RESUMO
BACKGROUND: Palifermin (trade name Kepivance®) is an amino-terminally truncated recombinant human keratinocyte growth factor 1 (KGF-1) with 140 residues that has been produced using Escherichia coli to prevent and treat oral mucositis following radiation or chemotherapy. In this study, an amino-terminally shortened KGF-1 variant with 135 residues was produced and purified in E. coli, and its cell proliferation activity was evaluated. RESULTS: We expressed soluble KGF-1 fused to thioredoxin (TRX) in the cytoplasmic fraction of E. coli to improve its production yield. However, three N-truncated forms (KGF-1 with 140, 138, and 135 residues) were observed after the removal of the TRX protein from the fusion form by cleavage of the human enterokinase light chain C112S (hEKL C112S). The shortest KGF-1 variant, with 135 residues, was expressed by fusion with TRX via the hEKL cleavage site in E. coli and purified at high purity (> 99%). Circular dichroism spectroscopy shows that purified KGF-1135 had a structure similar to that of the KGF-1140 as a random coiled form, and MCF-7 cell proliferation assays demonstrate its biological activity. CONCLUSIONS: We identified variations in N-terminus-truncated KGF-1 and selected the most stable form. Furthermore, by a simple two-step purification, highly purified KGF-1135 was obtained that showed biological activity. These results demonstrate that KGF-1135 may be considered an alternative protein to KGF-1.
Assuntos
Escherichia coli , Fator 7 de Crescimento de Fibroblastos , Humanos , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Recombinant human keratinocyte growth factor 2 (KGF-2), also known as repifermin, is used in various therapeutic applications. However, KGF-2 production has not been optimized for facilitating large-scale production. Therefore, we attempted to attain high-level production of bioactive KGF-2. KGF-2 was fused with 6HFh8 (6HFh8-KGF-2) at the tobacco etch virus protease cleavage site. The 6HFh8-KGF-2 was expressed in Escherichia coli with high expression levels of approximately 33% and 20% of soluble protein in flask culture and 5 L fermentation, respectively. 6HFh8-KGF-2 was purified via nickel affinity chromatography. To maintain a stable form of KGF-2, the conditions of the cleavage reaction were optimized based on the isoelectric point. KGF-2 was purified via ion-exchange chromatography to high purity (>99%) with an optimal purification yield (91%). Circular dichroism spectroscopy demonstrated that purified KGF-2 had a secondary structure and thermal stability similar to that of commercial KGF-2. Bioactivity assays indicated that purified KGF-2 could induce MCF-7 cell proliferation in the same manner as commercial KGF-2. These results demonstrate that bioactive KGF-2 was overexpressed in E. coli and purified to high quality. Our findings indicated that bioactive KGF-2 can be produced in large quantities in E. coli.
Assuntos
Escherichia coli , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Fator 10 de Crescimento de Fibroblastos/metabolismo , Células MCF-7 , FermentaçãoRESUMO
The autophagy-mediated lysosomal pathway plays an important role in conferring stress tolerance to tumor cells during cellular stress such as increased metabolic demands. Thus, targeted disruption of this function and inducing lysosomal cell death have been proved to be a useful cancer therapeutic approach. In this study, we reported that octyl syringate (OS), a novel phenolic derivate, was preferentially cytotoxic to various cancer cells but was significantly less cytotoxic to non-transformed cells. Treatment with OS resulted in non-apoptotic cell death in a caspase-independent manner. Notably, OS not only enhanced accumulation of autophagic substrates, including lapidated LC3 and sequestosome-1, but also inhibited their degradation via an autophagic flux. In addition, OS destabilized the lysosomal function, followed by the intracellular accumulation of the non-digestive autophagic substrates such as bovine serum albumin and stress granules. Furthermore, OS triggered the release of lysosomal enzymes into the cytoplasm that contributed to OS-induced non-apoptotic cell death. Finally, we demonstrated that OS was well tolerated and reduced tumor growth in mouse xenograft models. Taken together, our study identifies OS as a novel anticancer agent that induces lysosomal destabilization and subsequently inhibits autophagic flux and further supports development of OS as a lysosome-targeting compound in cancer therapy. ⢠Octyl syringate, a phenolic derivate, is preferentially cytotoxic to various cancer cells. ⢠Octyl syringate destabilizes the lysosomal function. ⢠Octyl syringate blocks the autophagic flux. ⢠Octyl syringate is a potential candidate compound for cancer therapy.
Assuntos
Antineoplásicos , Neoplasias , Camundongos , Animais , Humanos , Apoptose , Antineoplásicos/farmacologia , Morte Celular , Autofagia , Lisossomos/metabolismo , Neoplasias/metabolismoRESUMO
Acute myeloid leukemia (AML) is an aggressive blood cancer with poor clinical outcomes. Emerging data suggest that mitochondrial oxidative phosphorylation (mtOXPHOS) plays a significant role in AML tumorigenesis, progression, and resistance to chemotherapies. However, how the mtOXPHOS is regulated in AML cells is not well understood. In this study, we investigated the oncogenic functions of ERRα in AML by combining in silico, in vitro, and in vivo analyses and showed ERRα is a key regulator of mtOXPHOS in AML cells. The increased ERRα level was associated with worse clinical outcomes of AML patients. Single cell RNA-Seq analysis of human primary AML cells indicated that ERRα-expressing cancer cells had significantly higher mtOXPHOS enrichment scores. Blockade of ERRα by pharmacologic inhibitor (XCT-790) or gene silencing suppressed mtOXPHOS and increased anti-leukemic effects in vitro and in xenograft mouse models.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Camundongos , Animais , Fosforilação Oxidativa , Apoptose , Mitocôndrias/metabolismo , Leucemia Mieloide Aguda/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Cancer-associated fibroblasts (CAFs) are key structural components of the tumor microenvironment and are closely associated with tumor invasion and metastasis. Lysophosphatidic acid (LPA) is a biolipid produced extracellularly and involved in tumorigenesis and metastasis. LPA has recently been implicated in the education and transdifferentiation of normal fibroblasts (NFs) into CAFs. However, little is known about the effects of LPA on CAFs and their participation in cancer cell invasion. In the present study, we identified a critical role of LPA-induced amphiregulin (AREG) secreted from CAFs in cancer invasiveness. CAFs secrete higher amounts of AREG than NFs, and LPA induces AREG expression in CAFs to augment their invasiveness. Strikingly, knocking out the AREG gene in CAFs attenuates cancer invasiveness and metastasis. Mechanistically, LPA induces Yes-associated protein (YAP) activation and Zinc finger E-box binding homeobox 1 (Zeb1) expression through the LPAR1 and LPAR3/Gi/Rho signaling axes, leading to AREG expression. Furthermore, we provide evidence that metformin, a biguanide derivative, significantly inhibits LPA-induced AREG expression in CAFs to attenuate cancer cell invasiveness. Collectively, the present data show that LPA induces AREG expression through YAP and Zeb1 in CAFs to promote cancer cell invasiveness, with the process being inhibited by metformin, providing potential biomarkers and therapeutic avenues to interdict cancer cell invasion.
RESUMO
In TNF signaling, ubiquitination of RIP1 functions as an early cell-death checkpoint, which prevents the spatial transition of the signaling complex from complex-I to death-inducing complex-II. Here, we report that ankyrin repeat domain 13a (ANKRD13a) acts as a novel component of complex-II to set a higher signal threshold for the cytotoxic potential of TNF. ANKRD13a deficiency is sufficient to turn the response to TNF from survival to death by promoting the formation of complex-II without affecting NF-κB activation. ANKRD13a binds to ubiquitinated-RIP1 via its UIM, and subsequently limits the association of FADD and caspase-8 with RIP1. Moreover, high ANKRD13a expression is inversely correlated with apoptotic phenotypes in ovarian cancer tissues and is associated with poor prognosis. Our work identifies ANKRD13a as a novel gatekeeper of the early cell-death checkpoint, which may function as part of an escape mechanism from cell death in some cancers.
Assuntos
Proteínas de Membrana , NF-kappa B , Complexo de Proteínas Formadoras de Poros Nucleares , Neoplasias Ovarianas , Proteínas de Ligação a RNA , Fator de Necrose Tumoral alfa , Apoptose/fisiologia , Caspase 8/metabolismo , Morte Celular/fisiologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Feminino , Humanos , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , UbiquitinaçãoRESUMO
The orphan nuclear receptor ESRRA (estrogen related receptor alpha) is critical in mitochondrial biogenesis and macroautophagy/autophagy function; however, the roles of ESRRA in intestinal function remain uncharacterized. Herein we identified that ESRRA acts as a key regulator of intestinal homeostasis by amelioration of colonic inflammation through activation of autophagic flux and control of host gut microbiota. Esrra-deficient mice presented with increased susceptibility to dextran sodium sulfate (DSS)-induced colitis with upregulation of intestinal inflammation. In addition, esrra-null mice had depressed AMP-activated protein kinase phosphorylation (AMPK), lower levels of TFEB (transcription factor EB), and accumulation of SQSTM1/p62 (sequestosome 1) with defective mitochondria in intestinal tissues. Esrra-deficient mice showed distinct gut microbiota composition and significantly higher microbial diversity than wild-type (WT) mice. Cohousing or fecal microbiota transplantation from WT mice to Esrra-deficient mice ameliorated DSS-induced colitis severity. Importantly, patients with ulcerative colitis (UC) had significantly decreased ESRRA expression in intestinal mucosal tissues that correlated with disease activity, suggesting clinical relevance of ESRRA in UC. Taken together, our results show that ESRRA contributes to intestinal homeostasis through autophagy activation and gut microbiota control to protect the host from detrimental inflammation and dysfunctional mitochondria.Abbreviations: ABX, antibotics; AMPK, AMP-activated protein kinase; ATP5A1, ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1; BECN1, beclin1, autophagy related, CCL, C-C motif chemokine ligand; CD, Crohn disease; CLDN, claudin; COX4I1, cytochrome c oxidase subunit 4I1; cKO, conditional knockout; cWT, conditional wild-type; CXCL, C-X-C motif chemokine ligand; DAI, disease activity index; DSS, dextran sodium sulfate; EGFP, enhanced green fluorescent protein; ESRR, estrogen related receptor; ESRRA, estrogen related receptor alpha; Esrra+/+, Esrra wild type; esrra-/-, esrra homozygous knockout; FMT, fecal microbiota transplantation; GABARAP, gamma-aminobutyric acid receptor associated protein; GSEA, gene set enrichment analysis; IBD, inflammatory bowel disease; IL, interleukin; KO, knockout; LAMP1, lysosomal-associated membrane protein 1; LCN2, lipocalin 2; LEfSe, linear discriminant analysis (LDA) effect size; LPS, lipopolysachharide; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; NDUFAB1, NADH: ubiquinone oxidoreductase subunit AB1; OCLN, occludin; OUT, operational taxonomic unit; OXPHOS, oxidative phosphorylation; PCoA, principal coordinate analysis; PPARGC1A, PPARG coactiva- tor 1 alpha; PRKAA, 5'-AMP-activated protein kinase catalytic subunit alpha; PTGS2/COX2, prostaglandin-endoperoxide synthase 2; RAB7, member RAS oncogene family; SDHB, succinate dehydrogenase complex, subunit B, iron sulfur (Ip); SQSTM1/p62, sequestosome 1; S100A9, S100 calcium binding protein A9 (calgranulin B); TCA, tricarboxylic acid; TFEB, transcription factor EB; TNF, tumor necrosis factor; UC, ulcerative colitis; UCP2, uncoupling protein 2 (mitochondrial, proton carrier); UQCRC1, ubiquinol-cytochrome c reductase core protein 1; UVRAG, UV radiation resistance associated gene; Vil1, villin; VPS11, VPS11, CORVET/HOPS core sub-unit; WT, wild type.
Assuntos
Autofagia , Microbioma Gastrointestinal , Animais , Autofagia/fisiologia , Sulfato de Dextrana/metabolismo , Sulfato de Dextrana/farmacologia , Estrogênios/metabolismo , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismoRESUMO
The ligand of Numb protein-X (LNX) family, also known as the PDZRN family, is composed of four discrete RING-type E3 ubiquitin ligases (LNX1, LNX2, LNX3, and LNX4), and LNX5 which may not act as an E3 ubiquitin ligase owing to the lack of the RING domain. As the name implies, LNX1 and LNX2 were initially studied for exerting E3 ubiquitin ligase activity on their substrate Numb protein, whose stability was negatively regulated by LNX1 and LNX2 via the ubiquitin-proteasome pathway. LNX proteins may have versatile molecular, cellular, and developmental functions, considering the fact that besides these proteins, none of the E3 ubiquitin ligases have multiple PDZ (PSD95, DLGA, ZO-1) domains, which are regarded as important protein-interacting modules. Thus far, various proteins have been isolated as LNX-interacting proteins. Evidence from studies performed over the last two decades have suggested that members of the LNX family play various pathophysiological roles primarily by modulating the function of substrate proteins involved in several different intracellular or intercellular signaling cascades. As the binding partners of RING-type E3s, a large number of substrates of LNX proteins undergo degradation through ubiquitin-proteasome system (UPS) dependent or lysosomal pathways, potentially altering key signaling pathways. In this review, we highlight recent and relevant findings on the molecular and cellular functions of the members of the LNX family and discuss the role of the erroneous regulation of these proteins in disease progression.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Citoplasma/metabolismo , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Ubiquitina-Proteína Ligases/químicaRESUMO
Morphological and functional changes in cells during the epithelial-mesenchymal transition (EMT) process are known to be regulated by alternative splicing. However, only a few splicing factors involved in EMT have been reported and their underlying mechanisms remain largely unknown. Here, we showed that an isoform of tight junction protein 1 (TJP1) lacking exon 20 (TJP1-α-) is predominantly expressed in tumor tissues and in A549 cells during transforming growth factor-ß (TGF-ß)-induced EMT. RBM47 promoted the inclusion of exon 20 of TJP1, the alternative exon encoding the α-domain, by which RBM47 recognizes to (U)GCAUG in the downstream intronic region of exon 20. We also found that the first RNA recognition motif (RRM) domain of RBM47 is critical in the regulation of alternative splicing and its recognition to pre-mRNA of TJP1. Furthermore, we demonstrated that the TJP1-α- isoform enhances the assembly of actin stress fibers, thereby promoting cellular migration in a wound healing assay. Our results suggest the regulatory mechanism for the alternative splicing of TJP1 pre-mRNA by RBM47 during EMT, providing a basis for studies related to the modulation of EMT via alternative splicing.
Assuntos
Transição Epitelial-Mesenquimal/genética , Proteínas de Ligação a RNA/fisiologia , Fibras de Estresse/metabolismo , Proteína da Zônula de Oclusão-1/genética , Células A549 , Actinas/metabolismo , Processamento Alternativo , Linhagem Celular Tumoral , Progressão da Doença , Células HEK293 , Humanos , Células MCF-7 , Neoplasias/genética , Neoplasias/patologia , Multimerização Proteica/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.
Assuntos
Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Transgenes , Animais , Células HEK293 , Humanos , TransfecçãoRESUMO
Primary cilia are microtubule-based, dynamic organelles characterized by continuous assembly and disassembly. The intraflagellar transport (IFT) machinery, including IFT88 in cilia, is involved in the maintenance of bidirectional motility along the axonemes, which is required for ciliogenesis and functional competence. Cancer cells are frequently associated with loss of primary cilia and IFT functions. However, there is little information on the role of IFT88 or primary cilia in the metabolic remodeling of cancer cells. Therefore, we investigated the cellular and metabolic effects of the loss-of-function (LOF) mutations of IFT88/primary cilia in thyroid cancer cells. IFT88-deficient 8505C thyroid cancer cells were generated using the CRISPR/Cas9 system, and RNA-sequencing analysis was performed. LOF of the IFT88 gene resulted in a marked defect in ciliogenesis and mitochondrial oxidative function. Gene expression patterns in IFT88-deficient thyroid cancer cells favored glycolysis and lipid biosynthesis. However, LOF of IFT88/primary cilia did not promote thyroid cancer cell proliferation, migration, and invasion. The results suggest that IFT88/primary cilia play a role in metabolic reprogramming in thyroid cancer cells.
Assuntos
Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Células Cultivadas , Cílios/genética , Cílios/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , FenótipoRESUMO
AIMS: CR6 interacting factor 1 (CRIF1) deficiency impairs mitochondrial oxidative phosphorylation complexes, contributing to increased mitochondrial and cellular reactive oxygen species (ROS) production. CRIF1 downregulation has also been revealed to decrease sirtuin 1 (SIRT1) expression and impair vascular function. Inhibition of SIRT1 disturbs oxidative energy metabolism and stimulates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-induced inflammation. Therefore, we hypothesized that both CRIF1 deficiency-induced mitochondrial ROS production and SIRT1 reduction play stimulatory roles in vascular inflammation. METHODS AND RESULTS: Plasma levels and mRNA expression of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6) were markedly elevated in endothelium-specific CRIF1-knockout mice and CRIF1-silenced endothelial cells, respectively. Moreover, CRIF1 deficiency-induced vascular adhesion molecule-1 (VCAM-1) expression was consistently attenuated by the antioxidant N-acetyl-cysteine and NF-κB inhibitor (BAY11). We next showed that siRNA-mediated CRIF1 downregulation markedly activated NF-κB. SIRT1 overexpression not only rescued CRIF1 deficiency-induced NF-κB activation but also decreased inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and VCAM-1 expression levels in endothelial cells. CONCLUSIONS: These results strongly suggest that CRIF1 deficiency promotes endothelial cell inflammation by increasing VCAM-1 expression, elevating inflammatory cytokines levels, and activating the transcription factor NF-κB, all of which were inhibited by SIRT1 overexpression.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Regulação para Baixo , Proteínas Nucleares/fisiologia , Sirtuína 1/genética , Animais , Proteínas de Ciclo Celular/genética , Ensaio de Imunoadsorção Enzimática , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Proteínas Nucleares/genética , Fosforilação Oxidativa , Transdução de SinaisRESUMO
We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. The donor construct was flanked with homology arms of about 1 kb at the 5' and 3' ends. Injected embryos (G0) were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have undergone the desired homologous recombination, as tested by PCR using primer pairs extending from genomic DNA outside the homology region to a site within the donor cassette. However, Southern blots revealed that no recombination had taken place. We conclude that recombination happened during PCR in vitro between the donor integrated elsewhere in the genome and the lnx2a locus. We conclude that PCR alone may be insufficient to verify homologous recombination in genome editing experiments in zebrafish.
Assuntos
Peixe-Zebra/genética , Animais , Artefatos , Southern Blotting , Edição de Genes , Recombinação Homóloga/genética , Reação em Cadeia da PolimeraseRESUMO
Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.
RESUMO
A specific small-molecule inhibitor of the TLR4 signaling complex upstream of the IKK would likely provide therapeutic benefit for NF-κB-mediated inflammatory disease. We previously identified brazilin as a selective upstream IKK inhibitor targeting the Myddosome complex. In this study, using a cell-based ubiquitination assay for IRAK1 and a chemical library comprising a series of structural analogues of brazilin, a novel small molecule, 2-hydroxy-5,6-dihydroisoindolo[1,2-a]isoquinoline-3,8-dione (IinQ), was identified as a selective and potent inhibitor of IRAK1-dependent NF-κB activation upon TLR4 ligation. In RAW264.7 macrophages, IinQ drastically suppressed activation of upstream IKK signaling events including membrane-bound IRAK1 ubiquitination and IKK phosphorylation by the TLR4 ligand, resulting in reduced expression of proinflammatory mediators including IL-6, TNF-α, and nitric oxide. Interestingly, IinQ did not suppress NF-κB activation via the TLR3 ligand, DNA damaging agents, or a protein kinase C activator, indicating IinQ is specific for TLR4 signaling. Analysis of upstream signaling events further confirmed that IinQ disrupts the MyD88-IRAK1-TRAF6 complex formation induced by LPS treatment, without affecting TLR4 oligomerization. Moreover, intravenous administration of IinQ significantly reduced lethality and attenuated systemic inflammatory responses in an in vivo mouse model of endotoxin shock following LPS challenge. Thus, IinQ represents a novel class of brazilin analogues with improved potency and specificity toward disruption of Myddosome complex formation in TLR4 signaling, indicating that IinQ may be a promising therapeutic candidate for the treatment of systemic inflammatory diseases.
Assuntos
Quinase I-kappa B/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Isoindóis/farmacologia , Isoquinolinas/farmacologia , Fator 88 de Diferenciação Mieloide/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Linhagem Celular , Feminino , Isoindóis/síntese química , Isoindóis/química , Isoquinolinas/síntese química , Isoquinolinas/química , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
The RNA-binding protein Rbfox3 is a well-known splicing regulator that is used as a marker for post-mitotic neurons in various vertebrate species. Although recent studies indicate a variable expression of Rbfox3 in non-neuronal tissues, including lung tissue, its cellular function in lung cancer remains largely unknown. Here, we report that the number of RBFOX3-positive cells in tumorous lung tissue is lower than that in normal lung tissue. As the transforming growth factor-ß (TGF-ß) signaling pathway is important in cancer progression, we investigated its role in RBFOX3 expression in A549 lung adenocarcinoma cells. TGF-ß1 treatment inhibited RBFOX3 expression at the transcriptional level. Further, RBFOX3 depletion led to a change in the expression levels of a subset of proteins related to epithelial-mesenchymal transition (EMT), such as E-cadherin and Claudin-1, during TGF-ß1-induced EMT. In immunofluorescence microscopic analysis, mesenchymal morphology was more prominent in RBFOX3-depleted cells than in control cells. These findings show that TGF-ß-induced RBFOX3 inhibition plays an important role in EMT and propose a novel role for RBFOX3 in cancer progression.
Assuntos
Adenocarcinoma/metabolismo , Antígenos Nucleares/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Mucosa Respiratória/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Antígenos Nucleares/genética , Caderinas/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Claudina-1/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Proteínas do Tecido Nervoso/genética , Splicing de RNA/genética , RNA Interferente Pequeno/genética , Mucosa Respiratória/patologia , Transdução de SinaisRESUMO
The transcription factor nuclear factor-kappa B (NF-κB) controls a number of essential cellular functions, including the immune response, cell proliferation, and apoptosis. NF-κB signaling must be engaged temporally and spatially and well orchestrated to prevent aberrant activation because loss of normal regulation of NF-κB is a major contributor to a variety of pathological diseases, including inflammatory diseases, autoimmune diseases, and cancers. Thus, understanding the molecular mechanisms controlling NF-κB activation is an important part of treatment of these relevant diseases. Although NF-κB transcriptional activity is largely regulated by nuclear translocation, post-translational modification of NF-κB signaling components, including phosphorylation, ubiquitination, acetylation, and methylation, has emerged as an important mechanism affecting activity. Many proteins have been shown to ubiquitinate and regulate NF-κB activation at the receptor signaling complex in response to a variety of ligands, such as tumor necrosis factor, interleukin-1, and Toll-like receptor ligands. In this review, we discuss our current knowledge of ubiquitination patterns and their functional role in NF-κB regulation.
Assuntos
NF-kappa B/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologia , Animais , Humanos , NF-kappa B/genéticaRESUMO
Death receptor (DR) ligation elicits two different modes of cell death (necroptosis and apoptosis) depending on the cellular context. By screening a plant extract library from cells undergoing necroptosis or apoptosis, we identified a water extract of Terminalia chebula (WETC) as a novel and potent dual inhibitor of DR-mediated cell death. Investigation of the underlying mechanisms of its anti-necroptotic and anti-apoptotic action revealed that WETC or its constituents (e.g., gallic acid) protected against tumor necrosis factor-induced necroptosis via the suppression of TNF-induced ROS without affecting the upstream signaling events. Surprisingly, WETC also provided protection against DR-mediated apoptosis by inhibition of the caspase cascade. Furthermore, it activated the autophagy pathway via suppression of mTOR. Of the WETC constituents, punicalagin and geraniin appeared to possess the most potent anti-apoptotic and autophagy activation effect. Importantly, blockage of autophagy with pharmacological inhibitors or genetic silencing of Atg5 selectively abolished the anti-apoptotic function of WETC. These results suggest that WETC protects against dual modes of cell death upon DR ligation. Therefore, WETC might serve as a potential treatment for diseases characterized by aberrantly sensitized apoptotic or non-apoptotic signaling cascades.
Assuntos
Morte Celular/efeitos dos fármacos , Extratos Vegetais/metabolismo , Receptores de Morte Celular/metabolismo , Terminalia/química , Glucosídeos/isolamento & purificação , Glucosídeos/metabolismo , Taninos Hidrolisáveis/isolamento & purificação , Taninos Hidrolisáveis/metabolismo , Extratos Vegetais/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Rbfox3, an RNA-binding fox protein, binds to the antibody to pan-neuronal marker, neuronal nuclei (NeuN). Rbfox3 is expressed in neural tissues across a wide range of species including mammals, birds, and amphibians. However, the molecular identity of Rbfox3 in the zebrafish is largely unknown. In this study, we cloned two zebrafish Rbfox3 genes, Rbfox3a and Rbfox3b. We also cloned the Rbfox3-d31 isoform, which excludes a 93-nucleotide alternative exon within the RNA-recognition motif in both, Rbfox3a and Rbfox3b. Multiple protein sequence alignment revealed that the amino acid sequence for residues 1-20 of the zebrafish Rbfox3, which is the epitope region of NeuN antibody, was different from that of other species. Therefore, NeuN antibody lost its function as a neuronal marker antibody in zebrafish. Reverse transcriptase-polymerase chain reaction showed that both Rbfox3-d31 transcripts were abundant in the early blastula stage, after which they dramatically reduced, suggesting that these isoforms exist mainly as maternal transcripts. In contrast, full-length Rbfox3 transcripts were detected from the 24 h post-fertilization embryo, expression was also maintained at a constant level. Furthermore, full-length Rbfox3-expressing cells were located within the central nervous system during later stages of the zebrafish embryo. Our study provides insight into the molecular structure of zebrafish Rbfox3 as a step towards genetic association studies investigating the developmental role of Rbfox3.
Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião não Mamífero/química , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.