Robert A. Rebres

A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Gα12 and Gα13 both singly and in combination, demonstrating the Gα13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gβ2 correlated with a reduced Ca2+ response to C5a. Insertion of a GFP transgene upstream of the Gβ2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.

Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway

Regulation of intracellular cyclic adenosine 3 ′,5 ′-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed Gs-dependent receptors for isoproterenol and prostaglandin E2. Whereas two ligands, uridine 5 ′-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via Gq/calcium and Gi, the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for Gs-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P2 receptor and the heterotrimeric G13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.

Membrane Raft Association of CD47 Is Necessary for Actin Polymerization and Protein Kinase C θ Translocation in Its Synergistic Activation of T Cells

CD47 is a ubiquitously expressed membrane protein with an extracellular Ig domain and a multiple membrane-spanning domain that can synergize with antigen to induce interleukin (IL)-2 secretion by T lymphocytes. Ligation of CD47 induced actin polymerization and increased protein kinase Cθ (PKCθ) association with the cytoskeleton independent of antigen receptor ligation, but ligation of mutant forms of the molecule missing either the Ig domain or the multiple membrane-spanning domain did not. Simultaneous ligation of CD47 and CD3 led to additive effects on F-actin and synergistic effects on PKCθ cytoskeletal association. Disruption of membrane rafts by removal of cholesterol with cyclodextrin blocked CD47-induced actin polymerization, and mutant forms of CD47 that localized poorly to rafts failed to effect cytoskeletal rearrangement. However, raft association alone was not sufficient, because a raft-localized CD47 Ig domain bound to the membrane by a glycan phosphoinositol anchor was unable to induce actin polymerization. A mutant form of CD47 without its Ig domain that did not induce actin polymerization or localize to rafts still enhanced T cell receptor (TCR)-dependent tyrosine phosphorylation of PLCγ and associated Ca2+ signaling but did not augment IL-2 secretion. Thus, CD47 synergy with TCR to increase [Ca2+] i is independent of actin and rafts but is insufficient to explain CD47 cooperation with TCR in IL-2 synthesis. Full synergy with TCR requires CD47 localization to membrane rafts where ligation leads to TCR-independent signals causing actin polymerization and PKCθ translocation.

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

Cross-talk between Gαi- and Gαq-linked G-protein-coupled receptors yields synergistic Ca2+ responses in a variety of cell types. Prior studies have shown that synergistic Ca2+ responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gαq activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca2+ synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gαq. Thus, both Gβγ and Gαq contribute to activation of PLCβ3 in cells for Ca2+ synergy. We conclude that Ca2+ synergy between Gαi-coupled and Gαq-coupled receptors requires the direct action of both Gβγ and Gαq on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent.

A dual receptor crosstalk model of G-protein-coupled signal transduction.

Macrophage cells that are stimulated by two different ligands that bind to G-protein-coupled receptors (GPCRs) usually respond as if the stimulus effects are additive, but for a minority of ligand combinations the response is synergistic. The G-protein-coupled receptor system integrates signaling cues from the environment to actuate cell morphology, gene expression, ion homeostasis, and other physiological states. We analyze the effects of the two signaling molecules complement factors 5a (C5a) and uridine diphosphate (UDP) on the intracellular second messenger calcium to elucidate the principles that govern the processing of multiple signals by GPCRs. We have developed a formal hypothesis, in the form of a kinetic model, for the mechanism of action of this GPCR signal transduction system using data obtained from RAW264.7 macrophage cells. Bayesian statistical methods are employed to represent uncertainty in both data and model parameters and formally tie the model to experimental data. When the model is also used as a tool in the design of experiments, it predicts a synergistic region in the calcium peak height dose response that results when cells are simultaneously stimulated by C5a and UDP. An analysis of the model reveals a potential mechanism for crosstalk between the Gαi-coupled C5a receptor and the Gαq-coupled UDP receptor signaling systems that results in synergistic calcium release.

Signaling and Cross-talk by C5a and UDP in Macrophages Selectively Use PLCβ3 to Regulate Intracellular Free Calcium

Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G protein-coupled receptors (GPCRs) in raising intracellular free Ca2+. To study signal integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca2+ response to GPCR ligands. We found a synergistic response to complement component 5a (C5a) in combination with uridine 5′-diphosphate (UDP), platelet activating factor (PAF), or lysophosphatidic acid (LPA). The C5a response was Gαi-dependent, whereas the UDP, PAF, and LPA responses were Gαq-dependent. Synergy between C5a and UDP, mediated by the C5a and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca2+ from intracellular stores as well as sustained Ca2+ levels. C5a and UDP synergized in generating inositol 1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) β. Macrophages expressed transcripts for three PLCβ isoforms (PLCβ2, PLCβ3, and PLCβ4), but GPCR ligands selectively used these isoforms in Ca2+ signaling. C5a predominantly used PLCβ3, whereas UDP used PLCβ3 but also PLCβ4. Neither ligand required PLCβ2. Synergy between C5a and UDP likewise depended primarily on PLCβ3. Importantly, the Ca2+ signaling deficiency observed in PLCβ3-deficient BMDM was reversed by re-constitution with PLCβ3. Neither phosphatidylinositol (PI) 3-kinase nor protein kinase C was required for synergy. In contrast to Ca2+, PI 3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI 3-kinase. PLCβ3 may thus provide a selective target for inhibiting Ca2+ responses to mediators of inflammation, including C5a, UDP, PAF, and LPA.

Novel CD47-dependent intercellular adhesion modulates cell migration

CD47 is a ubiquitously expressed plasma membrane protein, also known as Integrin Associated Protein, that modulates cell adhesion both through alteration of the avidity of integrin binding and through interaction with its own ligands, the extracellular matrix protein thrombospondin (TSP) and the plasma membrane response regulator SIRPα1. We now show that CD47 expression on fibroblasts can induce intercellular adhesion resulting in cell aggregation in the absence of active integrins, SIRPα1 binding, and detectable TSP. CD47‐expressing cells preferentially bind to other CD47‐expressing cells, and intercellular adhesion requires stimulation by serum or a CD47‐binding peptide from TSP. Cell–cell adhesion is inhibited by pertussis toxin and C. difficile toxin B, and both adherent and aggregating CD47‐expressing fibroblasts have more rac in the GTP bound state than CD47‐deficient cells. Spontaneous migration of Jurkat lymphocytes through a fibroblast monolayer is decreased by fibroblast expression of CD47, consistent with an increased barrier function of the CD47 expressing cells. The lymphocyte chemoattractant SDF‐1α stimulates migration of Jurkat cells through this monolayer only if both the lymphocytes and fibroblasts express CD47, and the inhibition of migration by a CD47‐interacting peptide from TSP similarly requires CD47 expression on both cell types. Thus, signaling dependent on both heterotrimeric and rho family GTPases can induce CD47 to participate in cell–cell interactions independent of known ligands that enhance intercellular adhesion and modulate cell migration.

Synergistic Ca2+ responses by Gαi- and Gαq-coupled GPCRs require a single PLCβ isoform that is sensitive to both Gβγ and Gαq

Cross-talk between Gαi- and Gαq-linked G-protein-coupled receptors yields synergistic Ca2+ responses in a variety of cell types. Prior studies have shown that synergistic Ca2+ responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gαq activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca2+ synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gαq. Thus, both Gβγ and Gαq contribute to activation of PLCβ3 in cells for Ca2+ synergy. We conclude that Ca2+ synergy between Gαi-coupled and Gαq-coupled receptors requires the direct action of both Gβγ and Gαq on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent.

Clostridium difficile toxin B differentially affects GPCR-stimulated Ca2+ responses in macrophages: independent roles for Rho and PLA2

Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca2+ responses to Gαi‐linked receptors, including the C5aR, but reduced responses to Gαq‐linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca2+ responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCβ isoform‐deficient BMDM, we found that ToxB inhibited Ca2+ signaling through PLCβ4 but enhanced signaling through PLCβ3. Effects of ToxB on GPCR Ca2+ responses correlated with GPCR use of PLCβ3 versus PLCβ4. ToxB inhibited UDP Ca2+ signaling without reducing InsP3 production or the sensitivity of cellular Ca2+ stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca2+coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca2+ signaling by C5a was prevented by inhibition of PLA2 or 5‐LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca2+ signaling by different GPCR‐linked PLCβ isoforms in macrophages.