ABSTRACT
Epilepsy is a common neurological disorder, which has been linked to mutations or deletions of RNA binding protein, fox-1 homolog (Caenorhabditis elegans) 3 (RBFOX3)/NeuN, a neuronal splicing regulator. However, the mechanism of seizure mediation by RBFOX3 remains unknown. Here, we show that mice with deletion of Rbfox3 in gamma-aminobutyric acid (GABA) ergic neurons exhibit spontaneous seizures and high premature mortality due to increased presynaptic release, postsynaptic potential, neuronal excitability, and synaptic transmission in hippocampal dentate gyrus granule cells (DGGCs). Attenuating early excitatory gamma-aminobutyric acid (GABA) action by administering bumetanide, an inhibitor of early GABA depolarization, rescued premature mortality. Rbfox3 deletion reduced hippocampal expression of vesicle-associated membrane protein 1 (VAMP1), a GABAergic neuron-specific presynaptic protein. Postnatal restoration of VAMP1 rescued premature mortality and neuronal excitability in DGGCs. Furthermore, Rbfox3 deletion in GABAergic neurons showed fewer neuropeptide Y (NPY)-expressing GABAergic neurons. In addition, deletion of Rbfox3 in NPY-expressing GABAergic neurons lowered intrinsic excitability and increased seizure susceptibility. Our results establish RBFOX3 as a critical regulator and possible treatment path for epilepsy.
Subject(s)
DNA-Binding Proteins , GABAergic Neurons , Nerve Tissue Proteins , Neuropeptide Y , Seizures , Vesicle-Associated Membrane Protein 1 , Animals , Bumetanide/pharmacology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dentate Gyrus/metabolism , GABA Antagonists/pharmacology , GABAergic Neurons/metabolism , Gene Deletion , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuropeptide Y/metabolism , Seizures/genetics , Seizures/metabolism , Vesicle-Associated Membrane Protein 1/genetics , Vesicle-Associated Membrane Protein 1/metabolism , gamma-Aminobutyric Acid/metabolismABSTRACT
RTL1/PEG11, which has been associated with anxiety disorders, is a retrotransposon-derived imprinted gene in the placenta. However, imprinting patterns and functions of RTL1 in the brain have not been well-investigated. We found Rtl1 was paternally, but not maternally, expressed in brain stem, thalamus, and hypothalamus of mice, and imprinting status of RTL1 was maintained in human brain. Paternal Rtl1 knockout (Rtl1m+/p-) mice had higher neonatal death rates due to impaired suckling, and low body weights beginning on embryonic day 16.5. High paternal expression of Rtl1 was detected in the locus coeruleus (LC) and Rtl1m+/p- mice showed an increased delay in time of onset for action potentials and inward currents with decreased neuronal excitability of LC neurons. Importantly, Rtl1m+/p- mice exhibited behaviors associated with anxiety, depression, fear-related learning and memory, social dominance, and low locomotor activity. Taken together, our findings demonstrate RTL1 is imprinted in brain, mediates emotional and social behaviors, and regulates excitability in LC neurons.
Subject(s)
Pregnancy Proteins , Retroelements , Animals , Anxiety/genetics , Anxiety Disorders/genetics , Female , Genomic Imprinting , Humans , Locus Coeruleus/metabolism , Mice , Neurons/metabolism , Pregnancy , Pregnancy Proteins/genetics , Pregnancy Proteins/metabolism , Social BehaviorABSTRACT
PURPOSE: Currently, there are no accurate markers for predicting potentially lethal prostate cancer (PC) before biopsy. This study aimed to develop urine tests to predict clinically significant PC (sPC) in men at risk. METHODS: Urine samples from 928 men, namely, 660 PC patients and 268 benign subjects, were analyzed by gas chromatography/quadrupole time-of-flight mass spectrophotometry (GC/Q-TOF MS) metabolomic profiling to construct four predictive models. Model I discriminated between PC and benign cases. Models II, III, and GS, respectively, predicted sPC in those classified as having favorable intermediate risk or higher, unfavorable intermediate risk or higher (according to the National Comprehensive Cancer Network risk groupings), and a Gleason sum (GS) of ≥ 7. Multivariable logistic regression was used to evaluate the area under the receiver operating characteristic curves (AUC). RESULTS: In Models I, II, III, and GS, the best AUCs (0.94, 0.85, 0.82, and 0.80, respectively; training cohort, N = 603) involved 26, 24, 26, and 22 metabolites, respectively. The addition of five clinical risk factors (serum prostate-specific antigen, patient age, previous negative biopsy, digital rectal examination, and family history) significantly improved the AUCs of the models (0.95, 0.92, 0.92, and 0.87, respectively). At 90% sensitivity, 48%, 47%, 50%, and 36% of unnecessary biopsies could be avoided. These models were successfully validated against an independent validation cohort (N = 325). Decision curve analysis showed a significant clinical net benefit with each combined model at low threshold probabilities. Models II and III were more robust and clinically relevant than Model GS. CONCLUSION: This urine test, which combines urine metabolic markers and clinical factors, may be used to predict sPC and thereby inform the necessity of biopsy in men with an elevated PC risk.
Subject(s)
Metabolome , Prostatic Neoplasms , Humans , Male , Biopsy , Neoplasm Grading , Prostate-Specific Antigen , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Prostatic Neoplasms/urine , Risk Factors , Early Detection of Cancer/methods , Urinalysis/methods , Urine/chemistryABSTRACT
Pompe disease (OMIM # 232300) is a glycogen storage disease caused by autosomal recessive mutations of the gene encoding alpha-1,4-glucosidase (GAA; EC 3.2.1.20). Despite the relatively effective employment of enzyme replacement therapy, some critical medical issues still exist in patients with this disease, including the persistence of abnormalities in the central nervous system (CNS), probably because of the inability of the recombinant GAA to pass through the blood-brain barrier. To address this issue, identification of more therapeutic agents that target the CNS of patients with Pompe disease may be required. In this study, we derived neuronal cells from Pompe disease-induced pluripotent stem cells (Pom-iPSCs) and proved that they are able to recapitulate the hallmark cellular and biochemical phenotypes of Pompe disease. Using the Pom-iPSC-derived neurons as an in vitro drug-testing model, we then identified three compounds, ebselen, wortmannin and PX-866, with therapeutic potential to alleviate Pompe disease-associated pathological phenotypes in the neurons derived from Pom-iPSCs. We confirmed that all three compounds were able to enhance the GAA activity in the Pom-iPSC-derived neurons. Moreover, they were able to enhance the GAA activity in several important internal organs of GAA-deficient mice when co-injected with recombinant human GAA, and we found that intraperitoneal injection of ebselen was able to promote the GAA activity of the GAA-heterozygous mouse brain. Our results prove the usefulness of Pom-iPSC-derived neuronal populations for identifying new compounds with therapeutic potential.
Subject(s)
Azoles/administration & dosage , Glycogen Storage Disease Type II/pathology , Induced Pluripotent Stem Cells/cytology , Neural Stem Cells/cytology , Organoselenium Compounds/administration & dosage , alpha-Glucosidases/metabolism , Animals , Azoles/pharmacology , Blood-Brain Barrier , Brain/metabolism , Cell Culture Techniques , Cells, Cultured , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Glycogen Storage Disease Type II/drug therapy , Glycogen Storage Disease Type II/metabolism , Gonanes/pharmacology , Humans , Induced Pluripotent Stem Cells/drug effects , Injections, Intraperitoneal , Isoindoles , Male , Mice , Neural Stem Cells/drug effects , Organoselenium Compounds/pharmacology , Wortmannin/pharmacology , alpha-Glucosidases/geneticsABSTRACT
BACKGROUND: Breast cancer amplified sequence 2 (BCAS2) plays crucial roles in pre-mRNA splicing and androgen receptor transcription. Previous studies suggested that BCAS2 is involved in double-strand breaks (DSB); therefore, we aimed to characterise its mechanism and role in prostate cancer (PCa). METHODS: Western blotting and immunofluorescence microscopy were used to assay the roles of BCAS2 in the DSBs of PCa cells and apoptosis in Drosophila, respectively. The effect of BCAS2 dosage on non-homologous end joining (NHEJ) and homologous recombination (HR) were assayed by precise end-joining assay and flow cytometry, respectively. Glutathione-S-transferase pulldown and co-immunoprecipitation assays were used to determine whether and how BCAS2 interacts with NBS1. The expression of BCAS2 and other proteins in human PCa was determined by immunohistochemistry. RESULTS: BCAS2 helped repair radiation-induced DSBs efficiently in both human PCa cells and Drosophila. BCAS2 enhanced both NHEJ and HR, possibly by interacting with NBS1, which involved the BCAS2 N-terminus as well as both the NBS1 N- and C-termini. The overexpression of BCAS2 was significantly associated with higher Gleason and pathology grades and shorter survival in patients with PCa. CONCLUSION: BCAS2 promotes two DSB repair pathways by interacting with NBS1, and it may affect PCa progression.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA End-Joining Repair/physiology , Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Prostatic Neoplasms/metabolism , Animals , Apoptosis/genetics , DNA/radiation effects , DNA Repair Enzymes/metabolism , Drosophila/genetics , Humans , Male , Neoplasm Grading , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathologyABSTRACT
BACKGROUND: The Taiwan Human Disease iPSC Service Consortium was established to accelerate Taiwan's growing stem cell research initiatives and provide a platform for researchers interested in utilizing induced pluripotent stem cell (iPSC) technology. The consortium has generated and characterized 83 iPSC lines: 11 normal and 72 disease iPSC lines covering 21 different diseases, several of which are of high incidence in Taiwan. Whether there are any reprogramming-induced recurrent copy number variant (CNV) hotspots in iPSCs is still largely unknown. METHODS: We performed genome-wide copy number variant screening of 83 Han Taiwanese iPSC lines and compared them with 1093 control subjects using an Affymetrix genome-wide human SNP array. RESULTS: In the iPSCs, we identified ten specific CNV loci and seven "polymorphic" CNV regions that are associated with the reprogramming process. Additionally, we established several differentiation protocols for our iPSC lines. We demonstrated that our iPSC-derived cardiomyocytes respond to pharmacological agents and were successfully engrafted into the mouse myocardium demonstrating their potential application in cell therapy. CONCLUSIONS: The CNV hotspots induced by cell reprogramming have successfully been identified in the current study. This finding may be used as a reference index for evaluating iPSC quality for future clinical applications. Our aim was to establish a national iPSC resource center generating iPSCs, made available to researchers, to benefit the stem cell community in Taiwan and throughout the world.
Subject(s)
Cell Differentiation , DNA Copy Number Variations , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/physiology , Adolescent , Adult , Aged , Aged, 80 and over , Cellular Reprogramming , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Taiwan , Young AdultABSTRACT
BACKGROUND: Dysregulation of pericellular proteolysis usually accounts for cancer cell invasion and metastasis. Isolation of a cell-surface protease system for lung cancer metastasis is an important issue for mechanistic studies and therapeutic target identification. METHODS: Immunohistochemistry of a tissue array (n = 64) and TCGA database (n = 255) were employed to assess the correlation between serine protease inhibitors (SPIs) and lung adenocarcinoma progression. The role of SPI in cell motility was examined using transwell assays. Pulldown and LC/MS/MS were performed to identify the SPI-modulated novel protease(s). A xenografted mouse model was harnessed to demonstrate the role of the SPI in lung cancer metastasis. RESULTS: Hepatocyte growth factor activator inhibitor-2 (HAI-2) was identified to be downregulated following lung cancer progression, which was related to poor survival and tumour invasion. We further isolated a serum-derived serine protease, plasmin, to be a novel target of HAI-2. Downregulation of HAI-2 promotes cell surface plasmin activity, EMT, and cell motility. HAI-2 can suppress plasmin-mediated activations of HGF and TGF-ß1, EMT and cell invasion. In addition, downregulated HAI-2 increased metastasis of lung adenocarcinoma via upregulating plasmin activity. CONCLUSION: HAI-2 functions as a novel inhibitor of plasmin to suppress lung cancer cell motility, EMT and metastasis.
Subject(s)
Adenocarcinoma of Lung/metabolism , Fibrinolysin/metabolism , Lung Neoplasms/metabolism , Membrane Glycoproteins/metabolism , A549 Cells , Adenocarcinoma of Lung/pathology , Adenocarcinoma of Lung/secondary , Animals , Cell Line, Tumor , Cell Movement , Disease Progression , Epithelial-Mesenchymal Transition , Fibrinolysin/antagonists & inhibitors , Hepatocyte Growth Factor/metabolism , Humans , Lung Neoplasms/pathology , Mice , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Transplantation , Transforming Growth Factor beta1/metabolismABSTRACT
Signal transducer and activator of transcription 3 (STAT3) had been involved in liver fibrogenesis. We aimed to explore the antifibrotic activities of sorafenib and its derivative SC-1 (devoid of Raf kinase inhibition activity) both in vivo and in vitro with special focus on the STAT3 pathway in hepatic stellate cells (HSCs). The clinical role of STAT3 in chronic hepatitis B (CHB) was also investigated. Experimental fibrosis mouse models were established by thioacetamide injection and bile duct ligation in Balb/C mice and treated with sorafenib and SC-1. Rat and human HSCs were used for mechanistic investigations. Forty CHB patients were enrolled to quantify the hepatic phospho-STAT3 (p-STAT3) levels and correlated with liver fibrosis. Both sorafenib and SC-1 ameliorated liver fibrosis in vivo and promoted HSC apoptosis in vitro. p-STAT3 and downstream signals were down-regulated after sorafenib and SC-1 treatment in HSC. STAT3 overexpression in HSC enhanced cell proliferation and undermined the apoptotic effects of sorafenib and SC-1, whereas STAT3-specific inhibition promoted HSC apoptosis. Sorafenib and SC-1 activated Src-homology protein tyrosine phosphatase-1 (SHP-1) and STAT3 inhibition followed. Of particular interest, in CHB patients with advanced liver fibrosis, p-STAT3 in HSC was significantly overexpressed and positively correlated with the severity of liver fibrosis and plasma IL-6 levels. In conclusion, sorafenib and SC-1 ameliorate liver fibrosis through STAT3 inhibition in HSC and STAT3 may potentially serve as a promising fibrotic biomarker and target in liver fibrosis. SHP-1 phosphatase-directed STAT3 inhibition may represent a previously unidentified strategy for antifibrotic drug discovery.
Subject(s)
Liver Cirrhosis/prevention & control , Niacinamide/analogs & derivatives , Phenylurea Compounds/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , Animals , Cell Line , Hepatic Stellate Cells/drug effects , Humans , Male , Mice , Mice, Inbred BALB C , Niacinamide/chemistry , Niacinamide/pharmacology , Phenylurea Compounds/chemistry , Rats , SorafenibABSTRACT
BACKGROUND: 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) is a potential anticancer drug that may activate soluble guanylyl cyclase (sGC) and increase the level of cyclic guanosine monophosphate (cGMP). The aim of this study was to explore the effects of YC-1 on lipid droplet accumulation and foam cell formation in macrophages. RESULTS: Human-oxidized low density lipoprotein (ox-LDL) was used to induce accumulation of lipid droplets in a murine macrophage cell line, RAW 264.7. Oil red O staining showed that treatment with 20 µM YC-1 for 24 h increased the area of intracellular lipid droplets in macrophages. The results of high content screening (HCS) with the AdipoRed™ assay further revealed that YC-1 enhanced ox-LDL-induced foam cell formation. This was evidenced by an increase in the total area of lipid droplets and the mean fluorescence intensity per cell. Inhibition of cGMP-dependent protein kinase (PKG) using KT5823 significantly reduced YC-1-enhanced lipid droplet formation in ox-LDL-induced macrophage foam cells. CONCLUSION: YC-1 induces lipid droplet formation in macrophages, possibly through the sGC/cGMP/PKG signaling pathway. This chemical should be tested with caution in future clinical trials.
Subject(s)
Cyclic GMP/metabolism , Indazoles/pharmacology , Lipid Droplets/metabolism , Lipoproteins, LDL/metabolism , Macrophages/metabolism , Second Messenger Systems/drug effects , Animals , Cell Line , MiceABSTRACT
UNLABELLED: Sorafenib is the first approved targeted therapeutic reagent for hepatocellular carcinoma (HCC). Here, we report that Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1) is a major target of sorafenib and generates a series of sorafenib derivatives to search for potent SHP-1 agonists that may act as better anti-HCC agents than sorafenib. Sorafenib increases SHP-1 activity by direct interaction and impairs the association between the N-SH2 domain and the catalytic protein tyrosine phosphatase domain of SHP-1. Deletion of the N-SH2 domain (dN1) or point mutation (D61A) of SHP-1 abolished the effect of sorafenib on SHP-1, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and apoptosis, suggesting that sorafenib may affect SHP-1 by triggering a conformational switch relieving its autoinhibition. Molecular docking of SHP-1/sorafenib complex confirmed our findings in HCC cells. Furthermore, novel sorafenib derivatives SC-43 and SC-40 displayed more potent anti-HCC activity than sorafenib, as measured by enhanced SHP-1 activity, inhibition of p-STAT3, and induction of apoptosis. SC-43 induced substantial apoptosis in sorafenib-resistant cells and showed better survival benefits than sorafenib in orthotopic HCC tumors. CONCLUSION: In this study, we identified SHP-1 as a major target of sorafenib. SC-43 and SC-40, potent SHP-1 agonists, showed better anti-HCC effects than sorafenib in vitro and in vivo. Further clinical investigation is warranted.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Niacinamide/analogs & derivatives , Phenylurea Compounds/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/drug effects , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Catalytic Domain , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Humans , Mice , Models, Molecular , Niacinamide/pharmacology , Niacinamide/therapeutic use , Phenylurea Compounds/therapeutic use , Protein Tyrosine Phosphatase, Non-Receptor Type 6/chemistry , Random Allocation , STAT3 Transcription Factor/antagonists & inhibitors , Sorafenib , Xenograft Model Antitumor AssaysABSTRACT
Depletion of ß-catenin impairs regeneration of the rapid turn-over gut epithelial cells, but appears dispensable for that of the slow turn-over mature hepatocytes in mice until 1 y of age. As the life span of mature murine hepatocytes is about 400 d, we studied conditional ß-catenin knockout mice (Alb-Cre;Ctnnb1(flx/flx)) until 20 mo of age to determine the function of ß-catenin in the postnatal liver. ß-catenin was absent from the hepatocytes of ß-catenin knockout mice 4 wk after delivery. From 9 mo of age, hepatocytes were gradually replaced by newly formed ß-catenin-positive hepatocytes, which constituted about 90% of hepatocytes at 18-20 mo of age. This process was accompanied by active proliferation of bile duct/ductule cells. ß-catenin-positive hepatocytes exhibited elevated proliferation activity and expression of progenitor cell markers, but lower albumin and Cre. This might explain their intact ß-catenin protein, and suggest their origins from hepatic progenitor cells. Liver tumors arose spontaneously from ß-catenin-positive cells, and tumorigenesis was accelerated by hepatitis B X protein. These results indicate ß-catenin critical for the regeneration of mature hepatocytes. Failure to regenerate mature hepatocytes results in proliferation of hepatic progenitor cells that are able to maintain liver function but are predisposed to form liver tumors.
Subject(s)
Hepatocytes/metabolism , Liver Neoplasms, Experimental/pathology , Stem Cells/cytology , beta Catenin/metabolism , Albumins/genetics , Animals , Liver Neoplasms, Experimental/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic , beta Catenin/geneticsABSTRACT
Mucopolysaccharidosis (MPS) type II is caused by a deficiency of iduronate-2-sulfatase and is characterized by the accumulation of glycosaminoglycans (GAGs). Without effective therapy, the severe form of MPS II causes progressive neurodegeneration and death. This study generated multiple clones of induced pluripotent stem cells (iPSCs) and their isogenic controls (ISO) from four patients with MPS II neurodegeneration. MPS II-iPSCs were successfully differentiated into cortical neurons with characteristic biochemical and cellular phenotypes, including axonal beadings positive for phosphorylated tau, and unique electrophysiological abnormalities, which were mostly rescued in ISO-iPSC-derived neurons. RNA sequencing analysis uncovered dysregulation in three major signaling pathways, including Wnt/ß-catenin, p38 MAP kinase, and calcium pathways, in mature MPS II neurons. Further mechanistic characterization indicated that the dysregulation in calcium signaling led to an elevated intracellular calcium level, which might be linked to compromised survival of neurons. Based on these dysregulated pathways, several related chemicals and drugs were tested using this mature MPS II neuron-based platform and a small-molecule glycogen synthase kinase-3ß inhibitor was found to significantly rescue neuronal survival, neurite morphology, and electrophysiological abnormalities in MPS II neurons. Our results underscore that the MPS II-iPSC-based platform significantly contributes to unraveling the mechanisms underlying the degeneration and death of MPS II neurons and assessing potential drug candidates. Furthermore, the study revealed that targeting the specific dysregulation of signaling pathways downstream of GAG accumulation in MPS II neurons with a well-characterized drug could potentially ameliorate neuronal degeneration.
Subject(s)
Glycogen Synthase Kinase 3 beta , Induced Pluripotent Stem Cells , Mucopolysaccharidosis II , Neurons , Induced Pluripotent Stem Cells/metabolism , Humans , Glycogen Synthase Kinase 3 beta/metabolism , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Neurons/metabolism , Neurons/pathology , Neurons/drug effects , Mucopolysaccharidosis II/pathology , Mucopolysaccharidosis II/metabolism , Mucopolysaccharidosis II/genetics , Cell Differentiation/drug effects , Wnt Signaling Pathway/drug effects , Signal Transduction/drug effects , Calcium Signaling/drug effects , Nerve Degeneration/pathology , Calcium/metabolismABSTRACT
PURPOSE: Biomarkers predicting clinically significant prostate cancer (sPC) before biopsy are currently lacking. This study aimed to develop a non-invasive urine test to predict sPC in at-risk men using urinary metabolomic profiles. MATERIALS AND METHODS: Urine samples from 934 at-risk subjects and 268 treatment-naïve PC patients were subjected to liquid chromatography/mass spectrophotometry (LC-MS)-based metabolomics profiling using both C18 and hydrophilic interaction liquid chromatography (HILIC) column analyses. Four models were constructed (training cohort [n=647]) and validated (validation cohort [n=344]) for different purposes. Model I differentiates PC from benign cases. Models II, III, and a Gleason score model (model GS) predict sPC that is defined as National Comprehensive Cancer Network (NCCN)-categorized favorable-intermediate risk group or higher (Model II), unfavorable-intermediate risk group or higher (Model III), and GS ≥7 PC (model GS), respectively. The metabolomic panels and predicting models were constructed using logistic regression and Akaike information criterion. RESULTS: The best metabolomic panels from the HILIC column include 25, 27, 28 and 26 metabolites in Models I, II, III, and GS, respectively, with area under the curve (AUC) values ranging between 0.82 and 0.91 in the training cohort and between 0.77 and 0.86 in the validation cohort. The combination of the metabolomic panels and five baseline clinical factors that include serum prostate-specific antigen, age, family history of PC, previously negative biopsy, and abnormal digital rectal examination results significantly increased AUCs (range 0.88-0.91). At 90% sensitivity (validation cohort), 33%, 34%, 41%, and 36% of unnecessary biopsies were avoided in Models I, II, III, and GS, respectively. The above results were successfully validated using LC-MS with the C18 column. CONCLUSIONS: Urinary metabolomic profiles with baseline clinical factors may accurately predict sPC in men with elevated risk before biopsy.
ABSTRACT
Pompe disease is caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene, which encodes GAA. Although enzyme replacement therapy has recently improved patient survival greatly, the results in skeletal muscles and for advanced disease are still not satisfactory. Here, we report the derivation of Pompe disease-induced pluripotent stem cells (PomD-iPSCs) from two patients with different GAA mutations and their potential for pathogenesis modeling, drug testing and disease marker identification. PomD-iPSCs maintained pluripotent features and had low GAA activity and high glycogen content. Cardiomyocyte-like cells (CMLCs) differentiated from PomD-iPSCs recapitulated the hallmark Pompe disease pathophysiological phenotypes, including high levels of glycogen and multiple ultrastructural aberrances. Drug rescue assessment showed that exposure of PomD-iPSC-derived CMLCs to recombinant human GAA reversed the major pathologic phenotypes. Furthermore, l-carnitine treatment reduced defective cellular respiration in the diseased cells. By comparative transcriptome analysis, we identified glycogen metabolism, lysosome and mitochondria-related marker genes whose expression robustly correlated with the therapeutic effect of drug treatment in PomD-iPSC-derived CMLCs. Collectively, these results demonstrate that PomD-iPSCs are a promising in vitro disease model for the development of novel therapeutic strategies for Pompe disease.
Subject(s)
Glycogen Storage Disease Type II/drug therapy , Glycogen Storage Disease Type II/pathology , Induced Pluripotent Stem Cells/pathology , Models, Biological , Adenine/analogs & derivatives , Adenine/pharmacology , Adenine/therapeutic use , Animals , Base Sequence , Biomarkers/metabolism , Carnitine/pharmacology , Carnitine/therapeutic use , Cell Differentiation/drug effects , Drug Monitoring , Fibroblasts/drug effects , Fibroblasts/pathology , Glycogen Storage Disease Type II/physiopathology , Humans , Induced Pluripotent Stem Cells/drug effects , Mice , Molecular Sequence Data , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Myocytes, Cardiac/ultrastructure , alpha-Glucosidases/pharmacology , alpha-Glucosidases/therapeutic useABSTRACT
UNLABELLED: The liver architecture plays an important role in maintaining hemodynamic balance, but the mechanisms that underlie this role are not fully understood. Hepsin, a type II transmembrane serine protease, is predominantly expressed in the liver, but has no known physiological functions. Here, we report that hemodynamic balance in the liver is regulated through hepsin. Deletion of hepsin (hepsin(-/-) ) in mice resulted in enlarged hepatocytes and narrowed liver sinusoids. Using fluorescent microbeads and antihepsin treatment, we demonstrated that metastatic cancer cells preferentially colonized the hepsin(-/-) mouse liver as a result of the retention of tumor cells because of narrower sinusoids. The enlarged hepatocytes expressed increased levels of connexin, which resulted from defective prohepatocyte growth factor (pro-HGF) processing and decreased c-Met phosphorylation in the livers of hepsin(-/-) mice. Treatment of hepsin(-/-) mice with recombinant HGF rescued these phenotypes, and treatment of wild-type mice with an HGF antagonist recapitulated the phenotypes observed in hepsin(-/-) mice. CONCLUSION: Our findings show that the maintenance of hepatic structural homeostasis occurs through HGF/c-Met/connexin signaling by hepsin, and hepsin-mediated changes in liver architecture significantly enhance tumor metastasis to the liver.
Subject(s)
Hepatocyte Growth Factor/metabolism , Hepatocytes/pathology , Liver Neoplasms/secondary , Liver/metabolism , Liver/pathology , Neoplasm Metastasis/pathology , Serine Endopeptidases/metabolism , Animals , Connexins/metabolism , Hemodynamics , Hepatocyte Growth Factor/pharmacology , Hepatocytes/drug effects , Hepatocytes/metabolism , Liver/drug effects , Male , Mice , Mice, Knockout , Microscopy, Electron, Transmission , Neoplasm Transplantation , Phosphorylation , Proto-Oncogene Proteins c-met/metabolism , Serine Endopeptidases/genetics , Signal TransductionABSTRACT
UNLABELLED: Alteration of cell surface proteolysis has been proposed to play a role in liver fibrosis, a grave complication of biliary atresia (BA). In this study we investigated the roles of hepatocyte growth factor activator inhibitor (HAI)-1 and -2 in the progression of BA. The expression levels of HAI-1 and -2 were significantly increased in BA livers compared with those in neonatal hepatitis and correlated with disease progression. In BA livers, HAI-1 and -2 were coexpressed in cells involved in ductular reactions. In other selective cholangiopathies, ductular cells positive for HAI-1 or HAI-2 also increased in number. Inflammatory cytokines, growth factors, and bile acids differentially up-regulated expression of HAI-1 and -2 transcripts in fetal liver cells and this induction could be antagonized by a cyclooxygenase-2 inhibitor. Conditioned media from cell lines stably overexpressing HAI-1 or HAI-2 enhanced the fibrogenic activity of portal fibroblasts and stellate cells, suggesting that both proteins might be involved in liver fibrosis. Because HAI-1 and -2 colocalized in ductular reactions sharing similar features to those observed during normal liver development, we sought to investigate the role of HAI-1 and -2 in cholangiopathies by exploring their functions in fetal liver cells. Knockdown of HAI-1 or HAI-2 promoted bidirectional differentiation of hepatoblast-derived cells. In addition, we showed that the hepatocyte growth factor activator, mitogen-activated protein kinase kinase 1, and phosphatidylinositol 3-kinase signaling pathways were involved in hepatic differentiation enhanced by HAI-2 knockdown. CONCLUSION: HAI-1 and -2 are overexpressed in the liver in cholangiopathies with ductular reactions and are possibly involved in liver fibrosis and hepatic differentiation; they could be investigated as disease markers and potential therapeutic targets.
Subject(s)
Cholestasis/pathology , Hepatitis/pathology , Liver Cirrhosis/pathology , Membrane Glycoproteins/genetics , Proteinase Inhibitory Proteins, Secretory/genetics , Animals , Cell Differentiation/physiology , Cell Line , Cholestasis/physiopathology , Female , Fibroblasts/cytology , Hepatic Stellate Cells/cytology , Hepatitis/congenital , Hepatitis/physiopathology , Hepatocytes/cytology , Humans , Infant , Infant, Newborn , Liver Cirrhosis/congenital , Male , Membrane Glycoproteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Proteinase Inhibitory Proteins, Secretory/metabolism , Rats , Signal Transduction/physiology , Stem Cells/cytology , Stem Cells/physiologyABSTRACT
The multiple cellular targets affected by proteasome inhibition implicate a potential role for bortezomib, a first-in-class proteasome inhibitor, in enhancing antitumor activities in hematologic malignancies. Here, we examined the antitumor activity and drug targets of bortezomib in leukemia cells. Human leukemia cell lines were used for in vitro studies. Drug efficacy was evaluated by apoptosis assays and associated molecular events assessed by Western Blot. Gene silencing was performed by small interference RNA. Drug was tested in vivo in xenograft models of human leukemia cell lines and in primary leukemia cells. Clinical samples were assessed by immunohistochemical staining. Bortezomib differentially induced apoptosis in leukemia cells that was independent of its proteasome inhibition. Cancerous inhibitor of protein phosphatase 2A, a cellular inhibitor of protein phosphatase 2A, mediated the apoptotic effect of bortezomib. Bortezomib increased protein phosphatase 2A activity in sensitive leukemia cells (HL-60 and KG-1), but not in resistant cells (MOLT-3 and K562). Bortezomib's downregulation of cancerous inhibitor of protein phosphatase 2A and phospho-Akt correlated with its drug sensitivity. Furthermore, cancerous inhibitor of protein phosphatase 2A negatively regulated protein phosphatase 2A activity. Ectopic expression of CIP2A up-regulated phospho-Akt and protected HL-60 cells from bortezomib-induced apoptosis, whereas silencing CIP2A overcame the resistance to bortezomib-induced apoptosis in MOLT3 and K562 cells. Importantly, bortezomib exerted in vivo antitumor activity in HL-60 xenografted tumors and induced cell death in some primary leukemic cells. Cancerous inhibitor of protein phosphatase 2A was expressed in leukemic blasts from bone marrow samples. Cancerous inhibitor of protein phosphatase 2A plays a major role in mediating bortezomib-induced apoptosis in leukemia cells.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Boronic Acids/pharmacology , Leukemia/metabolism , Protein Phosphatase 2/metabolism , Pyrazines/pharmacology , Animals , Autoantigens/genetics , Autoantigens/metabolism , Bortezomib , Cell Line, Tumor , DNA Replication/drug effects , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Enzyme Activation/drug effects , Gene Expression Regulation, Leukemic/drug effects , HL-60 Cells , Humans , Intracellular Signaling Peptides and Proteins , K562 Cells , Leukemia/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Transcription, Genetic , Xenograft Model Antitumor AssaysABSTRACT
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is highly expressed in embryonic stem cells (ESCs) and its role in maintenance of pluripotency has been suggested previously. In epithelial cancer cells, activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. However, their role in somatic cell reprogramming is still unknown. Here we demonstrate that EpCAM and its associated protein, Cldn7, play a critical role in reprogramming. Quantitative RT-PCR analysis of Oct4, Sox2, Klf4, and c-Myc (OSKM) infected mouse embryonic fibroblasts (MEFs) indicated that EpCAM and Cldn7 were up-regulated during reprogramming. Analysis of numbers of alkaline phosphatase- and Nanog-positive clones, and the expression level of pluripotency-related genes demonstrated that inhibition of either EpCAM or Cldn7 expression resulted in impairment in reprogramming efficiency, whereas overexpression of EpCAM, EpCAM plus Cldn7, or EpCAM intercellular domain (EpICD) significantly enhanced reprogramming efficiency in MEFs. Furthermore, overexpression of EpCAM or EpICD significantly repressed the expression of p53 and p21 in the reprogramming MEFs, and both EpCAM and EpICD activated the promoter activity of Oct4. These observations suggest that EpCAM signaling may enhance reprogramming through up-regulation of Oct4 and possible suppression of the p53-p21 pathway. In vitro and in vivo characterization indicated that the EpCAM-reprogrammed iPSCs exhibited similar molecular and functional features to the mouse ESCs. In summary, our studies provide additional insight into the molecular mechanisms of reprogramming and suggest a more effective means of induced pluripotent stem cell generation.