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author:("Wu, anqing")
1.  A critical role for phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac exchanger 1 in endothelial junction disruption and vascular hyperpermeability 
Circulation research  2012;111(12):1517-1527.
Rationale
The small GTPase Rac is critical to vascular endothelial functions, yet its regulation in endothelial cells remains unclear. Understanding the upstream pathway may delineate Rac activation mechanisms and its role in maintaining vascular endothelial barrier integrity.
Objective
By investigating P-Rex1, one of the Rac-specific guanine nucleotide exchange factors (GEFs) previously known for G protein-coupled receptor (GPCR) signaling, we sought to determine whether Rac-GEF is a nodal for signal integration and potential target for drug intervention.
Methods and Results
Using gene deletion and siRNA silencing approach, we investigated the role of P-Rex1 in lung microvascular endothelial cells (HLMVECs). TNF-α exposure led to disruption of endothelial junctions, and silencing P-Rex1 protected junction integrity. TNF-α stimulated Rac activation and ROS production in a P-Rex1-dependent manner. Removal of P-Rex1 significantly reduced ICAM-1 expression, PMN transendothelial migration and leukocyte sequestration in TNF-α challenged mouse lungs. The P-Rex1 knockout mice were also refractory to lung vascular hyper-permeability and edema in a LPS-induced sepsis model.
Conclusions
These results demonstrate for the first time that P-Rex1 expressed in endothelial cells is activated downstream of TNF-α, which is not a GPCR agonist. Our data identify P-Rex1 as a critical mediator of vascular barrier disruption. Targeting P-Rex1 may effectively protect against TNF-α and LPS-induced endothelial junction disruption and vascular hyper-permeability.
doi:10.1161/CIRCRESAHA.112.273078
PMCID: PMC3677960  PMID: 22965143
Vascular permeability; acute lung injury; reactive oxygen species; small GTPases; guanine nucleotide exchange factors; pulmonary edema; endothelial dysfunction
2.  Clathrin and AP2 are required for PtdIns(4,5)P2-mediated formation of LRP6 signalosomes 
The Journal of Cell Biology  2013;200(4):419-428.
PtdIns(4,5)P2 promotes the assembly of LRP6 signalosomes at the cell surface via the recruitment of AP2 and clathrin.
Canonical Wnt signaling is initiated by the binding of Wnt proteins to their receptors, low-density lipoprotein-related protein 5 and 6 (LRP5/6) and frizzled proteins, leading to phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P2) production, signalosome formation, and LRP phosphorylation. However, the mechanism by which PtdIns(4,5)P2 regulates the signalosome formation remains unclear. Here we show that clathrin and adaptor protein 2 (AP2) were part of the LRP6 signalosomes. The presence of clathrin and AP2 in the LRP6 signalosomes depended on PtdIns(4,5)P2, and both clathrin and AP2 were required for the formation of LRP6 signalosomes. In addition, WNT3A-induced LRP6 signalosomes were primarily localized at cell surfaces, and WNT3A did not induce marked LRP6 internalization. However, rapid PtdIns(4,5)P2 hydrolysis induced artificially after WNT3A stimulation could lead to marked LRP6 internalization. Moreover, we observed WNT3A-induced LRP6 and clathrin clustering at cell surfaces using super-resolution fluorescence microscopy. Therefore, we conclude that PtdIns(4,5)P2 promotes the assembly of LRP6 signalosomes via the recruitment of AP2 and clathrin and that LRP6 internalization may not be a prerequisite for Wnt signaling to β-catenin stabilization.
doi:10.1083/jcb.201206096
PMCID: PMC3575536  PMID: 23400998
3.  A MODEL OF GAG:MIP-2:CXCR2 INTERFACES AND ITS FUNCTIONAL EFFECTS 
Biochemistry  2012;51(28):5642-5654.
MIP-2/CXCL2 is a murine chemokine related to human chemokines that possess the Glu-Leu-Arg (ELR) activation motif and activates CXCR2 for neutrophil chemotaxis. We determined the structure of MIP-2 to 1.9Å resolution and created a model with its receptor murine CXCR2 based on the coordinates of human CXCR4. Chemokine-induced migration of cells through specific G protein-coupled receptors is regulated by glycosaminoglycans (GAGs) that oligomerize chemokines. MIP-2 GAG-binding residues were identified that interact with heparin disaccharide I-S by NMR spectroscopy. A model a GAG:MIP-2:CXCR2 complex that supports a 2:2 complex between chemokine and receptor was created. Mutants of these disaccharide-binding residues were made and tested for heparin binding, in vitro neutrophil chemotaxis, and in vivo neutrophil recruitment to the mouse peritoneum and lung. The mutants have a 10-fold decrease in neutrophil chemotaxis in vitro. There is no difference in neutrophil recruitment between wild-type MIP-2 and mutants in the peritoneum but all activity of the mutants is lost in the lung supporting the concept that GAG regulation of chemokines is tissue-dependent.
doi:10.1021/bi3001566
PMCID: PMC3511906  PMID: 22686371
4.  The Structural Basis of DKK-Mediated Inhibition of Wnt/LRP Signaling 
Science signaling  2012;5(224):pe22.
Low-density lipoprotein receptor–related proteins 5 and 6 (LRP5/6) mediate canonical Wnt–β-catenin signaling by forming a complex with the co-receptor Frizzled, which binds to Wnt proteins. Dickkopf (DKK)–related proteins inhibit the Wnt signaling pathway by directly binding to the ectodomains of LRP5/6. However, the mechanism for DKK-mediated antagonism has not been fully understood as of yet. Crystal structures of the LRP6 ectodomain in complex with DKK1, along with mutagenesis studies, provide considerable insights into the molecular basis for DKK-mediated inhibition and Wnt signaling through LRP5/6.
doi:10.1126/scisignal.2003028
PMCID: PMC3465688  PMID: 22589387
5.  Role for the guanine nucleotide exchange factor P-Rex1 in platelet secretion and aggregation 
Objective
Recent studies have shown a role for Rac1 in regulating platelet functions, but how Rac1 is activated in platelets remains unclear. PIP3-dependent Rac exchanger 1 (P-Rex1) was originally identified in neutrophils that regulates phagocyte functions. We sought to examine whether P-Rex1 plays a role in platelet activation.
Methods and Results
Western blotting showed P-Rex1 expression in mouse and human platelets. Mice lacking P-Rex1 exhibited prolonged bleeding time and increased re-bleeding. When challenged with low doses of the G protein-coupled receptor (GPCR) agonists U46619 and thrombin, P-Rex1-/- platelets displayed significantly reduced secretion and aggregation compared to WT platelets. Increasing the concentration of these agonists could overcome the defect. Platelet aggregation induced by collagen, a non-GPCR agonist, was also compromised in the absence of P-Rex1. Along with these phenotypic changes were impaired Rac1 activation, reduced ATP secretion, decreased phosphorylation of Akt, JNK and p38 MAPK in P-Rex1-/- platelets upon agonist stimulation.
Conclusion
These results demonstrate for the first time the presence of P-Rex1 in platelets and its role in platelet secretion as well as aggregation induced by low-dose agonists for GPCR and by collagen.
doi:10.1161/ATVBAHA.111.243675
PMCID: PMC3288658  PMID: 22207728
platelets; P-Rex1; Rac1; secretion; aggregation
6.  PRR5L degradation promotes MTORC2-mediated PKCδ phosphorylation and cell migration downstream of Gα12 
Nature Cell Biology  2012;14(7):686-696.
Mammalian target of rapamycin complex (MTORC) 2 phosphorylates AGC protein kinases including PKC and regulates cellular functions including cell migration. However, its regulation remains poorly understood. Here we show that LPA induces two phases of PKCδ hydrophobic motif (HM) phosphorylation. The late phase is mediated by Gα12, which specifically activates ARAF, leading to upregulation of the expression of an E3 ubiquitin ligase RFFL and subsequent ubiquitination and degradation of PRR5L. Destabilization of PRR5L, a suppressor of mTORC2-mediated HM phosphorylation of PKCδ, but not AKT, results in PKCδ HM phosphorylation and activation. This Gα12-mediated pathway is critically important for fibroblast migration and pulmonary fibrosis development. Thus, our study unravels a signaling pathway for mTORC2 regulation and fibroblast migration.
doi:10.1038/ncb2507
PMCID: PMC3389271  PMID: 22609986
7.  A PLCβ/PI3Kγ-GSK3 signaling pathway regulates cofilin phosphatase slingshot2 and neutrophil polarization and chemotaxis 
Developmental cell  2011;21(6):1038-1050.
SUMMARY
Neutrophils, in response to a chemoattractant gradient, undergo dynamic F actin remodeling, a process important for their directional migration or chemotaxis. However, signaling mechanisms for chemoattractants to regulate the process are incompletely understood. Here, we characterized chemoattractant-activated signaling mechanisms that regulate cofilin dephosphorylation and actin cytoskeleton reorganization and are critical for neutrophil polarization and chemotaxis. In neutrophils, chemoattractants induced phosphorylation and inhibition of GSK3 via both PLCβ-PKC and PI3Kγ-AKT pathways, leading to the attenuation of GSK3-mediated phosphorylation and inhibition of the cofilin phosphatase slingshot2 and an increase in dephosphorylated, active cofilin. The relative contribution of this GSK3-mediated pathway to neutrophil chemotaxis regulation depended on neutrophil polarity preset by integrin-induced polarization of PIP5K1C. Therefore, our study characterizes a signaling mechanism for chemoattractant-induced actin cytoskeleton remodeling and elucidates its context-dependent role in regulating neutrophil polarization and chemotaxis.
doi:10.1016/j.devcel.2011.10.023
PMCID: PMC3241930  PMID: 22172670
Neutrophil; Chemotaxis; polarization; Chemoattractant signaling; Slingshot; PLC; GSK3
8.  Phospholipase C-β3 regulates FcεRI-mediated mast cell activation by recruiting the protein phosphatase SHP-1 
Immunity  2011;34(6):893-904.
Summary
Mast cells are major effectors in high-affinity IgE receptor (FcεRI)-dependent allergic reactions. Here we show that phospholipase C (PLC)-β3 is crucial for FcεRI-mediated mast cell activation. Plcb3−/− mice showed blunted FcεRI-dependent late-phase, but not acute, anaphylactic responses and airway inflammation. Accordingly, FcεRI stimulation of Plcb3−/− mast cells exhibited reduced cytokine production, but normal degranulation. Reduced cytokine production in Plcb3−/− cells could be accounted for by increased activity of the negative regulatory Src family kinase Lyn and reduced activities of the positive regulatory protein kinases MAPKs. Mechanistically, PLC-β3 constitutively interacts with FcεRI, Lyn and SHP-1 (protein phosphatase). SHP-1 likely recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM. Consistent with PLC-β3- and SHP-1-mediated repression of Lyn activity by dephosphorylation at Tyr396, FcεRI-mediated phenotypes were similar in Plcb3−/− and SHP-1-mutant mast cells. Thus, we have defined a PLC-β3- and SHP-1-mediated signaling pathway for FcεRI-mediated cytokine production.
doi:10.1016/j.immuni.2011.04.010
PMCID: PMC3124618  PMID: 21683628
9.  Identification Of Small Molecule TRABID Deubiquitinase Inhibitors By Computation-Based Virtual Screen 
BMC Chemical Biology  2012;12:4.
Background
Wnt/β-catenin-mediated gene transcription plays important roles in a wide range of biological and pathophysiological processes including tumorigenesis where β-catenin-mediated transcription activity frequently elevates. TRABID, a deubiquitinase, was shown to have a positive Wnt/β-catenin-mediated gene transcription and hence holds a promise as a putative anti-cancer target.
Results
In this study, we used a combination of structure based virtual screening and an in vitro deubiquitinase (DUB) assay to identify several small molecules that inhibit TRABID DUB activity. However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription. In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.
Conclusions
TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.
doi:10.1186/1472-6769-12-4
PMCID: PMC3475094  PMID: 22584113
10.  Pathway-selective suppression of chemokine receptor signaling in B cells by LPS through downregulation of PLC-β2 
Cellular & molecular immunology  2010;7(6):428-439.
Lymphocyte activation leads to changes in chemokine receptor expression. There are limited data, however, on how lymphocyte activators can alter chemokine signaling by affecting downstream pathways. We hypothesized that B cell-activating agents might alter chemokine responses by affecting downstream signal transducers, and that such effects might differ depending on the activator. We found that activating mouse B cells using either anti-IgM or lipopolysaccharide (LPS) increased the surface expression of CCR6 and CCR7 with large increases in chemotaxis to their cognate ligands. By contrast, while anti-IgM also led to enhanced calcium responses, LPS-treated cells showed only small changes in calcium signaling as compared with cells that were freshly isolated. Of particular interest, we found that LPS caused a reduction in the level of B-cell phospholipase C (PLC)-β2 mRNA and protein. Data obtained using PLC-β2−/− mice showed that the β2 isoform mediates close to one-half the chemokine-induced calcium signal in resting and anti-IgM-activated B cells, and we found that calcium signals in the LPS-treated cells were boosted by increasing the level of PLC-β2 using transfection, consistent with a functional effect of downregulating PLC-β2. Together, our results show activator-specific effects on responses through B-cell chemokine receptors that are mediated by quantitative changes in a downstream signal-transducing protein, revealing an activity for LPS as a downregulator of PLC-β2, and a novel mechanism for controlling chemokine-induced signals in lymphocytes.
doi:10.1038/cmi.2010.46
PMCID: PMC3343318  PMID: 20871625
B cells; calcium; chemokine; chemotaxis; GPCR
11.  PI3Kγ Deletion Reduces Variability in the In Vivo Osteolytic Response Induced by Orthopaedic Wear Particles 
Journal of Orthopaedic Research  2011;29(11):1649-1653.
Orthopedic wear particles activate a number of intracellular signaling pathways associated with inflammation in macrophages and we have previously shown that the phosphoinositol-3-kinase (PI3K)/Akt pathway is one of the signal transduction pathways that mediates the in vitro activation of macrophages by orthopedic wear particles. Since PI3Kγ is primarily responsible for PI3K activity during inflammation, we hypothesized that PI3Kγ mediates particle-induced osteolysis in vivo. Our results do not strongly support the hypothesis that PI3Kγ regulates the overall amount of particle-induced osteolysis in the murine calvarial model. However, our results strongly support the conclusion that variability in the amount of particle-induced osteolysis between individual mice is reduced in the PI3Kγ−/− mice. These results suggest that PI3Kγ contributes to osteolysis to different degrees in individual mice and that the mice, and patients, that are most susceptible to osteolysis may be so, in part, due to an increased contribution from PI3Kγ.
doi:10.1002/jor.21440
PMCID: PMC3338193  PMID: 21538508
aseptic loosening; titanium; PI3K
12.  PLCβ is Critical for T-cell Chemotaxis 
Chemokines acting through G-protein-coupled receptors play an essential role in the immune response. Phosphatidylinositol-3-kinase and phospholipase-C are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3Kγ, but not PLCβ, in Gαi-coupled receptor-mediated neutrophil chemotaxis. We compared the chemotactic response of peripheral T-cells derived from wild type mice with mice containing loss-of-function mutations of either PI3Kγ, or both of the two predominant lymphocyte PLCβ isoforms (PLCβ2 and PLCβ3). Loss of PI3Kγ did not significantly impair T-cell migration, whereas loss of PLCβ2β3 did. PI3K pharmacologic inhibition suggests that an isoform other than PI3Kγ contributes to T-cell migration. Intracellular calcium chelation decreased the chemotactic response of wild type lymphocytes, which was not impaired by pharmacologic inhibition of PKC isoforms. SDF1α-induced calcium efflux was undetectable in PLCβ2β3-null lymphocytes suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCβ and isoforms of PI3K, other than PI3Kγ, play a critical role in T-lymphocyte chemotaxis.
PMCID: PMC3228861  PMID: 17675482
T-cells; chemotaxis; signal transduction
13.  Integrin-Induced PIP5K1C Kinase Polarization Regulates Neutrophil Polarization, Directionality, and in vivo Infiltration 
Immunity  2010;33(3):340-350.
Neutrophils are important in innate immunity and acute inflammatory responses. However, the regulation of their recruitment to sites of inflammation has not been well characterized. Here, we investigated the kinase PIP5K1C and showed that PIP5K1C-deficiency impaired neutrophil recruitment due to an adhesion defect. PIP5K1C regulated the adhesion through facilitating RhoA GTPase and integrin activation by chemoattractants. Integrins could induce an isoform of PIP5K1C, PIP5K1C-90, polarization in neutrophils through intracellular vesicle transport independently of exogenous chemoattractant. PIP5K1C-90 polarization was required for polarized RhoA activation at uropods and provided an initial directional cue for neutrophil polarization on the endothelium. Importantly, the polarization was also required for circumventing the inhibition of lamellipodium formation by RhoA so that neutrophils could form leading edges required for transendothelial migration. Because integrins are not known to regulate neutrophil polarization, our study revealed a previously underappreciated role of integrin signaling in neutrophil regulation.
doi:10.1016/j.immuni.2010.08.015
PMCID: PMC2947797  PMID: 20850356
14.  Synaptotagmin-mediated vesicle fusion regulates cell migration 
Nature immunology  2010;11(6):495-502.
Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release.
doi:10.1038/ni.1878
PMCID: PMC2951881  PMID: 20473299
15.  GSK3: a multifaceted kinase in Wnt signaling 
Trends in biochemical sciences  2009;35(3):161-168.
GSK3 is one of the few signaling mediators that play central roles in a diverse range of signaling pathways, including those activated by Wnts, hedgehog, growth factors, cytokines, and G protein-coupled ligands. Although the inhibition of GSK3-mediated β-catenin phosphorylation is known to be the key event in Wnt-β-catenin signaling, the mechanisms which underlie this event remain incompletely understood. The recent demonstration of GSK3 involvement in Wnt receptor phosphorylation illustrates the multifaceted roles that GSK3 plays in Wnt-β-catenin signaling. In this review, we will summarize these recent results and offer explanations, hypotheses, and models to reconcile some of these observations.
doi:10.1016/j.tibs.2009.10.002
PMCID: PMC2834833  PMID: 19884009
16.  Tumor Suppression by Phospholipase C-β3 via SHP-1-Mediated Dephosphorylation of Stat5 
Cancer cell  2009;16(2):161-171.
SUMMARY
Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP3), phospholipase C (PLC) is implicated in promoting cell growth. However, we found that PLC-β3-deficient mice develop myeloproliferative disease (MPD), lymphoma, and other tumors. The mutant mice have increased numbers of hematopoietic stem cells (HSC) with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on Stat5 and can be antagonized by the protein phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-β3 deficiency was suggested in mouse lymphomas in PLC-β3−/− and in Eμ-myc;PLC-β3+/− mice and human Burkitt's lymphoma cells. The same mechanism for malignant transformation seems to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-β3 is likely a tumor suppressor.
doi:10.1016/j.ccr.2009.05.018
PMCID: PMC2744338  PMID: 19647226
17.  Cell stimulation with optically manipulated microsources 
Nature methods  2009;6(12):905.
Molecular gradients are important for various biological processes including the polarization of tissues and cells during embryogenesis and chemotaxis. Investigations of these phenomena require control over the chemical microenvironment of cells. We present a technique to set up molecular concentration patterns that are chemically, spatially and temporally flexible. Our strategy uses optically manipulated microsources, which steadily release molecules. Our technique enables the control of molecular concentrations over length scales down to about 1 µm and timescales from fractions of a second to an hour. We demonstrate this technique by manipulating the motility of single human neutrophils. We induced directed cell polarization and migration with microsources loaded with the chemoattractant formyl-methionine-leucine-phenylalanine. Furthermore, we triggered highly localized retraction of lamellipodia and redirection of polarization and migration with microsources releasing cytochalasin D, an inhibitor of actin polymerization.
doi:10.1038/nmeth.1400
PMCID: PMC2813772  PMID: 19915561
18.  Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity 
Cell  2009;138(3):525-536.
Summary
Modulation of intracellular chloride concentration ([Cl−]i) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl− exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl−]I; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl−]i and have implications for control of cell volume and neuronal function.
doi:10.1016/j.cell.2009.05.031
PMCID: PMC2811214  PMID: 19665974
19.  The canonical Wnt signaling antagonist DKK2 is an essential effector of PITX2 function during normal eye development 
Developmental biology  2008;317(1):310-324.
Summary
Local control of cell signaling activity and integration of inputs from multiple signaling pathways are central for normal development but the underlying mechanisms remain poorly understood. Here we show that Dkk2, encoding an antagonist of canonical Wnt signaling, is an essential downstream target of the PITX2 homeodomain transcription factor in neural crest during eye development. Canonical Wnt signaling is ectopically activated in central ocular surface ectoderm and underlying mesenchyme in Pitx2 and Dkk2 deficient mice. General ocular surface ectoderm identity is maintained during development in Dkk2 deficient mice but peripheral fates, including conjunctival goblet cells and eyelash follicles, are ectopically permitted within more central structures and eyelids are hypomorphic. Loss of DKK2 results in ectopic blood vessels within the periocular mesenchyme and PITX2 expression remains persistently high, providing evidence for a negative feedback loop. Collectively, these data suggest that activation of Dkk2 by PITX2 provides a mechanism to locally suppress canonical Wnt signaling activity during eye development, a paradigm that may be a model for achieving local or transient inhibition of pathway activity elsewhere during embryogenesis. We further propose a model placing PITX2 as an essential integration node between retinoic acid and canonical Wnt signaling during eye development.
doi:10.1016/j.ydbio.2008.02.030
PMCID: PMC2387126  PMID: 18367164
homeobox; canonical Wnt signaling; mouse
20.  Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation 
Science (New York, N.Y.)  2008;321(5894):1350-1353.
The canonical Wnt-β-catenin signaling pathway is initiated by induction of phosphorylation of one of the Wnt receptors, low density lipoprotein receptor-related protein (LRP) 5/6, at Thr1479 and Ser1490. We identified, by screening a human kinase siRNA library, phosphatidylinositol 4-kinase type II (PI4KII) α and phosphatidylinositol-4-phosphate 5-kinase type I (PIP5KI) as required for Wnt3a-induced LRP6 phosphorylation at Ser1490 in mammalian cells and confirmed that these kinases are important for Wnt signaling in Xenopus embryos. Wnt3a stimulates the formation of phosphatidylinositol 4,5-bisphosphates [PtdIns (4,5)P2] through frizzled (Fz) and dishevelled (Dvl), the latter of which directly interacted with and activated PIP5KI. PtdIns (4,5)P2 in turn regulated phosphorylation of LRP6 at Thr1479 and Ser1490. Therefore, our study reveals a new signaling mechanism for Wnt to regulate LRP6 phosphorylation.
doi:10.1126/science.1160741
PMCID: PMC2532521  PMID: 18772438
21.  Phospholipase C β3 deficiency leads to macrophage hypersensitivity to apoptotic induction and reduction of atherosclerosis in mice 
Atherosclerosis is an inflammatory disease that is associated with monocyte recruitment and subsequent differentiation into lipid-laden macrophages at sites of arterial lesions, leading to the development of atherosclerotic plaques. PLC is a key member of signaling pathways initiated by G protein–coupled ligands in macrophages. However, the role of this enzyme in the regulation of macrophage function is not known. Here, we studied macrophages from mice lacking PLC β2, PLC β3, or both PLC isoforms and found that PLC β3 is the major functional PLC β isoform in murine macrophages. Although PLC β3 deficiency did not affect macrophage migration, adhesion, or phagocytosis, it resulted in macrophage hypersensitivity to multiple inducers of apoptosis. PLC β3 appeared to regulate this sensitivity via PKC-dependent upregulation of Bcl-XL. The significance of PLC β signaling in vivo was examined using the apoE-deficient mouse model of atherosclerosis. Mice lacking both PLC β3 and apoE exhibited fewer total macrophages and increased macrophage apoptosis in atherosclerotic lesions, as well as reduced atherosclerotic lesion size when compared with mice lacking only apoE. These results demonstrate what we believe to be a novel role for PLC activity in promoting macrophage survival in atherosclerotic plaques and identify PLC β3 as a potential target for treatment of atherosclerosis.
doi:10.1172/JCI33139
PMCID: PMC2129238  PMID: 18079968
22.  Prostate-Specific Membrane Antigen Regulates Angiogenesis by Modulating Integrin Signal Transduction†  
Molecular and Cellular Biology  2006;26(14):5310-5324.
The transmembrane peptidase prostate-specific membrane antigen (PSMA) is universally upregulated in the vasculature of solid tumors, but its functional role in tumor angiogenesis has not been investigated. Here we show that angiogenesis is severely impaired in PSMA-null animals and that this angiogenic defect occurs at the level of endothelial cell invasion through the extracellular matrix barrier. Because proteolytic degradation of the extracellular matrix is a critical component of endothelial invasion in angiogenesis, it is logical to assume that PSMA participates in matrix degradation. However, we demonstrate a novel and more complex role for PSMA in angiogenesis, where it is a principal component of a regulatory loop that is tightly modulating laminin-specific integrin signaling and GTPase-dependent, p21-activated kinase 1 (PAK-1) activity. We show that PSMA inhibition, knockdown, or deficiency decreases endothelial cell invasion in vitro via integrin and PAK, thus abrogating angiogenesis. Interestingly, the neutralization of β1 or the inactivation of PAK increases PSMA activity, suggesting that they negatively regulate PSMA. This negative regulation is mediated by the cytoskeleton as the disruption of interactions between the PSMA cytoplasmic tail and the anchor protein filamin A decreases PSMA activity, integrin function, and PAK activation. Finally, the inhibition of PAK activation enhances the PSMA/filamin A interaction and, thus, boosts PSMA activity. These data imply that PSMA participates in an autoregulatory loop, wherein active PSMA facilitates integrin signaling and PAK activation, leading to both productive invasion and downregulation of integrin β1 signaling via reduced PSMA activity. Therefore, we have identified a novel role for PSMA as a true molecular interface, integrating both extracellular and intracellular signals during angiogenesis.
doi:10.1128/MCB.00084-06
PMCID: PMC1592718  PMID: 16809768
23.  IgE- and IgE+Ag-mediated mast cell migration in an autocrine/paracrine fashion 
Blood  2005;105(8):3222-3229.
Mast cells are the major effector cells for immediate hypersensitivity and chronic allergic reactions. These cells accumulate in mucosal tissues of allergic reactions, where immunoglobulin E (IgE) is produced locally. Here we provide evidence that, in addition to antigen that can attract IgE-bound mast cells, the type of IgE molecules that efficiently activate mast cells can promote the migration of mast cells in the absence of antigen. IgEand IgE+Ag-mediated migration involves an autocrine/paracrine secretion of soluble factors including adenosine, leukotriene B4, and several chemokines. Their secretion depends on 2 tyrosine kinases, Lyn and Syk, and they are agonists of G-protein–coupled receptors and signal through phosphatidylinositol 3-kinase γ, leading to mast cell migration. In mouse experiments, naive mast cells are attracted to IgE, and IgE-sensitized mast cells are attracted to antigen. Therefore, IgE and antigen are implicated in mast cell accumulation at allergic tissue sites with local high IgE levels.
doi:10.1182/blood-2004-11-4205
PMCID: PMC1464406  PMID: 15637135
24.  Inhibition of Pkhd1 Impairs Tubulomorphogenesis of Cultured IMCD CellsV⃞ 
Molecular Biology of the Cell  2005;16(9):4398-4409.
Fibrocystin/polyductin (FPC), the gene product of PKHD1, is responsible for autosomal recessive polycystic kidney disease (ARPKD). This disease is characterized by symmetrically large kidneys with ectasia of collecting ducts. In the kidney, FPC predominantly localizes to the apical domain of tubule cells, where it associates with the basal bodies/primary cilia; however, the functional role of this protein is still unknown. In this study, we established stable IMCD (mouse inner medullary collecting duct) cell lines, in which FPC was silenced by short hairpin RNA inhibition (shRNA). We showed that inhibition of FPC disrupted tubulomorphogenesis of IMCD cells grown in three-dimensional cultures. Pkhd1-silenced cells developed abnormalities in cell-cell contact, actin cytoskeleton organization, cell-ECM interactions, cell proliferation, and apoptosis, which may be mediated by dysregulation of extracellular-regulated kinase (ERK) and focal adhesion kinase (FAK) signaling. These alterations in cell function in vitro may explain the characteristics of ARPKD phenotypes in vivo.
doi:10.1091/mbc.E04-11-1019
PMCID: PMC1196347  PMID: 15975909
25.  The LRP5 High-Bone-Mass G171V Mutation Disrupts LRP5 Interaction with Mesd 
Molecular and Cellular Biology  2004;24(11):4677-4684.
The mechanism by which the high-bone-mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates canonical Wnt signaling was investigated. The mutation was previously shown to reduce DKK1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for DKK-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperone protein for LRP5/6 that is required for transport of the coreceptors to cell surfaces, resulting in fewer LRP5 molecules on the cell surface. Although the reduction in the number of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm. Together with the observation that osteoblast cells produce autocrine canonical Wnt, Wnt7b, and that osteocytes produce paracrine DKK1, we think that the G171V mutation may cause an increase in Wnt activity in osteoblasts by reducing the number of targets for paracrine DKK1 to antagonize without affecting the activity of autocrine Wnt.
doi:10.1128/MCB.24.11.4677-4684.2004
PMCID: PMC416395  PMID: 15143163

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