ABSTRACT
Diphthamide biosynthesis protein 1 (DPH1) is biochemically involved in the first step of diphthamide biosynthesis, a post-translational modification of eukaryotic elongation factor 2 (EEF2). Earlier studies showed that DPH1, also known as ovarian cancer-associated gene 1 (OVCA1), is involved in ovarian carcinogenesis. However, the role of DPH1 in hepatocellular carcinoma (HCC) remains unclear. To investigate the impact of DPH1 in hepatocellular carcinogenesis, we performed data mining from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset. We found that reduced DPH1 levels were associated with advanced stages and poor survival of patients with HCC. Also, we generated hepatocyte-specific Dph1-deficient mice and showed that diphthamide-deficient EEF2 resulted in a reduced translation elongation rate in the hepatocytes and led to mild liver damage with fatty accumulation. After N-diethylnitrosamine (DEN)-induced acute liver injury, p53-mediated pericentral hepatocyte death was increased, and compensatory proliferation was reduced in Dph1-deficient mice. Consistent with these effects, Dph1 deficiency decreased the incidence of DEN-induced pericentral-derived HCC and revealed a protective effect against p53 loss. In contrast, Dph1 deficiency combined with Trp53- or Trp53/Pten-deficient hepatocytes led to increased tumor loads associated with KRT19 (K19)-positive periportal-like cell expansion in mice. Further gene set enrichment analysis also revealed that HCC patients with lower levels of DPH1 and TP53 expression had enriched gene-sets related to the cell cycle and K19-upregulated HCC. Additionally, liver tumor organoids obtained from 6-month-old Pten/Trp53/Dph1-triple-mutant mice had a higher frequency of organoid re-initiation cells and higher proliferative index compared with those of the Pten/Trp53-double-mutant. Pten/Trp53/Dph1-triple-mutant liver tumor organoids showed expression of genes associated with stem/progenitor phenotypes, including Krt19 and Prominin-1 (Cd133) progenitor markers, combined with low hepatocyte-expressed fibrinogen genes. These findings indicate that diphthamide deficiency differentially regulates hepatocellular carcinogenesis, which inhibits pericentral hepatocyte-derived tumors and promotes periportal progenitor-associated liver tumors. © 2022 The Pathological Society of Great Britain and Ireland.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Animals , Carcinogenesis/genetics , Carcinoma, Hepatocellular/genetics , Histidine/analogs & derivatives , Liver Neoplasms/genetics , Mice , Tumor Suppressor Protein p53/geneticsABSTRACT
The primary cilium is a sensory organelle that receives specific signals from the extracellular environment important for vertebrate development and tissue homeostasis. Lamins, the major components of the nuclear lamina, are required to maintain the nuclear structure and are involved in most nuclear activities. In this study, we show that deficiency in lamin A/C causes defective ciliogenesis, accompanied by increased cytoplasmic accumulation of actin monomers and increased formation of actin filaments. Disruption of actin filaments by cytochalasin D rescues the defective ciliogenesis in lamin A/C-depleted cells. Moreover, lamin A/C-deficient cells display lower levels of nesprin 2 and defects in recruiting Arp2, myosin Va, and tau tubulin kinase 2 to the basal body during ciliogenesis. Collectively, our results uncover a functional link between nuclear lamina integrity and ciliogenesis and implicate the malfunction of primary cilia in the pathogenesis of laminopathy.
Subject(s)
Lamin Type A , Nuclear Lamina , Actins , Cell Nucleus , Cilia , Lamin Type A/genetics , Lamins/geneticsABSTRACT
This study aimed to shed light on the molecular and cellular mechanisms responsible for initiation and progression of liver malignancies by examining the role of phosphatase and tensin homolog on chromosome 10 (Pten) in liver tumor progression in miR-122a (Mir122a)-null mice. We generated and monitored liver tumor initiation in Mir122a-null Pten heterozygous (Mir122a-/-;Pten+/- and Mir122a-/-;Alb-Cre;Ptenfx/+) mice and compared the results with those in Mir122a-/- mice. Both Mir122a-/-;Pten+/- and Mir122a-/-;Alb-Cre;Ptenfx/+ mice developed visible liver tumor nodules at 6 months of age. In premalignant livers of Mir122a-/-;Pten+/- mice, decreased PTEN and increased phosphorylated AKT were specifically observed in periportal cells, associated with inflammatory and fibrotic microenvironments. Furthermore, IL-1ß and tumor necrosis factor-α levels significantly increased in Mir122a-/-;Pten+/- premalignant livers at 6 months of age. Oval cells expressing A6, epithelial cell adhesion molecule, keratin (K) 8, K19, and SRY (sex determining region Y)-box 9 (SOX9) were present in both Mir122a-/- and Mir122a-/-;Pten+/- livers. Interestingly, a hybrid hepatocyte-like population with intermediate levels of K8, HNF4α, and SOX9 was located proximally to the oval cells in Mir122a-/-;Pten+/- livers. Lineage-tracing experiments revealed that these intermediate levels of K8 hepatocyte-like cells may be the cells of origin for Mir122a-/-;Pten+/- liver tumors. These findings suggest that inflammatory microenvironments in the periportal area of Mir122a-null mice may locally cause Pten down-regulation and expand tumor-initiating cells, causing hepatocellular carcinoma.
Subject(s)
Gene Expression Regulation, Neoplastic , Hepatocytes/pathology , Liver Neoplasms, Experimental/pathology , MicroRNAs/physiology , Neoplastic Stem Cells/pathology , PTEN Phosphohydrolase/physiology , Tumor Microenvironment/immunology , Animals , Female , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Hepatocytes/metabolism , Keratin-8/genetics , Keratin-8/metabolism , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/immunology , Liver Neoplasms, Experimental/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/metabolism , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolismABSTRACT
The X-linked DEAD-box RNA helicase DDX3 (DDX3X) is a multifunctional protein that has been implicated in gene regulation, cell cycle control, apoptosis, and tumorigenesis. However, the precise physiological function of Ddx3x during development remains unknown. Here, we show that loss of Ddx3x results in an early post-implantation lethality in male mice. The size of the epiblast marked by Oct3/4 is dramatically reduced in embryonic day 6.5 (E6.5) Ddx3x-/Y embryos. Preferential paternal X chromosome inactivation (XCI) in extraembryonic tissues of Ddx3x heterozygous (Ddx3x-/+) female mice with a maternally inherited null allele leads to placental abnormalities and embryonic lethality during development. In the embryonic tissues, Ddx3x exhibits developmental- and tissue-specific differences in escape from XCI. Targeted Ddx3x ablation in the epiblast leads to widespread apoptosis and abnormal growth, which causes embryonic lethality in the Sox2-cre/+;Ddx3xflox/Y mutant around E11.5. The observation of significant increases in γH2AX and p-p53Ser15 indicates DNA damage, which suggests that loss of Ddx3x leads to higher levels of genome damage. Significant upregulation of p21WAF1/Cip1 and p15Ink4b results in cell cycle arrest and apoptosis in Ddx3x-deficient cells. These results have uncovered that mouse Ddx3x is essential for both embryo and extraembryonic development.
Subject(s)
Embryonic Development/genetics , Placentation/genetics , RNA Helicases/genetics , Transcriptional Activation/genetics , Animals , Apoptosis/genetics , Cyclin-Dependent Kinase Inhibitor p15/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , DEAD-box RNA Helicases , DNA Damage/genetics , Embryo, Mammalian , Female , Gene Expression Regulation, Developmental , Heterozygote , Humans , Mice , Pregnancy , RNA Helicases/biosynthesis , X Chromosome Inactivation/geneticsABSTRACT
OVCA1/DPH1 (OVCA1) encodes a component of the diphthamide biosynthesis pathway and is located on chromosome 17p13.3. Deletions in this region are associated with Miller-Dieker syndrome (MDS). Ovca1/Dph1 (Ovca1)-null mice exhibit multiple developmental defects, including cleft palate, growth restriction and perinatal lethality, suggesting a role in the craniofacial abnormalities associated with MDS. Conditional ablation of Ovca1 in neural crest cells, but not in cranial paraxial mesoderm, also results in cleft palate and shortened lower jaw phenotypes, similar to Ovca1-null embryos. Expression of transgenic myc-tagged Ovca1 in craniofacial structures can partially rescue the cleft palate and shortened mandible of Ovca1-null embryos. Interestingly, Ovca1-null mutants are resistant to conditional expression of diphtheria toxin subunit A in both neural crest cell and paraxial mesoderm derivatives. However, OVCA1-dependent diphthamide biosynthesis is essential for neural crest cell-derived craniofacial development but that is dispensable for paraxial mesodermal-derived craniofacial structures in mammals. These findings suggest that OVCA1 deficiency in the neural crest contributes to the craniofacial abnormalities in patients with MDS. Also, our findings provide new insights into the molecular and cellular mechanisms that lead to the craniofacial defects of MDS.
Subject(s)
Classical Lissencephalies and Subcortical Band Heterotopias/genetics , Classical Lissencephalies and Subcortical Band Heterotopias/pathology , Genetic Association Studies , Phenotype , Tumor Suppressor Proteins/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Chromosome Mapping , Cleft Palate/embryology , Cleft Palate/genetics , Cleft Palate/pathology , Disease Models, Animal , Embryo, Mammalian , Gene Expression , Genetic Linkage , Genotype , Male , Mice , Mice, Knockout , Minor Histocompatibility Antigens , Mutation , Neural Crest/embryology , Neural Crest/metabolism , Organogenesis/genetics , Wnt1 Protein/geneticsABSTRACT
Prostate glands comprise two major epithelial cell types: luminal and basal. Luminal cells have long been considered the cellular origin of prostate cancer (CaP). However, recent evidence from a prostate regeneration assay suggests that prostate basal cells can also give rise to CaP. Here, we characterize Pten-deficient prostate lesions arising from keratin 5-expressing basal cells in a temporally controlled system in mice. Pten-deficient prostate lesions arising from basal cells exhibited luminal phenotypes with higher invasiveness, and the cell fate of Pten-deficient basal cells was traced to neoplastic luminal cells. After temporally ablating Pten in keratin 8-expressing luminal cells, luminal-derived Pten-deficient prostate tumors exhibited slower disease progression, compared with basal-derived tumors, within 13 weeks after Pten ablation. Cellular proliferation was significantly increased in basal-derived versus luminal-derived Pten-deficient prostate lesions. Increased tumor invasion into the smooth muscle layer and aberrantly regulated aggressive signatures (Smad4 and Spp1) were identified exclusively in basal-derived Pten-deficient lesions. Interestingly, p63-expressing cells, which represent basal stem and progenitor cells of basal-derived Pten-deficient prostate lesions, were significantly increased, relative to cells of the luminal-derived prostate lesion. Furthermore, castration did not suppress cellular proliferation of either basal-derived or luminal-derived Pten-deficient prostate tumors. Taken together, our data suggest that, although prostate malignancy can originate from both basal and luminal populations, these two populations differ in aggressive potential.
Subject(s)
Cell Differentiation , Gene Deletion , PTEN Phosphohydrolase/metabolism , Prostate/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Androgens/deficiency , Animals , Biomarkers, Tumor/metabolism , Castration , Cell Differentiation/genetics , Cell Lineage , Cell Proliferation , Disease Progression , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genes, Neoplasm , Humans , Keratin-5 , Keratin-8/metabolism , Male , Mice , Mice, Transgenic , Muscle, Smooth/pathology , Neoplasm Invasiveness , PTEN Phosphohydrolase/deficiency , Prostate/metabolism , Prostatic Neoplasms/surgery , Regeneration , Tumor Suppressor Proteins/metabolismABSTRACT
Coronin 1B has been shown to be critical for cell motility and various actin-dependent processes. To understand its role more extensively, the expression and transcriptional regulation of Coro1b gene during mouse development were explored. Coronin 1B is ubiquitously expressed in the whole embryo but nevertheless shows distinct expression pattern in developing heart. In addition to the localization in endocardium, Coronin 1B is specifically expressed in the endocardial cushion and epicardium where cardiac EMT processes take place as the heart develops. Promoter deletion analysis identified the positions between -1038 and -681 is important for Coro1b basal promoter activity. In addition to a correlation of Coronin 1B localization with Wt1 expression in the epicardium, we also identified putative Wt1 binding sequences within Coro1b promoter. Direct binding of Wt1 to GC-rich sequences within the Coro1b promoter is required for the regulation of Coro1b gene expression. In accordance with the motility defect found in Coronin 1B-knockdown cells, a modest decrease in expression of Coronin 1B in the remaining epicardium of Wt1(EGFPCre/EGFPCre) mutant embryos was observed. These findings seem to shed light on the role of Wt1 during cell migration and suggest that, at least in part, this involves transcriptional control of Coro1b gene expression.
Subject(s)
Gene Expression Regulation, Developmental , Microfilament Proteins/metabolism , Pericardium/metabolism , WT1 Proteins/metabolism , Amino Acid Sequence , Animals , Cell Movement , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryonic Development , GC Rich Sequence , Genes, Reporter , HeLa Cells , Humans , Immunohistochemistry , Mice , Mice, Knockout , Microfilament Proteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , NIH 3T3 Cells , Organ Specificity , Pericardium/embryology , Promoter Regions, Genetic , Protein Binding , Transcriptional Activation , WT1 Proteins/geneticsABSTRACT
Sarcopenia, a prevalent muscle disease characterized by muscle mass and strength reduction, is associated with impaired skeletal muscle regeneration. However, the influence of the biomechanical properties of sarcopenic skeletal muscle on the efficiency of the myogenic program remains unclear. Herein, we established a mouse model of sarcopenia and observed a reduction in stiffness within the sarcopenic skeletal muscle in vivo. To investigate whether the biomechanical properties of skeletal muscle directly impact the myogenic program, we established an in vitro system to explore the intrinsic mechanism involving matrix stiffness control of myogenic differentiation. Our findings identify the microtubule motor protein, kinesin-1, as a mechano-transduction hub that senses and responds to matrix stiffness, crucial for myogenic differentiation and muscle regeneration. Specifically, kinesin-1 activity is positively regulated by stiff matrices, facilitating its role in transporting mitochondria and enhancing translocation of the glucose transporter GLUT4 to the cell surface for glucose uptake. Conversely, the softer matrices significantly suppress kinesin-1 activity, leading to the accumulation of mitochondria around nuclei and hindering glucose uptake by inhibiting GLUT4 membrane translocation, consequently impairing myogenic differentiation. The insights gained from the in-vitro system highlight the mechano-transduction significance of kinesin-1 motor proteins in myogenic differentiation. Furthermore, our study confirms that enhancing kinesin-1 activity in the sarcopenic mouse model restores satellite cell expansion, myogenic differentiation, and muscle regeneration. Taken together, our findings provide a potential target for improving muscle regeneration in sarcopenia.
Subject(s)
Kinesins , Regeneration , Sarcopenia , Animals , Kinesins/metabolism , Mice , Sarcopenia/metabolism , Sarcopenia/pathology , Muscle, Skeletal/metabolism , Mice, Inbred C57BL , Cell Differentiation , Muscle Development , Male , Glucose Transporter Type 4/metabolism , Extracellular Matrix/metabolism , Mitochondria/metabolism , Biomechanical Phenomena , Glucose/metabolismABSTRACT
The role of ß-catenin in thymocyte development has been extensively studied, however, the function of ß-catenin in thymic epithelial cells (TECs) remains largely unclear. Here, we demonstrate a requirement for ß-catenin in keratin 5 (K5)-expressing TECs, which comprise the majority of medullary TECs (mTECs) and a progenitor subset for cortical TECs (cTECs) in the young adult thymus. We found that conditionally ablated ß-catenin in K5(+)-TECs and their progeny cells resulted in thymic atrophy. The composition of TECs was also aberrantly affected. Percentages of K5(hi)K8(+)-TECs, K5(+)K8(-)-TECs and UEA1(+)-mTECs were significantly decreased and the percentage of K5(lo)K8(+)-TECs and Ly51(+)-cTECs were increased in ß-catenin-deficient thymi compared with that in the control thymi. We also observed that ß-catenin-deficient TEC lineage could give rise to K8(+)-cTECs more efficiently than wild-type TECs using lineage-tracing approach. Importantly, the expression levels of several transcription factors (p63, FoxN1 and Aire), which are essential for TEC differentiation, were altered in ß-catenin-deficient thymi. Under the aberrant differentiation of TECs, development of all thymocytes in ß-catenin-deficient thymi was impaired. Interleukin-7 (IL-7) and chemokines (Ccl19, Ccl25 and Cxcl12) levels were also downregulated in the thymic stromal cells in the mutants. Finally, introducing a BCL2 transgene in lymphoid lineages, which has been shown to rescue IL-7-deficient thymopoiesis, partially rescued the thymic atrophy and thymocyte development defects caused by induced ablation of ß-catenin in K5(+)-TECs. Collectively, these findings suggest that ß-catenin is required for the differentiation of TECs, thereby contributing to thymocyte development in the postnatal thymus.
Subject(s)
Epithelium/metabolism , Precursor Cells, T-Lymphoid/immunology , T-Lymphocytes/immunology , Thymocytes/immunology , Thymus Gland/pathology , beta Catenin/metabolism , Animals , Atrophy/genetics , Cells, Cultured , Cytokines/metabolism , Epithelium/immunology , Genes, bcl-2/genetics , Keratin-5/genetics , Keratin-5/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Thymus Gland/growth & development , Transcription, Genetic/genetics , beta Catenin/geneticsABSTRACT
ß(2)-glycoprotein I (ß(2)-GPI) is a plasma glycoprotein with diverse functions, but the impact and molecular effects of ß(2)-GPI on vascular biology are as yet unclear. Based on the limited information available on the contribution of ß(2)-GPI to endothelial cells, we investigated the effect of ß(2)-GPI on cell growth and migration in human aortic endothelial cells (HAECs). The regulation of ß(2)-GPI as part of intracellular signaling in HAECs was also examined. Vascular endothelial growth factor (VEGF) is a pro-angiogenic factor that may regulate endothelial functions. We found that ß(2)-GPI dose-dependently inhibited VEGF-induced endothelial cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyl tetrazolium bromide assay and cell counts. Using wound healing and Boyden chamber assays, ß(2)-GPI remarkably reduced VEGF-increased cell migration at the physiological concentration. Furthermore, ß(2)-GPI suppressed VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and Akt. These results suggest that ß(2)-GPI plays an essential role in the down-regulation of VEGF-induced endothelial responses and may be a useful component for anti-angiogenic therapy.
Subject(s)
Cell Movement , Endothelial Cells/physiology , Protein Serine-Threonine Kinases/metabolism , Vascular Endothelial Growth Factor A/physiology , Vascular Endothelial Growth Factor Receptor-2/metabolism , beta 2-Glycoprotein I/physiology , Aorta/cytology , Cell Proliferation , Cells, Cultured , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Gene Expression , Humans , Neovascularization, Pathologic/metabolism , Phosphorylation , Protein Processing, Post-Translational , Vascular Endothelial Growth Factor Receptor-2/geneticsABSTRACT
OBJECTIVE: Septal defects and coronary vessel anomalies are common congenital heart defects, yet their ontogeny and the underlying genetic mechanisms are not well understood. Here, we investigated the role of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII, NR2F2) in cardiac organogenesis. METHODS AND RESULTS: We analyzed embryos deficient in COUP-TFII and observed a spectrum of cardiac defects, including atrioventricular septal defect, thin-walled myocardium, and abnormal coronary morphogenesis. We show by expression analysis that COUP-TFII is expressed in the endocardium and the epicardium but not in the myocardium of the ventricle. Using endothelial-specific COUP-TFII mutants and molecular approaches, we show that COUP-TFII deficiency resulted in endocardial cushion hypoplasia. This was attributed to the reduced growth and survival of atrioventricular cushion mesenchymal cells and defective epithelial-mesenchymal transformation (EMT) in the underlying endocardium. In addition, the endocardial EMT defect was accompanied by downregulation of Snai1, one of the master regulators of EMT, and upregulation of vascular endothelial-cadherin. Furthermore, we show that although COUP-TFII does not play a major role in the formation of epicardial cell cysts, it is critically important for the formation of epicardium. Ablation of COUP-TFII impairs epicardial EMT and coronary plexus formation. CONCLUSIONS: Our results reveal that COUP-TFII plays cell-autonomous roles in the endocardium and the epicardium for endocardial and epicardial EMT, which are required for proper valve and coronary vessel formation during heart development.
Subject(s)
COUP Transcription Factor II/metabolism , Coronary Vessels/embryology , Coronary Vessels/metabolism , Endocardium/embryology , Endocardium/metabolism , Heart Defects, Congenital/embryology , Heart Defects, Congenital/metabolism , Heart/embryology , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , COUP Transcription Factor II/deficiency , COUP Transcription Factor II/genetics , Cadherins/genetics , Cadherins/metabolism , Cell Movement , Coronary Vessels/pathology , Endocardial Cushions/embryology , Endocardial Cushions/metabolism , Endocardium/pathology , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Developmental , Gestational Age , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Morphogenesis , Myocardium/metabolism , Myocardium/pathology , Neovascularization, Physiologic , Pericardium/embryology , Pericardium/metabolism , Pericardium/pathology , Snail Family Transcription Factors , Tissue Culture Techniques , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Beige adipocytes are thermogenic with high expression of uncoupling protein 1 in the white adipose tissue (WAT), accompanied by angiogenesis. Previous studies showed that Smad4 is important for angiogenesis. Here we studied whether endothelial Smad4-mediated angiogenesis is involved in WAT beiging. Inducible knockout of endothelial cell (EC) selective Smad4 (Smad4 iEC-KO) was achieved by using the Smad4 Floxp/floxp and Tie2 CreERT2 mice. Beige fat induction achieved by cold or adrenergic agonist, and angiogenesis were attenuated in WAT of Smad4 iEC-KO mice, with the less proliferation of ECs and adipogenic precursors. RNA sequencing of human ECs showed that Smad4 is involved in angiogenesis-related pathways. Knockdown of SMAD4 attenuated the upregulation of VEGFA, PDGFA, and angiogenesis in vitro. Treatment of human ECs with palmitic acid-induced Smad1/5 phosphorylation and the upregulation of core endothelial genes. Our study shows that endothelial Smad4 is involved in WAT beiging through angiogenesis and the expansion of adipose precursors into beige adipocytes.
ABSTRACT
Arteries and veins are anatomically, functionally and molecularly distinct. The current model of arterial-venous identity proposes that binding of vascular endothelial growth factor to its heterodimeric receptor--Flk1 and neuropilin 1 (NP-1; also called Nrp1)--activates the Notch signalling pathway in the endothelium, causing induction of ephrin B2 expression and suppression of ephrin receptor B4 expression to establish arterial identity. Little is known about vein identity except that it involves ephrin receptor B4 expression, because Notch signalling is not activated in veins; an unresolved question is how vein identity is regulated. Here, we show that COUP-TFII (also known as Nr2f2), a member of the orphan nuclear receptor superfamily, is specifically expressed in venous but not arterial endothelium. Ablation of COUP-TFII in endothelial cells enables veins to acquire arterial characteristics, including the expression of arterial markers NP-1 and Notch signalling molecules, and the generation of haematopoietic cell clusters. Furthermore, ectopic expression of COUP-TFII in endothelial cells results in the fusion of veins and arteries in transgenic mouse embryos. Thus, COUP-TFII has a critical role in repressing Notch signalling to maintain vein identity, which suggests that vein identity is under genetic control and is not derived by a default pathway.
Subject(s)
Cell Differentiation , DNA-Binding Proteins/metabolism , Membrane Proteins/antagonists & inhibitors , Receptors, Steroid/metabolism , Signal Transduction , Transcription Factors/metabolism , Veins/cytology , Veins/metabolism , Animals , Arteries/cytology , Arteries/metabolism , COUP Transcription Factor II , COUP Transcription Factors , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Gene Expression Regulation , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Models, Biological , Neuropilin-1/metabolism , Organ Specificity , Receptors, Notch , Receptors, Steroid/deficiency , Receptors, Steroid/genetics , Transcription Factors/deficiency , Transcription Factors/geneticsABSTRACT
We report the expression of the mouse Mpped1 in the telencephalon through embryonic stages to adulthood. Using Northern blotting analysis and RNA in situ hybridization (ISH), our data show that Mpped1 is specifically expressed in the brain and is enriched in the cortical plate of the developing telencephalon. Postnatally, the expression of Mpped1 is reduced in the cerebral cortex relative to its levels in the embryonic dorsal telencephalon. Also, Mpped1 expression is sustained in the hippocampal CA1 region. Examination of the expression of Mpped1 and other cortical layer markers by ISH in a malformed beta-catenin null dorsal telencephalon show that the Mpped1-, Cux2-, and Rorbeta-expressing superficial cortical layers are reduced and form patchy patterns, and the Tbr-1-expressing deep-layer neurons are incorrectly located on superficial layers, indicative of a migration defect of cortical neurons in the absence of beta-catenin.
Subject(s)
Telencephalon/metabolism , beta Catenin/genetics , Animals , Blotting, Northern , CA1 Region, Hippocampal , Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Embryo, Mammalian , Female , Genes , Mice , Mice, Congenic , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Transgenic , Neurons/metabolism , Pregnancy , beta Catenin/metabolismABSTRACT
During differentiation, skeletal muscle develops mature multinucleated muscle fibers, which could contract to exert force on a substrate. Muscle dysfunction occurs progressively in patients with muscular dystrophy, leading to a loss of the ability to walk and eventually to death. The synthetic glucocorticoid dexamethasone (Dex) has been used therapeutically to treat muscular dystrophy by an inhibition of inflammation, followed by slowing muscle degeneration and stabilizing muscle strength. Here, in mice with muscle injury, we found that Dex significantly promotes muscle regeneration via promoting kinesin-1 motor activity. Nevertheless, how Dex promotes myogenesis through kinesin-1 motors remains unclear. We found that Dex directly increases kinesin-1 motor activity, which is required for the expression of a myogenic marker (muscle myosin heavy chain 1/2), and also for the process of myoblast fusion and the formation of polarized myotubes. Upon differentiation, kinesin-1 mediates the recruitment of integrin ß1 onto microtubules allowing delivery of the protein into focal adhesions. Integrin ß1-mediated focal adhesion signaling then guides myoblast fusion towards a polarized morphology. By imposing geometric constrains via micropatterns, we have proved that cell adhesion is able to rescue the defects caused by kinesin-1 inhibition during the process of myogenesis. These discoveries reveal a mechanism by which Dex is able to promote myogenesis, and lead us towards approaches that are more efficient in improving skeletal muscle regeneration.
ABSTRACT
BACKGROUND: Cre/loxP-mediated genetic modification is the most widely used conditional genetic approach used in the mouse. Engineered Cre and the mutated ligand-binding domain of estrogen receptor fusion recombinase (CreERT) allow temporal control of Cre activity. RESULTS: In this study, we have generated two distinct transgenic mouse lines expressing CreERT, which show 4-hydroxytamoxifen (4-OHT)-inducible and spontaneous (4-OHT-independent) Cre activities, referred to Tg(BK5-CreERT)I and Tg(BK5-CreERT)S, respectively. The transgenic construct is driven by the bovine Keratin 5 promoter, which is active in the basal epithelial lineage of stratified and pseudo-stratified epithelium across multiple organs. Despite the difference in 4-OHT dependency, the Tg(BK5-CreERT)I and Tg(BK5-CreERT)S mouse lines shared similar Cre-mediated recombination among various organs, except for unique mammary epithelial Cre activity in Tg(BK5-CreERT)S females. CONCLUSION: These two new transgenic mouse lines for the analysis of basal epithelial function and for the genetic modification have been created allowing the identification of these cell lineages and analysis of their differentiation during embryogenesis, during perinatal development and in adult mice.
Subject(s)
Epithelial Cells/physiology , Integrases/metabolism , Keratin-5/genetics , Mice, Transgenic , Promoter Regions, Genetic , Animals , Cattle , Embryo, Mammalian/anatomy & histology , Embryo, Mammalian/physiology , Epithelial Cells/cytology , Estrogen Antagonists/metabolism , Female , Gene Targeting , Integrases/genetics , Male , Mice , Mice, Inbred C57BL , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tamoxifen/analogs & derivatives , Tamoxifen/metabolism , Tissue DistributionABSTRACT
The pleiotropic roles of DEAD-box helicase 3, X-linked (DDX3X), including its functions in transcriptional and translational regulation, chromosome segregation, DNA damage, and cell growth control, have highlighted the association between DDX3X and tumorigenesis. However, mRNA transcripts and protein levels of DDX3X in patient specimens have shown the controversial correlations of DDX3X with hepatocellular carcinoma (HCC) prevalence. In this study, generation of hepatocyte-specific Ddx3x-knockout mice revealed that loss of Ddx3x facilitates liver tumorigenesis. Loss of Ddx3x led to profound ductular reactions, cell apoptosis, and compensatory proliferation in female mutants at 6 weeks of age. The sustained phosphorylation of histone H2AX (γH2AX) and significant accumulation of DNA single-strand breaks and double-strand breaks in liver indicated that the replicative stress occurred in female mutants. Further chromatin immunoprecipitation analyses demonstrated that DDX3X bound to promoter regions and regulated the expression of DNA repair factors, DDB2 and XPA, to maintain genome stability. Loss of Ddx3x led to decreased levels of DNA repair factors, which contributed to an accumulation of unrepaired DNA damage, replication stress, and eventually, spontaneous liver tumors and DEN-induced HCCs in Alb-Cre/+;Ddx3xflox/flox mice. IMPLICATIONS: These data identify an important role of DDX3X in the regulation of DNA damage repair to protect against replication stress in liver and HCC development and progression.
Subject(s)
DEAD-box RNA Helicases/deficiency , DNA Damage , Liver Neoplasms, Experimental/enzymology , Animals , Carcinogenesis , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Diethylnitrosamine , Female , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, TransgenicABSTRACT
The bone marrow (BM) niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained. Previous studies have indicated that genetic mutations in various components of the niche can affect hematopoiesis and promote hematologic abnormalities, but the impact of abnormal BM endothelial cells (BMECs), a crucial niche component, on hematopoiesis remains incompletely understood. To dissect how genetic alterations in BMECs could affect hematopoiesis, we have employed a novel inducible Tie2-CreERT2 mouse model, with a tdTomato fluorescent reporter, to introduce an oncogenic KRasG12D mutation specifically in the adult endothelial cells. Tie2-CreERT2;KRasG12D mice had significantly more leukocytes and myeloid cells in the blood with mostly normal BM HSPC populations and developed splenomegaly. Genotyping polymerase chain reaction revealed KRasG12D activation in BMECs but not hematopoietic cells, confirming that the phenotype is due to the aberrant BMECs. Competitive transplant assays revealed that BM cells from the KRasG12D mice contained significantly fewer functional hematopoietic stem cells, and immunofluorescence imaging showed that the hematopoietic stem cells in the mutant mice were localized farther away from BM vasculature and closer to the endosteal area. RNA sequencing analyses found an inflammatory gene network, especially tumor necrosis factor α, as a possible contributor. Together, our results implicate an abnormal endothelial niche in compromising normal hematopoiesis.
Subject(s)
Gene Expression Regulation, Enzymologic , Hematopoiesis , Hematopoietic Stem Cells/metabolism , Mutation, Missense , Proto-Oncogene Proteins p21(ras)/biosynthesis , Signal Transduction , Stem Cell Niche , Amino Acid Substitution , Animals , Female , Hematopoietic Stem Cells/pathology , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Male , Mice , Mice, Mutant Strains , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) plays pivotal roles in cell growth, cell differentiation, and cell fate determination. Although genome-wide studies have identified COUP-TFII binding on gene sets mainly involved in neural crest cell (NCC) development and craniofacial morphogenesis, the direct functional connection between COUP-TFII and NCCs in vivo has not been well characterized. In this study, we show that COUP-TFII is expressed in the subpopulation of NCCs and its derivatives, and targeted ablation of COUP-TFII in mouse NCCs results in markedly shortened and bifurcated tympanic rings, which in turn disturb the caudal direction of external acoustic meatus invagination. However, formation of the manubrium of the malleus (MM) in Wnt1-Cre/+;COUP-TFII flox/flox mice is not perturbed, suggesting that the rostral half of the tympanic ring is sufficient to support proper MM development. Interestingly, we found that loss of COUP-TFII up-regulates Sox9 in the tympanic ring primordium and affects the distribution of preosteoblasts before mesenchymal condensation. Together, our results demonstrate that COUP-TFII plays an essential role in regulating the patterning of the NCC-derived tympanic ring.
Subject(s)
Body Patterning/genetics , COUP Transcription Factor II/metabolism , Cell Differentiation/genetics , Neural Crest/growth & development , Temporal Bone/growth & development , Animals , COUP Transcription Factor II/genetics , Cell Proliferation/genetics , Embryo, Mammalian , Gene Expression Regulation, Developmental/physiology , Malleus/growth & development , Mice, Transgenic , Neural Crest/cytology , Osteoblasts/metabolism , SOX9 Transcription Factor/metabolismABSTRACT
Studies indicate that the presence of cancer stem cells (CSCs) is responsible for poor prognosis of hepatocellular carcinoma (HCC) patients. In this study, the functional role of DDX3 in regulation of hepatic CSCs was investigated. Our results demonstrated that reduced DDX3 expression was not only inversely associated with tumor grade, but also predicted poor prognosis of HCC patients. Knockdown of DDX3 in HCC cell line HepG2 induced stemness gene signature followed by occurrence of self-renewal, chemoreisistance, EMT, migration as well as CSC expansion, and most importantly, DDX3 knockdown promotes tumorigenesis. Moreover, we found positive correlations between DDX3 level and expressions of tumor-suppressive miR-200b, miR-200c, miR-122 and miR-145, but not miR-10b and miR-519a, implying their involvement in DDX3 knockdown-induced CSC phenotypes. In addition, DDX3 reduction promoted up-regulation of DNA methyltransferase 3A (DNMT3A), while neither DNMT3B nor DNMT1 expression was affected. Enriched DNMT3A binding along with hypermethylation on promoters of these tumor-suppressive miRNAs reflected their transcriptional repressions in DDX3-knockdown cells. Furthermore, individual restoration of these tumor-suppressive miRNAs represses DDX3 knockdown-induced CSC phenotypes. In conclusion, our study suggested that DDX3 prevents generation of CSCs through epigenetically regulating a subset of tumor-suppressive miRNAs expressions, which strengthens tumor suppressor role of DDX3 in HCC.