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: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: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: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: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:Myeloid lineage cells (MLCs) such as macrophages are known to play a key role in postischemic neovascularization. However, the role of MLC-derived reactive oxygen species in this process and their specific chemical identity remain unknown.Transgenic mice with MLC-specific overexpression of catalase (Tg(Cat-MLC) mice) were created on a C57BL/6 background. Macrophage catalase activity was increased 3.4-fold compared with wild-type mice. After femoral artery ligation, laser Doppler perfusion imaging revealed impaired perfusion recovery in Tg(Cat-MLC) mice. This was associated with fewer collateral vessels, as assessed by microcomputed tomography angiography, and decreased capillary density. Impaired functional recovery of the ischemic limb was also evidenced by a 50% reduction in spontaneous running activity. The deficient neovascularization was associated with a blunted inflammatory response, characterized by decreased macrophage infiltration of ischemic tissues, and lower mRNA levels of inflammatory markers, such as tumor necrosis factor-?, osteopontin, and matrix mettaloproteinase-9. In vitro macrophage migration was impaired in Tg(Cat-MLC) mice, suggesting a role for H(2)O(2) in regulating the ability of macrophages to infiltrate ischemic tissues.MLC-derived H(2)O(2) plays a key role in promoting neovascularization in response to ischemia and is a necessary factor for the development of ischemia-induced inflammation.
Project description:TERT, the protein component of telomerase, serves to maintain telomere function through the de novo addition of telomere repeats to chromosome ends, and is reactivated in 90% of human cancers. In normal tissues, TERT is expressed in stem cells and in progenitor cells, but its role in these compartments is not fully understood. Here we show that conditional transgenic induction of TERT in mouse skin epithelium causes a rapid transition from telogen (the resting phase of the hair follicle cycle) to anagen (the active phase), thereby facilitating robust hair growth. TERT overexpression promotes this developmental transition by causing proliferation of quiescent, multipotent stem cells in the hair follicle bulge region. This new function for TERT does not require the telomerase RNA component, which encodes the template for telomere addition, and therefore operates through a mechanism independent of its activity in synthesizing telomere repeats. These data indicate that, in addition to its established role in extending telomeres, TERT can promote proliferation of resting stem cells through a non-canonical pathway.
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.
Project description:Aneuploidy is associated with a variety of diseases such as cancer and microcephaly. Although many studies have addressed the consequences of a non-euploid genome in cells, little is known about their overall consequences in tissue and organism development. Here we use two different mutant conditions to address the consequences of aneuploidy during tissue development and homeostasis in Drosophila. We show that aneuploidy causes brain size reduction due to a decrease in the number of proliferative neural stem cells (NSCs), but not through apoptosis. Instead, aneuploid NSCs present an extended G1 phase, which leads to cell cycle exit and premature differentiation. Moreover, we show that this response to aneuploidy is also present in adult intestinal stem cells but not in the wing disc. Our work highlights a neural and intestine stem cell-specific response to aneuploidy, which prevents their proliferation and expansion.