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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33771931

RESUMO

The lipid composition of the primary cilia membrane is emerging as a critical regulator of cilia formation, maintenance and function. Here, we show that conditional deletion of the phosphoinositide 5'-phosphatase gene Inpp5e, mutation of which is causative of Joubert syndrome, in terminally developed mouse olfactory sensory neurons (OSNs), leads to a dramatic remodeling of ciliary phospholipids that is accompanied by marked elongation of cilia. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], which is normally restricted to the proximal segment redistributed to the entire length of cilia in Inpp5e knockout mice with a reduction in phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] and elevation of phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] in the dendritic knob. The redistribution of phosphoinositides impaired odor adaptation, resulting in less efficient recovery and altered inactivation kinetics of the odor-evoked electrical response and the odor-induced elevation of cytoplasmic Ca2+. Gene replacement of Inpp5e through adenoviral expression restored the ciliary localization of PI(4,5)P2 and odor response kinetics in OSNs. Our findings support the role of phosphoinositides as a modulator of the odor response and in ciliary biology of native multi-ciliated OSNs.

2.
Front Immunol ; 12: 671756, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33953730

RESUMO

Neutrophils, the most abundant circulating leukocytes in humans have key roles in host defense and in the inflammatory response. Agonist-activated phosphoinositide 3-kinases (PI3Ks) are important regulators of many facets of neutrophil biology. PIP3 is subject to dephosphorylation by several 5' phosphatases, including SHIP family phosphatases, which convert the PI3K product and lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) into PI(3,4)P2, a lipid second messenger in its own right. In addition to the leukocyte restricted SHIP1, neutrophils express the ubiquitous SHIP2. This study analyzed mice and isolated neutrophils carrying a catalytically inactive SHIP2, identifying an important regulatory function in neutrophil chemotaxis and directionality in vitro and in neutrophil recruitment to sites of sterile inflammation in vivo, in the absence of major defects of any other neutrophil functions analyzed, including, phagocytosis and the formation of reactive oxygen species. Mechanistically, this is explained by a subtle effect on global 3-phosphorylated phosphoinositide species. This work identifies a non-redundant role for the hitherto overlooked SHIP2 in the regulation of neutrophils, and specifically, neutrophil chemotaxis/trafficking. It completes an emerging wider understanding of the complexity of PI3K signaling in the neutrophil, and the roles played by individual kinases and phosphatases within.

3.
Sci Signal ; 14(676)2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785611

RESUMO

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca2+ mobilization. In DCs, Ca2+ mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP3). Here, we showed that the IP3 receptor 3 (IP3R3) and ITPKB, a kinase that converts IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4), were both necessary for Ca2+ mobilization and NFAT activation in mouse and human DCs. A pool of IP3R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca2+ mobilization through plasma membrane-localized IP3R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.

4.
Adv Biol Regul ; : 100760, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-33060052

RESUMO

INPP5K (Inositol Polyphosphate 5-Phosphatase K, or SKIP (for Skeletal muscle and Kidney enriched Inositol Phosphatase) is a member of the phosphoinositide 5-phosphatases family. Its protein structure is comprised of a N-terminal catalytic domain which hydrolyses both PtdIns(4,5)P2 and PtdIns(3,4,5)P3, followed by a SKICH domain at the C-terminus which is responsible for protein-protein interactions and subcellular localization of INPP5K. Strikingly, INPP5K is mostly concentrated in the endoplasmic reticulum, although it is also detected at the plasma membrane, in the cytosol and the nucleus. Recently, mutations in INPP5K have been detected in patients with a rare form of autosomal recessive congenital muscular dystrophy with cataract, short stature and intellectual disability. INPP5K functions extend from control of insulin signaling, endoplasmic reticulum stress response and structural integrity, myoblast differentiation, cytoskeleton organization, cell adhesion and migration, renal osmoregulation, to cancer. The goal of this review is thus to summarize and comment recent and less recent data in the literature on INPP5K, in particular on the structure, expression, intracellular localization, interactions and functions of this specific member of the 5-phosphatases family.

5.
Development ; 147(3)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31964774

RESUMO

Sonic hedgehog (Shh) signal transduction specifies ventral cell fates in the neural tube and is mediated by the Gli transcription factors that play both activator (GliA) and repressor (GliR) roles. Cilia are essential for Shh signal transduction and the ciliary phosphatidylinositol phosphatase Inpp5e is linked to Shh regulation. In the course of a forward genetic screen for recessive mouse mutants, we identified a functional null allele of inositol polyphosphate-5-phosphatase E (Inpp5e), ridge top (rdg), with expanded ventral neural cell fates at E10.5. By E12.5, Inpp5erdg/rdg embryos displayed normal neural patterning and this correction over time required Gli3, the predominant repressor in neural patterning. Inpp5erdg function largely depended on the presence of cilia and on smoothened, the obligate transducer of Shh signaling, indicating that Inpp5e functions within the cilium to regulate the pathway. These data indicate that Inpp5e plays a more complicated role in Shh signaling than previously appreciated. We propose that Inpp5e attenuates Shh signaling in the neural tube through regulation of the relative timing of GliA and GliR production, which is important in understanding how the duration of Shh signaling regulates neural tube patterning.


Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais/genética , Alelos , Animais , Padronização Corporal/genética , Embrião de Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Tubo Neural/metabolismo , Monoéster Fosfórico Hidrolases/genética , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteína Gli3 com Dedos de Zinco/genética , Proteína Gli3 com Dedos de Zinco/metabolismo
6.
J Immunol ; 204(2): 360-374, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31836657

RESUMO

Balanced activity of kinases and phosphatases downstream of the BCR is essential for B cell differentiation and function and is disturbed in chronic lymphocytic leukemia (CLL). In this study, we employed IgH.TEµ mice, which spontaneously develop CLL, and stable EMC CLL cell lines derived from these mice to explore the role of phosphatases in CLL. Genome-wide expression profiling comparing IgH.TEµ CLL cells with wild-type splenic B cells identified 96 differentially expressed phosphatase genes, including SH2-containing inositol phosphatase (Ship2). We found that B cell-specific deletion of Ship2, but not of its close homolog Ship1, significantly reduced CLL formation in IgH.TEµ mice. Treatment of EMC cell lines with Ship1/2 small molecule inhibitors resulted in the induction of caspase-dependent apoptosis. Using flow cytometry and Western blot analysis, we observed that blocking Ship1/2 abrogated EMC cell survival by exerting dual effects on the BCR signaling cascade. On one hand, specific Ship1 inhibition enhanced calcium signaling and thereby abrogated an anergic response to BCR stimulation in CLL cells. On the other hand, concomitant Ship1/Ship2 inhibition or specific Ship2 inhibition reduced constitutive activation of the mTORC1/ribosomal protein S6 pathway and downregulated constitutive expression of the antiapoptotic protein Mcl-1, in both EMC cell lines and primary IgH.TEµ CLL cells. Importantly, also in human CLL, we found overexpression of many phosphatases including SHIP2. Inhibition of SHIP1/SHIP2 reduced cellular survival and S6 phosphorylation and enhanced basal calcium levels in human CLL cells. Taken together, we provide evidence that SHIP2 contributes to CLL pathogenesis in mouse and human CLL.


Assuntos
Linfócitos B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética
7.
Adv Biol Regul ; 76: 100651, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31519471

RESUMO

Opsismodysplasia (OPS) is a rare but severe autosomal recessive skeletal chondrodysplasia caused by inactivating mutations in the Inppl1/Ship2 gene. The molecular mechanism leading from Ship2 gene inactivation to OPS is currently unknown. Here, we used our Ship2Δ/Δ mouse expressing reduced amount of a catalytically-inactive SHIP2 protein and a previously reported SHIP2 inhibitor to investigate growth plate development and mineralization in vivo, ex vivo and in vitro. First, as observed in OPS patients, catalytic inactivation of SHIP2 in mouse leads to reduced body length, shortening of long bones, craniofacial dysmorphism, reduced height of the hyperthrophic chondrocyte zone and to defects in growth plate mineralization. Second, intrinsic Ship2Δ/Δ bone defects were sufficient to induce the characteristic OPS alterations in bone growth, histology and mineralization ex vivo. Third, expression of osteocalcin was significantly increased in SHIP2-inactivated chondrocyte cultures whereas production of mineralized nodules was markedly decreased. Targeting osteocalcin mRNA with a specific shRNA increased the production of mineralized nodules. Fourth, levels of p-MEK and p-Erk1/2 were significantly increased in SHIP2-inactivated chondrocytes in response to serum and IGF-1, but not to FGF2, as compared to control chondrocytes. Treatment of chondrocytes and bones in culture with a MEK inhibitor partially rescued the production of mineralized nodules, the size of the hypertrophic chondrocyte zone and bone growth, raising the possibility of a treatment that could partially reduce the phenotype of this severe condition. Altogether, our results indicate that Ship2Δ/Δ mice represent a relevant model for human OPS. They also highlight the important role of SHIP2 in chondrocytes during endochondral ossification and its different differentiation steps. Finally, we identified a role of osteocalcin in mineralized nodules production and for the MEK-Erk1/2 signaling pathway in the OPS phenotype.

8.
Dev Cell ; 51(6): 759-774.e5, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31846650

RESUMO

Appropriate axonal growth and connectivity are essential for functional wiring of the brain. Joubert syndrome-related disorders (JSRD), a group of ciliopathies in which mutations disrupt primary cilia function, are characterized by axonal tract malformations. However, little is known about how cilia-driven signaling regulates axonal growth and connectivity. We demonstrate that the deletion of related JSRD genes, Arl13b and Inpp5e, in projection neurons leads to de-fasciculated and misoriented axonal tracts. Arl13b deletion disrupts the function of its downstream effector, Inpp5e, and deregulates ciliary-PI3K/AKT signaling. Chemogenetic activation of ciliary GPCR signaling and cilia-specific optogenetic modulation of downstream second messenger cascades (PI3K, AKT, and AC3) commonly regulated by ciliary signaling receptors induce rapid changes in axonal dynamics. Further, Arl13b deletion leads to changes in transcriptional landscape associated with dysregulated PI3K/AKT signaling. These data suggest that ciliary signaling acts to modulate axonal connectivity and that impaired primary cilia signaling underlies axonal tract defects in JSRD.


Assuntos
Anormalidades Múltiplas/metabolismo , Axônios/metabolismo , Cerebelo/anormalidades , Cílios/metabolismo , Anormalidades do Olho/genética , Doenças Renais Císticas/metabolismo , Retina/anormalidades , Anormalidades Múltiplas/genética , Animais , Cerebelo/metabolismo , Modelos Animais de Doenças , Anormalidades do Olho/metabolismo , Doenças Renais Císticas/genética , Camundongos , Mutação/genética , Neurogênese/fisiologia , Retina/metabolismo
10.
PLoS Genet ; 14(1): e1007195, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29381707

RESUMO

Rasa3 is a GTPase activating protein of the GAP1 family which targets R-Ras and Rap1. Although catalytic inactivation or deletion of Rasa3 in mice leads to severe hemorrhages and embryonic lethality, the biological function and cellular location of Rasa3 underlying these defects remains unknown. Here, using a combination of loss of function studies in mouse and zebrafish as well as in vitro cell biology approaches, we identify a key role for Rasa3 in endothelial cells and vascular lumen integrity. Specific ablation of Rasa3 in the mouse endothelium, but not in megakaryocytes and platelets, lead to embryonic bleeding and death at mid-gestation, recapitulating the phenotype observed in full Rasa3 knock-out mice. Reduced plexus/sprouts formation and vascular lumenization defects were observed when Rasa3 was specifically inactivated in mouse endothelial cells at the postnatal or adult stages. Similar results were obtained in zebrafish after decreasing Rasa3 expression. In vitro, depletion of Rasa3 in cultured endothelial cells increased ß1 integrin activation and cell adhesion to extracellular matrix components, decreased cell migration and blocked tubulogenesis. During migration, these Rasa3-depleted cells exhibited larger and more mature adhesions resulting from a perturbed dynamics of adhesion assembly and disassembly which significantly increased their life time. These defects were due to a hyperactivation of the Rap1 GTPase and blockade of FAK/Src signaling. Finally, Rasa3-depleted cells showed reduced turnover of VE-cadherin-based adhesions resulting in more stable endothelial cell-cell adhesion and decreased endothelial permeability. Altogether, our results indicate that Rasa3 is a critical regulator of Rap1 in endothelial cells which controls adhesions properties and vascular lumen integrity; its specific endothelial cell inactivation results in occluded blood vessels, hemorrhages and early embryonic death in mouse, mimicking thus the Rasa3-/- mouse phenotype.


Assuntos
Permeabilidade Capilar/genética , Adesão Celular/genética , Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Proteínas rap1 de Ligação ao GTP/fisiologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Embrião de Mamíferos , Embrião não Mamífero , Feminino , Proteínas Ativadoras de GTPase/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Megacariócitos/fisiologia , Camundongos , Camundongos Knockout , Transdução de Sinais , Peixe-Zebra , Proteínas rap1 de Ligação ao GTP/genética
12.
Diabetes ; 66(11): 2808-2821, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28830894

RESUMO

Shc homology 2-containing inositol 5' phosphatase-2 (SHIP2) is a lipid phosphatase that inhibits insulin signaling downstream of phosphatidylinositol 3-kinase (PI3K); its role in vascular function is poorly understood. To examine its role in endothelial cell (EC) biology, we generated mice with catalytic inactivation of one SHIP2 allele selectively in ECs (ECSHIP2Δ/+). Hyperinsulinemic-euglycemic clamping studies revealed that ECSHIP2Δ/+ was resistant to insulin-stimulated glucose uptake in adipose tissue and skeletal muscle compared with littermate controls. ECs from ECSHIP2Δ/+ mice had increased basal expression and activation of PI3K downstream targets, including Akt and endothelial nitric oxide synthase, although incremental activation by insulin and shear stress was impaired. Insulin-mediated vasodilation was blunted in ECSHIP2Δ/+ mice, as was aortic nitric oxide bioavailability. Acetylcholine-induced vasodilation was also impaired in ECSHIP2Δ/+ mice, which was exaggerated in the presence of a superoxide dismutase/catalase mimetic. Superoxide abundance was elevated in ECSHIP2Δ/+ ECs and was suppressed by PI3K and NADPH oxidase 2 inhibitors. These findings were phenocopied in healthy human ECs after SHIP2 silencing. Our data suggest that endothelial SHIP2 is required to maintain normal systemic glucose homeostasis and prevent oxidative stress-induced endothelial dysfunction.


Assuntos
Endotélio Vascular/metabolismo , Resistência à Insulina/fisiologia , NADPH Oxidase 2/metabolismo , Estresse Oxidativo/fisiologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Animais , Aorta , Células Cultivadas , Células Endoteliais , Regulação da Expressão Gênica/fisiologia , Técnica Clamp de Glucose , Intolerância à Glucose , Camundongos , Camundongos Knockout , NADPH Oxidase 2/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Vasoconstrição/fisiologia
13.
Kidney Int ; 92(1): 125-139, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28302370

RESUMO

The microvillus brush border on the renal proximal tubule epithelium allows the controlled reabsorption of solutes that are filtered through the glomerulus and thus participates in general body homeostasis. Here, using the lipid 5-phosphatase Ship2 global knockout mice, proximal tubule-specific Ship2 knockout mice, and a proximal tubule cell model in which SHIP2 is inactivated, we show that SHIP2 is a negative regulator of microvilli formation, thereby controlling solute reabsorption by the proximal tubule. We found increased PtdIns(4,5)P2 substrate and decreased PtdIns4P product when SHIP2 was inactivated, associated with hyperactivated ezrin/radixin/moesin proteins and increased Rho-GTP. Thus, inactivation of SHIP2 leads to increased microvilli formation and solute reabsorption by the renal proximal tubule. This may represent an innovative therapeutic target for renal Fanconi syndrome characterized by decreased reabsorption of solutes by this nephron segment.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/enzimologia , Túbulos Renais Proximais/enzimologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Animais , Glicemia/metabolismo , Células Epiteliais/ultraestrutura , Feminino , Genótipo , Glicosúria/metabolismo , Túbulos Renais Proximais/ultraestrutura , Células LLC-PK1 , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvilosidades/enzimologia , Complexos Multiproteicos , Fenótipo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/deficiência , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Reabsorção Renal , Suínos , Fatores de Tempo , Proteínas rho de Ligação ao GTP/metabolismo
14.
Cell ; 168(1-2): 264-279.e15, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-28086093

RESUMO

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.


Assuntos
Ciclo Celular , Cílios/metabolismo , Actinas/metabolismo , Animais , Rim/citologia , Rim/metabolismo , Camundongos , Células NIH 3T3 , Fosfatidilinositol 4,5-Difosfato , Monoéster Fosfórico Hidrolases/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo
15.
J Immunol ; 197(9): 3481-3489, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27694492

RESUMO

Kawasaki disease (KD) is a multisystem vasculitis that predominantly targets the coronary arteries in children. Phenotypic similarities between KD and recurrent fever syndromes point to the potential role of inflammasome activation in KD. Mutations in NLRP3 are associated with recurrent fever/autoinflammatory syndromes. We show that the KD-associated genetic polymorphism in inositol-triphosphate 3-kinase C (ITPKC) (rs28493229) has important functional consequences, governing ITPKC protein levels and thereby intracellular calcium, which in turn regulates NLRP3 expression and production of IL-1ß and IL-18. Analysis of transcript abundance, protein levels, and cellular response profiles from matched, serial biospecimens from a cohort of genotyped KD subjects points to the critical role of ITPKC in mediating NLRP3 inflammasome activation. Treatment failure in those with the high-risk ITPKC genotype was associated with the highest basal and stimulated intracellular calcium levels and with increased cellular production of IL-1ß and IL-18 and higher circulating levels of both cytokines. Mechanistic studies using Itpkc-deficient mice in a disease model support the genomic, cellular, and clinical findings in affected children. Our findings provide the mechanism behind the observed efficacy of rescue therapy with IL-1 blockade in recalcitrant KD, and we identify that regulation of calcium mobilization is fundamental to the underlying immunobiology in KD.


Assuntos
Vasos Coronários/patologia , Síndrome de Linfonodos Mucocutâneos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Pré-Escolar , Estudos de Coortes , Vasos Coronários/metabolismo , Genótipo , Humanos , Inflamassomos/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome de Linfonodos Mucocutâneos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Polimorfismo de Nucleotídeo Único , Risco , Resultado do Tratamento
16.
Adv Biol Regul ; 62: 1-10, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27036498

RESUMO

Inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) is the last identified member of the inositol 1,4,5-trisphosphate 3-kinases family which phosphorylates inositol 1,4,5-trisphosphate into inositol 1,3,4,5-tetrakisphosphate. Although expression and function of the two other family members ITPKA and ITPKB are rather well characterized, similar information is lacking for ITPKC. Here, we first defined the expression of Itpkc mRNA and protein in mouse tissues and cells using in situ hybridization and new antibodies. Surprisingly, we found that cells positive for ITPKC in the studied tissues express either a multicilium (tracheal and bronchial epithelia, brain ependymal cells), microvilli forming a brush border (small and large intestine, and kidney proximal tubule cells) or a flagellum (spermatozoa), suggesting a role for ITPKC either in the development or the function of these specialized cellular structures. Given this surprising expression, we then analyzed ITPKC function in multiciliated tracheal epithelial cells and sperm cells using our Itpkc knock-out mouse model. Unfortunately, no significant difference was observed between control and mutant mice for any of the parameters tested, leaving the exact in vivo function of this third Ins(1,4,5)P3 3-kinase still open.


Assuntos
Cílios/enzimologia , Células Epiteliais/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Mensageiro/genética , Mucosa Respiratória/enzimologia , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Cílios/ultraestrutura , Células Epiteliais/citologia , Expressão Gênica , Hibridização In Situ , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Intestino Grosso/enzimologia , Intestino Delgado/enzimologia , Túbulos Renais Proximais/enzimologia , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Cultura Primária de Células , RNA Mensageiro/metabolismo , Mucosa Respiratória/citologia , Espermatozoides/enzimologia
17.
Dev Cell ; 34(4): 400-409, 2015 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-26305592

RESUMO

Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling.


Assuntos
Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Hedgehog/metabolismo , Fosfatidilinositóis/metabolismo , Transdução de Sinais , Animais , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Modelos Biológicos , Células NIH 3T3 , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases , Transporte Proteico , Proteínas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo
18.
Dev Cell ; 34(3): 338-50, 2015 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-26190144

RESUMO

Ciliary transport is required for ciliogenesis, signal transduction, and trafficking of receptors to the primary cilium. Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) have been associated with ciliary dysfunction; however, its role in regulating ciliary phosphoinositides is unknown. Here we report that in neural stem cells, phosphatidylinositol 4-phosphate (PI4P) is found in high levels in cilia whereas phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is not detectable. Upon INPP5E inactivation, PI(4,5)P2 accumulates at the ciliary tip whereas PI4P is depleted. This is accompanied by recruitment of the PI(4,5)P2-interacting protein TULP3 to the ciliary membrane, along with Gpr161. This results in an increased production of cAMP and a repression of the Shh transcription gene Gli1. Our results reveal the link between ciliary regulation of phosphoinositides by INPP5E and Shh regulation via ciliary trafficking of TULP3/Gpr161 and also provide mechanistic insight into ciliary alterations found in Joubert and MORM syndromes resulting from INPP5E mutations.


Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Anormalidades Múltiplas/genética , Animais , Movimento Celular/genética , Células Cultivadas , Doenças Cerebelares/genética , Cerebelo/anormalidades , AMP Cíclico/biossíntese , Embrião de Mamíferos/metabolismo , Anormalidades do Olho/genética , Oftalmopatias/genética , Hipocampo/embriologia , Deficiência Intelectual/genética , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Doenças Renais Císticas/genética , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Obesidade/genética , Doenças do Pênis/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transporte Proteico/genética , Retina/anormalidades , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco
19.
Hum Mol Genet ; 24(9): 2578-93, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25631876

RESUMO

Primary cilia are complex subcellular structures that play key roles during embryogenesis by controlling the cellular response to several signaling pathways. Defects in the function and/or structure of primary cilia underlie a large number of human syndromes collectively referred to as ciliopathies. Often, ciliopathies are associated with mental retardation (MR) and malformation of the corpus callosum. However, the possibility of defects in other forebrain axon tracts, which could contribute to the cognitive disorders of these patients, has not been explored. Here, we investigate the formation of the corticothalamic/thalamocortical tracts in mice mutant for Rfx3, which regulates the expression of many genes involved in ciliogenesis and cilia function. Using DiI axon tracing and immunohistochemistry experiments, we show that some Rfx3(-/-) corticothalamic axons abnormally migrate toward the pial surface of the ventral telencephalon (VT). Some thalamocortical axons (TCAs) also fail to leave the diencephalon or abnormally project toward the amygdala. Moreover, the Rfx3(-/-) VT displays heterotopias containing attractive guidance cues and expressing the guidance molecules Slit1 and Netrin1. Finally, the abnormal projection of TCAs toward the amygdala is also present in mice carrying a mutation in the Inpp5e gene, which is mutated in Joubert Syndrome and which controls cilia signaling and stability. The presence of identical thalamocortical malformations in two independent ciliary mutants indicates a novel role for primary cilia in the formation of the corticothalamic/thalamocortical tracts by establishing the correct cellular environment necessary for its development.


Assuntos
Padronização Corporal/genética , Córtex Cerebral/metabolismo , Proteínas de Ligação a DNA/genética , Telencéfalo/metabolismo , Tálamo/metabolismo , Fatores de Transcrição/genética , Animais , Embrião de Mamíferos , Homozigoto , Imuno-Histoquímica , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases/genética , Fatores de Transcrição de Fator Regulador X , Telencéfalo/embriologia , Telencéfalo/patologia , Tálamo/embriologia , Tálamo/patologia , Proteína Gli3 com Dedos de Zinco
20.
Adv Biol Regul ; 57: 153-61, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25294679

RESUMO

RASA3 (or GTPase Activating Protein III, R-Ras GTPase-activating protein, GAP1(IP4BP)) is a GTPase activating protein of the GAP1 subfamily which targets Ras and Rap1. RASA3 was originally purified from pig platelet membranes through its intrinsic ability to bind inositol 1,3,4,5-tetrakisphosphate (I(1,3,4,5)P4) with high affinity, hence its first name GAP1(IP4BP) (for GAP1 subfamily member which binds I(1,3,4,5)P4). RASA3 was thus the first I(1,3,4,5)P4 receptor identified and cloned. The in vitro and in vivo functions of RASA3 remained somewhat elusive for a long time. However, recently, using genetically-modified mice and cells derived from these mice, the function of RASA3 during megakaryopoiesis, megakaryocyte adhesion and migration as well as integrin signaling has been reported. The goal of this review is thus to summarize and comment recent and less recent data in the literature on RASA3, in particular on the in vivo function of this specific GAP1 subfamily member.


Assuntos
Movimento Celular/fisiologia , Proteínas Ativadoras de GTPase , Receptores Citoplasmáticos e Nucleares , Transdução de Sinais/fisiologia , Trombopoese/fisiologia , Animais , Adesão Celular/fisiologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Fosfatos de Inositol , Camundongos , Camundongos Transgênicos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo
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