RESUMO
OBJECTIVES: Recently, necroptosis has attracted increasing attention in arthritis research; however, it remains unclear whether its regulation is involved in osteoarthritis (OA) pathogenesis. Since receptor-interacting protein kinase-3 (RIP3) plays a pivotal role in necroptosis and its dysregulation is involved in various pathological processes, we investigated the role of the RIP3 axis in OA pathogenesis. METHODS: Experimental OA was induced in wild-type or Rip3 knockout mice by surgery to destabilise the medial meniscus (DMM) or the intra-articular injection of adenovirus carrying a target gene (Ad-Rip3 and Ad-Trim24 shRNA). RIP3 expression was examined in OA cartilage from human patients; Trim24, a negative regulator of RIP3, was identified by microarray and in silico analysis. Connectivity map (CMap) and in silico binding approaches were used to identify RIP3 inhibitors and to examine their direct regulation of RIP3 activation in OA pathogenesis. RESULTS: RIP3 expression was markedly higher in damaged cartilage from patients with OA than in undamaged cartilage. In the mouse model, adenoviral RIP3 overexpression accelerated cartilage disruption, whereas Rip3 depletion reduced DMM-induced OA pathogenesis. Additionally, TRIM24 knockdown upregulated RIP3 expression; its downregulation promoted OA pathogenesis in knee joint tissues. The CMap approach and in silico binding assay identified AZ-628 as a potent RIP3 inhibitor and demonstrated that it abolished RIP3-mediated OA pathogenesis by inhibiting RIP3 kinase activity. CONCLUSIONS: TRIM24-RIP3 axis perturbation promotes OA chronicity by activating RIP3 kinase, suggesting that the therapeutic manipulation of this pathway could provide new avenues for treating OA.
Assuntos
Proteínas de Transporte/metabolismo , Osteoartrite/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Necroptose/fisiologia , Proteínas Nucleares/metabolismo , Osteoartrite/patologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into mature cells of various cell types. Although the differentiation process of MSCs requires lineage-specific transcription factors, the exact molecular mechanism that determines MSCs differentiation is not clearly addressed. Here, we demonstrate a Smad4-Taz axis as a new intrinsic regulator for adipo-osteogenic differentiation of MSCs and show that this function of Smad4 is independent of the transforming growth factor-ß signal. Smad4 directly bound to the Taz protein and facilitated nuclear localization of Taz through its nuclear localization signal. Nuclear retention of Taz by direct binding to Smad4 increased expression of osteogenic genes through enhancing Taz-runt-related transcription factor 2 (Runx2) interactions in the C3H10T1/2 MSC cell line and preosteoblastic MC3T3-E1 cells, whereas it suppressed expression of adipogenic genes through promoting Taz-peroxisome proliferator-activated receptor-γ (PPARγ) interaction in C3H10T1/2 and preadipogenic 3T3-L1 cells. A reciprocal role of the Smad4 in osteogenic and adipogenic differentiation was also observed in human adipose tissue-derived stem cells (hASCs). Consequently, Smad4 depletion in C3H10T1/2 and hASCs reduced nuclear retention of Taz and thus caused the decreased interaction with Runx2 or PPARγ, resulting in delayed osteogenesis or enhanced adipogenesis of the MSC. Therefore, these findings provide insight into a novel function of Smad4 to regulate the balance of MSC lineage commitment through reciprocal targeting of the Taz protein in osteogenic and adipogenic differentiation pathways. Stem Cells 2019;37:368-381.
Assuntos
Adipogenia , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Transdução de Sinais , Proteína Smad4/metabolismo , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Diferenciação Celular , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Proteína Smad4/genética , Transativadores/genética , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
Autophagy begins with the formation of autophagosomes, a process that depends on the activity of the serine/threonine kinase ULK1 (hATG1). Although earlier studies indicated that ULK1 activity is regulated by dynamic polyubiquitination, the deubiquitinase involved in the regulation of ULK1 remained unknown. In this study, we demonstrate that ubiquitin-specific protease 20 (USP20) acts as a positive regulator of autophagy initiation through stabilizing ULK1. At basal state, USP20 binds to and stabilizes ULK1 by removing the ubiquitin moiety, thereby interfering with the lysosomal degradation of ULK1. The stabilization of basal ULK1 protein levels is required for the initiation of starvation-induced autophagy, since the depletion of USP20 by RNA interference inhibits LC3 puncta formation, a marker of autophagic flux. At later stages of autophagy, USP20 dissociates from ULK1, resulting in enhanced ULK1 degradation and apoptosis. Taken together, our findings provide the first evidence that USP20 plays a crucial role in autophagy initiation by maintaining the basal expression level of ULK1.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia , Ubiquitina Tiolesterase/metabolismo , Animais , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Linhagem Celular , Sobrevivência Celular , Expressão Gênica , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Ligação Proteica , Estabilidade Proteica , Proteólise , Interferência de RNA , RNA Interferente Pequeno/genética , Ubiquitina Tiolesterase/genética , UbiquitinaçãoRESUMO
p62/sequestosome-1 is a scaffolding protein involved in diverse cellular processes such as autophagy, oxidative stress, cell survival and death. It has been identified to interact with atypical protein kinase Cs (aPKCs), linking these kinases to NF-κB activation by tumor necrosis factor α (TNFα). The diverse functions of p62 are regulated through post-translational modifications of several domains within p62. Among the enzymes that mediate these post-translational modifications, little is known about the deubiquitinating enzymes (DUBs) that remove ubiquitin chains from p62, compared to the E3 ligases involved in p62 ubiquitination. In this study, we first demonstrate a role of ubiquitin-specific protease USP20 in regulating p62 stability in TNFα-mediated NF-κB activation. USP20 specifically binds to p62 and acts as a positive regulator for NF-κB activation by TNFα through deubiquitinating lysine 48 (K48)-linked polyubiquitination, eventually contributing to cell survival. Furthermore, depletion of USP20 disrupts formation of the atypical PKCζ-RIPK1-p62 complex required for TNFα-mediated NF-κB activation and significantly increases the apoptosis induced by TNFα plus cycloheximide or TNFα plus TAK1 inhibitor. These findings strongly suggest that the USP20-p62 axis plays an essential role in NF-κB-mediated cell survival induced by the TNFα-atypical PKCζ signaling pathway.
Assuntos
Lisina/metabolismo , Proteína Sequestossoma-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina Tiolesterase/metabolismo , Benzamidas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Regulação da Expressão Gênica , Células HEK293 , Células HT29 , Células HeLa , Humanos , NF-kappa B/metabolismo , Piperazinas/farmacologia , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Piridinas/farmacologia , Pirróis/farmacologia , Proteína Sequestossoma-1/química , Transdução de Sinais , Ubiquitina Tiolesterase/genéticaRESUMO
Mouse bone marrow-derived clonal mesenchymal stem cells (mcMSCs), which were originated from a single cell by a subfractionation culturing method, are recognized as new paradigm for stem cell therapy featured with its homogenous cell population. Next to proven therapeutic effects against pancreatitis, in the current study we demonstrated that mcMSCs showed significant therapeutic effects in dextran sulfate sodium (DSS)-induced experimental colitis model supported with anti-inflammatory and restorative activities. mcMSCs significantly reduced the disease activity index (DAI) score, including weight loss, stool consistency, and intestinal bleeding and significantly increased survival rates. The pathological scores were also significantly improved with mcMSC. We have demonstrated that especial mucosal regeneration activity accompanied with significantly lowered level of apoptosis as beneficiary actions of mcMSCs in UC models. The levels of inflammatory cytokines including TNF-α, IFN-γ, IL-1ß, IL-6, and IL-17 were all significantly concurrent with significantly repressed NF-κB activation compared to the control group and significantly decreased infiltrations of responsible macrophage and neutrophil. Conclusively, our findings provide the rationale that mcMSCs are applicable as a potential source of cell-based therapy in inflammatory bowel diseases, especially contributing either to prevent relapse or to accelerate healing as solution to unmet medical needs in IBD therapy.
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A facile, scalable route to new nanocomposites that are based on carbon nanotubes/heteroatom-doped carbon (CNT/HDC) core-sheath nanostructures is reported. These nanostructures were prepared by the adsorption of heteroatom-containing ionic liquids on the walls of CNTs, followed by carbonization. The design of the CNT/HDC composite allows for combining the electrical conductivity of the CNTs with the catalytic activity of the heteroatom-containing HDC sheath layers. The CNT/HDC nanostructures are highly active electrocatalysts for the oxygen reduction reaction and displayed one of the best performances among heteroatom-doped nanocarbon catalysts in terms of half-wave potential and kinetic current density. The four-electron selectivity and the exchange current density of the CNT/HDC nanostructures are comparable with those of a Pt/C catalyst, and the CNT/HDC composites were superior to Pt/C in terms of long-term durability and poison tolerance. Furthermore, an alkaline fuel cell that employs a CNT/HDC nanostructure as the cathode catalyst shows very high current and power densities, which sheds light on the practical applicability of these new nanocomposites.
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A poly(p-phenylene)-based multiblock polymer is developed with an oligomeric chain extender and cerium (CE-sPP-PPES + Ce3+) to realize better performance and durability in proton exchange membrane fuel cells. The membrane performance is evaluated in single cells at 80 °C and at 100% and 50% relative humidity (RH). The accelerated stability test is conducted 90 °C and 30% RH, during which linear sweep voltammetry and hydrogen permeation detection are monitored periodically. Results demonstrate that the proton conductivity of the pristine hydrocarbon membranes is superior to that of PFSA membranes, and the hydrogen crossover is significantly lower. In addition, a composite membrane containing cerium performs similarly to a pristine membrane, particularly at low RH levels. Adding cerium to CE-sPP-PPES + Ce3+ membranes improves their chemical durability significantly, with an open circuit voltage decay rate of only 89 µV/h for 1000 h. The hydrogen crossover is maintained across accelerated stability tests, as confirmed by hydrogen detection and crossover current density. The short-circuit resistance indicates that membrane thinning is less likely to occur. Collectively, these results demonstrate that a hydrocarbon membrane with cerium is a potential alternative for fuel cell applications.
RESUMO
We here demonstrate that SERTAD1 is an adaptor protein responsible for the regulation of lysine 63 (K63)-linked NLRP3 polyubiquitination by the Cullin1 E3 ubiquitin ligase upon inflammasome activation. SERTAD1 specifically binds to NLRP3 but not to other inflammasome sensors. This endogenous interaction increases after inflammasome activation, interfering with the interaction between NLRP3 and Cullin1. Interleukin (IL)-1ß and IL-18 secretion, as well as the cleavage of gasdermin D, are decreased in SERTAD1 knockout bone-marrow-derived macrophages, together with reduced formation of the NLRP3 inflammasome complex. Additionally, SERTAD1-deficient mice show attenuated severity of monosodium-uric-acid-induced peritonitis and experimental autoimmune encephalomyelitis. Analysis of public datasets indicates that expression of SERTAD1 mRNA is significantly increased in the patients of autoimmune diseases. Thus, our findings uncover a function of SERTAD1 that specifically reduces Cullin1-mediated NLRP3 polyubiquitination via direct binding to NLRP3, eventually acting as a crucial factor to regulate the initiation of NLRP3-mediated inflammasome activation.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Humanos , Camundongos , Inflamassomos/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
A pleiotropic immunoregulatory cytokine, TGF-ß, signals via the receptor-regulated SMADs: SMAD2 and SMAD3, which are constitutively expressed in normal cells. Here, we show that selective repression of SMAD3 induces cDC differentiation from the CD115+ common DC progenitor (CDP). SMAD3 was expressed in haematopoietic cells including the macrophage DC progenitor. However, SMAD3 was specifically down-regulated in CD115+ CDPs, SiglecH- pre-DCs, and cDCs, whereas SMAD2 remained constitutive. SMAD3-deficient mice showed a significant increase in cDCs, SiglecH- pre-DCs, and CD115+ CDPs compared with the littermate control. SMAD3 repressed the mRNA expression of FLT3 and the cDC-related genes: IRF4 and ID2. We found that one of the SMAD transcriptional corepressors, c-SKI, cooperated with phosphorylated STAT3 at Y705 and S727 to repress the transcription of SMAD3 to induce cDC differentiation. These data indicate that STAT3 and c-Ski induce cDC differentiation by repressing SMAD3: the repressor of the cDC-related genes during the developmental stage between the macrophage DC progenitor and CD115+ CDP.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA , Células Dendríticas , Proteínas Proto-Oncogênicas , Fator de Transcrição STAT3 , Proteína Smad3 , Animais , Camundongos , Células Dendríticas/metabolismo , Células Dendríticas/citologia , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad2/genética , Proteína Smad3/metabolismo , Proteína Smad3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
BACKGROUND: Multinucleation is a hallmark of osteoclast formation and has a unique ability to resorb bone matrix. During osteoclast differentiation, the cytoskeleton reorganization results in the generation of actin belts and eventual bone resorption. Tetraspanins are involved in adhesion, migration and fusion in various cells. However, its function in osteoclast is still unclear. In this study, we identified Tm4sf19, a member of the tetraspanin family, as a regulator of osteoclast function. MATERIALS AND METHODS: We investigate the effect of Tm4sf19 deficiency on osteoclast differentiation using bone marrow-derived macrophages obtained from wild type (WT), Tm4sf19 knockout (KO) and Tm4sf19 LELΔ mice lacking the large extracellular loop (LEL). We analyzed bone mass of young and aged WT, KO and LELΔ mice by µCT analysis. The effects of Tm4sf19 LEL-Fc fusion protein were accessed in osteoclast differentiation and osteoporosis animal model. RESULTS: We found that deficiency of Tm4sf19 inhibited osteoclast function and LEL of Tm4sf19 was responsible for its function in osteoclasts in vitro. KO and LELΔ mice exhibited higher trabecular bone mass compared to WT mice. We found that Tm4sf19 interacts with integrin αvß3 through LEL, and that this binding is important for cytoskeletal rearrangements in osteoclast by regulating signaling downstream of integrin αvß3. Treatment with LEL-Fc fusion protein inhibited osteoclast function in vitro and administration of LEL-Fc prevented bone loss in an osteoporosis mouse model in vivo. CONCLUSION: We suggest that Tm4sf19 regulates osteoclast function and that LEL-Fc may be a promising drug to target bone destructive diseases caused by osteoclast hyper-differentiation.
Assuntos
Doenças Ósseas , Reabsorção Óssea , Osteoporose , Tetraspaninas , Animais , Camundongos , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular , Integrina alfaVbeta3/metabolismo , Osteoclastos , Osteoporose/genética , Osteoporose/metabolismo , Tetraspaninas/genética , Tetraspaninas/metabolismoRESUMO
Angiopoietin-like protein 2 (Angptl2) levels are increased by obesity and obesity-related pathological conditions, and it is considered to be an important adipocyte-derived inflammatory mediator. In contrast, the multifunctional cytokine TGF-ß1 has been reported to be augmented in obesity of rodents and humans, but inhibits adipocyte differentiation in vitro. Here we demonstrate that TGF-ß1 induces expression of the Angptl2 gene through a Smad3-dependent pathway in RAW264.7 macrophage cells, primary peritoneal macrophages, and differentiated 3T3-L1 adipocytes. Transcriptional induction of the Angptl2 gene by TGF-ß1 was dependent on the Smad3 protein which binds to the Smad Binding Element (SBE) region located on the Angptl2 promoter. Macrophages with Smad3 knocked down by small interfering RNA showed reduction of TGF-ß1-induced Angptl2 expression. These findings may provide insight into the molecular mechanisms of the increased expression of Angptl2 and TGF-ß1 in obesity.
Assuntos
Angiopoietinas/genética , Regulação da Expressão Gênica , Obesidade/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células 3T3-L1 , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Animais , Linhagem Celular , Doença Crônica , Humanos , Imunoprecipitação , Inflamação/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the most lethal type of cancer and the third leading cause of cancer death with the lowest 5-year survival rate. Heterogeneity, difficulty in diagnosis, and rapid metastatic progression are the causes of high mortality in pancreatic cancer. Recent studies have shown that Protein arginine methyltransferase 5 (PRMT5) is overexpressed in pancreatic cancers, and these patients have a worse prognosis. Recently, PRMT5 as an anti-cancer target has gained considerable interest. In this study, we investigated whether inhibition of PRMT5 activity was synergistic with blockade of TGF-ß1 signaling, which plays an important role in the construction of the desmoplastic matrix in pancreatic cancer and induces therapeutic vulnerability. Compared with T1-44, a selective inhibitor of PRMT5 activity, the combination of T1-44 with the TGF-ß1 signaling inhibitor Vactosertib significantly reduced tumor size and surrounding tissue invasion and significantly improved long-term survival. RNA sequencing analysis of mouse tumors revealed that the combination of T1-44 and Vactosertib significantly altered the expression of genes involved in cancer progression, such as cell migration, extracellular matrix, and apoptotic processes. In particular, the expression of Btg2, known as a tumor suppressor factor in various cancers, was markedly induced by combination treatment. Ectopic overexpression of Btg2 inhibited the EMT response, blocking cell migration, and promoted cancer cell death. These data demonstrate that the combination therapy of T1-44 with Vactosertib is synergistic for pancreatic cancer, suggesting that this novel combination therapy has value in the treatment strategy of patients with pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Camundongos , Fator de Crescimento Transformador beta1/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Inibidores Enzimáticos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias PancreáticasRESUMO
The signaling pathways governing acetaminophen (APAP)-induced liver injury have been extensively studied. However, little is known about the ubiquitin-modifying enzymes needed for the regulation of APAP-induced liver injury. Here, we examined whether the Pellino3 protein, which has E3 ligase activity, is needed for APAP-induced liver injury and subsequently explored its molecular mechanism. Whole-body Peli3-/- knockout (KO) and adenovirus-mediated Peli3 knockdown (KD) mice showed reduced levels of centrilobular cell death, infiltration of immune cells, and biomarkers of liver injury, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), upon APAP treatment compared to wild-type (WT) mice. Peli3 deficiency in primary hepatocytes decreased mitochondrial and lysosomal damage and reduced the mitochondrial reactive oxygen species (ROS) levels. In addition, the levels of phosphorylation at serine 9 in the cytoplasm and mitochondrial translocation of GSK3ß were decreased in primary hepatocytes obtained from Peli3-/- KO mice, and these reductions were accompanied by decreases in JNK phosphorylation and mitochondrial translocation. Pellino3 bound more strongly to GSK3ß compared with JNK1 and JNK2 and induced the lysine 63 (K63)-mediated polyubiquitination of GSK3ß. In rescue experiments, the ectopic expression of wild-type Pellino3 in Peli3-/- KO hepatocytes restored the mitochondrial translocation of GSK3ß, but this restoration was not obtained with expression of a catalytically inactive mutant of Pellino3. These findings are the first to suggest a mechanistic link between Pellino3 and APAP-induced liver injury through the modulation of GSK3ß polyubiquitination.
Assuntos
Acetaminofen , Doença Hepática Crônica Induzida por Substâncias e Drogas , Animais , Camundongos , Acetaminofen/efeitos adversos , Fosforilação , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Fígado/metabolismo , Hepatócitos/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Bone morphogenetic proteins (BMPs) that belong to the transforming growth factor-ß (TGF-ß) superfamily cytokines, play crucial roles in hematopoiesis. However, roles of Smad6 in hematopoiesis remained unknown in contrast to the other inhibitory Smad (I-Smad), Smad7. Here we show that Smad6 inhibits erythropoiesis in human CD34(+) cord blood hematopoietic stem cells (HSCs). Smad6 was specifically expressed in CD34(+) cord blood HSCs, which was correlated with the expression of BMP2/4/6/7 and BMP type I receptor (BMPRI). BMP-specific receptor-regulated Smads (R-Smads), Smad1 and Smad5 in cooperation with Smad4 induced transcription of the Smad6 gene. Instead of affecting cell cycle, apoptosis, self-renewal, and stemness of CD34(+) cells, Smad6 knockdown enhanced, whereas Smad6 overexpression suppressed erythropoiesis in stem cell culture and colony formation assay. Consistently, Smad6 suppressed the expression of the genes essential for erythropoiesis, such as Kruppel-like factor 1 (erythroid) (KLF1/EKLF) and GATA binding protein 2 (GATA-2). Promoter analyses showed that Smad6 repressed Smad5/4-induced transcription of the Klf1 gene. Thus, our data suggest that Smad6 indirectly maintains stemness by preventing spontaneous erythropoiesis in HSCs.
Assuntos
Eritropoese/genética , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Proteína Smad6/metabolismo , Antígenos CD34/análise , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 7/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Células Cultivadas , Sangue Fetal/citologia , Fator de Transcrição GATA2/genética , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Regiões Promotoras Genéticas , Proteína Smad6/genética , Transcrição GênicaRESUMO
Suppressors of cytokine signaling (SOCS) have emerged as potential regulators of macrophage function. We have investigated mechanisms of SOCS3 action on type 2 macrophage (M2) differentiation induced by glucocorticoid using human monocytic cell lines and mouse bone marrow-derived macrophages. Treatment of THP1 monocytic cells with dexamethasone (Dex) induced ROS generation and M2 polarization promoting IL-10 and TGF-ß production, while suppressing IL-1ß, TNF-α and IL-6 production. SOCS3 over-expression reduced, whereas SOCS3 ablation enhanced IL-10 and TGF-ß induction with concomitant regulation of ROS. As a mediator of M2 differentiation, glucocorticoid-induced leucine zipper (GILZ) was down-regulated by SOCS3 and up-regulated by shSOCS3. The induction of GILZ and IL-10 by Dex was dependent on ROS and p38 MAPK activity. Importantly, GILZ ablation led to the inhibition of ROS generation and anti-inflammatory cytokine induction by Dex. Moreover, GILZ knock-down negated the up-regulation of IL-10 production induced by shSOCS3 transduction. Our data suggest that SOCS3 targets ROS- and p38-dependent GILZ expression to suppress Dex-induced M2 polarization.
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Site-specific ubiquitination can regulate the functions of Rab proteins in membrane trafficking. Previously we showed that site-specific monoubiquitination on Rab5 downregulates its function. Rab7 acts in the downstream of Rab5. Although site-specific ubiquitination of Rab7 can affect its function, it remains elusive how the ubiquitination is involved in modulation of the function of Rab7 at molecular level. Here, we report molecular basis for the regulation of Rab7 by site-specific monoubiquitination. Rab7 was predominantly monoubiquitinated at multiple sites in the membrane fraction of cultured cells. Two major ubiquitination sites (K191 and K194), identified by mutational analysis with single K mutants, were responsible for membrane localization of monoubiquitinated Rab7. Using small-angle X-ray scattering, we derived structural models of site-specifically monoubiquitinated Rab7 in solution. Structural analysis combined with molecular dynamics simulation corroborated that the ubiquitin moieties on K191 and K194 are key determinants for exclusion of Rab7 from the endosomal membrane. Ubiquitination on the two major sites apparently mitigated colocalization of Rab7 with ORF3a of SARS-CoV-2, potentially deterring the egression of SARS-CoV-2. Our results establish that the regulatory effects of a Rab protein through site-specific monoubiquitination are commonly observed among Rab GTPases while the ubiquitination sites differ in each Rab protein.
Assuntos
SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , proteínas de unión al GTP Rab7/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , UbiquitinaçãoRESUMO
BACKGROUND & AIMS: Galectin-3 is a beta-galactoside-binding protein that increases gastric cancer cell motility in response to integrin signaling and is highly expressed in gastric tumor cells. Galectin-3 induces cytoskeletal remodeling to increase cell motility, but the mechanisms of this process are not understood. We investigated the effects of galectin-3 on fascin-1, an actin-bundling protein. METHODS: We collected malignant and normal tissues from gastric cancer patients and examined the expression levels of galectin-3 and fascin-1. We silenced galectin-3 expression in human gastric cancer cell lines using small interfering RNA and lenti-viral constructs and determined the effects on fascin-1 expression, cell motility, and invasion. RESULTS: Malignant gastric tissues expressed high levels of galectin-3 and fascin-1, compared with normal gastric tissues. Silencing of galectin-3 resulted in altered cancer cell morphology, reduced fascin-1 expression, decreased cell motility, and reduced malignant cell invasion. Galectin-3 overexpression reversed these effects. Silencing of fascin-1 also reduced cell motility and caused changes in cell shape, as did silencing of galectin-3. Furthermore, galectin-3 silencing inhibited the interaction between glycogen synthase kinase (GSK)-3beta, beta-catenin, and T-cell factor (TCF) 4, and the binding of beta-catenin/TCF-4 to the fascin-1 promoter. Nuclear localization of GSK-3beta and beta-catenin were not detected when galectin-3 was silenced. Overexpression of mutated galectin-3 (with mutations in the GSK-3beta binding and phosphorylation motifs) did not increase fascin-1 levels, in contrast to overexpression of wild-type galectin-3. CONCLUSIONS: Galectin-3 increases cell motility by up-regulating fascin-1 expression. Galectin-3 might be a potential therapeutic target for the prevention and treatment of gastric cancer progression.
Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular , Galectina 3/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neoplasias Gástricas/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Forma Celular , Galectina 3/genética , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Proteínas dos Microfilamentos/genética , Mutação , Invasividade Neoplásica , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/secundário , Fatores de Transcrição TCF/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição , Transfecção , Regulação para Cima , beta Catenina/metabolismoRESUMO
BACKGROUND: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been demonstrated to be useful for molecular profiling of common solid tumors. Using recently developed MALDI matrices for lipid profiling, we evaluated whether direct tissue MALDI MS analysis on proteins and lipids may classify human breast cancer samples according to the intrinsic subtype. METHODS: Thirty-four pairs of frozen, resected breast cancer and adjacent normal tissue samples were analyzed using histology-directed, MALDI MS analysis. Sinapinic acid and 2,5-dihydroxybenzoic acid/α-cyano-4-hydroxycinnamic acid were manually deposited on areas of each tissue section enriched in epithelial cells to identify lipid profiles, and mass spectra were acquired using a MALDI-time of flight instrument. RESULTS: Protein and lipid profiles distinguish cancer from adjacent normal tissue samples with the median prediction accuracy of 94.1%. Luminal, HER2+, and triple-negative tumors demonstrated different protein and lipid profiles, as evidenced by permutation P values less than 0.01 for 0.632+ bootstrap cross-validated misclassification rates with all classifiers tested. Discriminatory proteins and lipids were useful for classifying tumors according to the intrinsic subtype with median prediction accuracies of 80.0-81.3% in random test sets. CONCLUSIONS: Protein and lipid profiles accurately distinguish tumor from adjacent normal tissue and classify breast cancers according to the intrinsic subtype.
Assuntos
Adenocarcinoma/classificação , Neoplasias da Mama/classificação , Carcinoma/classificação , Lipídeos/análise , Proteínas de Neoplasias/análise , Adenocarcinoma/metabolismo , Adulto , Idoso , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Few studies have examined the role of BAG2 in malignancies. We investigated the prognostic value of BAG2-expression in cancer-associated fibroblasts (CAFs) and tumor cells in predicting metastasis-free survival in patients with breast cancer. Tissue-microarray was constructed using human breast cancer tissues obtained by surgical resection between 1992 and 2015. BAG2 expression was evaluated by immunohistochemistry in CAFs or the tumor cells. BAG2 expression in the CAFs and cytoplasm of tumor cells was classified as positive and negative, and low and high, respectively. BAG2-CAF was evaluated in 310 patients and was positive in 67 (21.6%) patients. Kaplan-Meier plots showed that distant metastasis-free survival (DMFS) was lesser in patients with BAG2(+) CAF than in patients with BAG2(-) CAF (p = 0.039). Additionally, we classified the 310 patients into two groups: 109 in either BAG2-high or BAG2(+) CAF and 201 in BAG2-low and BAG2(-) CAF. DMFS was significantly reduced in patients with either BAG2-high or BAG2(+) CAF than in the patients of the other group (p = 0.005). Multivariable analysis demonstrated that DMFS was prolonged in patients with BAG2(-) CAF or BAG2-low. Evaluation of BAG2 expression on both CAFs and tumor cells could help in determining the risk of metastasis in breast cancer.
RESUMO
Transforming growth factor-beta1 (TGF-beta1) performs diverse cellular functions, including anti-inflammatory activity. The inhibitory Smad (I-Smad) Smad6 was previously shown to play an important role in TGF-beta1-induced negative regulation of Interleukin-1/Toll-like receptor (IL-1R/TLR) signaling through binding to Pellino-1, an adaptor protein of interleukin-1 receptor associated kinase 1(IRAK1). However, it is unknown whether Smad7, the other inhibitory Smad, also has a role in regulating IL-1R/TLR signaling. Here, we demonstrate that endogeneous Smad7 and Smad6 simultaneously bind to discrete regions of Pellino-1 upon TGF-beta1 treatment, via distinct regions of the Smad MH2 domains. In addition, the Smad7-Pellino-1 interaction abrogated NF-kappaB activity by blocking formation of the IRAK1-mediated IL-1R/TLR signaling complex, subsequently causing reduced expression of pro-inflammatory genes. Double knock-down of endogenous Smad6 and Smad7 genes by RNA interference further reduced the anti-inflammatory activity of TGF-beta1 than when compared with single knock-down of Smad7. These results provide evidence that the I-Smads, Smad6 and Smad7, act as critical mediators for effective TGF-beta1-mediated suppression of IL-1R/TLR signaling, by simultaneous binding to discrete regions of Pellino-1.