Biomedical subjects

Dianqing Wu

Publications and source records attributed to Dianqing Wu.

At least 19 recordsLinked to original sources

PubMed2007

Signaling requirements for translocation of P-Rex1, a key Rac2 exchange factor involved in chemoattractant-stimulated human neutrophil function.

PI 3,4,5-trisphosphate [PI(3,4,5)P3; PIP3]-dependent Rac exchanger 1 (P-Rex1) is a Rac-specific guanine nucleotide exchange factor abundant in neutrophils and myeloid cells. As a selective catalyst for Rac2 activation, P-Rex1 serves as an important regulator of human neutrophil NADPH oxidase activity and chemotaxis in response to a variety of extracellular stimuli. The exchange activity of P-Rex1 is synergistically activated by the binding of PIP3 and betagamma subunits of heterotrimeric G proteins in vitro, suggesting that the association of P-Rex1 with membranes is a prerequisite for cellular activation. However, the spatial regulation of endogenous P-Rex1 has not been well defined, particularly in human neutrophils activated through G protein-coupled receptors. Upon stimulation of neutrophil chemoattractant receptors, we observed that P-Rex1 translocated from cytoplasm to the leading edge of polarized cells in a G protein betagamma subunit- and PIP3-dependent manner, where it colocalized with F-actin and its substrate, Rac2. Redistribution of P-Rex1 to the leading edge was also dependent on tyrosine kinase activity and was modulated by cell adhesion. Furthermore, we observed that activation of cAMP-dependent protein kinase A (PKA), which phosphorylates and inactivates P-Rex1, inhibited its translocation. Our data indicate that endogenous P-Rex1 translocates to areas of Rac2 and cytoskeletal activation at the leading edge in response to chemoattractant stimuli in human neutrophils and that this translocation can be negatively modulated by activation of PKA and by cell adhesion.

Cell Adhesion↗

PubMed2006

Modulation of cell adhesion and motility in the immune system by Myo1f.

Although class I myosins are known to play a wide range of roles, the physiological function of long-tailed class I myosins in vertebrates remains elusive. We demonstrated that one of these proteins, Myo1f, is expressed predominantly in the mammalian immune system. Cells from Myo1f-deficient mice exhibited abnormally increased adhesion and reduced motility, resulting from augmented exocytosis of beta2 integrin-containing granules. Also, the cortical actin that co-localizes with Myo1f was reduced in Myo1f-deficient cells. In vivo, Myo1f-deficient mice showed increased susceptibility to infection by Listeria monocytogenes and an impaired neutrophil response. Thus, Myo1f directs immune cell motility and innate host defense against infection.

Actins↗

PubMed2006

Differential targeting of Gbetagamma-subunit signaling with small molecules.

G protein betagamma subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gbetagamma subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gbetagamma subunit functions. Several compounds bound to Gbetagamma subunits with affinities from 0.1 to 60 muM and selectively modulated functional Gbetagamma-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

Analgesics↗

PubMed2006

Characterization and application of a new optical probe for membrane lipid domains.

In this article, we characterize the fluorescence of an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. In large unilamellar lipid vesicles (LUVs), its emission spectrum shifts up to 30 nm to the blue with increasing cholesterol concentration. Independently, it displays a comparable blue shift in liquid-ordered relative to liquid-disordered phases. The cumulative effect is a 60-nm difference in emission spectra for cholesterol containing LUVs in the liquid-ordered state versus cholesterol-free LUVs in the liquid-disordered phase. Given these optical properties, we use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and nonlinear optical microscopy. The dye shows green and red fluorescence in liquid-ordered and -disordered domains, respectively. We propose that this reflects the relative rigidity of the molecular packing around the dye molecules in the two phases. We also observe a sevenfold stronger second harmonic generation signal in the liquid-disordered domains, consistent with a higher concentration of the dye resulting from preferential partitioning into the disordered phase. The efficacy of the dye for reporting lipid domains in cell membranes is demonstrated in polarized migrating neutrophils.

Animals↗

PubMed2006

Methods for studying neutrophil chemotaxis.

Chemotaxis is a fascinating biological process, through which a cell migrates along a shallow chemoattractant gradient that is less than 5% difference between the anterior and posterior of the cell. Chemotaxis is composed of two independent, but interrelated processes--motility and directionality--both of which are regulated by extracellular stimuli and chemoattractants; small GTPases have been shown to be involved. In this chapter, the methods that are used to prepare mouse neutrophils and study their chemotactic behaviors and morphological and biochemical changes in response to chemoattractant stimulation are described.

Animals↗

PubMed2006

Prostate-specific membrane antigen regulates angiogenesis by modulating integrin signal transduction.

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 beta(1) 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 beta(1) 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.

Animals↗

PubMed2005

Beta-catenin regulates myogenesis by relieving I-mfa-mediated suppression of myogenic regulatory factors in P19 cells.

Wnt/beta-catenin signaling plays a critical role in embryonic myogenesis. Here we show that, in P19 embryonic carcinoma stem cells, Wnt/beta-catenin signaling initiates the myogenic process depends on beta-catenin-mediated relief of I-mfa (inhibitor of MyoD Family a) suppression of myogenic regulatory factors (MRFs). We found that beta-catenin interacted with I-mfa and that the interaction was enhanced by Wnt3a. In addition, we found that the interaction between beta-catenin and I-mfa was able to attenuate the interaction of I-mfa with MRFs, relieve I-mfa-mediated suppression of the transcriptional activity and cytosolic sequestration of MRFs, and initiate myogenesis in a P19 myogenic model system that expresses exogenous myogenin. This work reveals a mechanism for the regulation of MRFs during myogenesis by elucidating a beta-catenin-mediated, but lymphoid enhancing factor-1/T cell factor independent, mechanism in regulation of myogenic fate specification and differentiation of P19 mouse stem cells.

Animals↗

PubMed2005

P-Rex1 is a primary Rac2 guanine nucleotide exchange factor in mouse neutrophils.

Leukocyte chemoattractants regulate many leukocyte functions, including leukocyte chemotaxis, via the Rho family of small GTPases that include RhoA, Cdc42, and Rac. Previous work has revealed mechanisms by which chemoattractants regulate RhoA and Cdc42 in mouse neutrophils, but the mechanisms for regulation of Rac remain unclear even though Rac is important for neutrophil functions. Here, we characterized P-Rex1, a Gbetagamma and PIP(3)-regulated guanine nucleotide exchange factor that was initially identified as a Rac activator in response to chemoattractants, for its roles in the regulation of Rac activity and neutrophil functions. We generated a mouse line in which the P-Rex1 gene is disrupted and found that P-Rex1 deficiency did not significantly affect Rac1 activation but diminished Rac2 activation in response to a chemoattractant fMLP in mouse neutrophils. This preference for Rac2 may partially result from the apparent higher affinity of P-Rex1 for Rac2 than for Rac1 because P-Rex1 was more readily immunoprecipitated with Rac2(S17N) than Rac1(S17N). In addition, P-Rex1 deficiency significantly attenuated fMLP-induced F actin formation and superoxide production without affecting LPS- or PMA-induced production. Furthermore, P-Rex1 deficiency caused a chemotactic defect that is primarily attributed to a reduction in the migration rate rather than directionality.

Actins↗

PubMed2005

Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation.

Human and mouse genetic and in vitro evidence has shown that canonical Wnt signaling promotes bone formation, but we found that mice lacking the canonical Wnt antagonist Dickkopf2 (Dkk2) were osteopenic. We reaffirmed the finding that canonical Wnt signaling stimulates osteogenesis, including the differentiation from preosteoblasts to osteoblasts, in cultured osteoblast differentiation models, but we also found that canonical Wnts upregulated the expression of Dkk2 in osteoblasts. Although exogenous overexpression of Dkk before the expression of endogenous canonical Wnt (Wnt7b) suppressed osteogenesis in cultures, its expression after peak Wnt7b expression induced a phenotype resembling terminal osteoblast differentiation leading to mineralization. In addition, osteoblasts from Dkk2-null mice were poorly mineralized upon osteogenic induction in cultures, and Dkk2 deficiency led to attenuation of the expression of osteogenic markers, which could be partially reversed by exogenous expression of Dkk2. Taken together with the finding that Dkk2-null mice have increased numbers of osteoids, these data indicate that Dkk2 has a role in late stages of osteoblast differentiation into mineralized matrices. Because expression of another Wnt antagonist, FRP3, differs from Dkk2 expression in rescuing Dkk2 deficiency and regulating osteoblast differentiation, the effects of Dkk2 on terminal osteoblast differentiation may not be entirely mediated by its Wnt signaling antagonistic activity.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Synaptically driven endocannabinoid release requires Ca2+-assisted metabotropic glutamate receptor subtype 1 to phospholipase Cbeta4 signaling cascade in the cerebellum.

Endocannabinoids mediate retrograde signaling and modulate synaptic transmission in various regions of the CNS. Depolarization-induced elevation of intracellular Ca2+ concentration causes endocannabinoid-mediated suppression of excitatory/inhibitory synaptic transmission. Activation of G(q/11)-coupled receptors including group I metabotropic glutamate receptors (mGluRs) also causes endocannabinoid-mediated suppression of synaptic transmission. However, precise mechanisms of endocannabinoid production initiated by physiologically relevant synaptic activity remain to be determined. To address this problem, we made whole-cell recordings from Purkinje cells (PCs) in mouse cerebellar slices and examined their excitatory synapses arising from climbing fibers (CFs) and parallel fibers (PFs). We first characterized three distinct modes to induce endocannabinoid release by analyzing CF to PC synapses. The first mode is strong activation of mGluR subtype 1 (mGluR1)-phospholipase C (PLC) beta4 cascade without detectable Ca2+ elevation. The second mode is Ca2+ elevation to a micromolar range without activation of the mGluR1-PLCbeta4 cascade. The third mode is the Ca2+-assisted mGluR1-PLCbeta4 cascade that requires weak mGluR1 activation and Ca2+ elevation to a submicromolar range. By analyzing PF to PC synapses, we show that the third mode is essential for effective endocannabinoid release from PCs by excitatory synaptic activity. Furthermore, our biochemical analysis demonstrates that combined weak mGluR1 activation and mild depolarization in PCs effectively produces 2-arachidonoylglycerol (2-AG), a candidate of endocannabinoid, whereas either stimulus alone did not produce detectable 2-AG. Our results strongly suggest that under physiological conditions, excitatory synaptic inputs to PCs activate the Ca2+-assisted mGluR1-PLCbeta4 cascade, and thereby produce 2-AG, which retrogradely modulates synaptic transmission to PCs.

Animals↗

PubMed2005

Inhibition of Pkhd1 impairs tubulomorphogenesis of cultured IMCD cells.

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.

Animals↗

PubMed2005

Regulation of PTEN by Rho small GTPases.

PTEN (phosphatase and tensin homologue) is a phosphatase that dephosphorylates both protein and phosphoinositide substrates. It is mutated in a variety of human tumours and has important roles in a diverse range of biological processes, including cell migration and chemotaxis. PTEN's intracellular localization and presumably activity are regulated by chemoattractants in Dictyostelium and mouse neutrophils. However, the mechanisms for its regulation remain elusive. Here we show that RhoA and Cdc42, members of the Rho family of small GTPases, regulate the intracellular localization of PTEN in leukocytes and human transfected embryonic kidney cells. In addition, active RhoA is able to stimulate the phospholipid phosphatase activity of PTEN in human embryonic kidney cells and leukocytes, and this regulation seems to require RhoA's downstream effector, RhoA-associated kinase (Rock). Furthermore, we have identified key residues on PTEN that are required for its regulation by the small GTPase, and show that small GTPase-mediated regulation of PTEN has a significant role in the regulation of chemotaxis.

Animals↗

PubMed2005

Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling.

The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass. In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293T cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by overexpression of Wnt co-receptor low density lipoprotein receptor-related protein (LRP) 5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transduction of canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b. Given the plethora of evidence indicating that canonical Wnt signaling stimulates osteogenesis, we believe that the high bone mass phenotype associated with the loss of sclerostin may be attributed, at least in part, to an increase in canonical Wnt signaling resulting from the reduction in sclerostin-mediated Wnt antagonism.

3T3 Cells↗

PubMed2005

PTEN as an effector in the signaling of antimigratory G protein-coupled receptor.

PTEN, a tumor suppressor phosphatase, is important in the regulation of cell migration and invasion. Physiological regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) by cell surface receptors has not been described. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P), which acts through the S1P2 receptor (S1P2R) G protein-coupled receptor (GPCR) to inhibit cell migration, utilizes PTEN as a signaling intermediate. S1P2R inhibition of cell migration is abrogated by dominant-negative PTEN expression. S1P was unable to efficiently inhibit the migration of Pten(DeltaloxP/DeltaloxP) mouse embryonic fibroblasts; however, the antimigratory effect was restored upon the expression of PTEN. S1P2R activation of Rho GTPase is not affected in Pten(DeltaloxP/DeltaloxP) cells, and dominant-negative Rho GTPase reversed S1P inhibition of cell migration in WT cells but not in Pten(DeltaloxP/DeltaloxP) cells, suggesting that PTEN acts downstream of the Rho GTPase. Ligand activation of the S1P2R receptor stimulated the coimmunoprecipitation of S1P2R and PTEN. Interestingly, S1P2R signaling increased PTEN phosphatase activity in membrane fractions. Furthermore, tyrosine phosphorylation of PTEN was stimulated by S1P2R signaling. These data suggest that the S1P2R receptor actively regulates the PTEN phosphatase by a Rho GTPase-dependent pathway to inhibit cell migration. GPCR regulation of PTEN maybe a general mechanism in signaling events of cell migration and invasion.

Animals↗

PubMed2005

The relative role of PLCbeta and PI3Kgamma in platelet activation.

Stimulation of platelet G protein-coupled receptors results in the cleavage of phosphatidylinositol 4,5-trisphosphate (PIP(2)) into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol by phospholipase C (PLCbeta). It also results in the phosphorylation of PIP2 by the gamma isoform of phosphatidylinositol 3-kinase (PI3Kgamma) to synthesize phosphatidylinositol 3,4,5-trisphosphate. To understand the role of PIP2 in platelet signaling, we evaluated knock-out mice lacking 2 isoforms of PLCbeta (PLCbeta2 and PLCbeta3) or lacking the G(betagamma)-activated isoform of PI3K (PI3Kgamma). Both knock-out mice were unable to form stable thrombi in a carotid injury model. To provide a functional explanation, knock-out platelets were studied ex vivo. PLCbeta2/beta3-/- platelets failed to assemble filamentous actin, had defects in both secretion and mobilization of intracellular calcium, and were unable to form stable aggregates following low doses of agonists. Platelets lacking PI3Kgamma disaggregated following low-dose adenosine diphosphate (ADP) and had a mildly impaired ability to mobilize intracellular calcium. Yet, they exhibited essentially normal actin assembly and secretion. Remarkably, both PLCbeta2/beta3-/- and PI3Kgamma-/- platelets spread more slowly upon fibrinogen. These results suggest substantial redundancy in platelet signaling pathways. Nonetheless, the diminished ability of knock-out platelets to normally spread after adhesion and to form stable thrombi in vivo suggests that both PLCbeta2/beta3 and PI3Kgamma play vital roles in platelet cytoskeletal dynamics.

Actins↗

PubMed2005

DNA-PKcs, but not TLR9, is required for activation of Akt by CpG-DNA.

CpG-DNA and its related synthetic CpG oligodeoxynucleotides (CpG-ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG-DNA-responsive serine/threonine kinases; however, the target protein of CpG-DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow-derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) results in defective phosphorylation and activation of Akt by CpG-DNA. Unexpectedly, loss of the Toll-like receptor 9 has a minimal effect on Akt activation in response to CpG-DNA. Further in vitro analysis using purified DNA-PK and recombinant Akt proteins reveals that DNA-PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA-PKcs associates with Akt upon CpG-DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA-PKcs in CpG-DNA signaling and define a CpG-DNA/DNA-PKcs/Akt pathway.

Active Transport, Cell Nucleus↗

PubMed2005

Mouse taste buds use serotonin as a neurotransmitter.

Synapses between gustatory receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the CNS. Several transmitter candidates have been proposed for these synapses, including serotonin (5-HT), glutamate, acetylcholine, ATP, peptides, and others, but, to date, none has been unambiguously identified. We used Chinese hamster ovary cells stably expressing 5-HT2C receptors as biodetectors to monitor 5-HT release from taste buds. When taste buds were depolarized with KCl or stimulated with bitter, sweet, or sour (acid) tastants, serotonin was released. KCl- and acid-induced 5-HT release, but not release attributable to sweet or bitter stimulation, required Ca2+ influx. In contrast, 5-HT release evoked by sweet and bitter stimulation seemed to be triggered by intracellular Ca2+ release. These experiments strongly implicate serotonin as a taste bud neurotransmitter and reveal unexpected transmitter release mechanisms.

Adenosine Triphosphate↗

PubMed2005

IgE- and IgE+Ag-mediated mast cell migration in an autocrine/paracrine fashion.

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. IgE- and 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 gamma, 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.

Animals↗