Prostaglandin E2 amplifies cytosolic phospholipase A2- and cyclooxygenase-2-dependent delayed prostaglandin E2 generation in mouse osteoblastic cells. Enhancement by secretory phospholipase A2

We used the MC3T3-E1 cell line, which originates from C57BL/6J mouse that is genetically type IIA secretory phospholipase A2 (sPLA2)-deficient, to reveal the type IIA sPLA2-independent route of the prostanglandin (PG) biosynthetic pathway. Kinetic and pharmacological studies showed that delayed PGE2 generation by this cell line in response to interleukin (IL)-1beta and tumor necrosis factor alpha (TNFalpha) was dependent upon cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2. Expression of these two enzymes was reduced by cPLA2 or COX-2 inhibitors and restored by adding exogenous arachidonic acid or PGE2, indicating that PGE2 produced by these cells acted as an autocrine amplifier of delayed PGE2 generation through enhanced cPLA2 and COX-2 expression. Exogenous addition or enforced expression of type IIA sPLA2 significantly increased IL-1beta/TNFalpha-initiated PGE2 generation, which was accompanied by increased expression of both cPLA2 and COX-2 and suppressed by inhibitors of these enzymes. Thus, our results revealed a particular cross-talk between the two PLA2 enzymes and COX-2 for delayed PGE2 biosynthesis by a type IIA sPLA2-deficient cell line. cPLA2 is responsible for initiating COX-2-dependent delayed PGE2 generation, and sPLA2, if introduced, enhances PGE2 generation by increasing cPLA2 and COX-2 expression via endogenous PGE2.     

Type II secretory phospholipase A2 associated with cell surfaces via C-terminal heparin-binding lysine residues augments stimulus-initiated delayed prostaglandin generation

Type II secretory phospholipase A2 (sPLA2) has been shown to be induced by a variety of proinflammatory stimuli and, therefore, has been implicated in the inflammatory process. In order to determine whether association of sPLA2 with cell surfaces via heparan sulfate proteoglycan is important for its effects on cellular functions, we have identified the critical domain in sPLA2 for heparin and cell surface binding and examined its role in cellular prostaglandin (PG) biosynthesis. Replacement of several conserved Lys residues in the C-terminal region of mouse and rat sPLA2s by Glu resulted in a marked reduction of their capacities to bind to heparin and mammalian cell surfaces without affecting their enzymatic activities toward dispersed phospholipid as a substrate. CHO cells stably transfected with wild-type sPLA2 released about twice as much arachidonic acid (AA) during culture for 10 h with fetal calf serum and interleukin-1beta than cells transfected with vector alone, whereas the ability to enhance AA release was impaired in sPLA2 mutants incapable of binding to cell surfaces. AA released by wild-type sPLA2-transfected CHO cells was metabolized to prostaglandin E2 via prostaglandin endoperoxide H synthase (PGHS)-2 after IL-1beta stimulation, revealing a particular functional linkage of sPLA2 to PGHS-2. In contrast, A23187-initiated immediate AA release over 30 min was not affected by sPLA2 overexpression. Taken together, these results suggest that sPLA2 expressed endogenously and anchored on cell surfaces via its C-terminal heparin-binding domain is involved in the PGHS-2-dependent delayed PG biosynthesis initiated by growth factors and cytokines during long term culture.     

Antiphospholipid antibodies inhibit prostaglandin release by decidual cells of early pregnancy: possible involvement of extracellular secretory phospholipase A2

OBJECTIVE: To investigate the effect of antiphospholipid antibodies on eicosanoid production by human decidual cells and the in vitro interaction between antiphospholipid antibodies and secretory phospholipase A2. DESIGN: Cultures of human decidual cells from early pregnancy. SETTING: All decidual specimens were obtained from the Obstetrics and Gynecology Department of the Catholic University, Rome, Italy. PATIENT(S): Patients were undergoing operative laparoscopy for extrauterine pregnancy, with a period of amenorrhea ranging from 6 to 9 weeks. INTERVENTION(S): Decidual samples were collected at laparoscopy by routine uterine curettage. MAIN OUTCOME MEASURE(S): Decidual cells were incubated with antiphospholipid antibodies, and eicosanoids (prostaglandin [PG] E2, PGF2alpha, and thromboxane B2) were assayed by RIA after 24 hours of culture. In vitro interactions between antiphospholipid antibodies and secretory phospholipase A2 were investigated with use of a modified ELISA for phospholipase A2. RESULT(S): Antiphospholipid antibodies reduced eicosanoid release from decidual cells in a dose-dependent fashion. In vitro assays showed that antiphospholipid antibodies bound secretory phospholipase A2 and that a competition occurred between antiphospholipid antibodies and secretory phospholipase A2 for the common substrate cardiolipin. CONCLUSION(S): In light of the critical role played by eicosanoids in decidual function, we suggest that an interaction between antiphospholipid antibodies and secretory phospholipase A2 occurring in vivo might impair important cellular communications at the decidual level in the antiphospholipid antibody syndrome.     

Group II phospholipase A2 as an autocrine growth factor mediating interleukin-1 action on mesangial cells

The effects of intravenous (3 mg/kg i.v.) and intraplantar (50 micrograms/50 microliters i.pl.) morphine were investigated on spinal c-Fos expression induced 2 h after intraplantar carrageenin (6 mg/150 microliters of saline) and on carrageenin (2 mg/150 microliters of saline) induced mechanical hyperalgesia, at day 4, in both naive and chronic morphine treated (80 mg/kg/day s.c. on days 1, 2 and 3) rats. In naive rats, i.v. and i.pl. morphine significantly decreased spinal c-Fos expression (64 +/- 4% and 44 +/- 4% reduction of control carrageenin c-Fos expression, P < 0.0001 for both, respectively) and mechanical hyperalgesia (maximal increase: 326 +/- 29%, P < 0.0001 and 87 +/- 5%, P < 0.005 of control carrageenin paw pressure vocalisation threshold (VTPP), respectively), which only developed in the carrageenin injected paw. Both treatments were ineffective in chronic morphine treated rats (92 +/- 9% and 106 +/- 6% of control carrageenin c-Fos expression; 33 +/- 17% and 30 +/- 15% increase of control carrageenin VTPP, respectively). Furthermore, only i.v. morphine increased the VTPP in the contralateral paw, in naive rats (maximal increase: 90 +/- 8%, P < 0.0001 of control carrageenin VTPP), its effects being significantly less pronounced than for the inflamed paw (P < 0.0001). These studies based on spinal c-Fos expression as an indirect marker of spinal nociceptive processes and on behavioural experiments clearly revealed that chronic treatment with systemic morphine induced tolerance to both its systemic and peripheral effects.     

Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein-coupled receptors transactivate growth factor receptors to transmit mitogenic effects. In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II-induced extracellular signal-regulated kinase (ERK) activation, c-fos gene expression, and DNA synthesis in cardiac fibroblasts. Ang II induced a rapid tyrosine phosphorylation of EGF-R in association with phosphorylation of Shc protein and ERK activation. Specific inhibition of EGF-R function by either a dominant-negative EGF-R mutant or selective tyrphostin AG1478 completely abolished Ang II-induced ERK activation. Induction of c-fos gene expression and DNA synthesis were also abolished by the inhibition of EGF-R function. Calmodulin or tyrosine kinase inhibitors, but not protein kinase C (PKC) inhibitors or downregulation of PKC, completely abolished transactivation of EGF-R by Ang II or the Ca2+ ionophore A23187. Epidermal growth factor (EGF) activity in concentrated supernatant from Ang II-treated cells was not detected, and saturation of culture media with anti-EGF antibody did not affect the Ang II-induced transactivation of EGF-R. Conditioned media in which cells were incubated with Ang II could not induce phosphorylation of EGF-R on recipient cells. Platelet-derived growth factor-beta receptor was not phosphorylated on Ang II stimulation, and Ang II-induced c-jun gene expression was not affected by tyrphostin AG1478. Our results demonstrated that in cardiac fibroblasts Ang II-induced ERK activation and its mitogenic signals are dominantly mediated by EGF-R transactivated in a Ca2+/calmodulin-dependent manner and suggested that the effects of Ang II on cardiac fibroblasts should be interpreted in association with the signaling pathways regulating cellular proliferation and/or differentiation by growth factors.     

Prostacyclin formation elicited by endothelin-1 in rat aorta is mediated via phospholipase D activation and not phospholipase C or A2

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also stimulates production of prostacyclin (PGI2) from arachidonic acid. The purpose of this study was to determine the contribution of phospholipases (PLs) A2, C, and/or D in ET-1-induced PGI2 formation in the rat aorta, measured as immunoreactive 6-ketoprostaglandin (PG) F1 alpha. ET-1 increased 6-keto-PGF1 alpha formation, which was not affected by a PLA2 inhibitor, 7,7-dimethyl eicosadienoic acid (DEDA). Furthermore, ET-1 failed to stimulate PLA2 activity measured in the cytosol (cPLA2), using phosphatidylcholine, L-a-1-palmitoyl-2-arachidonyl[14C] as a substrate. However, the adrenergic agonist norepinephrine increased 6-keto-PGF1 alpha formation, which was attenuated by DEDA, and enhanced PLA2 activity. ET-1 enhanced PLC activity, as indicated by increased inositol phosphate production, which was prevented by a PLC inhibitor, U-73122. However, ET-1-induced 6-keto-PGF1 alpha production was not altered by U-73122. An inhibitor of PLD activation, C2-ceramide, attenuated ET-1-induced PLD activity, as indicated by the production of phosphatidylethanol. Furthermore, ET-1-induced 6-keto-PGF1 alpha formation was inhibited by C2-ceramide as well as by ethanol treatment. Moreover, inhibitors of phosphatidate phosphohydrolase (propranolol) and diacylglycerol lipase (RHC-80267), attenuated ET-1-induced 6-keto-PGF1 alpha formation. Finally, ET-1-induced activation of PLD was not attenuated by a selective PKC inhibitor, bisindolylmaleimide I. These data suggest a novel pathway for ET-1-induced PGI2 formation in the rat aorta involving activation of PLD but not cPLA2 and independent of PLC or PKC activation.     

Requirement of calcium influx for hydrolytic action of membrane phospholipids by cytosolic phospholipase A2 rather than mitogen-activated protein kinase activation in Fc epsilon RI-stimulated rat peritoneal mast cells

The mechanism by which cytosolic phospholipase A2 (cPLA2) is not responsible for eicosanoid production in rat peritoneal mast cells upon antigen stimulation [Ishimoto et al. (1996) J. Biochem. 120, 616-623] was investigated in the mast cells stimulated by cross-linking of the IgE receptor or with thapsigargin. Stimulation with thapsigargin, but not with antigen, resulted in apparent lysophosphatidylcholine (lysoPC) formation. Antigen stimulation significantly increased the activities of mitogen-activated protein (MAP) kinase and cPLA2. These activities were further potentiated by phorbol ester. The antigen elicited a rapid and transient increase in intracellular Ca2+ concentration, while thapsigargin produced a slow and sustained increase. Furthermore, a combination of antigen and thapsigargin rapidly increased and prolonged the intracellular Ca2+ concentration. Under these conditions, lysoPC was apparently generated, whereas it was not in response to antigen alone. These results suggest that a prolonged increase in the intracellular Ca2+ concentration is required for cPLA2 to associate with membranes, thus leading to hydrolysis of membrane phospholipids by the enzyme.     

2F1 antigen, the mouse homolog of the rat "mast cell function-associated antigen", is a lectin-like type II transmembrane receptor expressed by natural killer cells

Inhibitory lectin-like receptors expressed on the surface of hematopoietic cells are critically involved in regulation of their effector functions. Here we report that a novel mAb specific for mouse NK cells, 2F1, recognizes the mouse homolog of the mast cell function-associated antigen (MAFA), an inhibitory lectin-like transmembrane receptor expressed on rat mast cells. The 2F1 antigen (2F1-Ag) and rat MAFA are structurally highly conserved and contain a cytoplasmic motif similar to the immunoreceptor tyrosine-based inhibitory motif that is presumably utilized for inhibitory signaling. We also identified a human homolog that is closely related to the rodent MAFA/2F1-Ag proteins. Like rat MAFA, 2F1-Ag is probably encoded by a single gene, which exhibits relatively little polymorphism. Strikingly, while rat MAFA is considered a mast cell antigen, we have been unable to detect cell surface expression of 2F1-Ag by mouse mast cell lines, bone marrow-derived mast cells, or peritoneal mast cells. Furthermore, mouse bone marrow-derived mast cells were devoid of 2F1-Ag mRNA. Instead, we find that approximately 40% of mouse NK cells express 2F1-Ag. Thus, MAFA/2F1-Ag may modulate immunological responses on at least two different cell types bridging the specific and innate immune system.     

Growth inhibition by transforming growth factor beta (TGF-beta) type I is restored in TGF-beta-resistant hepatoma cells after expression of TGF-beta receptor type II cDNA

The growth of human hepatoma Hep 3B cells is potently inhibited by TGF-beta 1 (ID50 = 0.2 ng/ml, 8 pM). A mutant cell line was derived that was not inhibited in growth by TGF-beta 1 at 5 ng/ml (200 pM) and that lacked TGF-beta receptor type II (TGF-beta RII) gene. Transfection of the cloned cDNA for human TGF-beta RII to this mutant cell line restored receptor expression as well as the inhibition in growth by TGF-beta 1. In both wild-type and mutant cells stably transfected with TGF-beta RII cDNA, TGF-beta RII coimmunoprecipitated with TGF-beta receptor type I in the presence of ligand. These experiments provide direct evidence for the role of TGF-beta RII in the inhibitory effect of TGF-beta on growth and suggest that TGF-beta RII acts by means of a heteromeric surface complex with TGF-beta receptor type I.     

Sustained activation of phospholipase D via adenosine A3 receptors is associated with enhancement of antigen- and Ca(2+)-ionophore-induced secretion in a rat mast cell line

The adenosine analog, N-ethylcarboxamidoadenosine (NECA), causes transient activation of phospholipase C and an enhancement of antigen-induced secretion in a rat mast cell (RBL-2H3) line via adenosine A3-receptors (Ramkumar et al., J. Biol. Chem. 268:16887, 1993) by a mechanism that is inhibited by bacterial toxins and potentiated by dexamethasone (Ali et al., J. Biol. Chem. 265:745-753, 1990). Here we show that NECA synergizes the secretory response to Ca(2+)-ionophore as well as to antigen. The ability of NECA to synergize the secretory responses persisted for 10 to 20 min, long after the early phospholipase C-mediated reactions to NECA had subsided. NECA caused, however, a dose-dependent sustained activation of phospholipase D, as indicated by the formation of [3H]phosphatidic acid, or in the presence of 0.3% ethanol, [3H]phosphatidylethanol. This activation was associated with a sustained increase in diglycerides, in protein kinase C activity and in the phosphorylation of myosin light chains by protein kinase C. The generation of diglycerides was enhanced in dexamethasone-treated cells and suppressed in cells that had been treated with cholera toxin or pertussis toxin. Collectively, the studies suggested that the generation of diglycerides via phospholipase D and the associated activation of protein kinase C were, by themselves, insufficient signals for secretion in RBL-2H3 cells, but that these reactions synergized responses to stimulants such as antigen or A23187 that caused substantial increases in [Ca2+]i.     

Regulation of transforming growth factor-beta type II receptor expression in human breast cancer MCF-7 cells by vitamin D3 and its analogues

In view of the tumor suppressor role of the transforming growth factor-beta (TGFbeta) type II receptor (RII), the identification and characterization of agents that can induce the expression of this receptor are of potential importance to the development of chemoprevention approaches as well as treatment of cancer. To date, the identification of exogenous agents that control RII expression has been rare. We demonstrated that proliferation of MCF-7 early passage cells (MCF-7 E), which express RII and are sensitive to TGFbeta growth inhibition activity, was significantly inhibited by vitamin D3 and its analogue EB1089. In contrast, proliferation of MCF-7 late passage cells (MCF-7 L), which have lost cell surface RII and are resistant to TGFbeta, was not affected by these two compounds. TGFbeta-neutralizing antibody was able to block the inhibitory effect on MCF-7 E cells by these compounds, indicating that treatment induced autocrine-negative TGFbeta activity. An RNase protection assay showed approximately a 3-fold induction of the RII mRNA, while a receptor cross-linking assay revealed a 3-4-fold induction of the RII protein. In contrast, there was no change in either RII mRNA or protein in the MCF-7 L cells.     

Phospholipase A2 type II binds to extracellular matrix biglycan: modulation of its activity on LDL by colocalization in glycosaminoglycan matrixes

We recently reported the presence of secretory, nonpancreatic phospholipase A2 type II (snpPLA2; EC 3.1.1.4) in human atherosclerotic arteries (Hurt-Camejo et al, Arterioscler Thromb Vasc Biol. 1997;17:300-309). SnpPLA2 may generate the proinflammatory products lysophospholipids and free fatty acids, thus contributing to atherogenesis when acting on low density lipoproteins (LDLs) retained in the arterial wall. Immunohistochemical studies showed that smooth muscle cells (SMCs) in human arterial tissue are the main sources of snpPLA2. In cultures of human arterial SMCs, snpPLA2 interacts with versican and smaller heparan/chondroitin sulfate proteoglycans (PGs) secreted as soluble components into the medium. In the present study, we investigated the binding of snpPLA2 to extracellular matrix (ECM) PGs produced by SMCs. The results show that snpPLA2 can bind to the ECM at physiological salt concentrations. ECM-bound snpPLA2 was active, hydrolyzing phosphatidylcholine-containing micelles. Soluble chondroitin-6-sulfate at concentrations >1 micromol/L, but not heparin or heparan sulfate, was able to release ECM-bound snpPLA2. The PG mainly involved in the binding of snpPLA2 was identified as biglycan. Perlecan was also present in the ECM synthesized by SMCs, but it contributed less to the binding of snpPLA2. Experiments with immobilized glycosaminoglycans indicated that snpPLA2 hydrolyzed 7-fold more LDL phospholipids when the lipoprotein and the enzyme were colocalized in a matrix with chondroitin-6-sulfate compared with one with heparin. These data suggest that retention of snpPLA2 in ECMs of different composition may modulate the enzymatic activity of snpPLA2 toward LDL. The results presented in this work support the hypothesis of the potential contribution of snpPLA2 to atherosclerosis.     

Insulin-like growth factors counteract the effect of interleukin 1 beta on type II phospholipase A2 expression and arachidonic acid release by rabbit articular chondrocytes

Interleukin 1 beta was found to stimulate arachidonic acid release, and the synthesis and secretion of type II phospholipase A2 by rabbit articular chondrocytes in vitro. Interleukin 1 beta had no effect on the level of cytosolic phospholipase A2 mRNA. Insulin-like growth factors, which help stabilize the cartilage matrix, reduced the effect of interleukin 1 beta on type II phospholipase A2 activity and mRNA level, and decreased the Interleukin 1 beta-stimulated arachidonic acid release to the basal values. This suggests that type II phospholipase A2 plays a key role in arachidonic acid release from rabbit articular chondrocytes and that insulin-like growth factors counteract the effect of interleukin 1 beta. They may therefore be considered as potential antiinflammatory agents.     

Evaluation of propolis (II): effects of Brazilian and Chinese propolis on histamine release from rat peritoneal mast cells induced by compound 48/80 and concanavalin A

Patients suffering from rheumatoid arthritis (RA) may experience a temporary reduction of disease symptoms during pregnancy. As indicated by the occurrence of RA-disease symptoms during pregnancy, three categories of patients were defined, namely, remission, relapse and unchanged. In all three categories changes in the plasma level and glycosylation of alpha1-acid glycoprotein (AGP) were determined longitudinally in comparison to those occurring in pregnancy of healthy women. In healthy pregnancy, we observed: (i) a peak in the plasma concentration at week 18 and a minimum at week 30; (ii) a continuous increase in the degree of branching of the glycans during the entire pregnancy period, and (iii) a decrease in the degree of alpha3-fucosylation of AGP-glycans with a minimum occurring at week 25. Comparable pregnancy-induced changes in glycosylation were found for two other acute-phase proteins alpha1-protease inhibitor (PI) and alpha1-antichymotrypsin (ACT). Increased oestrogen levels, known to occur during pregnancy, may be one of the factors that induce these changes, because the increased branching and decreased alpha3-fucosylation is in agreement with our earlier findings regarding an involvement of this hormone in the regulation of acute phase protein glycosylation in oestrogen-treated males as well as females. In all three clinical categories in RA, pregnancy also induced a continuous increase in the degree of branching of the glycans of AGP. However, similar changes in concentration and fucosylation were only found during remission of the disease symptoms. In the relapse and unchanged categories in RA, the degree of fucosylation and the plasma concentration of AGP remained constant throughout pregnancy. This indicates a relationship between changes in alpha3-fucosylation of AGP and RA disease activity.     

The epidermal growth factor/cAMP-inducible ectoCa(2+)-ATPase of human hepatoma Li-7A cells is similar to rat liver ectoATPase/hepatocyte cell adhesion molecule (cell-CAM 105)

Human hepatoma Li-7A cells exhibit two cell surface ATPase (ectoATPase) activities distinguishable by their different biochemical properties. The activity of the minor ectoATPase, ectoCa(2+)-ATPase, is enhanced severalfold when Li-7A cells are treated simultaneously by epidermal growth factor (EGF) and cAMP elevating agents (Knowles, A. F., 1990, Arch. Biochem. Biophys. 283, 114-119). Here we report that the human ectoCa(2+)-ATPase is biochemically similar to the major rat hepatocyte ectoATPase/cell adhesion molecule (cell-CAM 105) with respect to response to divalent ions and sulfhydryl reagents. Furthermore, the binding of rat liver ectoATPase antibody increased markedly in EGF/cholera toxin/hydrocortisone-treated Li-7A cells compared to untreated cells. Western blot analysis revealed cross-reactivity of the antibody with a 125-kDa protein. Partial purification of ectoCa(2+)-ATPase from EGF/cholera toxin/hydrocortisone-treated Li-7A cells confirmed that enrichment of the 125-kDa protein correlated with an increase in ATPase activity. We conclude that EGF and increased levels of cAMP lead to increased synthesis of the ectoCa(2+)-ATPase in Li-7A cells. The present demonstration of similarity between the ectoCa(2+)-ATPase and a rat liver cell adhesion molecule, cell-CAM 105, contributes significantly to an understanding of the implication of down-regulation of ectoCa(2+)-ATPase during hepatocyte-hepatoma transformation.