Project description:Vasopressin-regulated expression and insertion of aquaporin-2 channels in the luminal membrane of renal principal cells is essential for urine concentration. Lithium affects urine concentrating ability, and approximately 20% of patients treated with lithium develop nephrogenic diabetes insipidus (NDI), a disorder characterized by polyuria and polydipsia. Lithium-induced NDI is caused by aquaporin-2 downregulation and a reduced ratio of principal/intercalated cells, yet lithium induces principal cell proliferation. Here, we studied how lithium-induced principal cell proliferation can lead to a reduced ratio of principal/intercalated cells using two-dimensional and three-dimensional polarized cultures of mouse renal collecting duct cells and mice treated with clinically relevant lithium concentrations. DNA image cytometry and immunoblotting revealed that lithium initiated proliferation of mouse renal collecting duct cells but also increased the G2/S ratio, indicating G2/M phase arrest. In mice, treatment with lithium for 4, 7, 10, or 13 days led to features of NDI and an increase in the number of principal cells expressing PCNA in the papilla. Remarkably, 30%-40% of the PCNA-positive principal cells also expressed pHistone-H3, a late G2/M phase marker detected in approximately 20% of cells during undisturbed proliferation. Our data reveal that lithium treatment initiates proliferation of renal principal cells but that a significant percentage of these cells are arrested in the late G2 phase, which explains the reduced principal/intercalated cell ratio and may identify the molecular pathway underlying the development of lithium-induced renal fibrosis.
Project description:Primary cilia are hair-like projections that protrude from most mammalian cells and mediate various extracellular signaling pathways. Pancreatic ductal adenocarcinoma (PDAC) cells are known to lose their primary cilia, but the relevance of this phenomenon remains unclear. In this study, we generated PDAC-originated Panc1 cells devoid of primary cilia by mutating a centriolar protein, centrosomal protein 164 (CEP164), which is required for ciliogenesis. CEP164 depletion enhanced the clonogenicity of Panc1 cells, along with chemically induced elimination of primary cilia, suggesting that a lack of these organelles promotes PDAC cells proliferation. In addition, the loss of CEP164 altered the cell cycle progression irrespective of absence of primary cilia. We found that CEP164 was co-localized with the GLI2 transcription factor at the mother centriole and controlled its activation, thus inducing Cyclin D-CDK6 expression. Furthermore, CEP164-mutated Panc1 cells were significantly tolerant to KRAS depletion-dependent growth inhibition. This study suggests that CEP164 deficiency is advantageous for PDAC cells proliferation due to not only lack of ciliation but also cilia-independent GLI2-Cyclin D/CDK6 activation, and that CEP164 is a potential therapeutic target for PDAC.
Project description:Soluble decorin affects the biology of several receptor tyrosine kinases by triggering receptor internalization and degradation. We found that decorin induced paternally expressed gene 3 (Peg3), an imprinted tumor suppressor gene, and that Peg3 relocated into autophagosomes labeled by Beclin 1 and microtubule-associated light chain 3. Decorin evoked Peg3-dependent autophagy in both microvascular and macrovascular endothelial cells leading to suppression of angiogenesis. Peg3 coimmunoprecipitated with Beclin 1 and LC3 and was required for maintaining basal levels of Beclin 1. Decorin, via Peg3, induced transcription of Beclin 1 and microtubule-associated protein 1 light chain 3 alpha genes, thereby leading to a protracted autophagic program. Mechanistically, decorin interacted with VEGF receptor 2 (VEGFR2) in a region overlapping with its natural ligand VEGFA, and VEGFR2 was required for decorin-evoked Beclin 1 and microtubule-associated protein 1 light chain 3 alpha expression as well as for Peg3 induction in endothelial cells. Moreover, decorin induced VEGFR2-dependent mitochondrial fragmentation and loss of mitochondrial membrane potential. Thus, we have unveiled a mechanism for a secreted proteoglycan in inducing Peg3, a master regulator of macroautophagy in endothelial cells.
Project description:The developmentally regulated GTP binding protein 2 (DRG2) is involved in the control of cell growth and differentiation. Here, we demonstrate that DRG2 regulates microtubule dynamics in HeLa cells. Analysis of live imaging of the plus-ends of microtubules with EB1-EGFP showed that DRG2 deficiency (shDRG2) significantly reduced the growth rate of HeLa cells. Depletion of DRG2 increased 'slow and long-lived' subpopulations, but decreased 'fast and short-lived' subpopulations of microtubules. Microtubule polymerization inhibitor exhibited a reduced response in shDRG2 cells. Using immunoprecipitation, we show that DRG2 interacts with tau, which regulates microtubule polymerization. Collectively, these data demonstrate that DRG2 may aid in affecting microtubule dynamics in HeLa cells.
Project description:Birt-Hogg-Dubé syndrome (BHDS), an autosomal dominant inheritance disease caused by folliculin (FLCN) mutations, is associated with lung cysts and spontaneous pneumothorax. The possibility of FLCN haploinsufficiency in pleural mesothelial cells (PMCs) contributing to development of pneumothorax has not yet been clarified. Electron microscopy revealed exposed intercellular boundaries between PMCs on visceral pleura and decreased electron density around the adherens junctions in BHDS. To characterize cellular function of PMCs in BHDS patients (BHDS-PMCs), during surgery for pneumothorax, we established the flow cytometry-based methods of isolating high-purity PMCs from pleural lavage fluid. BHDS-PMCs showed impaired cell attachment and a significant decrease in proliferation and migration, but a significant increase in apoptosis compared with PMCs from primary spontaneous pneumothorax (PSP) patients (PSP-PMCs). Microarray analysis using isolated PMCs revealed a significant alteration in the expression of genes belonging to Gene Ontology terms "cell-cell adhesion junction" and "cell adhesion molecule binding". Gene set enrichment analysis demonstrated that CDH1, encoding E-cadherin, was identified in the down-regulated leading edge of a plot in BHDS-PMCs. AMPK and LKB1 activation were significantly impaired in BHDS-PMCs compared with PSP-PMCs. Our findings indicate that FLCN haploinsufficiency may affect the E-cadherin-LKB1-AMPK axis and lead to abnormal cellular function in BHDS-PMCs.
Project description:In Drosophila, normal and transformed cells compete with each other for survival in a process called cell competition. However, it is not known whether comparable phenomena also occur in mammals. Scribble is a tumor suppressor protein in Drosophila and mammals. In this study we examine the interface between normal and Scribble-knockdown epithelial cells using Madin-Darby Canine Kidney (MDCK) cells expressing Scribble short hairpin RNA (shRNA) in a tetracycline-inducible manner. We observe that Scribble-knockdown cells undergo apoptosis and are apically extruded from the epithelium when surrounded by normal cells. Apoptosis does not occur when Scribble-knockdown cells are cultured alone, suggesting that the presence of surrounding normal cells induces the cell death. We also show that death of Scribble-knockdown cells occurs independently of apical extrusion. Finally, we demonstrate that apoptosis of Scribble-knockdown cells depends on activation of p38 mitogen-activated protein kinase (MAPK). This is the first demonstration that an oncogenic transformation within an epithelium induces cell competition in a mammalian cell culture system.
Project description:Enhancer of zeste homolog 2 (EZH2) is the catalytic core of polycomb repressive complex 2 (PRC2), which primarily methylates lysine 27 on histone H3 (H2K27me3), generating transcriptionally suppressed heterochromatin. Since EZH2 suppresses expression of genes involved in dentin formation, we examined the role of EZH2 in tooth development. Intriguingly, microCT analysis of teeth from mice with conditional Ezh2 knockout in uncommitted mesenchymal cells showed hyper-mineralization of enamel, which is produced by the epithelial-lineage cells, ameloblasts. Scanning electron microscopy analysis and nano-indentation of the incisor enamel from knockout mice revealed smaller inter-rod spaces and higher hardness compared to wild type enamel, respectively. Interestingly, expression of the calcium channel subunit gene, Orai2, was decreased compared to its competitor, Orai1, both in knockout mouse incisors and the ex vivo culture of ameloblasts with the surrounding tissues under EZH2 inhibition. Moreover, histological analysis of incisor from knockout mice showed decreased ameloblastin and expedited KLK4 expression in the ameloblasts. These observations suggest that EZH2 depletion in dental mesenchymal cells reduces enamel matrix formation and increases enamel protease activity from ameloblasts, resulting in enamel hyper-mineralization. This study demonstrates the significant role of the suppressive H3K27me3 mark for heterochromatin on enamel formation.
Project description:Osteoarthritis (OA) is characterised by an irreversible degeneration of articular cartilage. Here we show that the BMP-antagonist Gremlin 1 (Grem1) marks a bipotent chondrogenic and osteogenic progenitor cell population within the articular surface. Notably, these progenitors are depleted by injury-induced OA and increasing age. OA is also caused by ablation of Grem1 cells in mice. Transcriptomic and functional analysis in mice found that articular surface Grem1-lineage cells are dependent on Foxo1 and ablation of Foxo1 in Grem1-lineage cells caused OA. FGFR3 signalling was confirmed as a promising therapeutic pathway by administration of pathway activator, FGF18, resulting in Grem1-lineage chondrocyte progenitor cell proliferation, increased cartilage thickness and reduced OA. These findings suggest that OA, in part, is caused by mechanical, developmental or age-related attrition of Grem1 expressing articular cartilage progenitor cells. These cells, and the FGFR3 signalling pathway that sustains them, may be effective future targets for biological management of OA.
Project description:Most animal cells contain a centrosome, which comprises a pair of centrioles surrounded by an ordered pericentriolar matrix (PCM). Although the role of this organelle in organizing the mitotic spindle poles is well established, its precise contribution to cell division and cell survival remains a subject of debate. By genetically ablating key components of centriole biogenesis in chicken DT40 B cells, we generated multiple cell lines that lack centrioles. PCM components accumulated in acentriolar microtubule (MT)-organizing centers but failed to adopt a higher-order structure, as shown by three-dimensional structured illumination microscopy. Cells without centrioles exhibited both a delay in bipolar spindle assembly and a high rate of chromosomal instability. Collectively, our results expose a vital role for centrosomes in establishing a mitotic spindle geometry that facilitates correct kinetochore-MT attachments. We propose that centrosomes are essential in organisms in which rapid segregation of a large number of chromosomes needs to be attained with fidelity.
Project description:Patients with chronic hepatitis C virus (HCV) infection frequently develop systemic iron overload, which exacerbates morbidity. Nevertheless, iron inhibits HCV replication in cell culture models and thereby exerts antiviral activity. We hypothesized that the cellular iron status is crucial for the establishment of HCV infection. We show that HCV infection of permissive Huh7.5.1 hepatoma cells promotes an iron deficient phenotype. Thus, HCV leads to increased iron regulatory protein (IRP) activity, accumulation of IRP2 and suppression of transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) in the host. These data suggest that HCV regulates cellular iron levels to bypass iron-mediated inhibition in viral replication.