RESUMEN
Nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a membrane protein that mediates cell adhesion. The role of Ninj1 during inflammatory response has been widely investigated in macrophages and endothelial cells. Ninj1 is expressed in various tissues, and the liver also expresses high levels of Ninj1. Although the hepatic upregulation of Ninj1 has been reported in human hepatocellular carcinoma and septic mice, little is known of its function during the pathogenesis of liver diseases. In the present study, the role of Ninj1 in liver inflammation was explored using lipopolysaccharide (LPS)/D-galactosamine (D-gal)-induced acute liver failure (ALF) model. When treated with LPS/D-gal, conventional Ninj1 knock-out (KO) mice exhibited a mild inflammatory phenotype as compared with wild-type (WT) mice. Unexpectedly, myeloid-specific Ninj1 KO mice showed no attenuation of LPS/D-gal-induced liver injury. Whereas, Ninj1 KO primary hepatocytes were relatively insensitive to TNF-α-induced caspase activation as compared with WT primary hepatocytes. Also, Ninj1 knock-down in L929 and AML12 cells and Ninj1 KO in HepG2 cells ameliorated TNF-α-mediated apoptosis. Consistent with in vitro results, hepatocyte-specific ablation of Ninj1 in mice alleviated LPS/D-gal-induced ALF. Summarizing, our in vivo and in vitro studies show that lack of Ninj1 in hepatocytes diminishes LPS/D-gal-induced ALF by alleviating TNF-α/TNFR1-induced cell death.
Asunto(s)
Moléculas de Adhesión Celular Neuronal , Galactosamina , Fallo Hepático Agudo , Factores de Crecimiento Nervioso , Animales , Apoptosis , Caspasas/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Células Endoteliales/metabolismo , Hepatocitos/metabolismo , Humanos , Lipopolisacáridos , Hígado/metabolismo , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/genética , Fallo Hepático Agudo/metabolismo , Ratones , Ratones Noqueados , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We investigated the effect of chitinase-3-like protein 1 (CHI3L1) on glucose metabolism and its underlying mechanisms in skeletal muscle cells, and evaluated whether the observed effects are relevant in humans. CHI3L1 was associated with increased glucose uptake in skeletal muscles in an AMP-activated protein kinase (AMPK)-dependent manner, and with increased intracellular calcium levels via PAR2. The improvement in glucose metabolism observed in an intraperitoneal glucose tolerance test on male C57BL/6J mice supported this association. Inhibition of the CaMKK was associated with suppression of CHI3L1-mediated glucose uptake. Additionally, CHI3L1 was found to influence glucose uptake through the PI3K/AKT pathway. Results suggested that CHI3L1 stimulated the phosphorylation of AS160 and p38 MAPK downstream of AMPK and AKT, and the resultant GLUT4 translocation. In primary myoblast cells, stimulation of AMPK and AKT was observed in response to CHI3L1, underscoring the biological relevance of CHI3L1. CHI3L1 levels were elevated in cells under conditions that mimic exercise in vitro and in exercised mice in vivo, indicating that CHI3L1 is secreted during muscle contraction. Finally, similar associations between CHI3L1 and metabolic parameters were observed in humans alongside genotype associations between CHI3L1 and diabetes at the population level. CHI3L1 may be a potential therapeutic target for the treatment of diabetes.
Asunto(s)
Proteína 1 Similar a Quitinasa-3 , Diabetes Mellitus/metabolismo , Glucosa/metabolismo , Músculo Esquelético , Proteínas Quinasas Activadas por AMP/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular , Proteína 1 Similar a Quitinasa-3/sangre , Proteína 1 Similar a Quitinasa-3/fisiología , Estudios de Asociación Genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos , Proteínas Proto-Oncogénicas c-akt/metabolismo , RatasRESUMEN
BACKGROUND: Neointimal hyperplasia and its related arterial stiffness are the crucial pathophysiological features in atherosclerosis and in-stent restenosis. Cluster of differentiation 147 (CD147), a member of the immunoglobulin super family that induces the expression of matrix metalloproteinase-9 (MMP-9) by dimerization, may play important roles in neointimal hyperplasia and may therefore be an effective target for the treatment of this condition. Here, we investigated whether a novel CD147 inhibitor SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) reduces neointimal hyperplasia and arterial stiffness in a rat model of partial carotid artery ligation. METHODS: Neointimal hyperplasia was induced in Sprague-Dawley rats by partial ligation of the right carotid artery combined with a high fat diet and vitamin D injection. Rats were subdivided into vehicle, SP-8356 (50 mg/kg), and rosuvastatin (10 mg/kg) groups. The drugs were administrated via intraperitoneal injections for 4 weeks. The elasticity of blood vessels was assessed by measuring pulse wave velocity using Doppler ultrasonography before sacrifice. Histomolecular analysis was carried out on harvested carotid arteries. RESULTS: SP-8356 significantly reduced MMP activity by inhibiting CD147 dimerization. SP-8356 reduced neointimal hyperplasia and prevented the deterioration of vascular elasticity. SP-8356 had a greater inhibitory effect on neointimal hyperplasia than did rosuvastatin. Furthermore, rosuvastatin did not improve vascular elasticity. SP-8356 increased the expression of smooth muscle myosin heavy chain (SM-MHC), but decreased the expression of collagen type III and MMP-9 in the neointimal region. In contrast to SP-8356, rosuvastatin did not alter the expression of SM-MHC or MMP-9. CONCLUSIONS: The ability of SP-8356 to reduce neointimal hyperplasia and improve arterial stiffness in affected carotid artery suggests that SP-8356 could be a promising therapeutic drug for vascular remodeling disorders involving neointimal hyperplasia and arterial stiffness.
Asunto(s)
Basigina/antagonistas & inhibidores , Monoterpenos Bicíclicos/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Arterias Carótidas/patología , Arterias Carótidas/fisiopatología , Neointima/patología , Rigidez Vascular/efectos de los fármacos , Animales , Basigina/metabolismo , Monoterpenos Bicíclicos/química , Compuestos Bicíclicos con Puentes/química , Línea Celular , Células Cultivadas , Colágeno Tipo III/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Descubrimiento de Drogas , Hiperplasia , Ligadura , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Músculo Liso Vascular/metabolismo , Fenotipo , Ratas Sprague-DawleyRESUMEN
Herein, we successfully generated transgenic pigs expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin (HA)-tagged-human heme oxygenase-1 (hHO-1) without Gal epitope. Healthy cloned pigs were produced by somatic cell nuclear transfer (SCNT) using the genetically modified cells. The genetic disruption of the GGTA1 genes and absence of expression of BS-IB4 lectin in tail-derived fibroblast of the SCNT-generated piglets were successfully confirmed. The expression of shTNFRI-Fc and HAhHO-1 was fully identified with protective effect against oxidative stress and apoptosis stimulation. Antibody-mediated complement-dependent cytotoxicity assay for examining the immuno-reactivity of transgenically derived pig cells showed that pigs lacking GGTA1 with the expression of double genes reduce the humoral barrier to xenotransplantation, more than pigs simply expressing double genes and the wild type. Through this approach, rapid production of a pig strain deficient in various genes may be expected to be applicable for xenotransplantation research without extensive breeding protocols.
Asunto(s)
Animales Modificados Genéticamente/genética , Galactosiltransferasas/genética , Hemo-Oxigenasa 1/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Animales , Apoptosis/genética , Epítopos/genética , Epítopos/inmunología , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Hemo-Oxigenasa 1/inmunología , Humanos , Técnicas de Transferencia Nuclear , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Porcinos , Trasplante HeterólogoRESUMEN
As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.
Asunto(s)
Animales Modificados Genéticamente/genética , Galactosiltransferasas/genética , Hemo-Oxigenasa 1/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Animales , Citidina Monofosfato/análogos & derivados , Citidina Monofosfato/genética , Femenino , Técnicas de Inactivación de Genes , Ácidos Neuramínicos , Embarazo , Porcinos/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Trasplante HeterólogoRESUMEN
Glucagon-like peptide-1 (GLP-1) plays a pivotal role in glucose homeostasis through its receptor GLP1R. Due to its multiple beneficial effects, GLP-1 has gained great attention for treatment of type 2 diabetes and obesity. However, little is known about the molecular mechanism underlying the interaction of GLP-1 with the heptahelical core domain of GLP1R conferring high affinity ligand binding and ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R, we determined that the evolutionarily conserved amino acid residue Arg(380) flanked by hydrophobic Leu(379) and Phe(381) in extracellular loop 3 (ECL3) may have an interaction with Asp(9) and Gly(4) of the GLP-1 peptide. The molecular modeling study showed that Ile(196) at transmembrane helix 2, Met(233) at ECL1, and Asn(302) at ECL2 of GLP1R have contacts with His(1) and Thr(7) of GLP-1. This study may shed light on the mechanism underlying high affinity interaction between the ligand and the binding pocket that is formed by these conserved residues in the GLP1R core domain.
Asunto(s)
Aminoácidos/química , Péptido 1 Similar al Glucagón/química , Estructura Terciaria de Proteína , Receptores de Glucagón/química , Secuencia de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Sitios de Unión/genética , Secuencia Conservada/genética , Evolución Molecular , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Mutación Puntual , Unión Proteica , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Non-metastatic cells 1 (NME1) or nm23 is the first metastasis suppressor gene discovered. It functions through various enzymatic activities and interacts with many intracellular proteins. The NME1 gene encodes two splicing variants, NME1 and NME1L. Most studies have focused on NME1 because of its abundance in cells. We previously reported NME1L-mediated suppression of NF-κB signaling by interacting with and inhibiting IKKß. In this study, we demonstrated that NME1L, but not NME1, mediated inhibition of cell proliferation, although both NME1 and NME1L were involved in suppressing metastasis. A reporter gene assay was performed to determine the growth signaling pathway regulated by NME1L but none of the growth factors tested could induce an NF-κB-dependent luciferase expression except TNFα. Interestingly, SRE-reporter gene activation by IGF1 was significantly downregulated, along with reduction of ERK phosphorylation in NME1L expressing cells, compared to vector or NME1 expressing cells. NME1L directly interacted with KSR1, which is a scaffold for Raf-1, MEK, and ERK, that regulates ERK activation. Hence, NME1L plays a crucial role in regulation of cell proliferation by inhibiting IGF1-stimulated ERK phosphorylation through N-terminal 25 amino acid-mediated interaction with KSR1.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Proteínas Quinasas/metabolismo , Movimiento Celular , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Células MCF-7 , Metástasis de la Neoplasia , Isoformas de Proteínas/metabolismoRESUMEN
Nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a cell surface molecule that can mediate homophilic adhesion and promote neurite outgrowth from cultured dorsal root ganglion (DRG) neurons. Interestingly, Ninj1 overexpressed in human cancer; however, its role in metastasis is not clear. This study showed that inhibition of Ninj1 promotes lung cancer metastasis through interleukin 6 (IL-6)/STAT3 signaling. Ninj1 levels were relatively low in highly motile lung cancer cells. While inhibition of Ninj1 enhanced cell migration in lung cancer cells, overexpression of Ninj1 significantly suppressed it. We found that inhibition of Ninj1 significantly increased expression and secretion of IL-6 in A549 cells. We also found that inhibition of IL-6 decreased intercellular adhesion molecule 1 (ICAM-1) expression. In addition, inhibition of Ninj1 significantly increased cell motility and invasiveness of lung cancer cells. In an in vivo model, we found that Ninj1 suppression did not affect tumor growth but induced significant increase in incidence of lung metastasis, and sizes and number of tumor nodules. Taken together, our data clearly demonstrate that Ninj1 suppresses migration, invasion and metastasis of lung cancer via inhibition of the IL-6 signaling pathway in vitro and in vivo.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Interleucina-6/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Factores de Crecimiento Nervioso/metabolismo , Transducción de Señal , Línea Celular Tumoral , Movimiento Celular/genética , Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-6/genética , Neoplasias Pulmonares/genética , Metástasis de la Neoplasia , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
In humans, numerous genes encode neuropeptides that comprise a superfamily of more than 70 genes in approximately 30 families and act mainly through rhodopsin-like G protein-coupled receptors (GPCRs). Two rounds of whole-genome duplication (2R WGD) during early vertebrate evolution greatly contributed to proliferation within gene families; however, the mechanisms underlying the initial emergence and diversification of these gene families before 2R WGD are largely unknown. In this study, we analyzed 25 vertebrate rhodopsin-like neuropeptide GPCR families and their cognate peptides using phylogeny, synteny, and localization of these genes on reconstructed vertebrate ancestral chromosomes (VACs). Based on phylogeny, these GPCR families can be divided into five distinct clades, and members of each clade tend to be located on the same VACs. Similarly, their neuropeptide gene families also tend to reside on distinct VACs. Comparison of these GPCR genes with those of invertebrates including Drosophila melanogaster, Caenorhabditis elegans, Branchiostoma floridae, and Ciona intestinalis indicates that these GPCR families emerged through tandem local duplication during metazoan evolution prior to 2R WGD. Our study describes a presumptive evolutionary mechanism and development pathway of the vertebrate rhodopsin-like GPCR and cognate neuropeptide families from the urbilaterian ancestor to modern vertebrates.
Asunto(s)
Evolución Molecular , Receptores Acoplados a Proteínas G/genética , Animales , Secuencia Conservada , Duplicación de Gen , Genoma , Humanos , Invertebrados , Neuropéptidos/genética , Filogenia , Rodopsina/genética , Sintenía , Vertebrados/genéticaRESUMEN
BACKGROUND: Despite the development of α1,3-galactosyl transferase-knockout (GTKO) pigs, acute humoral xenograft rejection caused by antibodies against non-Gal antigens, along with complement activation, are hurdles that need to be overcome. Among non-Gal antigens, N-glycolylneuraminic acid (Neu5Gc) is considered to play an important role in xenograft rejection in human. METHODS: We generated human embryonic kidney 293 (HEK293) cells that expressed xenogeneic Neu5Gc (HEK293-pCMAH) or α1,3Gal (HEK293-pGT) antigen and investigated the degree of human antibody binding and complement-dependent cytotoxicity (CDC) against these antigens using 100 individual human sera. RESULTS: Both IgM and IgG bound to α1,3Gal, while only IgG bound to Neu5Gc. Of the ABO blood groups, the degree of IgG binding to α1,3Gal was highest for blood group A. The degree of CDC against HEK293-pCMAH cells was significantly lower than that against HEK293-pGT cells. However, CDC against HEK293-pCMAH cells was significantly higher than that against control HEK293 cells. In addition, the severity of CDC against HEK293-pCMAH cells positively correlated with that against GTKO pig aortic endothelial cells (PAECs), suggesting that Neu5Gc is the main antigen in GTKO PAECs. Similar to antibody-binding activity, only IgG binding correlated with CDC against HEK293-pCMAH cells. The most common subclass of IgGs against Neu5Gc was IgG1, which typically induces strong complement activation. CONCLUSIONS: We showed that IgG-mediated CDC was detected in Neu5Gc-overexpressed HEK293 cells incubated with human sera; however, this antibody reactivity to Neu5Gc was highly variable among individuals. Our results suggest that additional modifications to the CMAH gene should be considered for widespread use of pig organs for human transplants.
Asunto(s)
Galactosa/inmunología , Rechazo de Injerto/inmunología , Inmunoglobulina G/inmunología , Ácidos Neuramínicos/inmunología , Trasplante Heterólogo , Animales , Animales Modificados Genéticamente , Activación de Complemento/inmunología , Células HEK293 , Humanos , Inmunoglobulina G/sangre , Ácidos Neuramínicos/metabolismo , Porcinos , Trasplante Heterólogo/métodosRESUMEN
IKKß functions as a principal upstream activator of the canonical NF-κB pathway by phosphorylating IκB, leading to its proteasomal degradation. Because IKKß is considered a therapeutic target, understanding its regulation may facilitate the design of efficient regulators of this molecule. Here, we report a novel IKKß-interacting molecule, NME1L, a splicing variant of the NME1 protein. NME1 has attracted attention in cancer research because of its antimetastatic activity and reduced expression in multiple aggressive types of cancer. However, the effect was just moderate but not dramatic in anti-cancer activities. We found that only NME1L interacts with IKKß. Exogenous expression of NME1L resulted in a potent decrease in TNFα-stimulated NF-κB activation, whereas knockdown of NME1/NME1L with shRNA enhanced activity of NF-κB. NME1L down-regulates IKKß signaling by blocking IKKß-mediated IκB degradation. When NME1L was introduced into highly metastatic HT1080 cells, the mobility was efficiently inhibited. Furthermore, in a metastasis assay, NME1L-expressing cells did not colonize the lung. Based on these results, NME1L is a potent antimetastatic protein and may be a useful weapon in the fight against cancers.
Asunto(s)
Empalme Alternativo , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Nucleósido Difosfato Quinasas NM23/biosíntesis , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Quinasa I-kappa B/genética , FN-kappa B/genética , Nucleósido Difosfato Quinasas NM23/genética , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologíaRESUMEN
BACKGROUND: With the introduction of the α1, 3-galactosyltransferase gene-knockout (GT-KO) pig and its pivotal role in preventing hyperacute rejection (HAR), coagulation remains a considerable obstacle yet to be overcome in order to provide long-term xenograft survival. Thrombomodulin (TBM) plays a critical anticoagulant and anti-inflammatory role in its part of the protein C pathway. Many studies have demonstrated the strong anticoagulant effects of TBM in xenotransplantation, but its complement regulatory effects have not been appropriately examined. Here, we investigate whether TBM can regulate complement activation as well as coagulation in response to xenogeneic stimuli. METHODS: We transfected porcine endothelial cells (MPN-3) with adenovirus vectors containing the human TBM gene (ad-hTBM), or a control gene containing GFP (ad-GFP). The expression level of ad-hTBM was measured by flow cytometry. To confirm the anticoagulant effect of TBM, coagulation time was measured after treatment with recalcified human plasma in ad-hTBM-transfected MPN-3, and a thrombin activity assay was performed after treatment with 50% human serum in ad-hTBM-infected MPN-3. RESULTS: Thrombin generation was significantly decreased in a dose-dependent manner in ad-TBM group, and coagulation time was increased in the ad-hTBM group when compared to the ad-GFP group. Complement-dependent serum toxicity assays were performed after treatment with 20% human serum or heat-inactivated human serum by LDH assay. Complement-dependent toxicity was significantly attenuated in the ad-hTBM group, but complement-independent toxicity was not attenuated in the ad-hTBM group. These results suggest that human thrombomodulin (hTBM) has complement regulatory effects as well as anticoagulant effects. To further investigate the mechanisms of complement regulation by hTBM, we deleted the EGF5, 6 domains that are involved in thrombin generation or the lectin-like domain involved in inflammation of TBM and functional tests were performed using these modified forms. We showed that the EGF5, 6 domain of TBM principally inhibits complement activation rather than the lectin domain. CONCLUSION: The EGF5, 6 domains of TBM appear to be the major domains for down-regulating the complement system rather than the lectin-like domain during xenogenic stimuli. The role of EGF5, 6 domains of hTBM may be due to inhibition of thrombin as thrombin can cleave C3a and C5a directly and hTBM may also be involved in complement regulation. Clearly then human TBM has complement regulatory effects as well as anticoagulant effects in xeno-immune response, and it is a promising target for attenuating xenograft rejection.
Asunto(s)
Coagulación Sanguínea/inmunología , Activación de Complemento/inmunología , Xenoinjertos/inmunología , Trombomodulina/inmunología , Animales , Animales Modificados Genéticamente , Línea Celular , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Humanos , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Porcinos , Porcinos Enanos , Trombina/metabolismo , Trombomodulina/química , Trombomodulina/genética , Transfección , Trasplante Heterólogo/efectos adversosRESUMEN
In humans, the secretin-like G protein-coupled receptor (GPCR) family comprises 15 members with 18 corresponding peptide ligand genes. Although members have been identified in a large variety of vertebrate and nonvertebrate species, the origin and relationship of these proteins remain unresolved. To address this issue, we employed large-scale genome comparisons to identify genome fragments with conserved synteny and matched these fragments to linkage groups in reconstructed early gnathostome ancestral chromosomes (GAC). This genome comparison revealed that most receptor and peptide genes were clustered in three GAC linkage groups and suggested that the ancestral forms of five peptide subfamilies (corticotropin-releasing hormone-like, calcitonin-like, parathyroid hormone-like, glucagon-like, and growth hormone-releasing hormone-like) and their cognate receptor families emerged through tandem local gene duplications before two rounds (2R) of whole-genome duplication. These subfamily genes have, then, been amplified by 2R whole-genome duplication, followed by additional local duplications and gene loss prior to the divergence of land vertebrates and teleosts. This study delineates a possible evolutionary scenario for whole secretin-like peptide and receptor family members and may shed light on evolutionary mechanisms for expansion of a gene family with a large number of paralogs.
Asunto(s)
Duplicación de Gen/genética , Genómica/métodos , Hormonas Peptídicas/genética , Receptores Acoplados a Proteínas G/genética , Secretina/genética , Evolución Molecular , HumanosRESUMEN
This study investigated the anti-inflammatory and protective properties of SP-8356, a synthetic derivative of (1S)-(-)-verbenone, in a mouse model of LPS-induced acute lung injury (ALI). By targeting intracellular signaling pathways and inflammatory responses, SP-8356 demonstrated a potent ability to attenuate deleterious effects of proinflammatory stimuli. Specifically, SP-8356 effectively inhibited the activation of crucial signaling molecules such as NF-κB and Akt, and subsequently dampened the expression of inflammatory cytokines in various lung cellular components. Intervention with SP-8356 treatment also preserved the structural integrity of the epithelial and endothelial barriers. By reducing immune cell infiltration into inflamed lung tissue, SP-8356 exerted a broad protective effect against ALI. These findings position SP-8356 as a promising therapeutic candidate for pulmonary inflammatory diseases that cause ALI.
Asunto(s)
Lesión Pulmonar Aguda , Monoterpenos Bicíclicos , Animales , Ratones , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , Transducción de Señal , Pulmón , FN-kappa B/metabolismo , Citocinas/metabolismo , Lipopolisacáridos/farmacologíaRESUMEN
Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) play important roles in insulin secretion through their receptors, GLP1R and GIPR. Although GLP-1 and GIP are attractive candidates for treatment of type 2 diabetes and obesity, little is known regarding the molecular interaction of these peptides with the heptahelical core domain of their receptors. These core domains are important not only for specific ligand binding but also for ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R/GIPR, we determined that evolutionarily conserved amino acid residues such as Ile(196) at transmembrane helix 2, Leu(232) and Met(233) at extracellular loop 1, and Asn(302) at extracellular loop 2 of GLP1R are responsible for interaction with ligand and receptor activation. Application of chimeric GLP-1/GIP peptides together with molecular modeling suggests that His(1) of GLP-1 interacts with Asn(302) of GLP1R and that Thr(7) of GLP-1 has close contact with a binding pocket formed by Ile(196), Leu(232), and Met(233) of GLP1R. This study may provide critical clues for the development of peptide and/or nonpeptide agonists acting at GLP1R.
Asunto(s)
Evolución Molecular , Modelos Moleculares , Receptores de Glucagón/química , Animales , Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/metabolismo , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón , Células HEK293 , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-ActividadRESUMEN
CXCL14 is a chemokine family member that is involved in various cellular responses in addition to immune cell activation. Although constitutive CXCL14 expression in normal epithelial cells may help protect against infection by activating immune systems, its expression in cancer cells has raised controversy regarding its possible role in tumorigenesis. However, the underlying mechanisms for this disparity remain unknown. Investigation of cellular CXCL14 binding properties might increase our understanding of the peptide's roles in tumorigenesis. In the present study, we found that CXCL14 binds to various cell types. Interestingly, binding to NCI-H460 cells was prevented by heparan sulfate and N-acetyl neuraminic acid. Next, we examined effect of CXCL14 binding in NCI-H460 and NCI-H23. CXCL14 enhanced proliferation and migration in NCI-H460 but had no effect on NCI-H23. A reporter gene assay with various transcription factor response elements revealed that only nuclear factor-κB (NF-κB) signaling was activated by CXCL14 in NCI-H460 cells, which was blocked by BAPTA-AM, TPCA-1, and brefeldin A. Exogenous expression of some glycoproteins such as syndecan-4, podoplanin, and CD43 in these cells enhanced CXCL14 binding and NF-κB activity. Collectively, these results demonstrate that CXCL14 binding to glycoproteins harboring heparan sulfate proteoglycans and sialic acids leads proliferation and migration of some cancer cells.
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Movimiento Celular/efectos de los fármacos , Quimiocinas CXC/farmacología , Glicoproteínas/metabolismo , Heparitina Sulfato/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ácido N-Acetilneuramínico/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Células HEK293 , Humanos , FN-kappa B/metabolismo , Unión Proteica/efectos de los fármacos , Receptores Fc/metabolismoRESUMEN
BACKGROUND: Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells has been regarded as an early mechanism of peritoneal fibrosis. A substantial and rapidly growing literature indicates that HO-1 provides the provenance for pathways that can interrupt virtually all major mechanisms of tissue injury. The effects of HO-1 expression on EMT, which plays a critical role in the development of peritoneal membrane (PM) fibrosis, are unknown and its roles in peritoneal fibrosis has not been studied, yet. METHODS: A piece of human omentum obtained from consenting patients undergoing elective abdominal surgery was used for study. We treated the human peritoneal mesothelial cells (HPMCs) with high glucose solution and HO-1 inducer (hemin, 10 µmol/L). To further investigate the pure effect of HO-1 on EMT of mesothelium, gene transfer of recombinant Adenovirus-harboring human HO-1 (Adv-HO-1 gene) to HPMCs was done. RESULTS: Exposure of HPMCs to HG solution resulted in an increase of the expression of mesenchymal markers such as α-smooth muscle actin (α-SMA) and was associated with a decrease in the expression of epithelial markers, E-cadherin. HO-1 protein expression was decreased in the same situation. Treatment of HPMCs with HO-1 inducer, hemin showed a dosage-dependent amelioration of HG induced changes in markers of EMT with increase of expression of HO-1. Human HO-1 gene transfection resulted in a significant increase in HO-1 expression and ameliorated HG-induced changes in expression of E-cadherin and α-SMA. CONCLUSION: Taken together, our results suggest that HO-1 has a critical role in the modulation of peritoneal fibrosis, and, more important, the suppression of EMT. This study is the first to show the beneficial effect of HO-1 on reversing EMT in MC.
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Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal/fisiología , Hemo-Oxigenasa 1/fisiología , Actinas/metabolismo , Cadherinas/metabolismo , Dependovirus/genética , Inducción Enzimática/efectos de los fármacos , Fibrosis , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Glucosa/farmacología , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/genética , Hemina/farmacología , Humanos , Inmunohistoquímica , Peritoneo/citología , Peritoneo/efectos de los fármacos , Peritoneo/patología , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Lysophosphatidic acid receptor 1 (LPAR1) is an emerging therapeutic target for numerous human diseases including fibrosis. However, the limited number of available core structures of LPAR1 antagonists has prompted the need for novel chemical templates. In this study, we conducted a high-throughput virtual screening to discover potential new scaffolds. We tested three existing crystal structures alongside an AlphaFold model to evaluate their suitability in structure-based virtual screening, finding that the crystal structures show superior performance compared with the predictive model. Furthermore, we also found that enhancing the precision in the screening process did not necessarily improve the enrichment of hits. From the screening campaign, we identified five structures that were validated using an LPAR1-dependent calcium flux assay. To gain a deeper insight into the protein-ligand interaction, we extensively analyzed the binding modes of these compounds using in silico techniques, laying the groundwork for the discovery of novel LPAR1 antagonists.
RESUMEN
CXCR3 regulates leukocyte trafficking, maturation, and various pathophysiological conditions. Alternative splicing generates three CXCR3 isoforms in humans. Previous studies investigated the roles of CXCR3 isoforms, and some biochemical data are not correlated with biological relevance analyses. RT-PCR analyses indicate that most cells express all three splicing variants, suggesting that they may mutually affect the chemokine binding and cellular responses of other splicing variants. Here, we performed an integrative analysis of the functional relations among CXCR3 splicing variants and their chemokine-dependent signaling using NanoBiT live cell protein interaction assays. The results indicated that the CXCR3 N-terminal region affected cell surface expression levels and ligand-dependent activation. CXCR3A was efficiently expressed in the plasma membrane and responded to I-TAC, IP-10, and MIG chemokines. By contrast, CXCR3B had low plasma membrane expression and mediated I-TAC-stimulated cellular responses. CXCR3Alt was rarely expressed on the cell surface and did not mediate any cell responses to the tested chemokines; however, CXCR3Alt negatively affected the plasma membrane expression of CXCR3A and CXCR3B and their chemokine-stimulated cellular responses. Jurkat cells express endogenous CXCR3, and exogenous CXCR3A expression enhanced chemotactic activity in response to I-TAC, IP-10, and MIG. By contrast, exogenous expression of CXCR3B and CXCR3Alt eliminated or reduced the CXCR3A-induced chemotactic activity. The PF-4 chemokine did not activate any CXCR3-mediated cellular responses. NanoBiT technology are useful to integrative studies of CXCR3-mediated cell signaling, and expand our knowledge of the cellular responses mediated by molecular interactions among the splicing variants, including cell surface expression, ligand-dependent receptor activation, and chemotaxis.
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Quimiocina CXCL10 , Transducción de Señal , Humanos , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Ligandos , Empalme Alternativo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores CXCR3/genética , Receptores CXCR3/metabolismoRESUMEN
Oxidative stress due to abnormal accumulation of reactive oxygen species (ROS) is an initiator of a large number of human diseases, and thus, the elimination and prevention of excessive ROS are important aspects of preventing the development of such diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is an essential transcription factor that defends against oxidative stress, and its function is negatively controlled by Kelch-like ECH-associated protein 1 (KEAP1). Therefore, activating NRF2 by inhibiting KEAP1 is viewed as a strategy for combating oxidative stress-related diseases. Here, we generated a cereblon (CRBN)-based proteolysis-targeting chimera (PROTAC), which we named SD2267, that induces the proteasomal degradation of KEAP1 and leads to NRF2 activation. As was intended, SD2267 bound to KEAP1, recruited CRBN, and induced the degradation of KEAP1. Furthermore, the KEAP1 degradation efficacy of SD2267 was diminished by MG132 (a proteasomal degradation inhibitor) but not by chloroquine (an autophagy inhibitor), which suggested that KEAP1 degradation by SD2267 was proteasomal degradation-dependent and autophagy-independent. Following KEAP1 degradation, SD2267 induced the nuclear translocation of NRF2, which led to the expression of NRF2 target genes and attenuated ROS accumulation induced by acetaminophen (APAP) in hepatocytes. Based on in vivo pharmacokinetic study, SD2267 was injected intraperitoneally at 1 or 3 mg/kg in APAP-induced liver injury mouse model. We observed that SD2267 degraded hepatic KEAP1 and attenuated APAP-induced liver damage. Summarizing, we described the synthesis of a KEAP1-targeting PROTAC (SD2267) and its efficacy and mode of action in vitro and in vivo. The results obtained suggest that SD2267 could be used to treat hepatic diseases related to oxidative stress.