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1.
Hum Mol Genet ; 29(1): 31-48, 2020 01 01.
Article in English | MEDLINE | ID: mdl-31625572

ABSTRACT

Polycystic kidney disease (PKD) results in the formation of renal cysts that can impair function leading to renal failure. DNA damage accumulates in renal epithelial cells in PKD, but the molecular mechanisms are unclear and are investigated here. Phosphoinositide 3-kinase (PI3K)/AKT signaling activates mammalian target of rapamycin complex 1 (mTORC1) and hyperactivation of mTORC1 is a common event in PKD; however, mTORC1 inhibitors have yielded disappointing results in clinical trials. Here, we demonstrate AKT and mTORC1 hyperactivation in two representative murine PKD models (renal epithelial-specific Inpp5e knockout and collecting duct-specific Pkd1 deletion) and identify a downstream signaling network that contributes to DNA damage accumulation. Inpp5e- and Pkd1-null renal epithelial cells showed DNA damage including double-stranded DNA breaks associated with increased replication fork numbers, multinucleation and centrosome amplification. mTORC1 activated CAD, which promotes de novo pyrimidine synthesis, to sustain cell proliferation. AKT, but not mTORC1, inhibited the DNA repair/replication fork origin firing regulator TOPBP1, which impacts on DNA damage and cell proliferation. Notably, Inpp5e- and Pkd1-null renal epithelial cell spheroid formation defects were rescued by AKT inhibition. These data reveal that AKT hyperactivation contributes to DNA damage accumulation in multiple forms of PKD and cooperates with mTORC1 to promote cell proliferation. Hyperactivation of AKT may play a causal role in PKD by regulating DNA damage and cell proliferation, independent of mTORC1, and AKT inhibition may be a novel therapeutic approach for PKD.


Subject(s)
DNA Damage/physiology , Polycystic Kidney Diseases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Cell Proliferation/genetics , Cell Proliferation/physiology , Cells, Cultured , DNA Damage/genetics , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Immunohistochemistry , Male , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Polycystic Kidney Diseases/genetics , Proto-Oncogene Proteins c-akt/genetics , Signal Transduction/genetics , Signal Transduction/physiology
2.
Hum Mol Genet ; 25(11): 2295-2313, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27056978

ABSTRACT

Polycystic kidney disease (PKD) is a common cause of renal failure with few effective treatments. INPP5E is an inositol polyphosphate 5-phosphatase that dephosphorylates phosphoinositide 3-kinase (PI3K)-generated PI(3,4,5)P3 and is mutated in ciliopathy syndromes. Germline Inpp5e deletion is embryonically lethal, attributed to cilia stability defects, and is associated with polycystic kidneys. However, the molecular mechanisms responsible for PKD development upon Inpp5e loss remain unknown. Here, we show conditional inactivation of Inpp5e in mouse kidney epithelium results in severe PKD and renal failure, associated with a partial reduction in cilia number and hyperactivation of PI3K/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment with an mTORC1 inhibitor improved kidney morphology and function, but did not affect cilia number or length. Therefore, we identify Inpp5e as an essential inhibitor of the PI3K/Akt/mTORC1 signaling axis in renal epithelial cells, and demonstrate a critical role for Inpp5e-dependent mTORC1 regulation in PKD suppression.


Subject(s)
Kidney/metabolism , Multiprotein Complexes/genetics , Phosphoric Monoester Hydrolases/genetics , Polycystic Kidney Diseases/genetics , TOR Serine-Threonine Kinases/genetics , Animals , Ciliopathies/drug therapy , Ciliopathies/genetics , Ciliopathies/pathology , Disease Models, Animal , Elafin/genetics , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Germ-Line Mutation , Humans , Kidney/drug effects , Kidney/pathology , Mechanistic Target of Rapamycin Complex 1 , Mice , Multiprotein Complexes/antagonists & inhibitors , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Polycystic Kidney Diseases/drug therapy , Polycystic Kidney Diseases/pathology , Proto-Oncogene Proteins c-akt/genetics , Sequence Deletion , Signal Transduction/drug effects , Sirolimus/administration & dosage , TOR Serine-Threonine Kinases/antagonists & inhibitors
3.
Blood ; 117(20): 5362-71, 2011 May 19.
Article in English | MEDLINE | ID: mdl-21421839

ABSTRACT

In a recessive ENU mutagenesis screen for embryonic lethality, we identified a mouse pedigree with a missense mutation of SHIP1 (SHIP1(el20)) leading to an amino acid substitution I641T in the inositol-5'-phosphatase domain that represses phosphatidylinositol-3-kinase signaling. Despite detectable expression of functional SHIP1 protein, the phenotype of homozygous SHIP1(el20/el20) mice was more severe than gene-targeted SHIP1-null (SHIP1(-/-)) mice. Compared with age-matched SHIP1(-/-) mice, 5-week-old SHIP1(el20/el20) mice had increased myeloid cells, serum IL-6 levels, marked reductions in lymphoid cells, and died by 7 weeks of age with infiltration of the lungs by activated macrophages. Bone marrow transplantation demonstrated that these defects were hematopoietic-cell-autonomous. We show that the el20 mutation reduces expression in SHIP1(el20/el20) macrophages of both SHIP1 and s-SHIP, an isoform of SHIP1 generated by an internal promoter. In contrast, SHIP1(-/-) macrophages express normal levels of s-SHIP. Compound heterozygous mice (SHIP1(-/el20)) had the same phenotype as SHIP1(-/-) mice, thus providing genetic proof that the more severe phenotype of SHIP1(el20/el20) mice is probably the result of concomitant loss of SHIP1 and s-SHIP. Our results suggest that s-SHIP synergizes with SHIP1 for suppression of macrophage activation, thus providing the first evidence for a role of s-SHIP in adult hematopoiesis.


Subject(s)
Macrophage Activation/genetics , Macrophage Activation/physiology , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/physiology , Amino Acid Substitution , Animals , Base Sequence , Bone Marrow Transplantation , DNA Primers/genetics , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Ethylnitrosourea , Female , Genes, Recessive , Hematopoiesis/genetics , Hematopoiesis/physiology , Homozygote , Inositol Polyphosphate 5-Phosphatases , Interleukin-6/biosynthesis , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Mutagenesis , Mutation, Missense , Phenotype , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoric Monoester Hydrolases/deficiency , Protein Isoforms/deficiency , Protein Isoforms/genetics , Protein Isoforms/physiology , Signal Transduction
4.
Subcell Biochem ; 58: 215-79, 2012.
Article in English | MEDLINE | ID: mdl-22403078

ABSTRACT

Phosphoinositide phosphatases comprise several large enzyme families with over 35 mammalian enzymes identified to date that degrade many phosphoinositide signals. Growth factor or insulin stimulation activates the phosphoinositide 3-kinase that phosphorylates phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] to form phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)], which is rapidly dephosphorylated either by PTEN (phosphatase and tensin homologue deleted on chromosome 10) to PtdIns(4,5)P(2), or by the 5-phosphatases (inositol polyphosphate 5-phosphatases), generating PtdIns(3,4)P(2). 5-phosphatases also hydrolyze PtdIns(4,5)P(2) forming PtdIns(4)P. Ten mammalian 5-phosphatases have been identified, which regulate hematopoietic cell proliferation, synaptic vesicle recycling, insulin signaling, and embryonic development. Two 5-phosphatase genes, OCRL and INPP5E are mutated in Lowe and Joubert syndrome respectively. SHIP [SH2 (Src homology 2)-domain inositol phosphatase] 2, and SKIP (skeletal muscle- and kidney-enriched inositol phosphatase) negatively regulate insulin signaling and glucose homeostasis. SHIP2 polymorphisms are associated with a predisposition to insulin resistance. SHIP1 controls hematopoietic cell proliferation and is mutated in some leukemias. The inositol polyphosphate 4-phosphatases, INPP4A and INPP4B degrade PtdIns(3,4)P(2) to PtdIns(3)P and regulate neuroexcitatory cell death, or act as a tumor suppressor in breast cancer respectively. The Sac phosphatases degrade multiple phosphoinositides, such as PtdIns(3)P, PtdIns(4)P, PtdIns(5)P and PtdIns(3,5)P(2) to form PtdIns. Mutation in the Sac phosphatase gene, FIG4, leads to a degenerative neuropathy. Therefore the phosphatases, like the lipid kinases, play major roles in regulating cellular functions and their mutation or altered expression leads to many human diseases.


Subject(s)
Breast Neoplasms/enzymology , Leukemia/enzymology , Oculocerebrorenal Syndrome/enzymology , PTEN Phosphohydrolase/metabolism , Phosphoric Monoester Hydrolases/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Diglycerides/metabolism , Female , Gene Expression Regulation , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Inositol Polyphosphate 5-Phosphatases , Leukemia/genetics , Leukemia/pathology , Oculocerebrorenal Syndrome/genetics , Oculocerebrorenal Syndrome/pathology , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoric Monoester Hydrolases/genetics , Second Messenger Systems
5.
Subcell Biochem ; 58: 281-336, 2012.
Article in English | MEDLINE | ID: mdl-22403079

ABSTRACT

Two classes of lipid phosphatases selectively dephosphorylate the 3 position of the inositol ring of phosphoinositide signaling molecules: the PTEN and the Myotubularin families. PTEN dephosphorylates PtdIns(3,4,5)P(3), acting in direct opposition to the Class I PI3K enzymes in the regulation of cell growth, proliferation and polarity and is an important tumor suppressor. Although there are several PTEN-related proteins encoded by the human genome, none of these appear to fulfill the same functions. In contrast, the Myotubularins dephosphorylate both PtdIns(3)P and PtdIns(3,5)P(2), making them antagonists of the Class II and Class III PI 3-kinases and regulators of membrane traffic. Both phosphatase groups were originally identified through their causal mutation in human disease. Mutations in specific myotubularins result in myotubular myopathy and Charcot-Marie-Tooth peripheral neuropathy; and loss of PTEN function through mutation and other mechanisms is evident in as many as a third of all human tumors. This chapter will discuss these two classes of phosphatases, covering what is known about their biochemistry, their functions at the cellular and whole body level and their influence on human health.


Subject(s)
Charcot-Marie-Tooth Disease/enzymology , Myopathies, Structural, Congenital/enzymology , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Second Messenger Systems , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/pathology , Gene Expression Regulation , Humans , Hydrolysis , Mutation , Myopathies, Structural, Congenital/genetics , Myopathies, Structural, Congenital/pathology , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol Phosphates/metabolism , Phosphorylation , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Substrate Specificity
6.
Sci Adv ; 9(13): eadd6911, 2023 03 31.
Article in English | MEDLINE | ID: mdl-37000875

ABSTRACT

Dynamic positioning of endothelial tip and stalk cells, via the interplay between VEGFR2 and NOTCH signaling, is essential for angiogenesis. VEGFR2 activates PI3K, which phosphorylates PI(4,5)P2 to PI(3,4,5)P3, activating AKT; however, PI3K/AKT does not direct tip cell specification. We report that PI(4,5)P2 hydrolysis by the phosphoinositide-5-phosphatase, INPP5K, contributes to angiogenesis. INPP5K ablation disrupted tip cell specification and impaired embryonic angiogenesis associated with enhanced DLL4/NOTCH signaling. INPP5K degraded a pool of PI(4,5)P2 generated by PIP5K1C phosphorylation of PI(4)P in endothelial cells. INPP5K ablation increased PI(4,5)P2, thereby releasing ß-catenin from the plasma membrane, and concurrently increased PI(3,4,5)P3-dependent AKT activation, conditions that licensed DLL4/NOTCH transcription. Suppression of PI(4,5)P2 in INPP5K-siRNA cells by PIP5K1C-siRNA, restored ß-catenin membrane localization and normalized AKT signaling. Pharmacological NOTCH or AKT inhibition in vivo or genetic ß-catenin attenuation rescued angiogenesis defects in INPP5K-null mice. Therefore, PI(4,5)P2 is critical for ß-catenin/DLL4/NOTCH signaling, which governs tip cell specification during angiogenesis.


Subject(s)
Adaptor Proteins, Signal Transducing , beta Catenin , Animals , Mice , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , beta Catenin/genetics , beta Catenin/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Endothelial Cells/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Neovascularization, Physiologic/genetics , Membrane Proteins/metabolism , RNA, Small Interfering/metabolism , Receptors, Notch/genetics , Receptors, Notch/metabolism
7.
Br J Radiol ; 94(1127): 20210620, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34357789

ABSTRACT

Recent trends in medical decision-making have moved from paternalistic doctor-patient relations to shared decision-making. Informed consent is fundamental to this process and to ensuring patients' ongoing trust in the health-care profession. It cannot be assumed that patients consent to the risk associated with medical exposures, unless they have been provided with the information to make that decision. This position is supported by both the legal and ethical framework around Radiation Protection detailed in this commentary.


Subject(s)
Clinical Decision-Making/ethics , Clinical Decision-Making/methods , Informed Consent/ethics , Physician-Patient Relations/ethics , Radiation Exposure/ethics , Radiology/ethics , Humans
8.
Front Cell Dev Biol ; 9: 634649, 2021.
Article in English | MEDLINE | ID: mdl-33996795

ABSTRACT

Primary cilia are evolutionary conserved microtubule-based organelles that protrude from the surface of most mammalian cells. Phosphoinositides (PI) are membrane-associated signaling lipids that regulate numerous cellular events via the recruitment of lipid-binding effectors. The temporal and spatial membrane distribution of phosphoinositides is regulated by phosphoinositide kinases and phosphatases. Recently phosphoinositide signaling and turnover has been observed at primary cilia. However, the precise localization of the phosphoinositides to specific ciliary subdomains remains undefined. Here we use superresolution microscopy (2D stimulated emission depletion microscopy) to map phosphoinositide distribution at the cilia transition zone. PI(3,4,5)P3 and PI(4,5)P2 localized to distinct subregions of the transition zone in a ring-shape at the inner transition zone membrane. Interestingly, the PI(3,4,5)P3 subdomain was more distal within the transition zone relative to PtdIns(4,5)P2. The phosphoinositide effector kinase pAKT(S473) localized in close proximity to these phosphoinositides. The inositol polyphosphate 5-phosphatase, INPP5E, degrades transition zone phosphoinositides, however, studies of fixed cells have reported recombinant INPP5E localizes to the ciliary axoneme, distant from its substrates. Notably, here using live cell imaging and optimized fixation/permeabilization protocols INPP5E was found concentrated at the cilia base, in a distribution characteristic of the transition zone in a ring-shaped domain of similar dimensions to the phosphoinositides. Collectively, this superresolution map places the phosphoinositides in situ with the transition zone proteins and reveals that INPP5E also likely localizes to a subdomain of the transition zone membrane, where it is optimally situated to control local phosphoinositide metabolism.

9.
Dev Cell ; 54(1): 75-91.e7, 2020 07 06.
Article in English | MEDLINE | ID: mdl-32485139

ABSTRACT

Epithelia are active materials where mechanical tension governs morphogenesis and homeostasis. But how that tension is regulated remains incompletely understood. We now report that caveolae control epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) increased steady-state tensile stresses in epithelial monolayers. As a result, loss of CAV1 in the epithelial cells surrounding oncogene-expressing cells prevented their apical extrusion. Epithelial tension in CAV1-depleted monolayers was increased by cortical contractility at adherens junctions. This reflected a signaling pathway, where elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P2) recruited the formin, FMNL2, to promote F-actin bundling. Steady-state monolayer tension and oncogenic extrusion were restored to CAV1-depleted monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P2, or disabling the interaction between FMNL2 and PtdIns(4,5)P2. Thus, caveolae can regulate active mechanical tension for epithelial homeostasis by controlling lipid signaling to the actin cytoskeleton.


Subject(s)
Caveolae/metabolism , Epithelial Cells/metabolism , Oncogene Proteins/metabolism , Actin Cytoskeleton/metabolism , Animals , Caco-2 Cells , Caveolin 1/metabolism , Epithelial Cells/ultrastructure , Formins/metabolism , HEK293 Cells , Humans , Male , Mice , Oncogene Proteins/genetics , Phosphatidylinositol 4,5-Diphosphate/metabolism , Stress, Mechanical
10.
Article in English | MEDLINE | ID: mdl-29468142

ABSTRACT

The Gram-negative bacterium, Helicobacter pylori, causes chronic gastritis, peptic ulcers, and gastric cancer in humans. Although the gastric epithelium is the primary site of H. pylori colonization, H. pylori can gain access to deeper tissues. Concurring with this notion, H. pylori has been found in the vicinity of endothelial cells in gastric submucosa. Endothelial cells play crucial roles in innate immune response, wound healing and tumorigenesis. This study examines the molecular mechanisms by which H. pylori interacts with and triggers inflammatory responses in endothelial cells. We observed that H. pylori infection of primary human endothelial cells stimulated secretion of the key inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8). In particular, IL-8, a potent chemokine and angiogenic factor, was secreted by H. pylori-infected endothelial cells to levels ~10- to 20-fold higher than that typically observed in H. pylori-infected gastric epithelial cells. These inflammatory responses were triggered by the H. pylori type IV secretion system (T4SS) and the T4SS-associated adhesin CagL, but not the translocation substrate CagA. Moreover, in contrast to integrin α5ß1 playing an essential role in IL-8 induction by H. pylori upon infection of gastric epithelial cells, both integrin α5ß1 and integrin αvß3 were dispensable for IL-8 induction in H. pylori-infected endothelial cells. However, epidermal growth factor receptor (EGFR) is crucial for mediating the potent H. pylori-induced IL-8 response in endothelial cells. This study reveals a novel mechanism by which the H. pylori T4SS and its adhesin subunit, CagL, may contribute to H. pylori pathogenesis by stimulating the endothelial innate immune responses, while highlighting EGFR as a potential therapeutic target for controlling H. pylori-induced inflammation.


Subject(s)
Bacterial Proteins/metabolism , Helicobacter Infections/microbiology , Helicobacter pylori/physiology , Host-Pathogen Interactions , Type IV Secretion Systems/metabolism , Adhesins, Bacterial/metabolism , Biomarkers , Cell Line , Cytokines/metabolism , Epithelial Cells/metabolism , Epithelial Cells/microbiology , ErbB Receptors/metabolism , Helicobacter Infections/metabolism , Humans , Inflammation Mediators/metabolism , NF-kappa B/metabolism , Phosphorylation , Protein Transport
11.
Cancer Cell ; 28(2): 155-69, 2015 Aug 10.
Article in English | MEDLINE | ID: mdl-26267533

ABSTRACT

Metastasis is the major cause of breast cancer mortality. Phosphoinositide 3-kinase (PI3K) generated PtdIns(3,4,5)P3 activates AKT, which promotes breast cancer cell proliferation and regulates migration. To date, none of the inositol polyphosphate 5-phosphatases that inhibit PI3K/AKT signaling have been reported as tumor suppressors in breast cancer. Here, we show depletion of the inositol polyphosphate 5-phosphatase PIPP (INPP5J) increases breast cancer cell transformation, but reduces cell migration and invasion. Pipp ablation accelerates oncogene-driven breast cancer tumor growth in vivo, but paradoxically reduces metastasis by regulating AKT1-dependent tumor cell migration. PIPP mRNA expression is reduced in human ER-negative breast cancers associated with reduced long-term outcome. Collectively, our findings identify PIPP as a suppressor of oncogenic PI3K/AKT signaling in breast cancer.


Subject(s)
Breast Neoplasms/genetics , Cell Proliferation/genetics , Phosphoric Monoester Hydrolases/genetics , Proto-Oncogene Proteins c-akt/genetics , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/genetics , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Inositol Polyphosphate 5-Phosphatases , Kaplan-Meier Estimate , Mice, Inbred BALB C , Mice, Knockout , Mice, Nude , Neoplasm Metastasis , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoric Monoester Hydrolases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Tumor Burden/genetics , Xenograft Model Antitumor Assays/methods
12.
IUBMB Life ; 58(8): 451-6, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16916781

ABSTRACT

Phosphoinositide signaling molecules control cellular growth, proliferation and differentiation, intracellular vesicle trafficking, and cytoskeletal rearrangement. The inositol polyphosphate 5-phosphatase family remove the D-5 position phosphate from PtdIns(3,4,5)P3, PtdIns(4,5)P2 and PtdIns(3,5)P2 forming PtdIns(3,4)P2, PtdIns(4)P and PtdIns(3)P respectively. This enzyme family, comprising ten mammalian members, exhibit seemingly non-redundant functions including the regulation of synaptic vesicle recycling, hematopoietic cell function and insulin signaling. Here we highlight recently established insights into the functions of two well characterized 5-phosphatases OCRL and SHIP2, which have been the subject of extensive functional studies, and the characterization of recently identified members, SKIP and PIPP, in order to highlight the diverse and complex functions of this enzyme family.


Subject(s)
Phosphatidylinositols/metabolism , Phosphoric Monoester Hydrolases/metabolism , Signal Transduction , Animals , Humans , Inositol Polyphosphate 5-Phosphatases , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Biological , Oculocerebrorenal Syndrome/enzymology , Oculocerebrorenal Syndrome/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/genetics , Protein Structure, Tertiary
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