scholarly journals Hematopoietic Stem and Progenitor Cells Manifest Direct Migration Towards Secreted DEK in a CXCR2- and G Protein Signaling-Dependent Manner

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1196-1196
Author(s):  
Maegan L. Capitano ◽  
Yasser Sammour ◽  
Maureen Legendre ◽  
Scott Cooper ◽  
David Markovitz ◽  
...  
Keyword(s):  
Cell Migration ◽  
G Protein ◽  
Pertussis Toxin ◽  
Conflicts Of Interest ◽  
Neutrophil Migration ◽  
Regulation Of Transcription ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
G Protein Coupled

DEK, a nuclear DNA-binding protein implicated in the regulation of transcription, chromatin architecture, and mRNA processing, is secreted by macrophages and acts as a proinflammatory molecule (Mor-Vanknin et al., 2006, Mol. Cell. Bio., 26: 9484). Recombinant (r)DEK functions as a chemotactic factor attracting neutrophils, CD8+ T lymphocytes and natural killer cells. Few cytokines/growth modulating proteins are known to be chemoattractants for hematopoietic stem (HSC) and progenitor (HPC) cells; stromal cell-derived factor-1 (SDF-1/CXCL12) being the most potent known protein with this capability. To test whether rDEK can serve as a chemotactic agent, transwell assays were performed utilizing lineage negative mouse bone marrow (BM) cells with neutrophils (Ly6G+ cells) as a positive control. Both SDF-1 and DEK induced directed migration of Lin-Sca1+cKit+ (LSK) BM cells at a dose of 100ng/mL, as determined by flow cytometry of input and migrated cells, with no significant migration occurring towards 100ng/mL of IL-8 or MIP-2. All four cytokines induced migration of Ly6G+ neutrophils. After examining the ability of LSK cells to migrate towards various doses of rDEK (0-200ng/mL), it was determined that LSK cells can migrate towards rDEK in a dose dependent manner with maximum chemoattraction potential (~20%) occurring at a dose of DEK equal to or greater than 50ng/mL. A checkerboard assay using LSK cells was performed to determine whether rDEK acted more as a chemotactic (directed cell movement) or a chemokinetic (random migration) agent. Checkerboard analysis demonstrated that DEK acted as a chemotactic molecule. Upon our recent discovery and report that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8 and binds to the chemokine receptor CXCR2 to regulate hematopoiesis (Capitano et al., 2019, J.C.I. 130: 2555-2570), we hypothesized that DEK may manifest its chemotactic actions through CXCR2, known previously to only bind and mediate the actions of the chemokines IL-8 and MIP-2. To examine this, we first confirmed expression of CXCR2 on the surface of HSC and HPC. Next, to determine if LSK migration towards DEK is dependent upon its ability to signal through CXCR2, LSK cells were pretreated with a neutralizing monoclonal antibody for CXCR2 immediately prior to being placed in a transwell chemotaxis assay utilizing 100ng/mL of rDEK in the bottom chamber. Neutralizing anti-CXCR2 antibodies inhibited migration of both LSK and Ly6G+ cells toward DEK; however, if LSK cells were pretreated with an isotype control or a neutralizing antibody towards CXCR4, migration towards DEK still occurred. To confirm that the neutralizing CXCR2 antibody did not inhibit migration in a non-specific manner, transwell assays were performed examining LSK cell migration towards SDF-1, IL-8, and MIP-2. LSK cells were still able to migrate towards SDF-1 except when CXCR4 was neutralized. No migration of LSK cells was observed when IL-8 or MIP-2 was utilized. When Ly6G+ neutrophils were used, CXCR2 neutralizing antibodies blocked migration of the Ly6G+ neutrophils towards DEK, IL-8 and MIP-2. Neutralizing CXCR4 only blocked Ly6G+ neutrophil migration towards SDF-1. CXCR2 is a G protein-coupled receptor and this interaction can be blocked using pertussis toxin which prevents G proteins from interacting with G protein-coupled receptors thus interfering with receptor signaling. Pretreatment of LSK cells with pertussis toxin significantly inhibited the migration of LSK cells towards DEK and SDF-1. To determine if DEK and SDF-1 could inhibit one another, checkboard assays were performed where either different concentrations of DEK was used in the top well and different concentrations of SDF-1 was used on the bottom. Starting at 100ng/mL, DEK in the top well inhibited LSK cell migration towards SDF-1. However, when SDF-1 was in the top well, regardless of the SDF-1 dose, SDF-1 always inhibited LSK cell migration towards DEK. These data suggest that DEK acts as a chemotactic agent for HSC and HPC in vitro but is not as strong of a signaling protein for migration when competing against SDF-1. Thus, DEK may be involved as a compensatory chemotactic agent for HSCs and HPCs, especially under certain inflammatory conditions and when SDF-1 signaling is reduced. Disclosures No relevant conflicts of interest to declare.

scholarly journals Extracellular DEK Is a Novel Chemotactic Agent of Lineage Negative, Sca-1 Positive, c-Kit Positive Bone Marrow Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1583-1583
Author(s):  
Maegan L. Capitano ◽  
Nirit Mor-Vaknin ◽  
Maureen Legendre ◽  
David Markovitz ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
G Protein ◽  
Pertussis Toxin ◽  
Neutralizing Antibodies ◽  
Conflicts Of Interest ◽  
Regulation Of Transcription ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
G Protein Coupled

Abstract DEK, a nuclear DNA-binding protein that has been implicated in the regulation of transcription, chromatin architecture, and mRNA processing, is known to be secreted by macrophages and act as a proinflammatory molecule (Mor-Vanknin et al., 2006, Mol. Cell. Bio., 26: 9484). Recombinant (r)DEK is known to function as a chemotactic factor that attracts neutrophils, CD8+ T lymphocytes and natural killer cells. Few cytokines are known to be chemoattractants for hematopoietic stem (HSC) and progenitor (HPC) cells, SDF-1 being the most potent of proteins with this capability. To test whether rDEK can serve as a chemotactic agent, transwell assays were performed utilizing lineage negative, sca-1 positive, c-kit positive (LSK) mouse bone marrow and neutrophils (Ly6G+ cells) as a positive control. Both SDF-1 and DEK induced migration of LSK cells at a dose of 100ng/mL, with no significant migration occurring towards 100ng/mL of IL-8 or MIP-2. All four cytokines induced migration of Ly6G+ cells. After examining the ability of LSK cells to migrate towards various doses of rDEK (0-200ng/mL), it was determined that LSK cells can migrate towards rDEK in a dose dependent manner with the maximum chemoattraction potential (~20%) occurring at a dose of DEK equal to or greater than 50ng/mL. A checkerboard assay using LSK cells was performed to determine whether rDEK acted more as a chemotactic (directed cell movement) or a chemokinetic (random migration) agent. Checkerboard analysis demonstrated that DEK acted as a chemotactic molecule. Upon our discovery that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, we hypothesized that DEK may manifest at least some of its actions through CXCR2, known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case we first confirmed expression of CXCR2 on the surface of HSC and HPC. Next, to determine if LSK migration towards DEK is dependent upon its ability to signal through CXCR2, LSK cells were pretreated with a neutralizing monoclonal antibody for CXCR2 immediately prior to being placed in a transwell chemotaxis assay utilizing 100ng/mL of rDEK in the bottom chamber. Neutralizing anti-CXCR2 antibodies inhibited migration of LSK and Ly6G+ cells toward DEK; however, if LSK cells were pretreated with an isotype control or a neutralizing antibody towards CXCR4, migration towards DEK still occurred. To confirm that the neutralizing CXCR2 antibody did not inhibit migration in a non-specific manner, transwell assays were performed examining LSK migration towards SDF-1, IL-8, and MIP-2. LSK cells were still able to migrate towards SDF-1 except when CXCR4 was neutralized. No migration was observed when IL-8 or MIP-2 was utilized. When Ly6G+ cells were used CXCR2 neutralizing antibodies blocked the migration of Ly6G+ cells towards DEK, IL-8 and MIP-2. Neutralizing CXCR4 only blocked Ly6G+ migration towards SDF-1. CXCR2 is known to be a G protein-coupled receptor and this interaction can be blocked through the use of pertussis toxin which prevents G proteins from interacting with G protein-coupled receptors thus interfering with receptor signaling. Pretreatment of LSK cells with pertussis toxin significantly inhibited the migration of LSK cells towards DEK and SDF-1. These data suggest that DEK acts as a chemotactic agent for HSC and HPC in vitro. Thus, DEK may be involved in migration and homing of HSCs and HPCs. Disclosures No relevant conflicts of interest to declare.

Essential Role for PU.1 in MEIS1 Activation and MLL Fusion Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3466-3466
Author(s):  
Jing Zhou ◽  
Bo Li ◽  
Jun Wu ◽  
Fuhong He ◽  
Qiang Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloid Leukemia ◽  
Essential Role ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Genetic Alterations ◽  
Regulation Of Transcription ◽  
Mouse Bone Marrow ◽  
Down Regulation ◽  
Hematopoietic Stem

Abstract Abstract 3466 Down-regulation of transcription factor PU.1, a key regulator of hematopoiesis, induces myeloid leukemia in mice, demonstrating a role of PU.1 as tumor suppressor. Recent studies, however, have also suggested that PU.1 is required for repopulation/self-renewal capacity of normal hematopoietic stem cells (HSCs), and presence of PU.1 activity may be necessary to favor growth of myeloid leukemia stem cells. To explore whether PU.1 could possibly act as an oncogene in the development of certain type of myeloid leukemia, we set to look for differential up-regulation of PU.1 among AML patients with distinct cytogenetic and genetic alterations in public databases. Consistent with recent molecular studies showing suppression of PU.1 expression by AML1-ETO and PML-RARa fusion proteins, PU.1 expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. In contrast, PU.1 expression level in MLL leukemia patients is significantly higher than that of other subgroups of AML. In addition, we found that a set of PU.1 direct target genes, as defined by genome wide location analysis of this factor, expresses at higher level in MLL leukemia patients comparing with those with t(8;21) and t(15;17) translocations, supporting an increased PU.1 activity in this subgroup of leukemia. In our effort to characterize the functional consequence of high expression of PU.1 in AML, we found that PU.1 plays an essential role in activation of MEIS1, an oncogene essential for MLL leukemia stem cell potential, and in development of MLL fusion leukemia. MEIS1, as PU.1, is differentially up-regulated in MLL leukemia patients, and expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. Among AML patients with higher level MEIS1 expression, a positive correlation was observed between expression of PU.1 and that of MEIS1. Using promoter reporter assay, electro mobility shift assay (EMSA) and chromatin immunoprecipiation (ChIP) analysis, we found that PU.1 directly binds to and activates MEIS1 promoter in vitro and in vivo. Analysis of a hypomorphic PU.1 mouse model indicated that PU.1 is required to maintain Meis1 expression in murine HSCs and progenitors, and knockdown of PU.1 in patient-derived MLL leukemia cell lines resulted in lower enrichment of PU.1 protein at MEIS1 promoter, accompanied by down-regulation of MEIS1 expression and decreased proliferation and survival of these cells. We are now examining whether the ability of MLL-AF9 fusion protein to drive leukemia is compromised in PU.1-deficient mouse HSC/HPCs, and whether introduction of exogenous Meis1 can compensate for the loss of PU.1 in the development of MLL-AF9 leukemia in mouse bone marrow transplantation model. Finally, we are also testing knock-down of PU.1 as a therapeutic approach to primary AMLs isolated from MLL leukemia patients. Collectively, our data indicate that PU.1 is required for the pathogenesis of MLL associated leukemia, at least partially, through direct activation of MEIS1. In veiw of the dependency of MEIS1 in MLL leukemic transformation, targeting PU.1 mediated MEIS1 gene activation could be an alternative or synergistic approach for MLL leukemia therapies aimed at inhibition of DOT1L and HOXA9. Disclosures: No relevant conflicts of interest to declare.

The G Protein-Coupled Receptor Associated Sorting Proteins, Gprasp2 and Armcx1 Are Putative Negative Regulators of HSC Engraftment and Repopulation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2386-2386 ◽  
Author(s):  
Maheen Ferdous ◽  
Miguel Ganuza ◽  
Per Holmfeldt ◽  
Trent Hall ◽  
Megan Walker ◽  
...  
Keyword(s):  
G Protein ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
G Protein Coupled Receptor ◽  
Negative Regulators ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Qrt Pcr ◽  
G Protein Coupled

Abstract Although hematopoietic stem cell transplantation (HSCT) is used routinely to cure hematologic disease, the efficacy of transplantation is limited by the paucity of HSC. One way to overcome this is to increase the efficiency of HSC engraftment. Thus, we executed a functional screen for novel regulators of HSCT. Murine HSC were lentivirally transduced with shRNAs targeting prioritized gene candidates prior to transplantation into cohorts of lethally irradiated recipient mice. In total, around 1300 mice were transplanted to assess a putative role for about 50 genes in HSCT. We thereby identified Gprasp2 and Armcx1 as putative negative regulators of HSCT. When transplanted at a 1:4 disadvantage relative to control, recipients of either Gprasp2 or Armcx1 shRNA-treated CD45.2 Lineage- Sca-1+ c-Kit+ (LSK) cells displayed 3.12 (p=0.024) and 2.8 (p=0.04) fold enhanced CD45.2 chimerism in peripheral blood (PB) at 16 weeks post-transplant, respectively, relative to mice transplanted with CD45.2 LSK cells treated with control shRNAs. Although loss of each gene did not favor a particular PB lineage, CD45.2 chimerism was enhanced in all bone marrow (BM) HSC and progenitor (HSPC) compartments in these recipients, correlating with their enhanced PB chimerism. qRT-PCR reveals that both murine Armcx1 and Gprasp2 are highly enriched for expression in LSK CD150+CD48- cells relative to all downstream hematopoietic progeny. Further, HemaExplorer, a bioinformatics database of human hematopoietic gene expression, suggests that GPRASP2 and ARMCX1 are also highly expressed in human HSC. This prediction is currently being validated by qRT-PCR. Interestingly, Gprasp2 and Armcx1 both belong to the G protein-coupled receptor associated sorting protein (GASP) gene family, which has never before been implicated in HSC function. The closely related GASP family member, Gprasp1, sorts G protein-coupled receptors (GPCR) to lysosomes for degradation. As Gprasp1 and Gprasp2 both contain GPCR-binding domains and ~70% amino acid sequence conservation in their C-termini, Gprasp2 may also regulate GPCR trafficking and degradation in HSC. Although Gprasp1 was not tested in our screen, qRT-PCR analysis reveals that it is also highly expressed by murine HSC relative to downstream progeny, suggesting that it too may play a role in HSC function. We are currently assessing this using Gprasp1-shRNAs and competitive transplantation. In contrast, Armcx1 lacks the GPCR binding domain and contains both a nuclear and mitochondrial localization signal and has been shown to localize to mitochondrial networks when expressed in HEK-293 cells, suggesting a role in mitochondrial/nuclear communication. To determine how loss of Gprasp2 and Armcx1 promotes HSC engraftment, we are currently employing transplantation and ex vivo culture assays to analyze the effect of their loss on cell cycle, apoptosis, migration, and adhesion of HSPC post-transplant. Our work may help elucidate the mechanisms underlying efficient engraftment, adhesion, and retention of HSPC in the BM niche, which in turn may shed light on novel pathways that could be targeted to promote the efficiency of HSCT in the clinic. Disclosures No relevant conflicts of interest to declare.

The G Protein-Coupled Receptor cysLT1 Is Expressed in Acute Myeloid Leukemia Mediating Cell Migration and Survival.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1933-1933
Author(s):  
Christina Rether ◽  
Sandra Hengstebeck ◽  
Xingkui Xue ◽  
Andreas Boehmler ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Migration ◽  
G Protein ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Dependent Manner ◽  
G Protein Coupled ◽  
Dose Dependent ◽  
Acute Myeloid

Abstract Previous studies have shown that the G protein-coupled receptor (GPCR) cysLT1, which recognizes inflammatory lipid mediators (cysteinyl-leukotrienes), is expressed in immature hematopoietic and tumor (eg. colon cancer) cells. In the present study, we analyzed leukemic blasts from 19 patients with newly diagnosed acute myeloid leukemia for analysis of expression and function of cysLT1. By RT-PCR, cysLT1 mRNA was found in all primary AML cells. Quantitative TaqMan PCR demonstrated high levels particularly in acute myeloblastic leukemia without maturation (FAB M1). There was also a positive correlation between the cysLT1 mRNA level and the expression of CD34 and CD-117 (c-kit), indicating that high cysLT1 expression corresponds to an AML phenotype resembling normal hematopoietic progenitor cells. CysLT1 was functionally active in AML blasts, as demonstrated by intracellular calcium fluxes and actin polymerization induced by the ligand LTD4. Similar to mRNA expression, strongest responses were seen in AML FAB M1. As LTD4 can be produced in the bone marrow by stromal cells and may contribute to bone marrow infiltration of AML, chemotaxis was analyzed. Surprisingly, already low concentrations (10nM) induced significant chemotaxis of AML blasts, while higher concentrations (up to 1 uM) were less effective in a dose-dependent manner. Incubation of myeloid leukemic cell lines (eg. KG1a) and primary AML blasts with the specific cysLT1 antagonist MK571 resulted in significantly reduced viability after 48 h in a dose-dependent manner (10nM-10uM), suggesting also an autocrine function of cysLT1 ligands. To explore signal transduction pathways involved in leukemic cell proliferation and chemotaxis, we found that in AML cell lines, LTD4 induced phosphorylation of Erk/MAP kinase, wich is related to proliferation, and Pyk2, which represents a focal adhesion kinase-like signaling molecule that links GPCRs with cell migration, while the Akt pathway was not involved. We conclude that cysLT1 is consistently expressed in acute myeloid leukemia, and mediates both chemotactic and proliferative responses. Therefore, cysLT1 antagonists, which are already used in the treatment of allergy, may improve the effectiveness of antileukemic therapy.

scholarly journals C1q-mediated chemotaxis by human neutrophils: involvement of gClqR and G-protein signalling mechanisms

1998 ◽  
Vol 330 (1) ◽  
pp. 247-254 ◽  
Author(s):  
E. A. Leonora LEIGH ◽  
Berhane GHEBREHIWET ◽  
P. S. Tim PERERA ◽  
N. Ian BIRD ◽  
Peter STRONG ◽  
...  
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
Oxidative Burst ◽  
Binding Protein ◽  
Neutrophil Migration ◽  
Human Neutrophils ◽  
Classical Pathway ◽  
Dependent Manner ◽  
Directed Movement ◽  
G Protein Coupled

C1q, the first component of the classical pathway of the complement system, interacts with various cell types and triggers a variety of cell-specific cellular responses, such as oxidative burst, chemotaxis, phagocytosis, etc. Different biological responses are attributed to the interaction of C1q with more than one putative cell-surface C1q receptor/C1q-binding protein. Previously, it has been shown that C1q-mediated oxidative burst by neutrophils is not linked to G-protein-coupled fMet-Leu-Phe-mediated response. In the present study, we have investigated neutrophil migration brought about by C1q and tried to identify the signal-transduction pathways involved in the chemotactic response. We found that C1q stimulated neutrophil migration in a dose-dependent manner, primarily by enhancing chemotaxis (directed movement) rather than chemokinesis (random movement). This C1q-induced chemotaxis could be abolished by an inhibitor of G-proteins (pertussis toxin) and PtdIns(3,4,5)P3 kinase (wortmannin and LY294002). The collagen tail of C1q appeared to mediate chemotaxis. gC1qR, a C1q-binding protein, has recently been reported to participate in C1q-mediated chemotaxis of murine mast cells and human eosinophils. We observed that gC1qR enhanced binding of free C1q to adherent neutrophils and promoted C1q-mediated chemotaxis of neutrophils by nearly seven-fold. Our results suggests C1q-mediated chemotaxis involves gC1qR as well as G-protein-coupled signal-transduction mechanisms operating downstream to neutrophil chemotaxis.

scholarly journals Inhibitory and Stimulatory Regulation of Rac and Cell Motility by the G12/13-Rho and Gi Pathways Integrated Downstream of a Single G Protein-Coupled Sphingosine-1-Phosphate Receptor Isoform

2003 ◽  
Vol 23 (5) ◽  
pp. 1534-1545 ◽  
Author(s):  
Naotoshi Sugimoto ◽  
Noriko Takuwa ◽  
Hiroyuki Okamoto ◽  
Sotaro Sakurada ◽  
Yoh Takuwa
Keyword(s):  
Cell Migration ◽  
G Protein ◽  
Pertussis Toxin ◽  
Kinase Inhibitors ◽  
Fiber Formation ◽  
Membrane Ruffling ◽  
Sphingosine 1 Phosphate ◽  
And Migration ◽  
G Protein Coupled ◽  
Stimulation Of

ABSTRACT The G protein-coupled receptors S1P2/Edg5 and S1P3/Edg3 both mediate sphingosine-1-phosphate (S1P) stimulation of Rho, yet S1P2 but not S1P3 mediates downregulation of Rac activation, membrane ruffling, and cell migration in response to chemoattractants. Specific inhibition of endogenous Gα12 and Gα13, but not of Gαq, by expression of respective C-terminal peptides abolished S1P2-mediated inhibition of Rac, membrane ruffling, and migration, as well as stimulation of Rho and stress fiber formation. Fusion receptors comprising S1P2 and either Gα12 or Gα13, but not Gαq, mediated S1P stimulation of Rho and also inhibition of Rac and migration. Overexpression of Gαi, by contrast, specifically antagonized S1P2-mediated inhibition of Rac and migration. The S1P2 actions were mimicked by expression of V14Rho and were abolished by C3 toxin and N19Rho, but not Rho kinase inhibitors. In contrast to S1P2, S1P3 mediated S1P-directed, pertussis toxin-sensitive chemotaxis and Rac activation despite concurrent stimulation of Rho via G12/13. Upon inactivation of Gi by pertussis toxin, S1P3 mediated inhibition of Rac and migration just like S1P2. These results indicate that integration of counteracting signals from the Gi- and the G12/13-Rho pathways directs either positive or negative regulation of Rac, and thus cell migration, upon activation of a single S1P receptor isoform.

scholarly journals The G Protein-Coupled Receptor Kinases (GRKs) in Chemokine Receptor-Mediated Immune Cell Migration: From Molecular Cues to Physiopathology

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 75
Author(s):  
Marta Laganà ◽  
Géraldine Schlecht-Louf ◽  
Françoise Bachelerie
Keyword(s):  
G Protein ◽  
Chemokine Receptor ◽  
Immune Cell ◽  
Neutrophil Migration ◽  
G Protein Coupled Receptor ◽  
Receptor Kinases ◽  
Immune Cell Migration ◽  
And Migration ◽  
G Protein Coupled ◽  
Gpcr Desensitization

Although G protein-coupled receptor kinases (GRKs) have long been known to regulate G protein-coupled receptor (GPCR) desensitization, their more recently characterized functions as scaffolds and signalling adapters underscore that this small family of proteins governs a larger array of physiological functions than originally suspected. This review explores how GRKs contribute to the complex signalling networks involved in the migration of immune cells along chemokine gradients sensed by cell surface GPCRs. We outline emerging evidence indicating that the coordinated docking of several GRKs on an active chemokine receptor determines a specific receptor phosphorylation barcode that will translate into distinct signalling and migration outcomes. The guidance cues for neutrophil migration are emphasized based on several alterations affecting GRKs or GPCRs reported to be involved in pathological conditions.

scholarly journals Inhibition by somatostatin of amylase secretion induced by calcium and cyclic AMP in rat pancreatic acini

1994 ◽  
Vol 304 (2) ◽  
pp. 531-536 ◽  
Author(s):  
H Ohnishi ◽  
T Mine ◽  
I Kojima
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Pancreatic Acini

It has recently been shown that somatostatin inhibits amylase secretion from isolated pancreatic acini by reducing cyclic AMP (cAMP) production [Matsushita, Okabayashi, Hasegawa, Koide, Kido, Okutani, Sugimoto and Kasuga (1993) Gastroenterology 104, 1146-1152]. To date, however, little is known as to the other mechanism(s) by which somatostatin inhibits amylase secretion in exocrine pancreas. To investigate the action of somatostatin independent of cAMP generation, we examined the effect of somatostatin in isolated rat pancreatic acini stimulated by 1 microM calcium ionophore A23187 and 1 mM 8-bromo-cyclic AMP (8Br-cAMP). Somatostatin inhibited amylase secretion evoked by a combination of A23187 and 8Br-cAMP in a dose-dependent manner. The maximum inhibition was obtained by 10(-7) M somatostatin, and at this concentration somatostatin inhibited the effect of A23187 and 8Br-cAMP by approximately 30%. In electrically permeabilized acini, an elevation of free calcium concentration resulted in an increase in amylase secretion and cAMP enhanced the secretion evoked by calcium. cAMP shifted the dose-response curve for calcium-induced secretion leftwards and elevated the peak value of secretion. Somatostatin inhibited the effect of cAMP on calcium-induced amylase secretion by shifting the dose-response curve to the right. To determine the involvement of a G-protein(s), we examined the effect of somatostatin in acini pretreated with pertussis toxin. Pretreatment of acini with pertussis toxin completely blocked somatostatin-inhibition of amylase-secretion evoked by A23187 and 8Br-cAMP. These results indicate that somatostatin decreases amylase secretion induced by cAMP and calcium by reducing the calcium sensitivity of exocytosis. A pertussis toxin-sensitive G-protein is also involved in this step.

scholarly journals Regulation of fibroblast procollagen production. Transforming growth factor-β1 induces prostaglandin E2 but not procollagen synthesis via a pertussis toxin-sensitive G-protein

1995 ◽  
Vol 307 (1) ◽  
pp. 63-68 ◽  
Author(s):  
R J McAnulty ◽  
R C Chambers ◽  
G J Laurent
Keyword(s):  
Growth Factor ◽  
Prostaglandin E2 ◽  
G Protein ◽  
Pertussis Toxin ◽  
Transforming Growth Factor ◽  
Pge2 Production ◽  
Lung Fibroblasts ◽  
Extracellular Matrix Protein ◽  
Dependent Manner ◽  
Tgf Beta

Transforming growth factor-beta 1 (TGF beta 1) initiates a series of signalling events resulting in diverse cellular responses including stimulation of extracellular matrix protein production. In this study we have investigated the role of pertussis toxin-sensitive G-proteins in mediating the effects of TGF beta 1 on fibroblast procollagen metabolism. TGF beta 1 stimulated human fetal lung fibroblast procollagen synthesis and production in a dose-dependent manner which was maximal at 0.5 ng/ml. TGF beta 1 also decreased the proportion of newly synthesized procollagen degraded intracellularly. Pertussis toxin, a G-protein inhibitor, further stimulated TGF beta 1-induced procollagen synthesis and production, but alone it had no effect on fibroblast procollagen metabolism. Addition of indomethacin also potentiated the TGF beta 1-induced increase in procollagen synthesis and production. The effects of pertussis toxin and indomethacin were not additive. Pertussis toxin and indomethacin did not affect the proportion of newly synthesized procollagen degraded intracellularly, either alone or in combination, by control cells. The TGF beta 1-induced decrease in intracellular procollagen degradation was maintained but not further affected by pertussis toxin or indomethacin. TGF beta 1 increased prostaglandin E2 (PGE2) compared with PGE2 production by control cells. Addition of pertussis toxin or indomethacin blocked the TGF beta 1-induced increase in PGE2 production. The TGF beta 1-induced increase in PGE2 preceded the increase in procollagen production. These results demonstrate that TGF beta 1-induced procollagen synthesis by lung fibroblasts is modulated by production of PGE2. Pertussis toxin and indomethacin block the production of PGE2 and enhance the effect of TGF beta 1 on procollagen synthesis. From these data we conclude that the effects of TGF beta 1 on PGE2 production but not procollagen synthesis are mediated via a receptor linked to a pertussis toxin-sensitive G-protein.

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