Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol

Owing at least in part to oxysterol components that can induce apoptosis, oxidized LDL (oxLDL) is cytotoxic to mammalian cells with receptors that can internalize it. Vascular cells possess such receptors, and it appears that the apoptotic response of vascular cells to the oxysterols borne by oxLDL is an important part of the atherogenic effects of oxLDL. Thus, an analysis of the signaling pathway of apoptotic induction by oxysterols is of value in understanding the development of atherosclerotic plaque. In a prior study, we demonstrated an induction of calcium ion flux into cells treated with 25-hydroxycholesterol (25-OHC) and showed that this response is essential for 25-OHC-induced apoptosis. One possible signal transduction pathway initiated by calcium ion fluxes is the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we demonstrate that activation of cPLA2 does occur in both macrophages and fibroblasts treated with 25-OHC or oxLDL. Activation is evidenced by 25-OHC-induced relocalization of cPLA2 to the nuclear envelope and arachidonic acid release. Loss of cPLA2 activity, either through genetic knockout in mice, or by treatment with a cPLA2 inhibitor, results in an attenuation of arachidonic acid release as well as of the apoptotic response to oxLDL in peritoneal macrophages or to 25-OHC in cultured fibroblast and macrophage cell lines.     

The anti-apoptotic effects of nordihydroguaiaretic acid: inhibition of cPLA(2) activation during TNF-induced apoptosis arises from inhibition of calcium signaling

Nordihydroguaiaretic acid (NDGA) is a plant lignan produced by Larrea tridentata, the creosote bush of the American southwest. In this report we examine the mechanism underlying the ability of NDGA to inhibit TNF-induced apoptosis. Our results show that NDGA blocks many key indicators of apoptosis. Caspase cleavage, mitochondrial inactivation, externalization of phosphatidyl serine, and (51)Cr-release were all blocked by low micromolar concentrations of NDGA. NDGA also inhibited the cPLA(2)-dependent release of (3)H-arachidonic acid. We investigated this activity and found that NDGA prevented the rise in intracellular calcium necessary for the apoptotic activation of cPLA(2). On the other hand, NDGA did not interfere with the TNF-induced phosphorylation of cPLA(2), indicating that NDGA does not block all TNF-dependent signaling. Finally, we asked whether the anti-apoptotic effect of NDGA could be attributed to its anti-oxidant activity. Comparison with the effects of butylated hydroxyanisole (BHA) did not completely support this hypothesis. While BHA strongly inhibited caspase activation and partially blocked the release of (51)Cr, it was unable to significantly block the calcium response or the release of (3)H-arachidonic acid associated with TNF-induced apoptosis. The anti-oxidant activity of NDGA may, therefore, explain some but not all of its anti-apoptotic activity.     

Erk-dependent cytosolic phospholipase A2 activity is induced by CD95 ligand cross-linking in the mouse derived Sertoli cell line TM4 and is required to trigger apoptosis in CD95 bearing cells

In the present study we demonstrated that CD95L cross-linking generated reverse signalling in the mouse derived Sertoli cell line TM4. Treatment of TM4 cells with mAb anti-CD95L induced activation of the cytosolic phospholipase A2 (cPLA2). Cytosolic PLA2 activation was controlled by the MAPK pathway as indicated by the ability of the specific MEK inhibitor, PD098059, to abolish cPLA2 activation. In addition, Western blot experiments showed a rapid increase in phosphorylated Erk1/2 following CD95L cross-linking, while no effect on the phosphorylation of other MAPK, p38 or JNK, was observed. CD95L cross-linking by mAb increased the levels of soluble CD95L and apoptotic activity of TM4 cell supernatants, which was blocked by co-incubation with the PLA2 inhibitor, AACOCF3 or PD098059. Finally, pre-treatment of TM4 cells with AACOCF3 or PD098059 completely abolished TM4-induced apoptosis of Jurkat T cells, thus indicating that the Erk/cPLA2 pathway is required for CD95L-induced apoptosis.     

cPLA2 activator peptide, PLAP, increases arachidonic acid release and apoptosis of vascular smooth muscle cells

Apoptosis of vascular smooth muscle cells (VSMCs) has recently drawn a lot of interest in various laboratories due to its importance in atherogenesis. We have shown previously that nitric-oxide (NO) can induce apoptosis of VSMCs and that the NO-induced apoptosis is accompanied by an increase in arachidonic acid release via cytoplasmic Ca(2+)-dependent phospholipase A(2) (cPLA(2)). We have demonstrated here that NO-induced activation of cPLA(2) leading to increased arachidonic acid release can be mimicked via direct activation of cPLA(2) with a cPLA(2) activator peptide, PLAP. The PLAP induced arachidonic acid release and apoptosis is inhibitable by a cPLA(2)-specific inhibitor, AACOCF(3), indicating the direct involvement of cPLA(2). In this study, activation of cPLA(2) appears to be preceded by activation and binding by PLAP indicating that the cPLA(2) functions are mediated via PLAP.     

Electromechanical effects of endothelin on ferret bronchial and tracheal smooth muscle

The effects of endothelin (ET) on transmembrane potential and isometric force were studied in ferret bronchial and tracheal smooth muscles. At rest, the muscle cells were electrically and mechanically quiescent. The mean resting potential for the bronchial cells was -70 +/- 1 mV (n = 25 cells/8 ferrets), and that of the tracheal cells was -60 +/- 1 mV (n = 7 cells/2 ferrets). ET depolarized and contracted both types of muscle cells in a concentration-dependent manner. At 1 nM ET, the bronchial muscle cells were significantly depolarized with concomitant force generation. In contrast, greater than 30 nM ET was required for the tracheal muscle cells to respond. The bronchial cells were further depolarized by 10 and 100 nM ET with electrical slow-wave activity present. The calcium channel antagonist verapamil substantially inhibited the contractions produced by 100 nM ET and abolished the slow-wave activity without affecting the base-line depolarization. Pretreatment of the bronchial muscle with 30 microM indomethacin did not affect the ET-induced contraction. These results suggest that ET modulates airway smooth muscle tone by direct activation and/or depolarization-induced activation of sarcolemmal calcium channels.     

Verapamil reversibly inhibits spontaneous parthenogenetic activation in aged rat eggs cultured in vitro

The present study was designed to investigate whether verapamil could inhibit spontaneous parthenogenetic activation in aged rat eggs cultured in vitro. Eggs collected from oviduct after 19 h post human chorionic gonadotropin (hCG) were arrested at the metaphase-II (M-II) stage and exhibited a first polar body. Culture of these aged eggs in calcium/magnesium (Ca(2+)/Mg(2+))-deficient and serum-free medium for 3 h induced exit from M-II, a morphological sign of spontaneous parthenogenetic activation in all eggs. However, verapamil reversibly inhibited spontaneous parthenogenetic activation in a dose-dependent manner. Further, lower doses (6.25, 12.5, and 25 muM) of verapamil induced egg survival, while higher doses (50 and 100 muM) were associated with the appearance of morphological apoptotic features such as shrinkage, membrane blebbing and cytoplasmic granulation prior to degeneration. The DNA fragmentation was induced [as evidenced by terminal deoxynucleotidyl transferase (TdT) nick-end labeling (TUNEL) positive staining] in eggs undergoing morphological apoptotic changes. On the other hand, caspase-3 inhibitor (1 muM) partially inhibited morphological apoptotic changes (44.34+/-3.53%) suggesting the involvement of both Ca(2+)and caspase-3-mediated apoptotic pathways. These findings suggest that verapamil reversibly inhibits spontaneous parthenogenetic activation and induces egg survival at lower doses, while higher doses induce cell death via apoptosis.     

Muscarinic cholinoceptor activation by pilocarpine triggers apoptosis in human skin fibroblast cells

The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChRs) on apoptosis in human skin fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with pilocarpine in the presence or absence of specific antagonists. Pilocarpine stimulates apoptosis, total inositol phosphates (InsP) accumulation and nitric oxide synthase (NOS) activity. All these effects were inhibited by atropine, mustard hydrochloride (4‐DAMP) and pirenzepine, indicating that M₁ and M₃ mAChRs are implicated in pilocarpine action. Pilocarpine apoptotic action is accompanied by caspase‐3 and JNK activation. The intracellular pathway leading to pilocarpine‐induced biological effects involved phospholipase C, calcium/calmodulin and extracellular calcium as U‐73122, W‐7, verapamil, BAPTA and BAPTA‐AM blocked pilocarpine effects. L ‐NMMA, a NOS inhibitor, had no effect, indicating that the enzyme does not participate in the apoptosis phenomenon. These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on the apoptotic human skin fibroblast process. J. Cell. Physiol. 222: 640–647, 2010. © 2009 Wiley‐Liss, Inc.     

Interferon-alpha-induced phosphorylation and activation of cytosolic phospholipase A2 is required for the formation of interferon-stimulated gene factor three.

Treatment of cells with interferon (IFN)-alpha caused phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). The protein tyrosine kinase Jak1 was found to be necessary for the activation of cPLA2. Jak1 could be co-immunoprecipitated with cPLA2 from cell extracts, indicating that a close physical interaction occurs between these two proteins. The induction of IFN-stimulated gene factor three (ISGF3) by IFN-alpha, is blocked by cPLA2 inhibitors in cell cultures and in cell-free reconstituted systems. However, these inhibitors do not block IFN-alpha or gamma-induced binding of STAT1 to the inverted repeat (IR) element of the IFN regulatory factor 1 (IRF-1) gene. Thus, cPLA2 activations occurs as an early event in the IFN-alpha response and is selectively involved in ISGF3-dependent gene activation.     

Estrogen induces phospholipase A2 activation through ERK1/2 to mobilize intracellular calcium in MCF-7 cells

The principal secreted estrogen, 17beta-estradiol rapidly activates signaling cascades that regulate important physiological processes including ion transport across membranes, cytosolic pH and cell proliferation. These effects have been extensively studied in the MCF-7 estrogen-responsive human breast carcinoma cell line. Here, we demonstrate that a physiological concentration of 17beta-estradiol caused a rapid, synchronous and transient increase in intracellular calcium concentration in a confluent monolayer of MCF-7 cells 2-3 min after treatment. This response was abolished when cells were pre-incubated with the phospholipase A(2) (PLA(2)) inhibitor quinacrine or with the cyclooxygenase inhibitor indomethacin. The translocation of GFP-cPLA(2)alpha to perinuclear membranes occurred 1-2 min after 17beta-estradiol treatment; this translocation was concurrent with the transient phosphorylation of cPLA(2)alpha at serine residue 505. The phosphorylation and translocation of cPLA(2) were sensitive to inhibition of the extracellular signal regulated kinase (ERK) signaling cascade and occurred simultaneously with a transient activation of ERK. The phosphorylation of cPLA(2) could be stimulated by membrane impermeable 17beta-estradiol conjugated to bovine serum albumen and was blocked by an antagonist of the classical estrogen receptor. Here we show, for the first time, that PLA(2) and the eicosanoid biosynthetic pathway are involved in the 17beta-estradiol induced rapid calcium responses of breast cancer cells.     

Sustained activation of p34(cdc2) is required for noscapine-induced apoptosis.

Mitotic arrest and subsequent apoptosis has been observed in many types of cells treated with anti-microtubule agents. However, the molecular mechanisms underlying the two events as well as their relationship are not well understood; on the contrary, there has been increasing evidence indicating that anti-microtubule agents might induce apoptosis via signaling pathways independent of mitosis. In this study, we found that apoptosis induced by noscapine, an anti-microtubule drug previously shown to cause both mitotic arrest and apoptotic cell death, was blocked by inhibiting p34(cdc2) activity with olomoucine in FM3A murine mammary carcinoma cells or by reducing the level and activity of p34(cdc2) in a mutant cell line FT210 derived from FM3A. Furthermore, transfection of the mutant FT210 cells with wild-type p34(cdc2) restored their ability to undergo mitotic arrest and then apoptosis in response to noscapine. Thus, we conclude that sustained activation of the p34(cdc2) kinase during mitotic arrest is required for subsequent apoptosis induced by noscapine, establishing a link between the two events.     

Effects of calcium, calmodulin, protein kinase C and protein tyrosine kinases on volume-activated taurine efflux in human erythroleukemia cells.

The effects of calcium, calmodulin, protein kinase C (PKC) and protein tyrosine kinase (PTK) modulators were examined on the volume-activated taurine efflux in the erythroleukemia cell line K562. Exposure to hypoosmotic solution significantly increased taurine efflux and intracellular calcium concentration ([Ca2+]i). The Ca2+ channel blockers La3+ (1 mM), verapamil (200 microM) and nifedipine (100 microM) inhibited the hypoosmotically-induced [Ca2+]i increase by more than 90%, while the volume-activated taurine efflux was inhibited by 61.3 +/- 9.5, 74.1 +/- 9.3 and 38.0 +/- 1.5%, respectively. Furthermore, the calmodulin inhibitors W7 (50 microM) and trifluoperazine (10 microM) and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62 (2 microM) significantly blocked the volume-activated taurine efflux by 93.4 +/- 2.7, 77.9 +/- 3.5 and 61.3 +/- 15.8%, respectively. In contrast, the PKC inhibitor staurosporine (200 nM) or the PKC activator phorbol 12-myristate 13-acetate (100 nM) did not have significant effects on the volume-activated taurine efflux. However, pretreatment with PTK inhibitors genistein, tyrphostin A25, and tyrphostin A47 blocked the volume-activated taurine efflux. These results suggest that the volume-activated taurine efflux in K562 cells may not directly involve Ca2+, but may require the presence of calmodulin and/or PTK.     

钙池排空操纵的外钙内流决定甘草诱导MGC-803细胞凋亡

 用 EGTA螯合胞外 Ca2 +和异搏定抑制钙通道 ,研究胞外 Ca2 +在甘草诱导 MGC- 80 3细胞中的作用 .流式细胞仪检测凋亡峰和 DNA ladder分析均表明 ,EGTA和异博定阻断细胞凋亡 .分别以 PI或 Rh1 2 3活染后的相应荧光强度表示细胞膜通透性和线粒性膜电位 (ΔΨm) .结果表明 ,细胞膜通透性增强和线粒体 ΔΨm 下降均为细胞凋亡的早期事件 ,EGTA和异博定均可抑制细胞膜通透性增强 ,但 EGTA促进线粒体 ΔΨm 下降 ,而异博定作用相反 .进一步经 PI和 Hoechst33342荧光双染后同时观察细胞膜通透性和细胞核形态 .结果表明 ,凋亡细胞均可 PI着色 ,EGTA和异博定完全阻断染色质凝聚 ,但不能完全抑制细胞膜通透性变化 .借助 Ca2 +探针 Fluo- 3/AM研究凋亡时胞内游离钙的时相变化 ,发现 Ca2 +升高也是细胞凋亡的早期事件 . EGTA和异博定轻微促进凋亡早期 Ca2 +升高 ,但抑制随后 Ca2 +的继续升高 .所有结果提示 ,钙池排空操纵的外 Ca2 +内流在甘草诱导 MGC- 80 3细胞凋亡中发挥决定性的作用 .     

Hyperosmotic stress induces phosphorylation of cytosolic phospholipase A(2) in HaCaT cells by an epidermal growth factor receptor-mediated process

Cytosolic phospholipase A(2) (cPLA(2)) is an enzyme involved in the formation of proinflammatory mediators by catalyzing the release of arachidonic acid, thereby mediating eicosanoid biosynthesis. Using HaCaT keratinocytes as a model system, we present experimental evidence that in these cells, cPLA(2) is constitutively phosphorylated and that the degree of phosphorylation dramatically increases in cells under hyperosmotic stress induced by sorbitol. In parallel, a rapid release of arachidonic acid followed by prostaglandin E(2) formation was detected. Elucidating the mechanism of cPLA(2) upregulation, we observed that it is mediated via epidermal growth factor receptor (EGFR) activation, since tyrphostin AG1478, a selective inhibitor of EGFR tyrosine kinase, completely inhibited cPLA(2) phosphorylation. Furthermore, addition of PD98059, which is an inhibitor of MEK1 activation, but not of SB203580, which is an inhibitor of p38 stress kinase, inhibited cPLA(2) phosphorylation, indicating that the ras-raf-MEK cascade is the major signalling pathway involved in cPLA(2) phosphorylation. In addition, depletion of the cells from intracellular calcium does not prevent sorbitol-elicited cPLA(2) phosphorylation, suggesting that this process is independent of the presence of calcium. Together, our results demonstrate that hyperosmotic stress phosphorylates cPLA(2) in human keratinocytes by an EGFR-mediated process.     

N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2.

The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.     

Head involution defective (Hid)-triggered apoptosis requires caspase-8 but not FADD (Fas-associated death domain) and is regulated by Erk in mammalian cells

The molecular machinery of apoptosis is evolutionarily conserved with some exceptions. One such example is the Drosophila proapoptotic gene Head involution defective (Hid), whose mammalian homologue is not known. Hid is apoptotic to mammalian cells, and we have examined the mechanism by which Hid induces death. We demonstrate for the first time a role for the extracellular signal-related kinase-1/2 (Erk-1/2) in the regulation of Hid function in mammalian cells. Bcl-2 and an inhibitor of caspase-9 blocked apoptosis, indicative of a role for the mitochondrion in this pathway, and we provide evidence for a role for caspase-8 in Hid-induced apoptosis. Thus, apoptosis was blocked by an inhibitor of caspase-8, deletion of caspase-8 rendered cells resistant to Hid-induced apoptosis, and Hid associated with caspase-8 in cell lysates. The Fas-associated death domain (FADD) was dispensable for the apoptotic function of Hid, indicating that Hid does not require extracellular death receptor signaling for the activation of caspase-8. In activated T cells, the cytokine interleukin-2 blocked caspase-8 processing and apoptosis, suggesting that survival cues from trophic factors may target a Hid-like intermediate present in mammalian cells. Thus, this study shows that Hid engages with conserved components of cellular death machinery and suggests that apoptotic paradigms characterized by FADD-independent activation of caspase-8 may involve a Hid-like molecule in mammalian cells.     

Identification of Neurotensin Receptors Associated with Calcium Channels and Prolactin Release in Rat Pituitary

Neurotensin (NT) is now reasonably well established as a neurotransmitter or neuromodulator candidate in the CNS. In the present study, we characterized the NT receptors in dispersed cells from the anterior lobe of rat pituitary and investigated the involvement of both cyclic AMP and calcium in the release of prolactin (PRL) induced by NT receptor stimulation. The [3H]NT binding to membranes from anterior pituitary dispersed cells was found saturable and stereospecific. Scatchard analysis of the data gave a straight line indicating a Bmax value of 121 +/- 11 fmol/mg protein and a KD value of 1.4 +/- 0.2 nM. The calculated IC50 values for [3H]NT binding were 5.8 nM for NT, 7.8 nM for L-Phe-NT, and 3,000 nM for the pharmacologically inactive form D-Phe-NT. NT, up to a concentration of 1 microM, did not affect the cyclic AMP generating system in homogenates of anterior pituitary from male or lactating female rats. The same pattern of results was obtained for cyclic AMP formation in intact cells. NT and its analogs stereospecifically enhanced the influx of calcium into dispersed cells from rat anterior pituitary. The effect was time- and dose-dependent. It appeared to be associated with neurotransmitter-operated calcium channels since: preincubation of the cells with tetrodotoxin did not affect the increase in calcium influx induced by NT; concentrations of verapamil that counteract the influx of calcium induced by potassium lacked the capacity to modify the influx of calcium induced by NT; and NT lost its capacity to release PRL in the absence of extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual role of the p38 MAPK/cPLA2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists

p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase A₂ (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions.