Gi Protein Inhibitor Pertussis Toxin Research Articles

Butyrate, Valerate, and Niacin Ameliorate Anaphylaxis by Suppressing IgE-Dependent Mast Cell Activation: Roles of GPR109A, PGE2, and Epigenetic Regulation.

Short-chain fatty acids (SCFAs) are produced by the intestinal microbiota during the fermentation of dietary fibers as secondary metabolites. Several recent studies reported that SCFAs modulate the development and function of immune-related cells. However, the molecular mechanisms by which SCFAs regulate mast cells (MCs) remain unclear. In the current study, we analyzed the function and gene expression of mouse MCs in the presence of SCFAs invitro and invivo. We found that the oral administration of valerate or butyrate ameliorated passive systemic anaphylaxis and passive cutaneous anaphylaxis in mice. The majority of SCFAs, particularly propionate, butyrate, valerate, and isovalerate, suppressed the IgE-mediated degranulation of bone marrow-derived MCs, which were eliminated by the Gi protein inhibitor pertussis toxin and by the knockdown of Gpr109a. A treatment with the HDAC inhibitor trichostatin A also suppressed IgE-mediated MC activation and reduced the surface expression level of FcεRI on MCs. Acetylsalicylic acid and indomethacin attenuated the suppressive effects of SCFAs on degranulation. The degranulation degree was significantly reduced by PGE2 but not by PGD2. Furthermore, SCFAs enhanced PGE2 release from stimulated MCs. The SCFA-mediated amelioration of anaphylaxis was exacerbated by COX inhibitors and an EP3 antagonist, but not by an EP4 antagonist. The administration of niacin, a ligand of GPR109A, alleviated the symptoms of passive cutaneous anaphylaxis, which was inhibited by cyclooxygenase inhibitors and the EP3 antagonist. We conclude that SCFAs suppress IgE-mediated activation of MCs invivo and invitro involving GPR109A, PGE2, and epigenetic regulation.

Apelin-13 upregulates Egr-1 expression in rat vascular smooth muscle cells through the PI3K/Akt and PKC signaling pathways

Previous studies have shown that Apelin-13 upregulates early growth response factor-1 (Egr-1) via the extracellular signal-regulated protein kinase (ERK) signaling pathway. Apelin-13 induces proliferation and migration of vascular smooth muscle cells (VSMCs) as well as the upregulation of osteopontin (OPN) via the upregulation of Egr-1. This study was designed to further explore the activity of Apelin-13 in VSMCs by investigating members of the mitogen-activated protein kinase (MAPK) family, in particular Jun kinase (JNK) and p38 mitogen-activated protein kinase (P38). We also examined whether the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) signaling pathways were involved in the regulation of Egr-1 by Apelin-13. We treated rat aortic VSMCs with Apelin-13 and examined the expression of JNK, p-JNK, P38, and p-P38 to investigate whether Apelin-13-mediated increases in Egr-1 occurred through the JNK and P38 signaling pathways. We then pretreated VSMCs with the Gi protein inhibitor pertussis toxin (PTX) and the Gq inhibitor YM254890, added Apelin-13 and looked for changes in Egr-1 expression. Finally, we pretreated with the PI3K inhibitor LY294002 and the PKC inhibitor GF109203X, and treated with Apelin-13. Our results showed that JNK and P38 did not participate in Apelin-13-mediated increase in Egr-1. Instead, Apelin-13 upregulation of Egr-1 was mediated by a PTX-sensitive Gi protein. Apelin-13 did increase ERK phosphorylation through the PI3K/Akt and PKC signaling pathways, resulting in changes in Egr-1 expression. These data provide important targets for future studies to modulate vascular remodeling.

Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxin-sensitive Gi-proteins in murine BV-2 microglia cells.

BackgroundActivation of metabotropic glutamate receptor 5 (mGluR5) by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators. In contrast, the consequences of mGluR5 inhibition are less well understood. Here, we used BV-2 cells, retaining key characteristics of primary mouse microglia, to examine whether mGluR5 inhibition by 2-methyl-6-(phenylethynyl)-pyridine (MPEP) enhances cellular stress and production of inflammatory mediators.MethodsBV-2 cells were treated with MPEP, followed by determination of cellular stress using fluorescent dyes and high-content imaging. The expression of inflammatory mediators, endoplasmic reticulum (ER)-stress markers and phosphorylated AMPKα was analyzed by quantitative PCR, ELISA and Western blotting. Additionally, phospholipase C (PLC) activity, cellular ATP content and changes in intracellular free Ca2+ ([Ca2+]i) were measured using luminescence and fluorescence assays.ResultsTreatment of BV-2 microglia with 100 μM MPEP increased intracellular reactive oxygen species (ROS), mitochondrial superoxide, mitochondrial mass as well as inducible nitric oxide synthase (iNOS) and IL-6 expression. Furthermore, MPEP reduced cellular ATP and induced AMPKα phosphorylation and the expression of the ER-stress markers CHOP, GRP78 and GRP96. The MPEP-dependent effects were preceded by a rapid concentration-dependent elevation of [Ca2+]i, following Ca2+ release from the ER, mainly via inositol triphosphate-induced receptors (IP3R). The MPEP-induced ER-stress could be blocked by pretreatment with the chemical chaperone 4-phenylbutyrate and the Ca2+ chelator BAPTA-AM. Pretreatment with the AMPK agonist AICAR partially abolished, whilst the inhibitor compound C potentiated, the MPEP-dependent ER-stress. Importantly, the PLC inhibitor U-73122 and the Gi-protein inhibitor pertussis toxin (PTX) blocked the MPEP-induced increase in [Ca2+]i. Moreover, pretreatment of microglia with AICAR, BAPTA-AM, U-73122 and PTX prevented the MPEP-induced generation of oxidative stress and inflammatory mediators, further supporting a role for Gi-protein-mediated activation of PLC.ConclusionsThe results emphasize the potential pathophysiological role of mGluR5 antagonism in mediating oxidative stress, ER-stress and inflammation through a Ca2+-dependent pathway in microglia. The induction of cellular stress and inflammatory mediators involves PTX-sensitive Gi-proteins and subsequent activation of PLC, IP3R and Ca2+ release from the ER.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0190-7) contains supplementary material, which is available to authorized users.

Gz- and not Gi-proteins are coupled to pre-junctional μ-opioid receptors in bovine airways

We investigated the signal transmission pathway by which activation of μ-opioid receptors attenuates acetylcholine (ACh) release in bovine trachealis. Electrical stimulation (ES)-induced [3H]-ACh release was determined in bovine tracheal smooth muscle strips pre-incubated with either the Gi-protein inhibitor pertussis toxin (PTX, 500ng/ml and 1μg/ml) or the Gz-protein specific inhibitor arachidonic acid (AA, 10−6M and 10−5M) and then treated with DAMGO (D-Ala2,N-MePhe4,Gly-ol5-enkephalin) 10−5M. Indomethacin 10−5M was used to block AA cascade. The inhibitory effect of DAMGO on ES-induced [3H]-ACh release was PTX-insensitive, but, by contrast, ablated by AA in a concentration-dependent manner. AA 10−5M alone reduced [3H]-ACh release, an effect that was prevented by iberiotoxin 10−7M, suggesting an involvement of Ca2+-activated K+-channels. Western blot analysis consistently showed immunoreactive bands against a specific antibody anti-Gz-α subunit at ∼40kDa, consistent with the presence of Gz-protein. The present findings suggest that in isolated bovine trachealis, activation of μ-opioid receptors inhibits ACh-release through a signal transmission pathway involving Gz-protein rather than Gi-protein.

Cross-Talk between Transforming Growth Factor–β1 and Muscarinic M2 Receptors Augments Airway Smooth Muscle Proliferation

Transforming growth factor-β₁ (TGF-β₁) is a central mediator in tissue remodeling processes, including fibrosis and airway smooth muscle (ASM) hyperplasia, as observed in asthma. The mechanisms underlying this response, however, remain unclear because TGF-β₁ exerts only weak mitogenic effects on ASM cells. In this study, we hypothesized that the mitogenic effect of TGF-β₁ on ASM is indirect and requires prolonged exposure to allow for extracellular matrix (ECM) deposition. To address this hypothesis, we investigated the effects of acute and prolonged treatment with TGF-β₁, alone and in combination with the muscarinic receptor agonist methacholine, on human ASM cell proliferation. Acutely, TGF-β₁ exerted no mitogenic effect. However, prolonged treatment (for 7 d) with TGF-β₁ increased ASM cell proliferation and potentiated the platelet-derived growth factor-induced mitogenic response. Muscarinic receptor stimulation with methacholine synergistically enhanced the effect of TGF-β₁. Interestingly, the integrin-blocking peptide Arg-Gly-Asp-Ser, as well as integrin α5β1 function-blocking antibodies, inhibited the effects of TGF-β₁ and its combination with methacholine on cell proliferation. Accordingly, prolonged treatment with TGF-β₁ increased fibronectin expression, which was also synergistically enhanced by methacholine. The synergistic effects of methacholine on TGF-β₁-induced proliferation were reduced by the long-acting muscarinic receptor antagonist tiotropium and the M₂ receptor subtype-selective antagonist gallamine, but not the M₃-selective antagonist DAU5884. In line with these findings, the irreversible Gi protein inhibitor pertussis toxin also prevented the potentiation of TGF-β₁-induced proliferation by methacholine. We conclude that prolonged exposure to TGF-β₁ enhances ASM cell proliferation, which is mediated by extracellular matrix-integrin interactions, and which can be enhanced by muscarinic M₂ receptor stimulation.

Sphingosine‐1‐phosphate modulates expression of vascular endothelial growth factor in human articular chondrocytes: a possible new role in arthritis

Although sphingosine-1-phosphate (S1P) is suggested to have an important role in arthritis, its function in chondrocytes remains unknown. In contrast, vascular endothelial growth factor (VEGF) has been speculated to contribute to the pathogenesis of osteoarthritis (OA), most likely by regulating angiogenesis. We here investigated the in vitro effect of S1P on VEGF expression in human articular chondrocytes from OA patients. Human articular cartilage samples were obtained from patients with OA under informed consent. Chondrocytes were isolated by an enzymatic procedure, grown in monolayer culture, and then stimulated with S1P in the presence or absence of mitogen-activated protein kinase (MAPK) inhibitors or the Gi protein inhibitor pertussis toxin (PTX). VEGF expression and secretion in culture supernatants were analyzed using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Although S1P did not enhance basal secretion of matrix metalloproteinase (MMP)-1 and MMP-13, it stimulated VEGF expression in human articular chondrocytes, both at the messenger RNA and protein levels. MAPK inhibitors SB203580 and PD98059 were not effective at suppressing VEGF induction; rather, blocking extracellular signal-regulated kinase (ERK) MAPK enhanced VEGF expression. The Gi protein inhibitor PTX partially attenuated S1P-induced VEGF secretion. Our results suggest that S1P may contribute to the regulation of VEGF expression in human chondrocytes. S1P may therefore play a unique role in the pathophysiology of OA by regulating VEGF expression in chondrocytes.

Inhibition of Chemokine (CXC Motif) Ligand 12/Chemokine (CXC Motif) Receptor 4 Axis (CXCL12/CXCR4)-mediated Cell Migration by Targeting Mammalian Target of Rapamycin (mTOR) Pathway in Human Gastric Carcinoma Cells

CXCL12/CXCR4 plays an important role in metastasis of gastric carcinoma. Rapamycin has been reported to inhibit migration of gastric cancer cells. However, the role of mTOR pathway in CXCL12/CXCR4-mediated cell migration and the potential of drugs targeting PI3K/mTOR pathway remains unelucidated. We found that CXCL12 activated PI3K/Akt/mTOR pathway in MKN-45 cells. Stimulating CHO-K1 cells expressing pEGFP-C1-Grp1-PH fusion protein with CXCL12 resulted in generation of phosphatidylinositol (3,4,5)-triphosphate, which provided direct evidence of activating PI3K by CXCL12. Down-regulation of p110β by siRNA but not p110α blocked phosphorylation of Akt and S6K1 induced by CXCL12. Consistently, p110β-specific inhibitor blocked the CXCL12-activated PI3K/Akt/mTOR pathway. Moreover, CXCR4 immunoprecipitated by anti-p110β antibody increased after CXCL12 stimulation and G(i) protein inhibitor pertussis toxin abrogated CXCL12-induced activation of PI3K. Further studies demonstrated that inhibitors targeting the PI3K/mTOR pathway significantly blocked the chemotactic responses of MKN-45 cells triggered by CXCL12, which might be attributed primarily to inhibition of mTORC1 and related to prevention of F-actin reorganization as well as down-regulation of active RhoA, Rac1, and Cdc42. Furthermore, rapamycin inhibited the secretion of CXCL12 and the expression of CXCR4, which might form a positive feedback loop to further abolish upstream signaling leading to cell migration. Finally, we found cells expressing high levels of cxcl12 were sensitive to rapamycin in its activity inhibiting migration as well as proliferation. In summary, we found that the mTOR pathway played an important role in CXCL12/CXCR4-mediated cell migration and proposed that drugs targeting the mTOR pathway may be used for the therapy of metastatic gastric cancer expressing high levels of cxcl12.

Neuropeptides Orexin A and B Are Funktionally Aktive in CD34+ Hematopoietic Stem and Progenitor Cells.

Abstract 4593Orexin receptors are involved in the regulation of sleep-wake-rhythm, food intake and energy homeostasis and it was still recently believed that their expression is restricted to the nervous system. But, during the last years orexin receptors have been detected in an increasing number of peripheral tissues. We have earlier found orexin receptor 1 and 2 expression on human CD34+ hematopoietic stem and progenitor cells. Still, the sources of their physiological ligands, the peptides orexin A and B, seemed so far to be restricted to the central nerve system. Ca2+-dependent signaling and activation of mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (ERK1/2) pathways are considered as main downstream signaling pathways of the orexin receptors.In this study, we investigated the signaling and functional role of orexin receptors in CD34+ hematopoietic stem and progenitor cells.Using confocal fluorescence microscopy and flow cytometry we found that stimulation of purified CD34+ cells with orexin A and B led to an increase of the intracellular calcium concentration due to both calcium influx and calcium release from intracellular stores. Of interest, incubation with orexin reduces the SDF-1β-induced calcium influx. Furthermore orexin receptor stimulation led to a decrease of the intracellular cAMP concentration. Following orexin receptor stimulation with orexin A and B, we observed an initial increase of ERK1/2 phosphorylation up to 30 minutes upon incubation with orexin followed by a decrease at several time points up to 8 hours in comparison to the unstimulated control.To investigate a potential impact on the functional properties of human CD34+ cells we performed proliferation and apoptosis assays, migration and adhesion assays as well as colony forming and long-term culture assays. Remarkably, stimulation with orexin A and B led to a significant higher proportion of early pluripotent hematopoietic progenitor (CFU-GEMM) colonies and a significant reduction of erythroid precursors. A more immature phenotype of orexin-stimulated CD34+ cells is also reflected by array-based gene expression profiling. Long-term culture assays revealed a significant higher frequency of LTC-IC indicating also a more immature phenotype of orexin-stimulated cells. In line, orexin receptor stimulation led to a significant increase of the proportion of Lin-, CD34+, CD38- HSC in the G0-phase of the cell cycle. Furthermore, stimulation with orexin A and B increased the number of apoptotic cells in the Lin-, CD34+, CD38- HSC fraction and the total hematopoietic stem and progenitor population determined by flowcytometric analysis of intracellular cleaved caspase 3 content. The adhesive capacity of CD34+ cells to fibronectin and collagen coated dishes and the migratory capacity was significantly decreased upon orexin receptor stimulation. Concurrent incubation with the selective Gi-protein inhibitor pertussis toxin abrogated these effects. Given the functional impact of the orexin system on CD34+ cells, we asked if orexins are secreted locally in the bone marrow or autocrine by CD34+ cells or if they are humorally transported to the bone marrow cavity. Using FACS analysis, immunfluorescent staining and western blotting we could detect prepro-Orexin in CD34+ cells and using ELISA orexin was found in the serum obtained by bone marrow biopsies and peripheral blood. Taken together, the phenotype of orexin-stimulated hematopoietic stem and progenitor cells suggest a mobilizing effect of the orexin receptor stimulation as well as an increased repopulation capacity which might be of relevance in clinical stem cell mobilization and transplantation and is currently verified in murine models. Disclosures:No relevant conflicts of interest to declare.

FcεRI-mediated mast cell migration: Signaling pathways and dependence on cytosolic free Ca 2+ concentration

IgE-sensitized rat basophilic leukemia (RBL)-2H3 mast cells have been shown to migrate towards antigen. In the present study we tried to identify the mechanism by which antigen causes mast cell migration. Antigen caused migration of RBL-2H3 cells at the concentration ranges of 1000-fold lower than those required for degranulation and the dose response was biphasic. This suggests that mast cells can detect very low concentration gradients of antigen (pg/ml ranges), which initiate migration until they degranulate near the origin of antigen, of which concentration is in the ng/ml ranges. Similar phenomenon was observed in human mast cells (HMCs) derived from CD34 + progenitors. As one mechanism of mast cell migration, we tested the involvement of sphingosine 1-phosphate (S1P). FcεRI-mediated cell migration was dependent on the production of S1P but independent of a S1P receptor or its signaling pathways as determined with S1P receptor antagonist VPC23019 and Gi protein inhibitor pertussis toxin (PTX). This indicated that the site of action of S1P produced by antigen stimulation was intracellular. However, S1P-induced mast cell migration was dependent on S1P receptor activation and inhibited by both VPC23019 and PTX. Cell migration towards antigen or extracellular S1P was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, while only migration towards antigen was inhibited by the inhibitors of sphingosine kinase and phospholipase C (PLC) and intracellular calcium chelator BAPTA. In summary, our data suggest that the high affinity receptor for IgE (FcεRI)-mediated mast cell migration is dependent on the production of S1P but independent of S1P receptors. Cell migration mediated by either FcεRI or S1P receptors involves activation of both PI3K and MAPK.

Cardiomyocyte S1P1 Receptor-mediated Extracellular Signal-related Kinase Signaling and Desensitization

We examined the ability of sphingosine-1-phosphate (S1P) to desensitize extracellular signal-related kinase (ERK), a mitogen-activated protein kinase linked to antiapoptotic responses in the heart. In isolated adult mouse cardiomyocytes, S1P (10 nM-5 microM) induced ERK phosphorylation in a time- and dose-dependent manner. S1P stimulation of ERK was completely inhibited by an S1P1/3 subtype receptor antagonist (VPC23019), by a Gi protein inhibitor (pertussis toxin) and by a mitogen-activated protein kinase/ERK kinase inhibitor (PD98059). A selective S1P3 receptor antagonist (CAY10444) had no effect on S1P-induced ERK activation. The selective S1P1 agonist SEW2871 also induced ERK phosphorylation. Activation of ERK by restimulation with 100 nM S1P was suppressed after 1 hour of preincubation with 100 nM S1P but recovered fully the next day, suggesting receptor recycling. Similar results were obtained in protein kinase C epsilon-null cardiomyocytes. Treatment with the nonselective S1P receptor agonist FTY720 for 1 hour also reduced phospho-ERK expression in response to subsequent S1P stimulation. In contrast to S1P, some desensitization to FTY720 persisted after overnight exposure. Cell death induced by hypoxia/reoxygenation was reduced by pretreatment with exogenous S1P. This enhanced survival was abrogated by pretreatment with PD98059, VPC23019, or pertussis toxin. Thus, exogenous S1P induces rapid and reversible S1P1-mediated ERK phosphorylation. S1P-induced adult mouse cardiomyocyte survival requires ERK activation mediated via an S1P1-Gi pathway.

Abstract 237: Acute Activation of eNOS by Lovastatin Involves Scavenger Receptor-B1

Statins (HMG CoA reductase inhibitors) have beneficial vascular effects independent of their ability to reduce cholesterol synthesis. We and others have previously shown that statins acutely activate endothelial cell NOS (eNOS). Our present study characterized signaling pathways by which statins activate NOS using cultured bovine aortic endothelial cells (BAEC) and COS 7 cells. It also examined involvement of scavenger receptor - B1 (SR-B1), which is expressed in endothelial cells and maintains caveolae cholesterol concentration and eNOS localization. BAEC were exposed to lovastatin (LOV, 10 −6 M) for 2–5 min after pretreatment with the end product of HMG CoA reductase (mevalonic acid, 5×10 −4 M), a Gi protein inhibitor (pertussis toxin, 10 −4 M), a PLC inhibitor (U-73122, 10 −5 M), a receptor tyrosine kinase (RTK) inhibitor (tyrphostine 51A, 2×10 −6 M) or intracellular and extracellular calcium chelators -BAPTA-AM (10 −5 M) and EGTA (10 −4 M), respectively, and levels of NO production were determined. LOV alone acutely increased NO production by 67%. NO production in response to LOV was not blocked by mevalonic acid indicating that it occurs independent of HMG CoA reductase inhibition. Inhibition of Gi protein, PLC, and RTK almost completely blocked the LOV-induced NO generation. Removal of extracellular Ca ++ prevented statin-induced NO production by 90%, while intracellular Ca ++ chelation with BAPTA-AM reduced NO production by 45%. COS 7 cells transfected with eNOS did not show increased NO production to LOV or HDL, indicating NOS activation occurs through an endothelial cell receptor. In COS 7 cells, co-transfected with eNOS and the endothelial SR-B1, LOV or HDL (10 −5 M), a known agonist of SR-B1, increased NO production by over 200%. In COS 7 cells transfected with eNOS alone or co-transfected with the bradykinin B2 receptor, neither LOV nor HDL increased NO production, indicating effects specific for the SR-B1 receptor. In conclusion, our study shows LOV acutely activates eNOS independently of HMG CoA reductase inhibition. eNOS activation appears to involve an endothelial Gi protein receptor and subsequent activation of PLC and RTK pathways, leading to entry of extracellular calcium. Furthermore, LOV can activate eNOS via the SR-B1 receptor.

The Chemokine Receptor CXCR6 and Its Ligand CXCL16 Are Expressed in Carcinomas and Inhibit Proliferation

The chemokine receptor CXCR6 and its ligand CXCL16 are involved in inflammation. Thus far, they were known to be expressed mainly by T cells and macrophages, respectively. However, we detected both in all of 170 human primary mammary carcinomas and at similar levels in all 8 human mammary carcinoma cell lines tested by microarray analysis. Expression was confirmed by reverse transcription-PCR and for the cell lines also by fluorescence-activated cell sorting analysis. CXCR6 and CXCL16 were also detected in several mouse and human mammary, colon, and pancreatic carcinoma cell lines. CXCL16 is a transmembrane protein from which the soluble chemokine can be cleaved off. The transmembrane form is present on the surface of the carcinoma cells. Surprisingly, suppression of either CXCR6 or CXCL16 led to greatly enhanced proliferation in vitro as well as in vivo, indicating that their interaction inhibits proliferation. This notion was verified using inhibitory antibodies and by introduction of CXCL16 into a rare CXCL16-negative cell line. The effect was mediated by the G protein-coupled receptor CXCR6 because it was blocked by the G(i) protein inhibitor pertussis toxin. In contrast, the soluble CXCL16 chemokine enhanced proliferation, and this was also mediated by CXCR6 but not via G(i) protein. It is remarkable that both CXCR6 and CXCL16 are expressed by all mammary carcinomas because cells that lose either acquire a growth advantage and should be selected during tumor progression. This suggests an unknown important role in tumor formation. Proteases, possibly macrophage derived, might convert inhibitory transmembrane CXCL16 into the stimulatory chemokine.

Insights into the Biology of Mobilized Cells through Innovative Treatment Schedules of the CXCR4 Antagonist AMD3100.

The CXCR4 antagonist AMD3100 mobilizes hematopoietic stem/progenitor cells (HSPC), but most available data are on combination with G-CSF, rather than its use as a single modality for HSPC mobilization and transplantation. Because of low efficacy of the standard single-dose regimen, we additionally tested in mice the effect of pulsed or continuous AMD3100-infusion: Repeat injections as little as 4 hours apart for up to 6 days did not result in CFU-C accumulation or curtail the size of AMD3100-responsive HSPC pools. In contrast to the 3-fold increase in circulating CFU-C after bolus or repeat injections, after 5 days of continuous AMD3100-infusion circulating CFU-C were increased 30-fold over baseline, from 301±41/mL to 10,687±757/mL compared to 1,088±75/mL after a bolus. These numbers were sustained at least until day 8 and, of interest, were not further augmented by injection of an AMD3100-bolus, suggesting satiating doses of AMD3100-infusion. Furthermore, at least equally efficient mobilization by AMD3100-bolus or -infusion was documented in splenectomized hosts, indicating that the contribution of spleen to mobilization by AMD3100 is negligible. Long-term repopulating cells in AMD3100-infusion-mobilized blood, assessed by limiting dilution transplantation, were also increased, to 273(205–362) CRU/mL, vs. 77(56–106) CRU/mL after bolus. Of interest, the number of HSPC mobilized by AMD3100-infusion was much higher than by standard twice-daily dosing of G-CSF, and AMD3100 effectively mobilized two G-CSF resistant/refractory mouse models (G-CSFR−/−, Plasminogen−/−), while not surprisingly, mice treated with the Gi protein inhibitor Pertussis toxin were resistant to AMD3100-induced mobilization. Subsequent studies addressed the phenotype and the in vitro and in vivo functional behavior of AMD3100-mobilized HSPC. The immunophenotype of AMD3100-mobilized c-kit+ cells differed significantly from that of steady-state bone marrow (BM) c-kit+ cells. Thus expression of several integrins was lower, while CD26 expression was significantly higher than on BM c-kit+ cells. AMD3100-infusion-mobilized blood and BM CFU-C migrated equally efficiently towards SDF-1 in vitro. In vivo marrow homing of AMD3100-infusion-mobilized blood CFU-C was >50% increased over BM CFU-C, similarly to what we recently showed for G-CSF-infusion-mobilized blood. These properties differ from those of in vitro AMD3100-incubated BM-HSPC, in which migration was blocked, and homing was normal or reduced. Kinetics of hematopoietic recovery after transplantation of 100 μl AMD3100- or G-CSF-infusion-mobilized blood to lethally irradiated hosts were similar except for delayed platelet recovery in AMD3100-mobilized blood recipients. Partial BrdU labeling experiments for up to 3 days before mobilization (resulting in 20–40% BrdU labeling of c-kit+ cells in BM) documented that labeled cells were consistently underrepresented (<5%) among AMD3100-mobilized c-kit+ cells. These data defend the notion that only non-cycling HSPC are found in mobilized blood (>80% of AMD3100-mobilized c-kit+ cells are in G0), but extend it to conclude that such a status precedes their exit from BM, rather than being a consequence of modification in the blood environment. In summary, we are presenting a novel efficacious mobilization scheme which fully supports transplantation demands in normal and G CSF-refractory donors. Furthermore, our data expand previous knowledge about size and turn-over of AMD3100-mobilizable HSPC pools.

The Interaction of CXCR4/SDF-1 and VLA-4 Regulates Adhesion and Transendothelial Migration in Waldenstrom Macroglobulinemia.

Background. Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM). Adhesion of malignant cells to the BM microenvironment induces proliferation and resistance to therapy. We previously showed that SDF-1/CXCR4 axis regulates migration and adhesion of WM cells in vitro indicating a potential role in homing. Homing of malignant cells to the BM niches requires active navigation through the endothelial cell barrier where tumor cells must adhere, roll and transmigrate. We hypothesized that CXCR4 and its interaction with VLA-4 is critical for this 3-step process.Methods. The level of CXCR4 and VLA-4 was determined using flow cytometry and RT-PCR in patient samples and WM cell lines (BCWM.1 and WM-WSU). The CXCR4 inhibitor AMD3100 (10–100uM, Sigma, MO), Gi protein inhibitor pertussis toxin, PTX (10–200ng/ml, Sigma, MO) were used to inhibit CXCR4 signaling. These studies were confirmed using CXCR4 knockdown using shRNA with lentivirus infection. Adhesion assay was done with either plates coated with the VLA-4 ligand fibronectin for adhesion of tumor cells in presence and absence of SDF1 (30nM), or coated with endothelial cells (HUVEC) and BMSCs for cell-cell adhesion experiments. Transmigration assay was performed using the transwell system with either SDF1 in the lower chamber to study migration towards SDF1, or with the upper chamber coated overnight with endothelial cells (HUVEC) and BMSCs to study transmigration. Experiments with HUVEC cells were performed in absence or presence of TNF-alpha that activates the HUVEC cells.Results. WM tumor cells from patients and cell line expressed high surface levels of CXCR4 (mean 70%) and VLA-4 (mean 95%). Adhesion of WM cells to fibronectin was significantly increased with SDF1 compared to BSA control, and AMD3100 20uM inhibited adhesion by 50% compared to control. Similar results were obtained with PTX 200ng/mL and using CXCR4 knockdown in WM cells. Furthermore, WM cells adhesion was greatly enhanced in co-culture with BMSCs and endothelial cells, and was significantly inhibited using AMD3100 and PTX. To further dissect the mechanisms of interaction of CXCR4 and VLA-4, we showed that AMD3100 inhibited surface expression of CXCR4 but not of VLA-4. Immunoprecipitation studies showed that CXCR4 and VLA-4 directly interact in response to SDF-1. Downstream signaling pathways showed that CXCR4 regulates migration and adhesion through the PI3K/Akt and ERK/MAPK pathways. We next studied whether CXCR4 was involved in transmigration through the endothelial barrier. We demonstrated that BCWM.1 cells transmigrated through the barrier of endothelial cells, that was increased when SDF1 was added to the lower chamber, and that AMD3100 inhibited the transmigration of BCWM.1 cells. Studies of the role of CXCR4 in rolling on endothelial cells is ongoing.Conclusion. We showed CXCR4/SDF1 axis regulates migration and adhesion of WM cells to the BM microenvironment indicating a potential role in homing. Moreover, we demonstrate that CXCR4/SDF1 axis is critical for transendothelial migration, a critical step of homing and egression of tumor cells in and out the BM niches.

The CXCR4/SDF-1 Axis Regulates Migration and Adhesion in Waldenstrom Macroglobulinemia.

Background: Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM), and lymphadenopathy in 20% of the patients, implying continuous trafficking of WM cells into and out of the BM and lymph nodes. The normal process of B-cell homing is regulated by cytokines, chemokines, and adhesion molecules. One of the most extensively studied chemokines in migration is stromal derived factor SDF-1 and its receptor CXCR4. Here we study the role of chemokine receptors, and the SDF-1/CXCR4 axis on migration and adhesion in WM.Methods: Flow cytometry for CXC and CC chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR2, CCR4, CCR5, CCR6 and CCR7), and adhesion molecules (VLA-4 and LFA-1) on WM cell lines (BCWM.1 and WM-WSU) and patient samples was performed. Migration was determined using the transwell migration assay (Costar, NY). Cells were placed in the upper chambers of the migration assay with 1% FCS medium in the presence of serial concentrations of SDF-1 in the lower chambers. After 4 hours of incubation, cells that migrated to the lower chambers were counted. Similarly, adhesion was determined using an adhesion assay (EMD Biosciences, San Diego, CA) with 96-well plated coated with fibronectin. Immunoblotting for proteins downstream of CXCR4 was performed. The CXCR4 inhibitor AMD3100 (10–100uM, Sigma, MO) and Gi protein inhibitor pertussis toxin PTX (10–200ng/ml, Sigma, MO) were used to inhibit CXCR4 signaling.Results: The following chemokine receptors were expressed on patient CD19+WM cells with over 30% expression: CXCR1 (mean 60%), CXCR2 (mean 47%), CXCR4 (mean 47%), CXCR5 (mean 69%), CCR4 (mean 54%) and CCR6 (mean 61%). Similar expression was observed on WM cell lines. We next determined the effect of SDF-1 on migration and signaling pathways in WM. SDF-1 (10–100nM) induced migration in a bell-shaped curve with 30nM inducing maximum migration (110% compared to control). SDF-1 30nM induced a rapid activation of signaling pathways downstream of CXCR4 including pERK1/2, pAKT, and pPKC at 1 min, with maximum activation at 5min. The CXCR4 inhibitor AMD3100 inhibited migration of BCWM.1 in the presence of 30nM SDF-1, with AMD3100 10uM inhibiting migration at 59% of control, and 20 to 50uM leading to a plateau in inhibition of migration at 54% of control. AMD3100 inhibited pERK and pPKC activation, downstream of CXCR4 in a dose-dependent fashion. Similar results were observed using PTX, with inhibition of migration of WM cells at 50% compared to control. To determine the role of SDF-1 on adhesion, we first demonstrated that WM cells from patients and cell lines expressed high levels of surface VLA-4 expression (mean 95% surface expression). WM cells had an increase in adhesion to fibronectin (VLA-4 ligand) compared to BSA control. AMD3100 10uM inhibited adhesion to fibronectin (63 % of control), indicating that the SDF-1/CXCR4 axis regulates adhesion.Conclusion: CXCR4 is highly expressed on WM cells and regulates migration and adhesion, indicating a potential role in regulating WM trafficking into the BM and lymph nodes. These studies provide the preclinical framework to study CXCR4 inhibitors in the regulation of homing and adhesion in WM.

Cardioprotective effects of acute and chronic opioid treatment are mediated via different signaling pathways

A 5-day exposure to morphine exerts a profound cardioprotective phenotype in murine hearts. In the present study, we examined mechanisms by which morphine generates this effect, exploring the roles of G(i) and G(s) proteins, PKA, PKC, and beta-adrenergic receptors (beta-AR) in acute and chronic opioid preconditioning. Langendorff-perfused hearts from placebo, acute morphine (AM; 10 micromol/l)-, or chronic morphine (CM)-treated mice (75-mg pellet, 5 days) underwent 25-min ischemia and 45-min reperfusion. After reperfusion, placebo-treated hearts exhibited marked contractile and diastolic dysfunction [rate-pressure product (RPP), 40 +/- 4% baseline; end-diastolic pressure (EDP), 33 +/- 3 mmHg], whereas AM hearts showed significant improvement in recovery of RPP and EDP (60 +/- 3% and 23 +/- 4 mmHg, respectively; P < 0.05 vs. placebo). Furthermore, CM hearts demonstrated a complete return of diastolic function and significantly greater recovery of contractile function (83 +/- 3%, P < 0.05 vs. both placebo and AM). Pretreatment with G(i) protein inhibitor pertussis toxin abolished AM protection while partially attenuating CM recovery (P < 0.05 vs. placebo). Treatment with G(s) inhibitor NF-449 did not affect AM preconditioning yet completely abrogated CM preconditioning. Similarly, PKA inhibition significantly attenuated the ischemia-tolerant state afforded by CM, whereas it was ineffective in AM hearts. PKC inhibition with chelerythrine was ineffective in CM hearts while completely abrogating AM preconditioning. Moreover, whereas beta(1)-AR blockade with CGP-20712A failed to alter recovery in CM hearts, the beta(2)-AR antagonist ICI-118,551 significantly attenuated postischemic recovery. These data describe novel findings whereby CM preconditioning is mediated by a PKC-independent pathway involving PKA, beta(2)-AR, and G(s) proteins, whereas AM preconditioning is mediated via G(i) proteins and PKC.

The Pro-inflammatory Mediator Leukotriene D4 Induces Phosphatidylinositol 3-Kinase and Rac-dependent Migration of Intestinal Epithelial Cells

Inflammatory bowel diseases are associated with increased risk of developing colon cancer. A possible role of the pro-inflammatory leukotriene D4 (LTD4) in this process has been implicated by the findings that LTD4 can signal increased proliferation and survival, both hallmarks of a cancer cell, in non-transformed intestinal epithelial cells. Here we make the novel finding that LTD4 can also signal increased motility in these cells. In parallel, we found that LTD4 induced a simultaneous transient 10-fold increase in Rac but not Cdc42 activity. These data were also supported by the ability of LTD4 to activate the Rac GDP/GTP exchange factor Vav2. Further, LTD4 triggered a 3-fold transient increase in phosphatidylinositol 3-kinase (PI3K) phosphorylation, a possible upstream activator of the Vav2/Rac signaling pathway. The activation of Rac was blocked by the PI3K inhibitors LY294002 and wortmannin and by transfection of a kinase-negative mutant of PI3K or a dominant-negative form of Vav2. Furthermore, Rac was found to co-localize with actin in LTD4-generated membrane ruffles that were formed by a PI3K-dependent mechanism. In accordance, the inhibition of the PI3K and Rac signaling pathway also blocked the LTD4-induced migration of the intestinal cells. The present data reveal that an inflammatory mediator such as LTD4 cannot only increase proliferation and survival of non-transformed intestinal epithelial cells but also, via a PI3K/Rac signaling pathway, trigger a motile response in such cells. These data demonstrate the capacity of inflammatory mediators to participate in the process by which inflammatory bowel conditions increase the risk for colon cancer development.

Tumor necrosis factor reduces cAMP production in rat microglia.

cAMP has been reported to exert a neuroprotective role in several in vivo and in vitro models of brain pathologies, mainly by regulating microglial activation and orienting these cells toward a neuroprotective phenotype. In order to elucidate the intracellular pathways regulated by tumor necrosis factor (TNF) in glial cells, I have studied the modulation of cAMP accumulation by TNF in microglia and astrocyte cultures obtained from the neonatal rat brain. Pre-treatment of microglia with TNF reduced in a dose- and time-dependent manner cAMP accumulation induced by forskolin (FSK), in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). The TNF inhibitory action was 90% reverted by a neutralizing polyclonal anti-TNF antibody and was not prevented by a 16 h pre-treatment of microglial cultures with the Gi protein inhibitor pertussis toxin (PTx). These results suggest that TNF acts at a step of the cAMP transduction pathway other than receptors, G proteins, and phosphodiesterases. The target of TNF appeared to be adenylyl cyclase, whose ability to synthesize cAMP was markedly reduced (up to 50%) in membranes prepared from TNF-treated microglial cells, both in basal conditions and after stimulation with FSK. TNF induced a time-dependent degradation of IkappaB-alpha in microglial cells that was reverted by two inhibitors of nuclear factor kappaB activation, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-CBZ-Leu-Leu-Leu-al (MG132). The same inhibitors also markedly prevented the reduction of FSK-evoked cAMP accumulation by TNF, suggesting the involvement of NFkappaB in the regulation of adenylyl cyclase by TNF in microglia. Conversely, cAMP accumulation in astrocytes was not affected by TNF. Based on these findings, it is proposed that the ability of TNF to inhibit cAMP synthesis in microglia may exacerbate its response and contribute to cell damage in neuroinflammation and neurodegeneration, possibly through enhanced release of proinflammatory and/or cytotoxic factors.

P2X receptor activation elicits transporter-mediated noradrenaline release from rat hippocampal slices.

This study was designed to test the hypothesis of whether activation of presynaptic P2X receptor-gated ion channels elicits noradrenaline release from central catecholaminergic terminals. ATP, alpha,beta-methylene-adenosine 5'-triphosphate (alpha,beta-methyleneATP), and ADP elicited concentration-dependent [3H]noradrenaline outflow from superfused rat hippocampal slices with the following rank order of agonist potency: alpha,beta-methyleneATP > ATP > ADP. Among P2 receptor antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (30 microM), 4,4',4",4"'-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid (100 nM), and 8,8'-[carbonybis(imino-3,1-phenylenecarbonylimino)]bis1,3,5-naphthalenetrisulphonic acid (10 microM) significantly inhibited the outflow of [3H]noradrenaline, evoked by ATP, whereas Brilliant Blue G (100 nM), 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetraammonium (10 microM), the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (250 nM), and the A2A receptor antagonist 3,7-dimethyl-1-propargylxanthine (250 nM) were ineffective. Pretreatment with the Gi protein inhibitor pertussis toxin (2.5 microg/ml) did not change the effect of ATP on [3H]noradrenaline outflow. In contrast, a decrease in extracellular pH from 7.4 to 6.6 significantly attenuated the response by ATP. When extracellular Na+ was replaced by choline chloride and in the presence of the noradrenaline uptake inhibitor desipramine (10 microM), the ATP-evoked [3H]noradrenaline outflow was almost completely abolished, indicating that its underlying mechanism is the sodium-dependent reversal of the noradrenaline transporter. Reverse transcription-polymerase chain reaction analysis revealed that mRNA encoding P2X1, P2X2, P2X3, P2X4, P2X6, P2X7 and P2Y1 receptor subunits were expressed in the brainstem containing catecholaminergic nuclei projecting to the hippocampus, whereas mRNA encoding P2X5, P2Y2, P2Y4, and P2Y6 receptors were absent. Taken together, these results indicate that noradrenergic terminals of the rat hippocampus are equipped with presynaptic facilitatory P2X receptors, displaying a pharmacological profile similar to homomeric P2X1 and P2X3 receptors.

Calcium-sensing receptor-mediated activation of phospholipase C-γ1 is downstream of phospholipase C-β and protein kinase C in MC3T3-E1 osteoblasts

Elevated extracellular calcium (Ca e) stimulates both chemotaxis and mitogenesis of MC3T3-E1 osteoblasts via a calcium-sensing receptor (CasR). Ca e-mediated chemotaxis of these bone-forming cells is dependent on phospholipase C (PLC) and blocked by the Gi-protein inhibitor pertussis toxin. In this study, we examine the signaling mechanisms by which the CasR stimulates PLC activity in MC3T3-E1 osteoblasts. We found that elevated Ca e stimulated PLC-γ1 tyrosine phosphorylation in a time-dependent and Ca e-concentration-dependent manner. The maximal increase in PLC-γ1 tyrosine phosphorylation was observed 3–5 min after increasing Ca e by 3.2 mmol/L from 1.8 mmol/L. Elevated Ca e also promoted a rapid increase in both inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3], a second messenger formed by PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate, and cytosolic free calcium ([Ca +2] i). The kinetics of the CasR-mediated increases in Ins(1,4,5)P 3 and [Ca +2] i and the sensitivity of the Ca e-stimulated elevation in [Ca +2] i to U73122 (a PLC inhibitor) together suggest that the osteoblast CasR is coupled via Gq to PLC-β. U73122 blocked the Ca e-promoted, but not PDGF-promoted, PLC-γ1 tyrosine phosphorylation, suggesting that the activation of PLC-β is upstream of PLC-γ1 activation. Inhibition of protein kinase C (PKC) disrupted Ca e-stimulated tyrosine phosphorylation of PLC-γ1. In addition, exposure to pertussis toxin or exogenous activation of protein kinase A (PKA) inhibited PLC-γ1 tyrosine phosphorylation in response to Ca e. The results indicate that: (a) the osteoblast CasR activates PLC-γ1 downstream of PLC-β in a PKC-dependent manner; (b) PKA is a negative regulator of Ca e-promoted PLC-γ1 phosphorylation; and (c) Gq and Gi are both involved in the CasR-mediated phosphorylation of PLC-γ1.