ABSTRACT
The long non-coding FGD5-AS1 (LncFGD5-AS1) has been reported to be a novel carcinogenic gene and participant in regulating tumor progression by sponging microRNAs (miRNAs). However, the pattern of expression and the biological role of FGD5-AS1 in hepatocellular carcinoma (HCC) remains largely unknown. The expression level of FGD5-AS1 in tumor tissues and cell lines was measured by RT-qPCR. CCK-8, EdU, flow cytometry, wound healing, and transwell chamber assays were performed to investigate the role of FGD5-AS1 in cell proliferation, apoptosis, migration, and invasion in HCC. Dual luciferase reporter, and RNA pull-down assays were performed to identify the regulatory interactions among FGD5-AS1, miR-873-5p and GTP-binding protein 4 (GTPBP4). We found that the expression of FGD5-AS1 was upregulated in HCC tissues and cell lines. Moreover, the knockdown of FGD5-AS1 suppressed cell proliferation, migration and invasion, and induced apoptosis in HCC cells. Further studies demonstrated that FGD5-AS1 could function as a competitive RNA by sponging miR-873-5p in HCC cells. Moreover, GTPBP4 was identified as direct downstream target of miR-873-5p in HCC cells and FGD5-AS1mediated the effects of GTPBP4 by competitively binding with miR-873-5p. Taken together, this study demonstrated the regulatory role of FGD5-AS1 in the progression of HCC and identified the miR-873-5p/GTPBP4 axis as the direct downstream pathway. It represents a promising novel therapeutic strategy for HCC patients.
Subject(s)
Carcinoma, Hepatocellular/metabolism , GTP-Binding Proteins/biosynthesis , Gene Expression Regulation, Neoplastic , Liver Neoplasms/metabolism , MicroRNAs/biosynthesis , Neoplasm Proteins/biosynthesis , Nuclear Proteins/biosynthesis , Oncogenes , RNA, Long Noncoding/metabolism , RNA, Neoplasm/metabolism , Up-Regulation , Carcinoma, Hepatocellular/genetics , GTP-Binding Proteins/genetics , Hep G2 Cells , Humans , Liver Neoplasms/genetics , MicroRNAs/genetics , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , RNA, Long Noncoding/genetics , RNA, Neoplasm/geneticsABSTRACT
Reprogramming of energy metabolism is a hallmarkofcancer, and the pentose phosphate pathway (PPP) is a major glucose metabolic pathway important for meeting the cellular demands of biosynthesis and anti-oxidant defense. Our previous study showed that phosphoinositide 3-kinase enhancer-activating Akt (PIKE-A) plays an important role in glioblastoma cell survival and growth under cellular energy stress condition. However, the crucial functions of PIKE-A in cancer energy metabolism are poorly understood.In the present study, we show that PIKE-A promotes DNA biosynthesis, NADPH production and inhibits reactive oxygen species (ROS) production, leading to increasing proliferation and growth of glioblastoma cell and suppressing cellular senescence. Mechanistically, PIKE-A binds to STAT3 and stimulates its phosphorylation mediated by tyrosine kinase Fyn, which enhances transcription of the rate-limitting enzyme glucose-6-phosphate dehydrogenase (G6PD) in the PPP. Finally, targeting PIKE-A-G6PD axis sensitizes glioblastoma to temozolomide (TMZ)treatment. This study reveals that STAT3 is a novel binding partner of PIKE-A which recruits Fyn to phosphorylate STAT3, contributing to the expression of G6PD, leading to promoting tumor growth and suppressing cellular senescence. Thus, the PIKE-A/STAT3/G6PD axis strongly links the PPP to carcinogenesis and may become a promising cancer therapeutic target.
Subject(s)
Cell Proliferation/physiology , GTP-Binding Proteins/biosynthesis , GTPase-Activating Proteins/biosynthesis , Glioblastoma/metabolism , Glucosephosphate Dehydrogenase/biosynthesis , Pentose Phosphate Pathway/physiology , STAT3 Transcription Factor/biosynthesis , Animals , Cell Line, Tumor , Gene Expression Regulation, Enzymologic , Gene Knockout Techniques/methods , Glioblastoma/pathology , Glucosephosphate Dehydrogenase/genetics , HEK293 Cells , Humans , Male , Mice , Mice, Nude , Phosphorylation/physiologyABSTRACT
Increasing evidence supports that N6-methyladenosine (m6A) mRNA modification may play an important role in regulating immune responses. Intestinal epithelial cells orchestrate gastrointestinal mucosal innate defense to microbial infection, but underlying mechanisms are still not fully understood. In this study, we present data demonstrating significant alterations in the topology of host m6A mRNA methylome in intestinal epithelial cells following infection by Cryptosporidium parvum, a coccidian parasite that infects the gastrointestinal epithelium and causes a self-limited disease in immunocompetent individuals but a life-threatening diarrheal disease in AIDS patients. Altered m6A methylation in mRNAs in intestinal epithelial cells following C. parvum infection is associated with downregulation of alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 and the fat mass and obesity-associated protein with the involvement of NF-кB signaling. Functionally, m6A methylation statuses influence intestinal epithelial innate defense against C. parvum infection. Specifically, expression levels of immune-related genes, such as the immunity-related GTPase family M member 2 and interferon gamma induced GTPase, are increased in infected cells with a decreased m6A mRNA methylation. Our data support that intestinal epithelial cells display significant alterations in the topology of their m6A mRNA methylome in response to C. parvum infection with the involvement of activation of the NF-кB signaling pathway, a process that modulates expression of specific immune-related genes and contributes to fine regulation of epithelial antimicrobial defense.
Subject(s)
Adenosine/analogs & derivatives , Cryptosporidiosis/immunology , Cryptosporidium parvum/immunology , Epithelium/immunology , Immunity, Innate , Intestinal Mucosa/immunology , RNA Processing, Post-Transcriptional , RNA, Messenger/immunology , Adenosine/physiology , AlkB Homolog 5, RNA Demethylase/antagonists & inhibitors , AlkB Homolog 5, RNA Demethylase/biosynthesis , AlkB Homolog 5, RNA Demethylase/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/biosynthesis , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Animals , CRISPR-Cas Systems , GTP Phosphohydrolases/biosynthesis , GTP Phosphohydrolases/genetics , GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/genetics , Gene Expression Regulation/immunology , Humans , Intestinal Mucosa/cytology , Methylation , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , RNA Interference , RNA, Small Interfering/geneticsABSTRACT
BACKGROUND & AIMS: Currently, only a few genetic variants explain the heritability of fatty liver disease. Quantitative trait loci (QTL) analysis of mouse strains has identified the susceptibility locus Ltg/NZO (liver triglycerides from New Zealand obese [NZO] alleles) on chromosome 18 as associating with increased hepatic triglycerides. Herein, we aimed to identify genomic variants responsible for this association. METHODS: Recombinant congenic mice carrying 5.3 Mbp of Ltg/NZO were fed a high-fat diet and characterized for liver fat. Bioinformatic analysis, mRNA profiles and electrophoretic mobility shift assays were performed to identify genes responsible for the Ltg/NZO phenotype. Candidate genes were manipulated in vivo by injecting specific microRNAs into C57BL/6 mice. Pulldown coupled with mass spectrometry-based proteomics and immunoprecipitation were performed to identify interaction partners of IFGGA2. RESULTS: Through positional cloning, we identified 2 immunity-related GTPases (Ifgga2, Ifgga4) that prevent hepatic lipid storage. Expression of both murine genes and the human orthologue IRGM was significantly lower in fatty livers. Accordingly, liver-specific suppression of either Ifgga2 or Ifgga4 led to a 3-4-fold greater increase in hepatic fat content. In the liver of low-fat diet-fed mice, IFGGA2 localized to endosomes/lysosomes, while on a high-fat diet it associated with lipid droplets. Pulldown experiments and proteomics identified the lipase ATGL as a binding partner of IFGGA2 which was confirmed by co-immunoprecipitation. Both proteins partially co-localized with the autophagic marker LC3B. Ifgga2 suppression in hepatocytes reduced the amount of LC3B-II, whereas overexpression of Ifgga2 increased the association of LC3B with lipid droplets and decreased triglyceride storage. CONCLUSION: IFGGA2 interacts with ATGL and protects against hepatic steatosis, most likely by enhancing the binding of LC3B to lipid droplets. LAY SUMMARY: The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease.
Subject(s)
Fatty Liver/genetics , GTP-Binding Proteins/genetics , Gene Expression Regulation , Hepatocytes/metabolism , Lipase/genetics , Lipid Metabolism/genetics , Microtubule-Associated Proteins/genetics , Animals , Autophagy , Disease Models, Animal , Fatty Liver/metabolism , Fatty Liver/pathology , Female , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/biosynthesis , Hep G2 Cells , Hepatocytes/pathology , Humans , Lipase/biosynthesis , Lipase/metabolism , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/biosynthesis , Phenotype , RNA/geneticsABSTRACT
OBJECTIVES: AF4/FMR2 family member 1 (AFF1), known as a central scaffolding protein of super elongation complex (SEC), regulates gene transcription. We previously reported that AFF1 inhibited osteogenic differentiation of human mesenchymal stromal/stem cells (hMSCs). However, its role in adipogenic differentiation has not been elucidated. MATERIALS AND METHODS: hMSCs and 3T3-L1 pre-adipocytes were cultured and induced for adipogenic differentiation. Small interfering RNAs (siRNAs) were applied to deplete AFF1 while lentiviruses expressing HA-Aff1 were used for overexpression. Oil Red O staining, triglyceride (TAG) quantification, quantitative real-time PCR (qPCR), Western blot analysis, immunofluorescence staining, RNA sequencing (RNA-seq) analysis and ChIP-qPCR were performed. To evaluate the adipogenesis in vivo, BALB/c nude mice were subcutaneously injected with Aff1-overexpressed 3T3-L1 pre-adipocytes. RESULTS: AFF1 depletion leads to an enhanced adipogenesis in both hMSCs and 3T3-L1 pre-adipocytes. Overexpression of Aff1 in 3T3-L1 cells results in the reduction of adipogenic differentiation and less adipose tissue formation in vivo. Mechanistically, AFF1 binds to the promoter region of Tgm2 gene and regulates its transcription. Overexpression of Tgm2 largely rescues adipogenic differentiation of Aff1-deficient cells. CONCLUSIONS: Our data indicate that AFF1 inhibits adipogenic differentiation by regulating the transcription of TGM2.
Subject(s)
Adipogenesis/genetics , DNA-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Gene Expression Regulation , Nuclear Proteins/metabolism , Transcriptional Elongation Factors/metabolism , Transglutaminases/genetics , 3T3-L1 Cells , Animals , Cells, Cultured , DNA-Binding Proteins/genetics , GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/metabolism , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Nuclear Proteins/genetics , Protein Glutamine gamma Glutamyltransferase 2 , Stem Cells/metabolism , Transcription, Genetic , Transcriptional Elongation Factors/genetics , Transglutaminases/biosynthesis , Transglutaminases/metabolismABSTRACT
Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers and has been listed as sixth most common human cancer. Due to late diagnosis and insufficient therapeutic response among patients, the survival rate remains very low accentuating the importance of early diagnostic markers. The study aimed to identify differentially expressed proteins in search for putative serum biomarkers and drug targets. Serum samples (n = 45) were depleted and resolved on two dimensional gel electrophoresis. Among differentially expressed proteins, two were identified using MALDI-TOF mass spectrometry. Gene expression levels of identified proteins were quantified in malignant and normal tissue using RT-qPCR. To validate serum Rabl3 expression, sandwich ELISA was performed. Proteomics analysis revealed two proteins which were found to be associated with oral cancer. The expression of GIMAP7 was found to be down regulated in serum of patients suffering from oral cancer while the expression of Rabl3 was found to be up-regulated. Gene expression analysis in malignant tissue and adjacent normal tissue revealed the same pattern. Quantitative ELISA was used to validate expression of Rabl3 in serum from oral cancer patients and healthy subjects which demonstrated significant up-regulation in cancer patients. Findings in current study demonstrate differential expression of novel putative biomarkers GIMAP7 and Rabl3 in oral cancer which suggests their potential role in oral cancer pathology and can be considered as predictive biomarkers.
Subject(s)
Biomarkers, Tumor/blood , GTP-Binding Proteins/blood , Mouth Neoplasms/pathology , Squamous Cell Carcinoma of Head and Neck/pathology , rab GTP-Binding Proteins/blood , GTP-Binding Proteins/biosynthesis , Humans , Mouth Neoplasms/blood , Proteomics/methods , Squamous Cell Carcinoma of Head and Neck/blood , rab GTP-Binding Proteins/biosynthesisABSTRACT
The multifunctional protein Transglutaminase type 2, is associated with cancer epithelial mesenchymal transition, invasiveness, stemness and drugs resistance. Several variant isoforms and non-coding RNAs are present in cancer and this report explored the expression of these transcripts of the TGM2 gene in cancer cell lines after induction with all-trans retinoic acid. The expression of truncated variants along with two long non-coding RNAs, was demonstrated. One of these is coded from the first intron and the Last Exon Variant is constituted by a sequence corresponding to the last three exons and the 3'UTR. Analysis of ChIP-seq data, from ENCODE project, highlighted factors interacting with intronic sequences, which could interfere with the progression of RNApol II at checkpoints, during the elongation process. Some relevant transcription factors, bound in an ATRA-dependent way, were found by RNA immunoprecipitation, notably GATA3 mainly enriched to Last Exon Variant non-coding RNA. The involvement of NMD in the regulation of the ratio among these transcripts was observed, as the prevalent recovering of Last Exon Variant to phUPF1-complexes, with decrease of the binding towards other selective targets. This study contributes to identify molecular mechanisms regulating the ratio among the variants and improves the knowledge about regulatory roles of the non-coding RNAs of the TGM2 gene.
Subject(s)
GTP-Binding Proteins/biosynthesis , RNA, Long Noncoding/metabolism , Transglutaminases/biosynthesis , Tretinoin/pharmacology , Chromatin Immunoprecipitation Sequencing , GATA3 Transcription Factor/metabolism , GTP-Binding Proteins/genetics , HL-60 Cells , Humans , Isoenzymes/biosynthesis , Isoenzymes/genetics , Nonsense Mediated mRNA Decay , Protein Glutamine gamma Glutamyltransferase 2 , RNA, Long Noncoding/genetics , Transcription, Genetic , Transglutaminases/geneticsABSTRACT
The apoptotic death of cardiomyocytes critically contributes to cardiac remodeling after myocardial infarction (MI). Circular RNAs (circRNAs) are important regulators for a variety of biological functions. Circ-Ttc3 represents one of the top highest expressed circRNAs in the heart; however, its role in MI remains unknown. Herein, we found that circ-Ttc3 was markedly upregulated in the ischemic myocardium and the cardiomyocytes subjected to hypoxic insult. Forced expression of circ-Ttc3 in cardiomyocytes counteracted hypoxia-induced ATP depletion and apoptotic death, in sharp contrast to circ-Ttc3 knockdown. Accordingly, experiments with AAV9-cTnt-mediated knockdown of cardiac circ-Ttc3 in a rat model of MI recapitulated the in vitro findings, and showed the deterioration of cardiac dysfunction after MI. Furthermore, we identified that circ-Ttc3 sponged an endogenous miR-15b-5p to sequester and inhibit its activity, leading to the increased Arl2 expression. Conversely, knockdown of Arl2 partially abolished the beneficial effects of circ-Ttc3 overexpression on ATP production and apoptosis of cardiomyocytes. Thus, our findings revealed the cardioprotective role of circ-Ttc3 in MI. The miR-15b-Arl2 regulatory cascade underlies the protection against MI-induced cardiomyocyte apoptosis by circ-Ttc3.
Subject(s)
Apoptosis , MicroRNAs/metabolism , Myocardial Infarction/metabolism , Myocytes, Cardiac/metabolism , RNA, Circular/metabolism , Signal Transduction , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Animals , Disease Models, Animal , GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/genetics , Gene Expression Regulation , Gene Knockdown Techniques , Male , MicroRNAs/genetics , Myocardial Infarction/genetics , Myocardial Infarction/pathology , Myocytes, Cardiac/pathology , RNA, Circular/genetics , Rats , Rats, Sprague-DawleyABSTRACT
AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is considered a proto-oncogene, but not much is known about AGAP2 gene expression regulation. To get some insight into this process, AGAP2 proximal promoter was cloned and characterised using reporter assays. We have identified SP1 as a transcription factor bound to AGAP2 promoter and required for AGAP2 expression in two different types of cancer cells (KU812, a chronic myeloid leukaemia cell line; and DU145, a prostate cancer cell line): silencing SP1 decreased AGAP2 protein levels. We have also found that all-trans retinoic acid (ATRA) treatment increased AGAP2 protein levels in both cell lines whilst curcumin treatment reduced ATRA-mediated AGAP2 increase. Furthermore, chromatin immunoprecipitation studies revealed the presence of RARα, RXRα and the lysine acetyl transferase PCAF in AGAP2 promoter. Our results provide a novel understanding of AGAP2 expression regulation that could be beneficial to those patients with cancers where AGAP2 is overexpressed.
Subject(s)
GTP-Binding Proteins/biosynthesis , GTPase-Activating Proteins/biosynthesis , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Leukemic , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Neoplasm Proteins/metabolism , Prostatic Neoplasms/metabolism , Retinoic Acid Receptor alpha/metabolism , Sp1 Transcription Factor/metabolism , Cell Line, Tumor , GTP-Binding Proteins/genetics , GTPase-Activating Proteins/genetics , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Neoplasm Proteins/genetics , Prostatic Neoplasms/genetics , Proto-Oncogene Mas , Retinoic Acid Receptor alpha/genetics , Sp1 Transcription Factor/geneticsABSTRACT
The gene TRIM23 (tripartite motif containing 23) is a member of the tripartite motif (TRIM) family whose expression putatively participates in many pathophysiological processes. Nonetheless, the role of TRIM23 in gastric cancer (GC) remains uncertain. Our study evaluated the expression of TRIM23 in GC tissues and cell lines, and investigated an association between TRIM23 and survival. In the present study, our results demonstrated that TRIM23 mRNA and protein were frequently over-expressed in GC cell lines and GC tissues. High level of TRIM23 protein correlated with tumor size, tumor-node-metastasis (TNM) stage, depth of invasion, lymph node metastasis (LNM), tumor differentiation, and nerve invasion. Compared with the low TRIM23 protein group, the high TRIM23 protein group was significantly associated with worse prognosis of GC patients. Consistently, the KM-plot database suggested that high TRIM23 mRNA expression was also linked to a poor prognosis in GC patients both in positive and negative subgroups of human epidermal growth factor receptor 2 (HER2). But in the HER2 positive subgroup, the advantages of the low TRIM23 expression on overall survival were much more statistically significant. The univariate analysis indicated that TRIM23 expression correlated with overall survival. The multivariate analysis showed that independent factors of prognosis in GC were lymph node metastasis, vascular invasion, and depth of invasion. In summary, TRIM23 may be associated with progression of GC, and may be considered a therapeutic target for GC patients.
Subject(s)
Biomarkers, Tumor/analysis , GTP-Binding Proteins/biosynthesis , Stomach Neoplasms/pathology , Adult , Aged , Asian People , Female , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Prognosis , Proportional Hazards Models , Stomach Neoplasms/mortalityABSTRACT
This study aimed to investigate the role of transglutaminase 2 (TG2) expressed in mast cells in substantia nigra (SN) in Parkinson's disease (PD) model or human PD patients. C57BL/6 mice received 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by ip injection to induce PD. Bone marrow-derived mast cells (BMMCs) were adoptively transferred to TG2 knockout (KO or TG2-/-) mice by iv injection 1â¯day before MPTP injection or stimulated by 1 methyl-4-phenylpyridinium (MMP+). KO-MPTP mice showed reduced expression of tyrosine hydroxylase (TH) and dopamine (DA) transporter (DAT) and loss of TH+ DA neurons, and expression of markers (c-kit, tryptase, FcεRI), mediators' release (histamine, leukotrienes, cytokines), and TG2 related to mast cells, and co-localization of DA neuronal cells and mast cells in SN tissues or release of mediators and TG2 activity in SN tissues and sera versus those in WT (wild type)-MPTP or BMâ¯+â¯KO-MPTP mice. KO-MPTP mice reversed the alterations of behavior. KO-BMMCs-transferred KO-MPTP (BMâ¯+â¯KO-MPTP) mice had restoration of all the responses versus the KO-MPTP mice. MPP+-stimulated BMMCs had increased mediators' release, which were inhibited by TG2 inhibitor (R2 peptide). All the mediators and TG2 activity were also increased in the sera of human PD patients. The data suggest that TG2 expressed in mast cells recruited into SN tissues might contribute to neuroinflammation, which is known as one of the important features in pathogenesis of PD, via up-regulating the release of various mediators.
Subject(s)
GTP-Binding Proteins/biosynthesis , Gene Expression Regulation, Enzymologic , Inflammation Mediators/metabolism , Mast Cells/metabolism , Parkinsonian Disorders/metabolism , Transglutaminases/biosynthesis , Aged , Animals , Cell Survival/physiology , Female , GTP-Binding Proteins/genetics , Humans , Male , Mast Cells/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Parkinsonian Disorders/pathology , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases/geneticsABSTRACT
Tissue transglutaminase is a ubiquitous and multifunctional protein that contributes to several processes such as apoptosis/survival, efferocytosis, inflammation and tissue repairing under physiological and pathological conditions. Several activities can be associated with well-established functional domains; in addition, four RNA alternative splice variants have been described, characterized by sequence divergences and residues deletion at the C-terminal domains. Tissue transglutaminase is recognized as the central player in the physiopathology of coeliac disease (CD) mainly through calcium-dependent enzymatic activities. It can be hypothesized that differential regulation of tissue transglutaminase splice variants expression in persons with CD contributes to pathology by altering the protein functionality. We characterized the expression pattern of RNA alternative splice variants by RT-PCR in peripheral cells from patients with CD under free gluten diet adhesion; we considered inflammatory parameters and specific antibodies as markers of the stage of disease. We found significant higher expression of both the full length and the shortest C-truncated splice variants in leucocytes from patients with CD in comparison with healthy individuals. As tissue transglutaminase expression and canonical enzymatic activity are linked to inflammation, we studied the RNA expression of inflammatory cytokines in peripheral leucocytes of persons with CD in relation with splice variants expression; interestingly, we found that recently diagnosed patients showed significant correlation between both the full length and the shortest alternative spliced variants with IL-1 expression. Our results points that regulation of alternative splicing of tissue transglutaminase could account for the complex physiopathology of CD.
Subject(s)
Alternative Splicing/genetics , Celiac Disease/genetics , Celiac Disease/pathology , GTP-Binding Proteins/genetics , Leukocytes/immunology , Transglutaminases/genetics , Adult , Aged , Diet, Gluten-Free , Female , GTP-Binding Proteins/biosynthesis , Humans , Interleukin-1/biosynthesis , Interleukin-1/genetics , Isoenzymes/biosynthesis , Isoenzymes/genetics , Male , Middle Aged , Protein Domains/genetics , Protein Glutamine gamma Glutamyltransferase 2 , RNA, Messenger/genetics , Transglutaminases/biosynthesis , Young AdultABSTRACT
BACKGROUND/AIM: The aim of this study was to investigate the role of transglutaminase 2 (TGM2) in colorectal cancer stem cells (CCSCs). MATERIALS AND METHODS: We used the TU12 cell line possessing CD133-expressing CCSCs. After isolating CD133 (-) and CD133 (+) CCSCs, we overexpressed and knocked-down TGM2 to investigate its role in human CCSCs. RESULTS: The expression level of TGM2 was 25-fold higher in tumorigenic cells than non-tumorigenic cells. We found that knockdown of TGM2 by specific RNA interference markedly inhibited cell growth and caused down-regulation of the stemness markers, CD133, SOX2, and ß-catenin. We further demonstrated that knockdown of TGM2 inhibited cell metastatic abilities by down-regulating N-cadherin and vimentin and up-regulating E-cadherin. These findings revealed that TGM2 expression is markedly increased in human colorectal cancer and that down-regulation of TGM2 in tumors may serve as a treatment for colorectal cancer patients. Therefore, this study indicate that TGM2 affects the metastatic potential and stemness of CCSCs by regulating EMT- and stemness-related proteins. CONCLUSION: The metastatic potential of CSCs arises from highly expressed TGM2.
Subject(s)
Colorectal Neoplasms/enzymology , GTP-Binding Proteins/biosynthesis , Neoplastic Stem Cells/enzymology , Transglutaminases/biosynthesis , AC133 Antigen/biosynthesis , Antigens, CD/metabolism , Biomarkers, Tumor , Cadherins/metabolism , Cell Line, Tumor , Cell Movement/physiology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Down-Regulation , Epithelial-Mesenchymal Transition , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Gene Knockdown Techniques , Humans , Immunohistochemistry , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplastic Stem Cells/pathology , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases/genetics , Transglutaminases/metabolism , Tumor Cells, Cultured , Vimentin/metabolismABSTRACT
Purpose: Tissue transglutaminase (TGM2) is elevated in glaucomatous trabecular meshwork (TM) tissues. We investigated whether increased expression of TGM2 increases extracellular matrix crosslinking in the TM, thereby increasing aqueous humor outflow resistance and elevating intraocular pressure (IOP) in mouse eyes. Methods: GTM3, primary human GTM 125-05, and cultured mouse TM cells were transduced with adenovirus serotype 5 expressing human transglutaminase 2 (Ad5.TGM2; multiplicity of infection [MOI]-75) and fixed for immunocytochemistry. To test the effect on IOP in living eyes, Ad5.TGM2 was injected intravitreally into one eye of BALB/cJ (n = 18) or C57BL/6J mice (n = 9). The uninjected contralateral eye and Ad5.GFP served as controls. Daytime conscious IOPs were measured twice per week. Aqueous outflow facility (C) was measured by constant flow infusion on completion of IOP measurements. Immunohistochemistry was performed on BALB/cJ mouse eyes to study TGM2 expression and activity. Results: The treatment of cultured TM cells with Ad5.TGM2 increased immunostaining of N-ε(γ-glutamyl) lysine crosslinks. Ad5.TGM2 injection significantly increased IOP in BALB/cJ (15.86 mm Hg [injected] vs. 10.70 mm Hg [control]) and in C57BL/6J mice (17.09 mm Hg [injected] vs. 12.01 mm Hg [control]). Mean aqueous outflow facility in the injected eyes of BALB/cJ (0.013 µL/min/mm Hg) and C57BL/6J mice (0.012 µL/min/mm Hg) was significantly lower than in the uninjected control eyes (BALB/cJ, 0.021 µL/min/mm Hg; C57BL/6J, 0.019 µL/min/mm Hg). The Ad5.TGM2 transduction of mouse eyes increased TGM2 expression in the TM region and increased N-ε(γ-glutamyl) lysine crosslinks. Conclusions: The increased expression of TGM2 in the TM increases N-ε(γ-glutamyl) lysine crosslinking in the TM, increases aqueous outflow resistance, and elevates IOP in mice. TGM2 may be at least partially responsible for ocular hypertension in POAG.
Subject(s)
Aqueous Humor/enzymology , GTP-Binding Proteins/genetics , Gene Expression Regulation , Glaucoma, Open-Angle/genetics , Intraocular Pressure , RNA/genetics , Trabecular Meshwork/enzymology , Transglutaminases/genetics , Animals , Blotting, Western , Cells, Cultured , GTP-Binding Proteins/biosynthesis , Glaucoma, Open-Angle/enzymology , Glaucoma, Open-Angle/pathology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Protein Glutamine gamma Glutamyltransferase 2 , Trabecular Meshwork/pathology , Transglutaminases/biosynthesisABSTRACT
G protein nucleolar 3 (GNL3), a nucleolar GTP-binding protein, is highly expressed in progenitor cells, stem cells, and various types of cancer cells. Therefore, it is considered to have an important role in cancer pathogenesis. GNL3 has been reported to play crucial roles in cell proliferation, cell cycle regulation, inhibition of differentiation, ribosome biogenesis, and the maintenance of stemness, genome stability and telomere integrity. Furthermore, GNL3 has recently been shown to be involved in cancer invasion and metastasis. However, the biological significance of GNL3 in the invasion and metastasis of colon cancer remains unclear. This study was performed to address this gap in knowledge. GNL3 expression was upregulated in colon cancer tissue specimens and correlated with tumor differentiation, invasion and metastasis. GNL3 overexpression promoted cell proliferation, invasion, migration and the epithelial-mesenchymal transition (EMT) in colon cancer cells. Moreover, inhibition of the EMT and the Wnt/ßcatenin signaling pathway induced by GNL3 knockdown was partially reversed by lithium chloride (LiCl). Based on these data, GNL3 promotes the EMT in colon cancer by activating the Wnt/ßcatenin signaling pathway. In summary, GNL3 is upregulated in colon cancer and plays an important role in tumor growth, invasion and metastasis. Strategies targeting GNL3 are potential treatments for colon cancer.
Subject(s)
Colonic Neoplasms/metabolism , GTP-Binding Proteins/biosynthesis , Nuclear Proteins/biosynthesis , Wnt Signaling Pathway , beta Catenin/metabolism , Caco-2 Cells , Cell Line, Tumor , Cell Movement/physiology , Cell Proliferation/physiology , Colonic Neoplasms/pathology , Epithelial-Mesenchymal Transition , HCT116 Cells , HT29 Cells , Humans , Neoplasm Invasiveness , Up-RegulationABSTRACT
RAB3A-interacting molecule (RIM) proteins are important regulators of transmitter release from active zones. At conventional chemical synapses, RIMs contribute substantially to vesicle priming and docking and their loss reduces the readily releasable pool of synaptic vesicles by up to 75%. The priming function of RIMs is mediated via the formation of a tripartite complex with Munc13 and RAB3A, which brings synaptic vesicles in close proximity to Ca2+ channels and the fusion site and activates Munc13. We reported previously that, at mouse photoreceptor ribbon synapses, vesicle priming is Munc13 independent. In this study, we examined RIM expression, distribution, and function at male and female mouse photoreceptor ribbon synapses. We provide evidence that RIM1α and RIM1ß are highly likely absent from mouse photoreceptors and that RIM2α is the major large RIM isoform present at photoreceptor ribbon synapses. We show that mouse photoreceptors predominantly express RIM2 variants that lack the interaction domain for Munc13. Loss of full-length RIM2α in a RIM2α mutant mouse only marginally perturbs photoreceptor synaptic transmission. Our findings therefore strongly argue for a priming mechanism at the photoreceptor ribbon synapse that is independent of the formation of a RIM-Munc13-RAB3A complex and thus provide further evidence for a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses in synaptic vesicle exocytosis.SIGNIFICANCE STATEMENT RAB3A-interacting molecules 1 and 2 (RIM1/2) are essential regulators of exocytosis. At conventional chemical synapses, their function involves Ca2+ channel clustering and synaptic vesicle priming and docking through interactions with Munc13 and RAB3A, respectively. Examining wild-type and RIM2 mutant mice, we show here that the sensory photoreceptor ribbon synapses most likely lack RIM1 and predominantly express RIM2 variants that lack the interaction domain for Munc13. Our findings demonstrate that the photoreceptor-specific RIM variants are not essential for synaptic vesicle priming at photoreceptor ribbon synapses, which represents a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses with respect to synaptic vesicle priming mechanisms.
Subject(s)
GTP-Binding Proteins/biosynthesis , Photoreceptor Cells, Vertebrate/metabolism , Synapses/metabolism , Animals , Cells, Cultured , Female , GTP-Binding Proteins/analysis , GTP-Binding Proteins/genetics , Gene Expression , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , NIH 3T3 Cells , Photoreceptor Cells, Vertebrate/chemistry , Synapses/chemistry , Synapses/geneticsABSTRACT
The mechanism of early recurrence of hepato-cellular carcinoma (HCC) is not well understood. To examine whether early intrahepatic metastasis of HCC can be determined by the reliable molecular characteristics of the primary HCC, we focused on early-stage tumors of primary and solitary HCC cases. Proteomic differences were investigated between two groups, 11 early (recurrence within 12 months) and 10 late (no recurrence within 48 months) HCC cases, using two-dimensional fluorescence difference gel electrophoresis. Overall, 10 upregulated and 9 downregulated proteins were identified from a total of 1623 protein spots detected in early recurrent HCC. Cluster analysis using the 19 proteins successfully divided the 21 HCC samples exactly into the two above groups. A multifunctional protein, transglutaminase 2 (TGM2), was upregulated in the early recurrence group. Immunohistochemistry revealed the frequent observation of TGM2-positive HCC cells in the early group, with a tendency of TGM2-positive staining in HCC cells adjacent to fibrous stroma. To examine whether two major TGM2-associated pathways, epithelial-mesenchymal transition (EMT) and integrin signaling, were activated in the early recurrence group of HCC, downstream molecules of TGM2 were measured. The mRNA level of EMT-related genes was highly positively correlated with TGM2 mRNA. However, E-cadherin (CDH1) mRNA and protein were not downregulated in correlation with TGM2 expression. The phosphorylation of FAK and Akt and the downregulation of PTEN were not associated with the quantity of TGM2. Therefore, TGM2 might contribute to early HCC recurrence through signaling pathways not related to EMT and integrin signaling. The proteomics of strictly classified HCCs would be useful for characterizing pro-metastatic HCC and for developing a new therapeutic target for treatment of metastasis.
Subject(s)
Carcinoma, Hepatocellular/genetics , GTP-Binding Proteins/genetics , Liver Neoplasms/genetics , Proteomics , Transglutaminases/genetics , Aged , Antigens, CD , Cadherins/genetics , Carcinoma, Hepatocellular/pathology , Epithelial-Mesenchymal Transition , Female , GTP-Binding Proteins/biosynthesis , Gene Expression Regulation, Neoplastic , Humans , Integrins/genetics , Liver Neoplasms/pathology , Male , Middle Aged , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Protein Glutamine gamma Glutamyltransferase 2 , Signal Transduction , Transglutaminases/biosynthesisABSTRACT
Amphotericin B (AmB), a polyene antibiotic, is reported to cause the microglial activation to induce nitric oxide (NO) production and proinflammatory cytokines expression, and change neurotrophic factors expression in cultured microglia (Motoyoshi et al. in Neurochem Int 52:1290-1296, 2008). On the other hand, tissue-type transglutaminase (TG2) is involved in connection to phagocytes with apoptotic cells. Engulfment of neurons by activated microglia is thought to cause neurodegenerative diseases but detail is unclear, and involvement of TG2 in phagocytosis has been reported in our previous study using lipopolysaccharide-stimulated BV-2 cells (Kawabe et al. in Neuroimmunomodulation 22(4):243-249, 2015). In the present study, we examined the changes of TG2 expression, phagocytosis and pinocytosis in BV-2 cells stimulated by AmB. AmB stimulation increased TG2 expression and TG activity. Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were also up-regulated by AmB stimulation in BV-2 cells. Blockade of TG activity by cystamine, an inhibitor of TGs, suppressed AmB-enhanced TG2 expression, TG activity, NO production, phagocytosis and pinocytosis. Excessive NO production from microglia and/or facilitation of phagocytosis might be involved in neuronal death. To control TG activity might make possible to protect neurons and care for CNS diseases.
Subject(s)
Amphotericin B/pharmacology , Endocytosis/physiology , GTP-Binding Proteins/biosynthesis , Gene Expression Regulation, Enzymologic , Microglia/enzymology , Transglutaminases/biosynthesis , Up-Regulation/physiology , Animals , Cell Line , Dose-Response Relationship, Drug , Endocytosis/drug effects , GTP-Binding Proteins/genetics , Mice , Microglia/drug effects , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases/genetics , Up-Regulation/drug effectsABSTRACT
Guanylate binding protein 2 (GBP2) is one member of GBP family. Recently, GBP2 has been proposed to be a novel target of anti-cancer drugs. However, the role of GBP2 in the traumatic brain injury (TBI) is very limited. In this study, we sought to define GBP2's role in brain injury. GBP2 protein levels were significantly increased in the brain 3 days after injury, suggesting a functional role for GBP2 in TBI. Neuronal cells overexpressing GBP2 exhibited up-regulation of co-location of GBP2 and NeuN following TBI, suggesting that GBP2 potentiates the neuron apoptosis. To confirm the role of GBP2 in neuron apoptosis process, we employed a highly potent inhibitor of GBP2 (GBP2 RNAi). In H2O2-stimulated PC12 cells, in vitro blockade of GBP2 activity using GBP2 RNAi markedly attenuated the neuron apoptosis number. GBP2 RNAi also inhibited the expression levels of active caspase3 and p-Stat1. Furthermore, we found the expression of p-Stat1 in line with GBP2 and GBP2 interacted with p-Stat1 following TBI. The Jak2 inhibitor, AG490 inhibited this interaction and decreased the active caspase3 expression as well as promoted the functional recovery. Taken together, these data suggest that GBP2 RNAi has a protective effect in a rat TBI. This study demonstrates that GBP2 is an important positive regulator of TBI and is a promising therapeutic target for brain injury.
Subject(s)
Apoptosis/physiology , Brain Injuries, Traumatic/metabolism , Cerebral Cortex/metabolism , GTP-Binding Proteins/biosynthesis , Neurons/metabolism , Up-Regulation/physiology , Animals , Brain Injuries, Traumatic/pathology , Cerebral Cortex/pathology , Male , Neurons/pathology , PC12 Cells , Rats , Rats, Sprague-DawleyABSTRACT
BACKGROUND: The structural elements of the vascular wall, namely, extracellular matrix and smooth muscle cells (SMCs), contribute to the overall stiffness of the vessel. In this study, we examined the crosslinking-dependent and crosslinking-independent roles of tissue transglutaminase (TG2) in vascular function and stiffness. METHODS AND RESULTS: SMCs were isolated from the aortae of TG2-/- and wild-type (WT) mice. Cell adhesion was examined by using electrical cell-substrate impedance sensing and PicoGreen assay. Cell motility was examined using a Boyden chamber assay. Cell proliferation was examined by electrical cell-substrate impedance sensing and EdU incorporation assays. Cell micromechanics were studied using magnetic torsion cytometry and spontaneous nanobead tracer motions. Aortic mechanics were examined by tensile testing. Vasoreactivity was studied by wire myography. SMCs from TG2-/- mice had delayed adhesion, reduced motility, and accelerated de-adhesion and proliferation rates compared with those from WT. TG2-/- SMCs were stiffer and displayed fewer cytoskeletal remodeling events than WT. Collagen assembly was delayed in TG2-/- SMCs and recovered with adenoviral transduction of TG2. Aortic rings from TG2-/- mice were less stiff than those from WT; stiffness was partly recovered by incubation with guinea pig liver TG2 independent of crosslinking function. TG2-/- rings showed augmented response to phenylephrine-mediated vasoconstriction when compared with WT. In human coronary arteries, vascular media and plaque, high abundance of fibronectin expression, and colocalization with TG2 were observed. CONCLUSIONS: TG2 modulates vascular function/tone by altering SMC contractility independent of its crosslinking function and contributes to vascular stiffness by regulating SMC proliferation and matrix remodeling.