Nociceptin/orphanin FQ and related peptides reduce the increase in plasma corticosterone elicited in mice by an intracerebroventricular injection

Nociceptin/orphanin FQ (=N/OFQ), the endogenous ligand of ORL1 receptor (=NOP), has been reported to induce, in rodents, after intracerebroventricular (i.c.v.) administration, anti-stress and anxiolytic effects. We have observed that the handling of mice followed by an i.c.v. injection of saline, induced a marked increase in the plasma corticosterone level (+250%) measured 30 minutes later. When N/OFQ was injected intracerebroventricularly, using a 1 microg dose, the increase in plasma corticosterone was significantly lower than in saline injected mice. N/OFQ(1-13)NH(2), known as a NOP receptor agonist, at the same 1 microg dose, also induced a lesser increase in plasma corticosterone level than a saline i.c.v. injection. The pseudopeptide [Phe(1)-psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2), defined either as an agonist or an antagonist of NOP receptor, at the 0.1 microg dose, behaved in a similar manner as N/OFQ, by decreasing the plasma corticosterone level. Finally, [Nphe(1)]N/OFQ(1-13)NH(2), although presumed to be a selective NOP receptor antagonist, also decreased the corticosterone level at the 0.1 microg dose. These observations suggest the implication of N/OFQ in the regulation of response to stress, through an action on the hypothalamo-pituitary-adrenocortical axis. Moreover, they evidence a similar effect of N/OFQ and N/OFQ(1-13)NH(2), but also of two other related peptides displaying antagonist properties on NOP receptors. These data suggest that several subtypes of N/OFQ receptors could exist.     

Regulation of cyclic AMP-dependent response element-binding protein (CREB) by the nociceptin/orphanin FQ in human dopaminergic SH-SY5Y cells

Nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for opioid receptor-like (ORL1) receptor, transduces signaling cascades implicated in MAPK, PKC, PLC, and calcium, etc. This study was designed to investigate the intracellular signaling mechanism of N/OFQ in human dopaminergic neuroblastoma SH-SY5Y cells. N/OFQ rapidly induced the phosphorylation of CREB, which was significantly suppressed by pretreatment of PKA inhibitor, but not by MAPK inhibitors. It also time-dependently increased the phosphorylation of MAPK, which was proven as ERKs, whereas it did not affect the PI3K activity. Interestingly, KT5720, a specific inhibitor of PKA, markedly suppressed the phosphorylation of MAPK by N/OFQ in SH-SY5Y cells. Furthermore, BAPTA-AM, an intracellular chelator of Ca(2+), completely abolished the phosphorylation of CREB as well as MAPK in N/OFQ-treated SH-SY5Y cells. Taken together, these results suggest that N/OFQ independently induces the activation of CREB prior to MAPK phosphorylation, which was also modulated by PKA. Furthermore, Ca(2+)-related signaling implicates in the phosphorylation processes of CREB and MAPK simultaneously.     

IL-4 modulates the histamine content of mast cells in a mast cell/fibroblast co-culture through a Stat6 signaling pathway in fibroblasts

IL-4 plays a crucial role in the pathogenesis of allergic diseases, such as the induction of IgE synthesis and the development of mast cells. To further understand the effect of IL-4 on mast cells in skin, we utilized a mast cell/fibroblast co-culture system as an in vitro model of dermal mast cells. IL-4 induced mast cell growth in the culture with fibroblasts. Immunoblot analysis revealed that IL-4 activated Stat6 in both mast cells and fibroblasts. The over-expression of dominant-negative Stat6 in fibroblasts in the presence of IL-4 decreased the histamine content per mast cell, but not the number of mast cells. In contrast, the over-expression of constitutively-active Stat6 in fibroblasts increased the histamine content per mast cell, indicating that the activation of Stat6 in fibroblasts supports the maturation of mast cells co-cultured with fibroblasts.     

Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: Involvement of RhoA,Ca/CaM,and ERK

Background

Extracellular matrix (ECM) components and intracellular pH (pH_i) may serve as regulators of cell migration in various cell types.

Methods

The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na⁺/H⁺ exchanger (NHE)-1 activity was evaluated by measuring pH_i and [²²Na⁺] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.

Results

ECM components (FN, laminin, fibrinogen, and collagen type I) increased [²²Na⁺] uptake, pH_i, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca^2 + influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca^2 +/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [²²Na⁺] uptake and pH_i. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.

Conclusions

FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca^2 +/CaM signaling pathways.

General significance

The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.     

Taurine increases cell proliferation and generates an increase in [Mg2+]i accompanied by ERK 1/2 activation in human osteoblast cells

Taurine has been reported to influence bone metabolism, and its specific transport system, the taurine transporter, is expressed in osteoblasts. The mean [Mg2+]i was 0.51+/-0.01 mM in normal culture media. Taurine caused an increase in [Mg(2+)]i by 0.72+/-0.04 mM in human osteoblast (HOB) cells. This increment in [Mg2+]i was inhibited significantly by PD98059, nifedipine, lidocaine, and imipramine. Taurine was also shown to stimulate the activation of ERK 1/2. This taurine-stimulated ERK 1/2 activation was inhibited by PD98059. In the present study, taurine was shown to increase cell proliferation and generate an increase in [Mg2+]i accompanied by ERK 1/2 activation in HOB cells.     

Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca²⁺. Here, we show that extracellular Ca²⁺ (Ca²⁺_o) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca²⁺_o compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca²⁺_o in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cβ (PLCβ) led to an abolition of the Ca²⁺_o-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca²⁺_o-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca²⁺_o concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca²⁺-rich environment.     

Blockade of murine T cell activation by antagonists of P2Y6 and P2X7 receptors

Extracellular nucleotides and their metabolites activate ionotropic P2X and metabotropic P2Y receptors on the surface of various types of cells. Here, we investigated the involvement of P2X and P2Y receptor-mediated signaling in TCR-dependent T cell activation. Murine T cells were activated by stimulation of TCR, and both CD25 expression and interleukin (IL)-2 production were observed in activated T cells. Ecto-nucleotidase apyrase and P2Y₆ antagonist MRS2578 significantly blocked the increases of both CD25 expression and IL-2 production, and P2X₇ antagonists A438079 and oxidized ATP inhibited IL-2 production rather than CD25 expression, suggesting the involvement of P2Y₆ and P2X₇ receptors in different processes of T cell activation. MRS2578 also blocked TCR-dependent elevation of cytosolic Ca²⁺ in T cells. The P2X₇ and P2Y₆ receptors were expressed in murine CD4 T cells. In conclusion, our results indicate that activation of P2Y₆ and P2X₇ receptors contributes to T cell activation via TCR.     

Effects of staurosporine on the capacitative regulation of the state of the Ca reserves in activated Jurkat T lymphocytes

Staurosporine (Stp) is an inhibitor of protein kinase C (PKC) that has been used to address the role of this enzyme in a variety of cells. However, Stp can also inhibit protein tyrosine kinases (PTK). We have investigated the effects of Stp on the InsP₃- (using mAb C305 directed against the β chain of the T cell receptor (TcR)/CD3 complex) and the thapsigargin (Tg)-dependent release and influx of Ca²⁺ in human (Jurkat) T cells. The addition of Stp (200 nM) during the sustained phase of the TcR-dependent Ca²⁺ response resulted in a rapid inhibition of the influx of Ca²⁺ that was not seen when Ca²⁺ mobilization was triggered by Tg (1 μM). When the cells were preincubated with Stp (200 nM), there was an inhibition of the mAb C305- but not the Tg-dependent Ca²⁺ response. The effect of Stp was not the result of the inhibition of PKC as shown by down-regulation of PKC and with the use of the specific PKC inhibitor bis-indolyl maleimide GF 109203X. The effect of Stp on the entry of Ca²⁺ in activated (mAb C305) Jurkat lymphocytes was dose-related and was not the result of a direct inhibition of plasma membrane Ca²⁺ channels based on an absence of effect on the Tg-dependent entry of Ca²⁺ and the use of Ca²⁺ channel blockers (econazole and Ni²⁺). These blockers terminated the influx of Ca²⁺ but the Tg-sensitive Ca²⁺ reserves were not refilled in marked contrast to the effect of Stp. Quantification of InsP₃ revealed that the addition of Stp resulted in an approximate 40% reduction in mAb C305-activated Jurkat cells. The effects of Stp can be explained as follows. Stp decreases the mAb C305-induced production of InsP₃ by inhibiting the TcR/CD3-dependent activation of PTK associated with the stimulation of phospholipase C-γ₁. A decrease in [InsP₃] without a return to baseline is sufficient to close the InsP₃ Ca²⁺ channel, endoplasmic Ca²⁺ ATPases use the incoming Ca²⁺ to refill the Ca²⁺ pools and that terminates the capacitative entry of Ca²⁺. A simple kinetic model reproduced the experimental data.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca-independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺ + K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.     

A C-NMR study on the influxes into the tricarboxylic acid cycle of a renal epithelial cell line, LLC-PK1/Cl4: the metabolism of [2-C]glycine, l-[3-C]alanine and l-[3-C]aspartic acid in renal epithelial cells

Perchloric acid extracts of LLC-PK₁/Cl₄ cells, a renal epithelial cell line, incubated with either [2-¹³C]glycine l-[3-¹³C]alanine, or d,l-[3-¹³C]aspartic acid were investigated by ¹³C-NMR spectroscopy. All amino acids, except labelled glycine, gave rise to glycolytic products and tricarboxylic acid cycle (TCA) intermediates. For the first time we also observed activity of γ-glutamyltransferase activity and glutathione synthetase activity in LLC-PK₁ cells, as is evident from enrichment of reduced glutathione. Time courseS showed that only 6% of the labelled glycine was utilized in 30 min, whereas 31% of l-alanine and 60% of l-aspartic acid was utilized during the same period. ¹³C-NMR was also shown to be a useful tool for the determination of amino acid uptake in LLC-PK₁ cells. These uptake experiments indicated that glycine alanine and aspartic acid are transported into Cl₄ cells via a sodium-dependent process. From the relative enrichment of the glutamate carbons, we calculated the activity of pyruvate dehydrogenase to be about 61% of when labelled l-alanine was the only carbon source for LLC-PK₁/Cl₄ cells. Experiments with labelled d,l-aspartic, however, showed that about 40% of C-3-enriched oxaloacetate (arising from a de-amination of aspartic acid) reached the pyruvate pool.     

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK-OV3 ovarian cancer cells and OVCAR-3 ovarian cancer cells were responsive to LPE. LPE-stimulated intracellular calcium concentration ([Ca(2+)](i)) increase was inhibited by U-73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca(2+)](i) increase by LPE, indicating the involvement of PTX-sensitive G-proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE-stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B-driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells.     

Histidine-rich protein Hpn from Helicobacter pylori forms amyloid-like fibrils in vitro and inhibits the proliferation of gastric epithelial AGS cells

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections.     

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca^2 + concentration ([Ca^2 +]_i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca^2 +]_i. Chelating Ca^2 + ions in the extracellular medium suppressed the intracellular Ca^2 + signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca^2 +- and P2X7-independent transport mechanism in macrophages.     

TRP expression pattern and the functional importance of TRPC3 in primary human T-cells

TRP proteins form ion channels which are activated following receptor stimulation. In T-cell lines, expression data of TRP proteins have been published. However, almost no data about TRP expression is available in primary human T-cells. Using RT-PCR and quantitative RT-PCR, we compare the expression of TRP mRNA in 1) human peripheral blood lymphocytes, which are a mix of mostly mono-nuclear blood lymphocytes but contain other leucocytes, 2) a pure human CD4⁺ T-helper cell population in the resting (= naïve) and activated (= effector) state, and 3) two commonly used CD4⁺ Jurkat T-cell lines, E6-1 and parental. To mimic physiological cell stimulation, we analyzed TRP expression in primary human cells in a quantitative way over several days following formation of an immunological synapse through stimulation with antibody-coated beads. The TRP expression profile of primary human T-cells was significantly different from Jurkat T-cells. Among the TRP mRNAs of the TRPC, TRPM, and TRPV family, we found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 in primary human CD4⁺ T-cells of all analyzed blood donors. Among these, TRPC3 and TRPM2 were strongly up-regulated following stimulation, but with different kinetics. We found that TRPC3 modulates Ca²⁺-dependent proliferation of primary CD4⁺ T-cells indicating that TRPC3 may be involved in Ca²⁺ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca²⁺ entry.     

Conjugated linoleic acid-induced apoptosis in mouse mammary tumor cells is mediated by both G protein coupled receptor-dependent activation of the AMP-activated protein kinase pathway and by oxidative stress

Conjugated linoleic acid (CLA) has shown chemopreventive activity in several tumorigenesis models, in part through induction of apoptosis. We previously demonstrated that the t10,c12 isomer of CLA induced apoptosis of TM4t mouse mammary tumor cells through both mitochondrial and endoplasmic reticulum (ER) stress pathways, and that the AMP-activated protein kinase (AMPK) played a critical role in the apoptotic effect. In the current study, we focused on the upstream pathways by which AMPK was activated, and additionally evaluated the contributing role of oxidative stress to apoptosis. CLA-induced activation of AMPK and/or induction of apoptosis were inhibited by infection of TM4t cells with an adenovirus expressing a peptide which blocks the interaction between the G protein coupled receptor (GPCR) and Gα_q, by the phospholipase C (PLC) inhibitor U73122, by the inositol trisphosphate (IP₃) receptor inhibitor 2-APB, by the calcium/calmodulin-dependent protein kinase kinase α (CaMKK) inhibitor STO-609 and by the intracellular Ca²⁺ chelator BAPTA-AM. This suggests that t10,c12-CLA may exert its apoptotic effect by stimulating GPCR through Gα_q signaling, activation of phosphatidylinositol-PLC, followed by binding of the PLC-generated IP₃ to its receptor on the ER, triggering Ca²⁺ release from the ER and finally stimulating the CaMKK–AMPK pathway. t10,c12-CLA also increased oxidative stress and lipid peroxidation, and antioxidants blocked its apoptotic effect, as well as the CLA-induced activation of p38 MAPK, a downstream effector of AMPK. Together these data elucidate two major pathways by which t10,c12-CLA induces apoptosis, and suggest a point of intersection of the two pathways both upstream and downstream of AMPK.     

Expression and functional role of formyl peptide receptor in human bone marrow-derived mesenchymal stem cells

We investigated the expression of formyl peptide receptor (FPR) and its functional role in human bone marrow-derived mesenchymal stem cells (MSCs). We analyzed the expression of FPR by using ligand-binding assay with radio-labeled N-formyl-met-leu-phe (fMLF), and found that MSCs express FPR. FMLF stimulated intracellular calcium increase, mitogen-activated protein kinases activation, and Akt activation, which were mediated by G(i) proteins. MSCs were chemotactically migrated to fMLF. FMLF-induced MSC chemotaxis was also completely inhibited by pertussis toxin, LY294002, and PD98059, indicating the role of G(i) proteins, phosphoinositide 3-kinase, and extracellular signal regulated protein kinase. N-terminal fragment of annexin-1, Anx-1(2-26), an endogenous agonist for FPR, also induced chemotactic migration of MSCs. Thus MSCs express functional FPR, suggesting a new (patho)physiological role of FPR and its ligands in regulating MSC trafficking during induction of injured tissue repair.