target to mitigate stroke-induced brain injury.
Assuntos
Isquemia Encefálica/metabolismo , Moléculas de Adesão Celular Neuronais/biossíntese , Proteínas da Matriz Extracelular/biossíntese , MicroRNAs/administração & dosagem , MicroRNAs/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Serina Endopeptidases/biossíntese , Animais , Isquemia Encefálica/patologia , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Células Cultivadas , Proteínas da Matriz Extracelular/antagonistas & inibidores , Marcação de Genes , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteína ReelinaRESUMO
BACKGROUND: Capillary pericytes (cPCs), the mural cells of microvessels, play an important role in the formation and maintenance of microvessels; however, little is known about the mechanisms of how cPCs regulate angiogenesis. To identify factors that modulate cPC function, genes whose levels were altered in cPCs during neovessel formation were identified through a microarray screen. METHODS AND RESULTS: Ninjurin1 (nerve injury-induced protein, Ninj1) was selected as a candidate factor for angiogenesis regulation. Ninj1 was expressed in capillary cells including endothelial cells (cECs) and was expressed at a higher level in cPCs. Hypoxia induced the gene expression of Ninj1 in addition of vascular endothelial growth factor (VEGF) in cPCs. When cPCs were co-incubated with a thoracic aorta in a three-dimensional Matrigel system, the length of the EC-tubes sprouting from the aorta was increased. Small interfering RNA-mediated downregulation of Ninj1 in cPCs enhanced these cPCs-mediated angiogenic effects, whereas overexpression of Ninj1 attenuated their effects. The production of angiogenic growth factors, such as VEGF and angiopoietin 1, by cPCs was enhanced by the downregulation of Ninj1, and reduced by the overexpression of Ninj1. CONCLUSIONS: Ninj1 is a novel regulator for the angiogenic effect of PCs. Specifically, Ninj1 negatively regulates the formation of neovessels, that is, the EC-tube, by reducing the trophic effects of cPCs.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Células Endoteliais/citologia , Neovascularização Fisiológica/fisiologia , Fatores de Crescimento Neural/fisiologia , Pericitos/citologia , Animais , Aorta Torácica , Capilares , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/biossíntese , Moléculas de Adesão Celular Neuronais/genética , Técnicas de Cultura de Células , Hipóxia Celular , Linhagem Celular Transformada , Linhagem da Célula , Técnicas de Cocultura , Colágeno , Combinação de Medicamentos , Perfilação da Expressão Gênica , Genes Reporter , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Isquemia/patologia , Laminina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Miócitos de Músculo Liso , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/genética , Técnicas de Cultura de Órgãos , Proteoglicanas , Interferência de RNA , RNA Interferente Pequeno/farmacologiaRESUMO
Hyaluronic acid (HA) has been implicated in the proliferation and metastasis of tumor cells. However, most previous studies were conducted on extracellular matrix or pericellular HA, and the role of circulating HA in vivo has not been studied. HA is rapidly cleared from the bloodstream. The scavenger receptor Stabilin-2 (Stab2) is considered a major clearance receptor for HA. Here we report a dramatic elevation in circulating HA levels in Stab2-deficient mice without any overt phenotype. Surprisingly, the metastasis of B16F10 melanoma cells to the lungs was markedly suppressed in the Stab2-deficient mice, whereas cell proliferation was not affected. Furthermore, administration of an anti-Stab2 antibody in Stab2(+) mice elevated serum HA levels and prevented the metastasis of melanoma to the lung, and also suppressed spontaneous metastasis of mammary tumor and human breast tumor cells inoculated in the mammary gland. Administration of the antibody or high-dose HA in mice blocked the lodging of melanoma cells to the lungs. Furthermore, HA at high concentrations inhibited the rolling/tethering of B16 cells to lung endothelial cells. These results suggest that blocking Stab2 function prevents tumor metastasis by elevating circulating HA levels. Stab2 may be a potential target in antitumor therapy.
Assuntos
Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Ácido Hialurônico/sangue , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Animais , Anticorpos/administração & dosagem , Anticorpos/farmacologia , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular Neuronais/metabolismo , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/secundário , Melanoma Experimental/sangue , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Metástase Neoplásica/imunologiaRESUMO
Cluster of differentiation 166 (CD166) is critical for liver cancer cell survival. Our previously study demonstrated that CD166 exerts its anti-apoptotic role through interaction with YAP in liver cancer. However, the interaction between CD166 and other cell surface molecules remains unclear in liver cancer cells. In the current study, we found that both mRNA and protein of CD44 expression was significantly inhibited by knocking-down CD166. Moreover, CD166 affected-CD44 expression is dependent of transcription via blocking NF-κB pathway. On the contrary, CD44 promoted up-regulation of CD166 mRNA and protein. And it may be through E3 ubiquitin ligases COP1 and UBC3 to regulate CD166 protein degradation. Collectively, these results suggest that CD166 and CD44 play important roles in liver cancer development. Therefore, CD166 may develop as a potential therapeutic molecule target for the treatment of liver cancer.
Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , NF-kappa B/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Fetais/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/patologia , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Transdução de Sinais , Fatores de Transcrição , Regulação para Cima , Proteínas de Sinalização YAPRESUMO
Adult hippocampal neurogenesis is thought to be essential for learning and memory, and has been implicated in the pathogenesis of several disorders. Although recent studies have identified key factors regulating neuroprogenitor proliferation in the adult hippocampus, the mechanisms that control the migration and integration of adult-born neurons into circuits are largely unknown. Reelin is an extracellular matrix protein that is vital for neuronal development. Activation of the Reelin cascade leads to phosphorylation of Disabled-1, an adaptor protein required for Reelin signaling. Here we used transgenic mouse and retroviral reporters along with Reelin signaling gain-of-function and loss-of-function studies to show that the Reelin pathway regulates migration and dendritic development of adult-generated hippocampal neurons. Whereas overexpression of Reelin accelerated dendritic maturation, inactivation of the Reelin signaling pathway specifically in adult neuroprogenitor cells resulted in aberrant migration, decreased dendrite development, formation of ectopic dendrites in the hilus, and the establishment of aberrant circuits. Our findings support a cell-autonomous and critical role for the Reelin pathway in regulating dendritic development and the integration of adult-generated granule cells and point to this pathway as a key regulator of adult neurogenesis. Moreover, our data reveal a novel role of the Reelin cascade in adult brain function with potential implications for the pathogenesis of several neurological and psychiatric disorders.
Assuntos
Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Proteínas da Matriz Extracelular/antagonistas & inibidores , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neurogênese/genética , Transdução de Sinais/genética , Fatores Etários , Envelhecimento/genética , Animais , Moléculas de Adesão Celular Neuronais/fisiologia , Linhagem Celular , Células Cultivadas , Proteínas da Matriz Extracelular/fisiologia , Inativação Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/fisiologia , Ratos , Ratos Sprague-Dawley , Proteína Reelina , Serina Endopeptidases/fisiologiaRESUMO
INTRODUCTION: Radical prostatectomy for prostate cancer can not only induce cavernous nerve injury (CNI) but also result in structural changes in the cavernous tissues. Nerve injury-induced protein 1, Ninjurin-1 (Ninj1), is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. AIM: The study aims to determine whether and how Ninj1 neutralizing antibody (Ninj1-Ab) restores erectile function in mice with CNI. METHODS: Twelve-week-old C57BL/6J mice were used and distributed into four groups: sham operation group and CNI groups receiving a single intracavernous injection of immunoglobulin G (IgG) control antibody, low-dose Ninj1-Ab (1.0 µg/20 µL), or high-dose Ninj1-Ab (2.5 µg/20 µL). MAIN OUTCOME MEASURES: One week after bilateral cavernous nerve crush, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis. RESULTS: The cavernous expression of Ninj1 protein was upregulated up to 7 days after CNI and returned to baseline levels thereafter. Local delivery of Ninj1-Ab significantly increased penile neuronal nitric oxide synthase and neurofilament contents, induced cavernous endothelial proliferation and phosphorylation of Akt and endothelial nitric oxide synthase, and decreased endothelial cell apoptosis in the CNI mice by upregulating angiopoietin-1 and downregulating angiopoietin-2. High-dose Ninj1-Ab induced profound restoration of erectile function in the CNI mice (91% of sham control values), whereas low-dose Ninj1-Ab elicited partial improvement. CONCLUSION: The dual neurotrophic and angiogenic effects of Ninj1 blockade may provide a good opportunity for treating erectile dysfunction resulting from radical prostatectomy.
Assuntos
Anticorpos Neutralizantes/farmacologia , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Disfunção Erétil/tratamento farmacológico , Fatores de Crescimento Neural/antagonistas & inibidores , Ereção Peniana/efeitos dos fármacos , Pênis/efeitos dos fármacos , Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Animais , Anticorpos Neutralizantes/administração & dosagem , Moléculas de Adesão Celular Neuronais/imunologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Estimulação Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Disfunção Erétil/metabolismo , Fibrose , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Compressão Nervosa , Fatores de Crescimento Neural/imunologia , Regeneração Nervosa/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Pênis/inervação , Pênis/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Tumor progression and metastasis are promoted by the remodeling of organized tissue architecture and engagement of molecular interactions that support tumor cell passage through endothelial barriers. Prostate tumor cells that secrete and turn over excessive quantities of pericellular hyaluronan (HA) exhibit accelerated growth kinetics and spontaneous lymph node metastasis in mice. The HA receptor for endocytosis (HARE) is an endocytic clearance receptor for HA in the liver that is also highly expressed in sinusoidal endothelium of lymph nodes and bone marrow, which are frequent sites of prostate cancer metastasis. In our study, we tested the hypothesis that HARE can act as an endothelial receptor for metastatic tumor cells with pericellular HA. In an orthotopic mouse model of prostate cancer, we delivered a monoclonal antibody against HARE that specifically blocks HA binding and internalization. This treatment fully blocked the formation of metastatic tumors in lymph nodes. No effects on primary tumor growth were observed and the antibody did not induce toxic outcomes in any other tissue. Our results implicate HARE for the first time in potentiation of tumor metastasis and suggest a novel mechanism by which tumor cell-associated HA could promote tissue-specific dissemination. "Published 2012 Wiley Periodicals, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America."