BACKGROUND: Cell cycle progression and leukemia development are tightly regulated processes in which even a small imbalance in the expression of cell cycle regulatory molecules and microRNAs (miRNAs) can lead to an increased risk of cancer/leukemia development. Here, we focus on the study of a ubiquitous, multifunctional, and oncogenic miRNA-hsa-miR-155-5p (miR-155, MIR155HG), which is overexpressed in malignancies including chronic lymphocytic leukemia (CLL). Nonetheless, the precise mechanism of how miR-155 regulates the cell cycle in leukemic cells remains the subject of extensive research. METHODS: We edited the CLL cell line MEC-1 by CRISPR/Cas9 to introduce a short deletion within the MIR155HG gene. To describe changes at the transcriptome and miRNome level in miR-155-deficient cells, we performed mRNA-seq/miRNA-seq and validated changes by qRT-PCR. Flow cytometry was used to measure cell cycle kinetics. A WST-1 assay, hemocytometer, and Annexin V/PI staining assessed cell viability and proliferation. RESULTS: The limited but phenotypically robust miR-155 modification impaired cell proliferation, cell cycle, and cell ploidy. This was accompanied by overexpression of the negative cell cycle regulator p21/CDKN1A and Cyclin D1 (CCND1). We confirmed the overexpression of canonical miR-155 targets such as PU.1, FOS, SHIP-1, TP53INP1 and revealed new potential targets (FCRL5, ISG15, and MX1). CONCLUSIONS: We demonstrate that miR-155 deficiency impairs cell proliferation, cell cycle, transcriptome, and miRNome via deregulation of the MIR155HG/TP53INP1/CDKN1A/CCND1 axis. Our CLL model is valuable for further studies to manipulate miRNA levels to revert highly aggressive leukemic cells to nearly benign or non-leukemic types.
- MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell * genetics pathology MeSH
- Cyclin D1 genetics metabolism MeSH
- Cyclin-Dependent Kinase Inhibitor p21 * genetics metabolism MeSH
- Cell Cycle Checkpoints * genetics MeSH
- Humans MeSH
- MicroRNAs * genetics metabolism MeSH
- Cell Line, Tumor MeSH
- Cell Proliferation genetics MeSH
- Heat-Shock Proteins MeSH
- Gene Expression Regulation, Leukemic MeSH
- Carrier Proteins genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The sodium/calcium exchanger (NCX) type 1 has been well described in various cancers, but little is known about the other two NCX types (NCX2 and NCX3). In this study, we used the selective blocker of NCX3 - YM-244769 to investigate changes in apoptosis induction, migration, proliferation, intracellular calcium and ATP in four cancer cell lines - DLD1, HeLa, MDA-MB-231 and JIMT1. In all four cell lines we observed a concentration-dependent increase in the number of apoptotic cells, as well as reduced migration and proliferation. Induction of hypoxic conditions did not alter the response of these cells to YM-244769 in any of the above-mentioned parameters. These results indicate the role of NCX3 in cancer cell migration, proliferation and apoptosis, as inhibition of NCX1 by the specific blocker SEA0400 had no significant effect on these parameters. However, we verified the effect of NCX3 inhibition by using CRISPR/Cas9 to generate clones in which the SLC8A3 (NCX3) gene was deleted, and we obtained the same results. In addition, mitochondrial respiration was impaired in the clones with NCX3 knocked-out, suggesting that NCX3 also play a role in bioenergetics. In conclusion, we have clearly shown that NCX3 plays an important anti-apoptotic, pro-migratory and proliferative role in the cancer cells by affecting mitochondrial bioenergetics, thus supporting their survival and fate.
- MeSH
- Apoptosis drug effects MeSH
- Humans MeSH
- Mitochondria metabolism drug effects MeSH
- Cell Line, Tumor MeSH
- Neoplasms * metabolism pathology genetics MeSH
- Cell Movement drug effects MeSH
- Cell Proliferation drug effects MeSH
- Sodium-Calcium Exchanger * metabolism genetics antagonists & inhibitors MeSH
- Calcium metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Through the agnostic screening of patients with uncharacterised disease phenotypes for an upregulation of type I interferon (IFN) signalling, we identified a cohort of individuals heterozygous for mutations in PTPN1, encoding the protein-tyrosine phosphatase 1B (PTP1B). We aimed to describe the clinical phenotype and molecular and cellular pathology of this new disease. METHODS: In this case series, we identified patients and collected clinical and neuroradiological data through collaboration with paediatric neurology and clinical genetics colleagues across Europe (Czechia, France, Germany, Italy, Slovenia, and the UK) and Israel. Variants in PTPN1 were identified by exome and directed Sanger sequencing. The expression of IFN-stimulated genes was determined by quantitative (q) PCR or NanoString technology. Experiments to assess RNA and protein expression and to investigate type 1 IFN signalling were undertaken in patient fibroblasts, hTERT-immortalised BJ-5ta fibroblasts, and RPE-1 cells using CRISPR-Cas9 editing and standard cell biology techniques. FINDINGS: Between Dec 20, 2013, and Jan 11, 2023, we identified 12 patients from 11 families who were heterozygous for mutations in PTPN1. We found ten novel or very rare variants in PTPN1 (frequency on gnomAD version 4.1.0 of <1·25 × 10:sup>-6). Six variants were predicted as STOP mutations, two involved canonical splice-site nucleotides, and two were missense substitutions. In three patients, the variant occurred de novo, whereas in nine affected individuals, the variant was inherited from an asymptomatic parent. The clinical phenotype was characterised by the subacute onset (age range 1-8 years) of loss of motor and language skills in the absence of seizures after initially normal development, leading to spastic dystonia and bulbar involvement. Neuroimaging variably demonstrated cerebral atrophy (sometimes unilateral initially) or high T2 white matter signal. Neopterin in CSF was elevated in all ten patients who were tested, and all probands demonstrated an upregulation of IFN-stimulated genes in whole blood. Although clinical stabilisation and neuroradiological improvement was seen in both treated and untreated patients, in six of eight treated patients, high-dose corticosteroids were judged clinically to result in an improvement in neurological status. Of the four asymptomatic parents tested, IFN signalling in blood was normal (three patients) or minimally elevated (one patient). Analysis of patient blood and fibroblasts showed that tested PTPN1 variants led to reduced levels of PTPN1 mRNA and PTP1B protein, and in-vitro assays demonstrated that loss of PTP1B function was associated with impaired negative regulation of type 1 IFN signalling. INTERPRETATION: PTPN1 haploinsufficiency causes a type 1 IFN-driven autoinflammatory encephalopathy. Notably, some patients demonstrated stabilisation, and even recovery, of neurological function in the absence of treatment, whereas in others, the disease appeared to be responsive to immune suppression. Prospective studies are needed to investigate the safety and efficacy of specific immune suppression approaches in this disease population. FUNDING: The UK Medical Research Council, the European Research Council, and the Agence Nationale de la Recherche.
- MeSH
- Child MeSH
- Haploinsufficiency * genetics MeSH
- Infant MeSH
- Humans MeSH
- Adolescent MeSH
- Mutation genetics MeSH
- Brain Diseases genetics MeSH
- Neuroinflammatory Diseases genetics MeSH
- Child, Preschool MeSH
- Protein Tyrosine Phosphatase, Non-Receptor Type 1 * genetics MeSH
- Check Tag
- Child MeSH
- Infant MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
A20/Tnfaip3, an early NF-κB response gene and key negative regulator of NF-κB signaling, suppresses proinflammatory responses. Its ubiquitinase and deubiquitinase activities mediate proteasomal degradation within the NF-κB pathway. This study investigated the involvement of A20 signaling alterations in podocytes in the development of kidney injury. The phenotypes of A20Δpodocyte (podocyte-specific knockout of A20) mice were compared with those of control mice at 6 months of age to identify spontaneous changes in kidney function. A20Δpodocyte mice presented elevated serum urea nitrogen and creatinine levels, along with increased accumulation of inflammatory cells-neutrophils and macrophages-within the glomeruli. Additionally, A20Δpodocyte mice displayed significant podocyte loss. Ultrastructural analysis of A20 podocyte-knockout mouse glomeruli revealed hypocellularity of the glomerular tuft, expansion of the extracellular matrix, podocytopenia associated with foot process effacement, karyopyknosis, micronuclei, and podocyte detachment. In addition to podocyte death, we also observed damage to intracapillary endothelial cells with vacuolation of the cytoplasm and condensation of nuclear chromatin. A20 expression downregulation and CRISPR-Cas9 genome editing targeting A20 in a podocyte cell line confirmed these findings in vitro, highlighting the significant contribution of A20 activity in podocytes to glomerular injury pathogenesis. Finally, we analyzed TNFAIP3 transcription levels alongside genes involved in apoptosis, anoikis, NF-κB regulation, and cell attachment in glomerular and tubular compartments of kidney biopsies of patients with various renal diseases.
- MeSH
- Cytoskeleton * metabolism MeSH
- Glomerulonephritis * pathology metabolism genetics MeSH
- Kidney Glomerulus pathology metabolism MeSH
- Humans MeSH
- Mice, Inbred C57BL MeSH
- Mice, Knockout * MeSH
- Mice MeSH
- NF-kappa B metabolism MeSH
- Podocytes * metabolism pathology MeSH
- Signal Transduction MeSH
- Tumor Necrosis Factor alpha-Induced Protein 3 * metabolism genetics MeSH
- Inflammation * pathology genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
During GABAergic synaptic transmission, G protein-coupled GABAB receptors (GBRs) activate K+ channels that prolong the duration of inhibitory postsynaptic potentials (IPSPs). We now show that KCTD16, an auxiliary GBR subunit, anchors hyperpolarization-activated cyclic nucleotide-gated (HCN) channels containing HCN2/HCN3 subunits to GBRs. In dopamine neurons of the VTA (DAVTA neurons), this interaction facilitates activation of HCN channels via hyperpolarization during IPSPs, counteracting the GBR-mediated late phase of these IPSPs. Consequently, disruption of the GBR/HCN complex in KCTD16-/- mice leads to prolonged optogenetic inhibition of DAVTA neuron firing. KCTD16-/- mice exhibit increased anxiety-like behavior in response to stress - a behavior replicated by CRISPR/Cas9-mediated KCTD16 ablation in DAVTA neurons or by intra-VTA infusion of an HCN antagonist in wild-type mice. Our findings support that the retention of HCN channels at GABAergic synapses by GBRs in DAVTA neurons provides a negative feedback mechanism that restricts IPSP duration and mitigates the development of anxiety.
- MeSH
- Dopaminergic Neurons * metabolism MeSH
- Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels * metabolism genetics MeSH
- Inhibitory Postsynaptic Potentials physiology drug effects MeSH
- Mice, Inbred C57BL MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Receptors, GABA-B * metabolism MeSH
- Ventral Tegmental Area * metabolism MeSH
- Anxiety * metabolism MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: The choroid plexus (ChP) is the secretory epithelial structure located in the brain ventricles. Choroid plexus tumors (CPTs) are rare neoplasms predominantly occurring in young patients with intensified malignancy in children. CPT treatment is hindered by insufficient knowledge of tumor pathology and the limited availability of valid models. METHODS: Genomic and transcriptomic data from CPT patients were analyzed to identify the putative pathological pathway. Cellular and molecular techniques were employed to validate bioinformatic results in CPT patient samples. Pharmacologic inhibition of Wnt/β-catenin signaling was assessed in CPT cells. Cell-based assays of ChP cell lines were performed following CRISPR-Cas9-derived knockout and overexpression of Wnt/β-catenin pathway genes. A 3D CPT model was generated through CRISPR-Cas9-derived knockout of APC. RESULTS: We discovered that Wnt/β-catenin signaling is activated in human CPTs, likely as a consequence of large-scale chromosomal instability events of the CPT genomes. We demonstrated that CPT-derived cells depend on autocrine Wnt/β-catenin signaling for survival. Constitutive Wnt/β-catenin pathway activation, either through knockout of the negative regulator APC or overexpression of the ligand WNT3A, induced tumorigenic properties in ChP 2D in vitro models. Increased activation of the Wnt/β-catenin pathway in ChP organoids, through treatment with a potent GSK3β inhibitor, reduced the differentiation of mature ChP epithelial cells. Remarkably, the depletion of APC was sufficient to induce the oncogenic transformation of ChP organoids. CONCLUSIONS: Our research identifies Wnt/β-catenin signaling as a critical driver of CPT tumorigenesis and provides the first 3D in vitro model for future pathological and therapeutic studies of CPT.
- MeSH
- beta Catenin metabolism genetics MeSH
- Carcinogenesis metabolism MeSH
- Humans MeSH
- Tumor Cells, Cultured MeSH
- Choroid Plexus Neoplasms * pathology metabolism genetics MeSH
- Choroid Plexus metabolism pathology MeSH
- Cell Proliferation MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Wnt Signaling Pathway * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The lateral line system enables fishes and aquatic-stage amphibians to detect local water movement via mechanosensory hair cells in neuromasts, and many species to detect weak electric fields via electroreceptors (modified hair cells) in ampullary organs. Both neuromasts and ampullary organs develop from lateral line placodes, but the molecular mechanisms underpinning ampullary organ formation are understudied relative to neuromasts. This is because the ancestral lineages of zebrafish (teleosts) and Xenopus (frogs) independently lost electroreception. We identified Bmp5 as a promising candidate via differential RNA-seq in an electroreceptive ray-finned fish, the Mississippi paddlefish (Polyodon spathula; Modrell et al., 2017, eLife 6: e24197). In an experimentally tractable relative, the sterlet sturgeon (Acipenser ruthenus), we found that Bmp5 and four other Bmp pathway genes are expressed in the developing lateral line, and that Bmp signalling is active. Furthermore, CRISPR/Cas9-mediated mutagenesis targeting Bmp5 in G0-injected sterlet embryos resulted in fewer ampullary organs. Conversely, when Bmp signalling was inhibited by DMH1 treatment shortly before the formation of ampullary organ primordia, supernumerary ampullary organs developed. These data suggest that Bmp5 promotes ampullary organ development, whereas Bmp signalling via another ligand(s) prevents their overproduction. Taken together, this demonstrates opposing roles for Bmp signalling during ampullary organ formation.
- MeSH
- Bone Morphogenetic Proteins * metabolism genetics MeSH
- Lateral Line System * embryology metabolism MeSH
- Fish Proteins metabolism genetics MeSH
- Fishes genetics MeSH
- Signal Transduction * MeSH
- Gene Expression Regulation, Developmental MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
The adaptive immune response critically hinges on the functionality of T cell receptors, governed by complex molecular mechanisms, including ubiquitination. In this study, we delved into the role of in T cell immunity, focusing on T cell-B cell conjugate formation and T cell activation. Using a CRISPR-Cas9 screening approach targeting deubiquitinases genes in Jurkat T cells, we identified BAP1 as a key positive regulator of T cell-B cell conjugate formation. Subsequent investigations into BAP1 knockout cells revealed impaired T cell activation, evidenced by decreased MAPK and NF-kB signaling pathways and reduced CD69 expression upon T cell receptor stimulation. Flow cytometry and qPCR analyses demonstrated that BAP1 deficiency leads to decreased surface expression of T cell receptor complex components and reduced mRNA levels of the co-stimulatory molecule CD28. Notably, the observed phenotypes associated with BAP1 knockout are specific to T cells and fully dependent on BAP1 catalytic activity. In-depth RNA-seq and mass spectrometry analyses further revealed that BAP1 deficiency induces broad mRNA and protein expression changes. Overall, our findings elucidate the vital role of BAP1 in T cell biology, especially in T cell-B cell conjugate formation and T cell activation, offering new insights and directions for future research in immune regulation.
- MeSH
- Lymphocyte Activation * immunology MeSH
- B-Lymphocytes * immunology metabolism MeSH
- Jurkat Cells MeSH
- Humans MeSH
- Tumor Suppressor Proteins * metabolism genetics MeSH
- Receptors, Antigen, T-Cell * metabolism MeSH
- Signal Transduction MeSH
- T-Lymphocytes * immunology metabolism MeSH
- Ubiquitin Thiolesterase * genetics metabolism deficiency MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Bordetella pertussis is the causative agent of whooping cough in humans, a disease that has recently experienced a resurgence. In contrast, Bordetella bronchiseptica infects the respiratory tract of various mammalian species, causing a range of symptoms from asymptomatic chronic carriage to acute illness. Both pathogens utilize type III secretion system (T3SS) to deliver the effector protein BteA into host cells. Once injected, BteA triggers a cascade of events leading to caspase 1-independent necrosis through a mechanism that remains incompletely understood. We demonstrate that BteA-induced cell death is characterized by the fragmentation of the cellular endoplasmic reticulum and mitochondria, the formation of necrotic balloon-like protrusions, and plasma membrane permeabilization. Importantly, genome-wide CRISPR-Cas9 screen targeting 19,050 genes failed to identify any host factors required for BteA cytotoxicity, suggesting that BteA does not require a single nonessential host factor for its cytotoxicity. We further reveal that BteA triggers a rapid and sustained influx of calcium ions, which is associated with organelle fragmentation and plasma membrane permeabilization. The sustained elevation of cytosolic Ca2+ levels results in mitochondrial calcium overload, mitochondrial swelling, cristolysis, and loss of mitochondrial membrane potential. Inhibition of calcium channels with 2-APB delays both the Ca2+ influx and BteA-induced cell death. Our findings indicate that BteA exploits essential host processes and/or redundant pathways to disrupt calcium homeostasis and mitochondrial function, ultimately leading to host cell death.IMPORTANCEThe respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica exhibit cytotoxicity toward a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of B. bronchiseptica is associated with enhanced expression of T3SS and BteA. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.
- MeSH
- Bacterial Proteins * metabolism genetics MeSH
- Bordetella bronchiseptica genetics metabolism drug effects MeSH
- Bordetella pertussis genetics pathogenicity metabolism drug effects MeSH
- Cell Death * drug effects MeSH
- Endoplasmic Reticulum metabolism drug effects MeSH
- Homeostasis * MeSH
- Host-Pathogen Interactions MeSH
- Humans MeSH
- Mitochondria metabolism drug effects MeSH
- Type III Secretion Systems metabolism genetics MeSH
- Calcium * metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.
