RESUMEN
It has been frequently reported that myostatin inhibition increases muscle mass, but decreases muscle quality (i.e., strength/muscle mass). Resistance exercise training (RT) and essential amino acids (EAAs) are potent anabolic stimuli that synergistically increase muscle mass through changes in muscle protein turnover. In addition, EAAs are known to stimulate mitochondrial biogenesis. We have investigated if RT amplifies the anabolic potential of myostatin inhibition while EAAs enhance muscle quality through stimulations of mitochondrial biogenesis and/or muscle protein turnover. Mice were assigned into ACV (myostatin inhibitor), ACV+EAA, ACV+RT, ACV+EAA +RT, or control (CON) over 4 weeks. RT, but not EAA, increased muscle mass above ACV. Despite differences in muscle mass gain, myofibrillar protein synthesis was stimulated similarly in all vs. CON, suggesting a role for changes in protein breakdown in muscle mass gains. There were increases in MyoD expression but decreases in Atrogin-1/MAFbx expression in ACV+EAA, ACV+RT, and ACV+EAA+RT vs. CON. EAA increased muscle quality (e.g., grip strength and maximal carrying load) without corresponding changes in markers of mitochondrial biogenesis and neuromuscular junction stability. In conclusion, RT amplifies muscle mass and strength through changes in muscle protein turnover in conjunction with changes in implicated signaling, while EAAs enhance muscle quality through unknown mechanisms.
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Aminoácidos Esenciales/administración & dosificación , Proteínas en la Dieta/administración & dosificación , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Miostatina/antagonistas & inhibidores , Condicionamiento Físico Animal , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Fuerza Muscular/fisiología , Músculo Esquelético/fisiología , Entrenamiento de FuerzaRESUMEN
Following the clinical success of immunotherapeutic antibodies, bispecific antibodies for cytotoxic effector cell redirection, tumor-targeted immunomodulation and dual immunomodulation, have received particular attentions. Here, we developed a novel bispecific antibody platform, termed Antibody-Like Cell Engager (ALiCE), wherein the Fc domain of each heavy chain of immunoglobulin G (IgG) is replaced by the VH and VL domains of an IgG specific to a second antigen while retaining the N-terminal Fab of the parent antibody. Because of specific interactions between the substituted VH and VL domains, the C-terminal stem Fv enables ALiCE to assemble autonomously into hetero-tetramers, thus simultaneously binding to two distinct antigens but with different avidities. This design strategy was used to generate ACE-05 (two anti-PD-L1 Fab × anti-CD3 Fv) and ACE-31 (two anti-CD3 Fab × anti-PD-L1 Fv), both of which bound PD-L1 and CD3. However, ACE-05 was more effective than ACE-31 in reducing off-target toxicity caused by the indiscriminate activation of T cells. Moreover, in cell-based assays and PBMC-reconstituted humanized mice harboring human non-small-cell lung cancer tumors, ACE-05 showed marked antitumor efficacy, causing complete tumor regression at a dose of 0.05 mg/kg body weight. The dual roles of ACE-05 in immune checkpoint inhibition and T-cell redirection, coupled with reduced off-target toxicity, suggest that ACE-05 may be a promising anti-cancer therapeutic agent. Moreover, the bispecific ALiCE platform can be further used for tumor-targeted or multiple immunomodulation applications.
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Anticuerpos Biespecíficos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Anticuerpos Biespecíficos/uso terapéutico , Complejo CD3 , Leucocitos Mononucleares , Ratones , Linfocitos TRESUMEN
This study was aimed at generating and investigating the efficacy of a novel monoclonal bispecific antibody (BsAb) for the combined inhibition of tumor necrosis factor-α (TNF-α) and CXCL10 as a treatment option for rheumatoid arthritis (RA). A novel BsAb targeting TNF-α and CXCL10 was generated by conjugating a single-chain variable fragment (scFv) of the anti-CXCL10 monoclonal antibody to the Fc region of adalimumab (ADA). The effects of the BsAb on the inflammatory response in the in vitro and in vivo development of arthritis and joint destruction were evaluated in human TNF transgenic (hTNF-Tg) mice, and K/BxN serum transfer arthritis models. The BsAb inhibited CXCL10-mediated CD8+ T cell migration. The binding affinity of the BsAb to TNF-α was comparable to that of ADA and suppressed TNF-α induced cell death and inhibited TNF-α induced ICAM-1 and VCAM-1 in RA fibroblast-like synoviocytes (FLSs). The BsAb decreased the expression of TNFSF11 and the production of IL-6 in RA-FLS cells stimulated with TNF-α and CXCL10. Treatment with the BsAb attenuated the development of arthritis in hTNF-Tg mice and suppressed LPS-induced bone erosion. In the K/BxN serum transfer model, BsAb effectively attenuated ankle swelling, synovial inflammation, cartilage damage, and bone destruction, reducing the activation of osteoclasts. The additional neutralization of TNF-α and CXCL10 from treatment with the novel BsAb was more effective than TNF-α inhibition alone in the in vitro and in vivo models of RA. Thus, the BsAb, targeting both TNF-α and CXCL10, may provide a new therapeutic opportunity for RA patients who fail to respond to the blockade of a single cytokine.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Artritis Experimental/terapia , Artritis Reumatoide/terapia , Quimiocina CXCL10/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Adalimumab , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Quimiocina CXCL10/antagonistas & inhibidores , Clonación Molecular , Cruzamientos Genéticos , Humanos , Fragmentos Fc de Inmunoglobulinas , Factores Inmunológicos , Inmunoterapia/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Anticuerpos de Cadena Única , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
OBJECTIVES: The aim of this study was to discover new potential biomarkers of breast cancer and investigate their cellular functions. MATERIALS AND METHODS: We analysed the gene expression profiles of matched pairs of breast tumour and normal tissues from 24 breast cancer patients. Tetracycline-inducible MAMDC2 expression system was established and used to evaluate cell proliferation in vitro and in vivo. MAMDC2-mediated signalling was determined using immunoblot analysis. RESULTS: We identified MAMDC2 as a down-regulated gene showing significant prognostic capability. Overexpression of MAMDC2 or treatment with MAMDC2-containing culture medium significantly inhibited the cell proliferation of T-47D cells. Furthermore, MAMDC2 expression reduced in vivo growth of T-47D xenograft tumours. MAMDC2 may exert its growth-inhibitory functions by attenuating the MAPK signalling pathway. CONCLUSION: We report that MAMDC2 has a tumour-suppressive role and, as a secretory protein, it might be useful as a biomarker for breast cancer treatment.
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Neoplasias de la Mama/genética , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Animales , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Mama/patología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Genes Supresores de Tumor , Glicosilación , Humanos , Ratones , Persona de Mediana EdadRESUMEN
This study was aimed at investigating the therapeutic effects of BITRAP, a bispecific fusion protein targeting TNF-α and IL-21, on the development of autoimmune arthritis in humans and mice. To verify the effects of BITRAP in human, peripheral blood mononuclear cells were cultured with BITRAP under IL-17-producing T (Th17) cell-polarizing conditions or osteoclast differentiation conditions. BITRAP treatment inhibited the production of IL-17 and vascular endothelial growth factor but increased the production of IL-10 in CD4+ T cells, as well as directly suppressed osteoclastogenesis. Collagen-induced arthritis (CIA) and IL-1R antagonist (IL-1Ra) knockout mice were treated with BITRAP. Following injection in CIA mice, BITRAP rapidly migrated into the inflamed joints and remained there for 72 hours. Application of BITRAP attenuated the severity of autoimmune arthritis in CIA and IL-1Ra knockout mice by reducing the numbers of inflammatory cytokine-expressing cells and Th17 cells and antibody secretion. Finally, BITRAP suppressed STAT3 phosphorylation, as well as production of IL-17 and TNF-α, in murine splenic CD4+ T cells. These findings suggest that BITRAP, a bispecific fusion protein targeting TNF-α and IL-21, may be an effective treatment to overcome the limitations of anti-TNF therapy for patients with rheumatoid arthritis.
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Artritis/tratamiento farmacológico , Interleucinas/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Factores de Coagulación Sanguínea , Linfocitos T CD4-Positivos , Fibroblastos , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Inmunoglobulinas/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Osteogénesis/efectos de los fármacos , Ingeniería de Proteínas , Proteínas Recombinantes , Células Th17 , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Proteolysis of delta-like 1 homolog (DLK1), a cell-surface transmembrane protein, produces an active soluble form of DLK1 (sDLK1). Both membrane-bound DLK1 and sDLK1 modulate multiple developmental processes including adipogenesis, osteogenesis, chondrogenesis and myogenesis. However, cancer-related functions of DLK1 have not yet been established. We thus evaluated the roles of extracellular sDLK1, comprising six EGF-like domains and juxtamembrane regions, in human pancreatic cancer MIA PaCa-2 cells in vitro and in vivo. We observed that sDLK1 exerted antitumor effects not only in cancer cell migration and anchorage-independent cell growth but also in in vivo tumor growth.
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Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Páncreas/metabolismo , Páncreas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Animales , Células 3T3 BALB , Proteínas de Unión al Calcio , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/patología , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/análisis , Proteínas de la Membrana/análisis , Ratones , Ratones Desnudos , Dominios Proteicos , ProteolisisRESUMEN
G protein-coupled receptors (GPCRs) are among the most significant therapeutic targets and some of them promote the growth and metastasis of cancer. Here, we show that an increase in the levels of GPR171 is crucial for lung cancer tumor progression in vitro and in vivo. Immunostaining of clinical samples indicated that GPR171 was overexpressed in 46.8% of lung carcinoma tissues. Depletion of GPR171 with an anti-GPR171 antibody decreased proliferation of lung carcinoma cells and attenuated tumor progression in a mouse xenograft model. Knockdown of GPR171 also inhibited migration and invasion of the lung cancer cell lines. Notably, inhibition of GPR171 synergistically enhanced the tumoricidal activity of an epidermal growth factor receptor (EGFR) inhibitor in lung cancer cells. These results indicate that GPR171 blockade is a promising antineoplastic strategy and provide a preclinical rationale for combined inhibition of GPR171 and EGFR.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Movimiento Celular , Proliferación Celular , Neoplasias Pulmonares/patología , Receptores Acoplados a Proteínas G/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Animales , Apoptosis , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Estadificación de Neoplasias , Pronóstico , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cyclin-G-associated kinase (GAK), the ubiquitously expressed J-domain protein, is essential for the chaperoning and uncoating of clathrin that is mediated by Hsc70 (also known as HSPA8). Adjacent to the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain through a PTEN-like domain. Knocking out GAK in fibroblasts caused inhibition of clathrin-dependent trafficking, which was rescued by expressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene in mice rescued the lethality and the histological defects caused by knocking out GAK in the liver or in the brain. Furthermore, when both GAK and auxilin (also known as DNAJC6), the neuronal-specific homolog of GAK, were knocked out in the brain, mice expressing the 62-kDa GAK fragment were viable, lived a normal life-span and had no major behavior abnormalities. However, these mice were about half the size of wild-type mice. Therefore, the PTEN-like domains of GAK and auxilin are not essential for Hsc70-dependent chaperoning and uncoating of clathrin, but depending on the tissue, these domains appear to increase the efficiency of these co-chaperones.
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Encéfalo/metabolismo , Clatrina/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Auxilinas/genética , Auxilinas/metabolismo , Clatrina/genética , Proteínas del Choque Térmico HSC70/genética , Ratones , Ratones Noqueados , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiologíaRESUMEN
Cellular prion protein (PrP(C)) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP(C) in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP(C) protein on human natural killer (NK) cells. Recombinant soluble PrP(C) protein was generated by fusion of human PrP(C) with the Fc portion of human IgG1 (PrP(C)-Fc). PrP(C)-Fc binds to the surface of human NK cells, particularly to CD56(dim) NK cells. PrP(C)-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP(C)-Fc facilitated the IL-15-induced proliferation of NK cells. PrP(C)-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP(C)-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP(C)-Fc protein activates human NK cells via the ERK and JNK signaling pathways.
Asunto(s)
Células Asesinas Naturales/inmunología , Activación de Linfocitos/fisiología , Proteínas PrPC/fisiología , Degranulación de la Célula/fisiología , Células Cultivadas , Granzimas/metabolismo , Humanos , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP QuinasasRESUMEN
The conversion of the properly folded prion protein, PrPc, to its misfolded amyloid form, PrPsc, occurs as the two proteins traffic along the endocytic pathway and PrPc is exposed to PrPsc. To determine the specific site of prion conversion, we knocked down various proteins in the endocytic pathway including Rab7a, Tsg101 and Hrs (also known as HGS). PrPsc was markedly reduced in two chronically infected cell lines by preventing the maturation of the multivesicular body, a process that begins in the early endosome and ends with the sorting of cargo to the lysosome. By contrast, knocking down proteins in the retromer complex, which diverts cargo away from the multivesicular body caused an increase in PrPsc levels. These results suggest that the multivesicular body is the major site for intracellular conversion of PrPc to PrPsc.
Asunto(s)
Cuerpos Multivesiculares/metabolismo , Priones/metabolismo , Animales , Encéfalo/metabolismo , Lisosomas/metabolismo , Ratones , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Priones/genética , Procesamiento Proteico-PostraduccionalRESUMEN
The temporal and spatial regulation of ß-1,3-glucan synthesis plays an important role in morphogenesis during fungal growth and development. Northern blot analysis showed that the transcription of fksA, the gene encoding ß-1,3-glucan synthase in Aspergillus nidulans, was cell-cycle-dependent and increased steadily over the duration of the vegetative period, but its overall expression during the asexual and sexual stages was fairly constant up until the time of transcription cessation. In an A. nidulans strain mutated in the eukaryotic bHLH-like APSES transcription factor stuA1, the transcriptional level of fksA, and consequently the content of alkali-insoluble cell wall ß-glucan, significantly increased at the conidial chain formation and maturation stage. Electrophoretic mobility shift assays revealed that StuA was bound to StREs (StuA Response Elements) on the fksA promoter region. Promoter analysis with sGFP-fusion constructs also indicated the negative regulation of fksA expression by StuA, especially during asexual development. Taken together, these data suggest that StuA plays an important role in cell wall biogenesis during the development of A. nidulans, by controlling the transcription level of fksA.
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Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucosiltransferasas/genética , Factores de Transcripción/metabolismo , Aspergillus nidulans/crecimiento & desarrollo , Aspergillus nidulans/metabolismo , Pared Celular/metabolismo , Regulación Fúngica de la Expresión Génica , Glucosiltransferasas/metabolismo , Regiones Promotoras Genéticas , Reproducción Asexuada , Factores de Transcripción/genética , beta-Glucanos/metabolismoRESUMEN
The Hsc70 cochaperone, G cyclin-associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin-dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin-mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin-dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication.
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Senescencia Celular , Centrosoma/metabolismo , Clatrina/metabolismo , Endocitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Complejo 2 de Proteína Adaptadora/genética , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Línea Celular , Clatrina/genética , Daño del ADN , Lisosomas/metabolismo , Ratones , Fenotipo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
We investigated regulation of human NK cell function by CD27 engagement using a recombinant soluble CD70 protein. CD27 was preferentially expressed on CD56(bright) NK cells, and soluble CD70 protein bound to CD27(+)CD56(bright) NK cells. While soluble CD70 protein enhanced IFN-γ secretion by CD56(bright) NK cells in the presence of IL-12, it augmented neither cytolytic activity nor proliferation of NK cells. Thus, we next asked if soluble CD70 protein could be used to induce non-cytolytic antiviral activity of NK cells using an in vitro hepatitis C virus (HCV) infection system. Soluble CD70 protein stimulated NK cells to suppress HCV replication by enhancing NK cell IFN-γ secretion without killing infected cells. Taken together, we demonstrate that CD27 engagement by a soluble CD70 protein enhances non-cytolytic antiviral activity of CD56(bright) NK cells by IFN-γ secretion. Thus, this soluble CD70 protein may be useful for the treatment of viral infections such as HCV infection.
Asunto(s)
Antivirales/farmacología , Ligando CD27/farmacología , Antígeno CD56/inmunología , Interferón gamma/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Antígeno CD56/genética , Expresión Génica , Hepacivirus/efectos de los fármacos , Hepacivirus/inmunología , Humanos , Interferón gamma/inmunología , Interleucina-12/farmacología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Cultivo Primario de Células , Proteínas Recombinantes/farmacología , Transducción de Señal , Solubilidad , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Replicación Viral/efectos de los fármacosRESUMEN
The PrP(C) is expressed in many types of immune cells including monocytes and macrophages, however, its function in immune regulation remains to be elucidated. In the present study, we examined a role for PrP(C) in regulation of monocyte function. Specifically, the effect of a soluble form of PrP(C) was studied in human monocytes. A recombinant fusion protein of soluble human PrP(C) fused with the Fc portion of human IgG1 (designated as soluble PrP(C)-Fc) bound to the cell surface of monocytes, induced differentiation to macrophage-like cells, and enhanced adherence and phagocytic activity. In addition, soluble PrP(C)-Fc stimulated monocytes to produce pro-inflammatory cytokines such as TNF-α, IL-1ß, and IL-6. Both ERK and NF-κB signaling pathways were activated in soluble PrP(C)-treated monocytes, and inhibitors of either pathway abrogated monocyte adherence and cytokine production. Taken together, we conclude that soluble PrP(C)-Fc enhanced adherence, phagocytosis, and cytokine production of monocytes via activation of the ERK and NF-κB signaling pathways.
RESUMEN
OBJECTIVE: To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). METHODS AND RESULTS: We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. CONCLUSIONS: Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.
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Lipoproteínas LDL/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Pinocitosis/fisiología , Transporte Biológico Activo/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Indoles/farmacología , Macrólidos/farmacología , Macrófagos/citología , Microscopía Inmunoelectrónica , Pinocitosis/efectos de los fármacos , Sulfonamidas/farmacologíaRESUMEN
Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. To investigate the effects of force-expressed wild-type and mutant myocilin and optineurin on neurite outgrowth in neuronal cells, we transiently transfected cells with pEGFP-N1 (mock control) as well as myocilin and optineurin plasmids including pMYOC(WT)-EGFP, pMYOC(P370L)-EGFP, pMYOC(1-367)-EGFP, pOPTN(WT)-EGFP, and pOPTN(E50K)-EGFP. PC12 cells transfected with pEGFP-N1 produced, as anticipated, long and extensive neuritis on nerve growth factor induction. The neurite length in those cells transfected with myocilin constructs was shortened and the number of neurites was also reduced. A similar inhibitory effect on neurite outgrowth was also elicited by myocilin transfection in RGC5 cells. In contrast, neither transfection of the optineurin constructs pOPTN(WT)-EGFP and pOPTN(E50K)-EGFP nor the myocilin and optineurin small-interfering RNA treatments induced significant alterations in neurite outgrowth. Transfection with the wild-type optineurin construct, but not with that of the wild-type myocilin, increased the apoptotic activity in cells. These results demonstrated that the two glaucoma genes, myocilin and optineurin, exhibited differential effects on neurite outgrowth. They may contribute to the development of neurodegenerative glaucoma via distinct mechanisms.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas del Ojo/genética , Glaucoma/genética , Glicoproteínas/genética , Neuritas/metabolismo , Adulto , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Colforsina/farmacología , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/farmacología , Endocitosis/efectos de los fármacos , Proteínas del Ojo/metabolismo , Proteínas del Ojo/farmacología , Fluoresceína-5-Isotiocianato/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas/farmacología , Humanos , Persona de Mediana Edad , Proteínas Mutantes/metabolismo , Neuritas/efectos de los fármacos , Células PC12 , ARN Interferente Pequeño/metabolismo , Ratas , Proteínas Recombinantes/farmacología , Soluciones , Coloración y Etiquetado , Adulto JovenRESUMEN
Myocilin is a gene linked to the most common form of glaucoma, a major blinding disease. The trabecular meshwork (TM), a specialized eye tissue, is believed to be involved, at least in part, in the development of glaucoma. The myocilin expression is known to be up-regulated by glucocorticoids in TM cells, and an altered myocilin level may be the culprit in conditions such as corticosteroid glaucoma. Wild type myocilin, when transfected into cultured human TM cells, induced a dramatic loss of actin stress fibers and focal adhesions. Myocilin transfectants displayed a heightened sensitivity to trypsin. Adhesion to fibronectin, collagens, and vitronectin was compromised. The fibronectin deposition and the levels of fibronectin protein and mRNA were also reduced in myocilin transfectants. The fibronectin deposition could be restored by treatment with lysophosphatidic acid, a Rho stimulator. Assays further revealed that upon myocilin overexpression, the activity of RhoA was diminished, whereas the cAMP level and the protein kinase A (PKA) activity were augmented. Myocilin protein did not affect actin polymerization. The collapse of actin stress fibers and increased trypsin sensitivity from myocilin transfection could be reverted by co-expression of constitutively active RhoA or by treatment with PKA inhibitor H-89. The PKA activity, however, was not modified by co-expression of either constitutively active or dominant negative RhoA. These results demonstrate that myocilin has a de-adhesive activity and triggers signaling events. cAMP/PKA activation and the downstream Rho inhibition are possible mechanisms by which myocilin in overabundance may lead to TM cell or tissue damage.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas del Citoesqueleto/fisiología , Proteínas del Ojo/fisiología , Glicoproteínas/fisiología , Transducción de Señal , Malla Trabecular/metabolismo , Proteínas de Unión al GTP rho/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Actinas/metabolismo , Adulto , Western Blotting , Adhesión Celular/genética , Adhesión Celular/fisiología , Células Cultivadas , Niño , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Activación Enzimática/efectos de los fármacos , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Persona de Mediana Edad , Modelos Biológicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Malla Trabecular/citología , TransfecciónRESUMEN
Myocilin (MYOC) and optineurin (OPTN) are two genes linked to glaucoma, a major blinding disease. To investigate the possible molecular interactions between MYOC and OPTN genes, we over-expressed MYOC and examined its effect on the level of endogenous OPTN in human trabecular meshwork (TM) cells and vice versa. We noted that over-expressing MYOC did not affect the OPTN level, whereas OPTN over-expression induced an up-regulation of the endogenous MYOC. This induction was also observed in other ocular and non-ocular cell types including PC12 cells. The endogenous levels of both OPTN and MYOC genes were in addition found increased when PC12 cells underwent differentiation upon treatment with nerve growth factor (NGF). Over-expression of OPTN resulted in prolonged turnover rate of MYOC mRNA but had little effect on the promoter activity of the MYOC gene. The over-expressed OPTN was localized in the cytoplasm, not translocated into the nucleus. These results indicate that interaction exists between OPTN and MYOC genes. Regulation of MYOC expression by OPTN is achieved primarily through control of the mRNA stability.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas del Ojo/genética , Glaucoma/genética , Glicoproteínas/genética , Factor de Transcripción TFIIIA/genética , Adolescente , Adulto , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Ojo/metabolismo , Expresión Génica , Glicoproteínas/metabolismo , Humanos , Proteínas de Transporte de Membrana , Persona de Mediana Edad , Regiones Promotoras Genéticas/genética , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Malla Trabecular/citología , Malla Trabecular/metabolismoRESUMEN
The trabecular meshwork (TM), an ocular tissue next to the cornea, is a major site for regulation of the aqueous humor outflow. Malfunctioning of this tissue is believed to be responsible for development of glaucoma, a major blinding disease. Myocilin is a gene directly linked to the most common form of glaucoma. Its protein product has been localized to both intra- and extra-cellular sites in TM cells. This study was to investigate the association of myocilin with mitochondria in TM cells. In vitro mitochondrial import assays showed that myocilin was imported to the TM mitochondria, targeting to mitochondrial membranes and/or the intermembrane space. The targeting was mediated mostly via the amino-terminal region of myocilin. When myocilin expression was induced either by treatment with dexamethasone or transfection with a myocilin construct, the mitochondrial membrane potential in TM cells, as assessed by JC-1 staining, was lowered. Subcellular fractionation and Western blot analyses confirmed that a portion of myocilin sedimented with the mitochondrial fractions. Upon anti-Fas treatment to provoke apoptosis, an increase of myocilin distribution in cytosolic fraction was observed, suggesting that myocilin was partially released from mitochondrial compartments. These results confirmed the association of myocilin with TM cell mitochondria and indicated that myocilin may have a proapoptotic role in TM cells.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas del Ojo/metabolismo , Glicoproteínas/metabolismo , Mitocondrias/metabolismo , Malla Trabecular/citología , Adolescente , Adulto , Western Blotting , Células Cultivadas , Preescolar , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/farmacología , Ojo/citología , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/química , Proteínas del Ojo/genética , Proteínas del Ojo/farmacología , Fibroblastos/metabolismo , Glicoproteínas/biosíntesis , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/farmacología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Persona de Mediana Edad , Membranas Mitocondriales/fisiología , Peso Molecular , Porinas/biosíntesis , Porinas/química , Fracciones Subcelulares/metabolismo , Radioisótopos de Azufre/metabolismoRESUMEN
Optineurin (OPTN) has recently been linked to glaucoma, a major cause of blindness worldwide. Mutations in OPTN such as Glu50-->Lys (E50K) have been reported in patients, particularly those with normal pressure glaucoma. Here, we show that the endogenous OPTN was not secreted in two ocular cell types, human trabecular meshwork and retinal pigment epithelial cells. It localized instead in the cytoplasm in a diffuse pattern without a distinct association with the Golgi apparatus. When overexpressed, however, wild-type OPTN-green fluorescent protein (GFP) formed foci especially around the Golgi, colocalizing partially with the common endocytic pathway marker transferrin receptor in both cell types. Fragmentation of the Golgi was also observed. On nocodazole treatment, the OPTN foci were dispersed into the cytoplasm. Overexpression of mutant OPTNE50K-GFP resulted in a greater number (P<0.0055) and size of the foci, compared with the wild type, and the Golgi alteration was potentiated. Cell loss observed in OPTN-expressing cultures was also more pronounced in OPTNE50K-GFP compared with that of wild-type OPTN-GFP counterparts (P<0.01). This study highlights a possible role of OPTN in vesicle trafficking and Golgi integrity. It also provides in-sights into the possible mechanisms why E50K would exhibit a propensity toward the development of glaucoma.