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Tissue factor (TF) is expressed in vascular and nonvascular tissues and functions in several pathways, including embryonic development, inflammation, and cell migration. Many risk factors for atherosclerosis, including hypertension, diabetes, obesity, and smoking, increase TF expression. To better understand the TF-related mechanisms in atherosclerosis, here we investigated the role of 12/15-lipoxygenase (12/15-LOX) in TF expression. 15( S )-hydroxyeicosatetraenoic acid (15( S )-HETE), the major product of human 15-LOXs 1 and 2, induced TF expression and activity in a time-dependent manner in the human monocytic cell line THP1. Moreover, TF suppression with neutralizing antibodies blocked 15( S )-HETE-induced monocyte migration. We also found that NADPH- and xanthine oxidase-dependent reactive oxygen species (ROS) production, calcium/calmodulin-dependent protein kinase IV (CaMKIV) activation, and interactions between nuclear factor of activated T cells 3 (NFATc3) and FosB proto-oncogene, AP-1 transcription factor subunit (FosB) are involved in 15( S )-HETE-induced TF expression. Interestingly, NFATc3 first induced the expression of its interaction partner FosB before forming the heterodimeric NFATc3-FosB transcription factor complex, which bound the proximal AP-1 site in the TF gene promoter and activated TF expression. We also observed that macrophages from 12/15-LOX -/- mice exhibit diminished migratory response to monocyte chemotactic protein 1 (MCP-1) and lipopolysaccharide compared with WT mouse macrophages. Similarly, compared with WT macrophages, monocytes from 12/15-LOX -/- mice displayed diminished trafficking, which was rescued by prior treatment with 12( S )-HETE, in a peritonitis model. These observations indicate that 15( S )-HETE-induced monocyte/macrophage migration and trafficking require ROS-mediated CaMKIV activation leading to formation of NFATc3 and FosB heterodimer, which binds and activates the TF promoter., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Previously, we have demonstrated that 15(S)-hydroxyeicosatetranoic acid (15(S)-HETE) induces CD36 expression involving STAT1. Many studies have shown that peroxisome proliferator-activated receptor (PPAR)-γ mediates CD36 expression. Therefore, we asked the question whether these transcriptional factors interact with each other in the regulation of CD36 expression by 15(S)-HETE. Here, we show that STAT1 interacts with PPARγ in the induction of CD36 expression and foam cell formation by 15(S)-HETE. In addition, using molecular biological approaches such as EMSA, supershift EMSA, ChIP, re-ChIP, and promoter-reporter gene assays, we demonstrate that the STAT1 and PPARγ complex binds to the STAT-binding site at -107 nucleotides in the CD36 promoter and enhances its activity. Furthermore, the interaction of STAT1 with PPARγ depends on STAT1 acetylation, which is mediated by p300. In addition, our findings show that reactive oxygen species-dependent Syk and Pyk2 stimulation is required for p300 tyrosine phosphorylation and activation. Together, these results demonstrate that an interaction between STAT1, p300, and peroxisome proliferator-activated receptor-γ is required for 15(S)-HETE-induced CD36 expression, oxidized low density lipoprotein uptake, and foam cell formation, critical events underlying the pathogenesis of atherosclerosis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

To understand the mechanisms by which 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) activates signal transducer and activator of transcription 3 (STAT3), we studied the role of epidermal growth factor receptor (EGFR). 15(S)-HETE stimulated tyrosine phosphorylation of EGFR in a time-dependent manner in vascular smooth muscle cells (VSMCs). Interference with EGFR activation blocked 15(S)-HETE-induced Src and STAT3 tyrosine phosphorylation, monocyte chemoattractant protein-1 (MCP-1) expression and VSMC migration. 15(S)-HETE also induced tyrosine phosphorylation of Janus kinase 2 (Jak2) in VSMCs, and its inhibition substantially reduced STAT3 phosphorylation, MCP-1 expression, and VSMC migration. In addition, Src formed a complex with EGFR and Jak2, and its inhibition completely blocked Jak2 and STAT3 phosphorylation, MCP-1 expression, and VSMC migration. 15(S)-HETE induced the production of H(2)O(2) via an NADPH oxidase-dependent manner and its scavengers, N-acetyl cysteine (NAC) and catalase suppressed 15(S)-HETE-stimulated EGFR, Src, Jak2, and STAT3 phosphorylation and MCP-1 expression. Balloon injury (BI) induced EGFR, Src, Jak2, and STAT3 phosphorylation, and inhibition of these signaling molecules attenuated BI-induced MCP-1 expression and smooth muscle cell migration from the medial to the luminal surface resulting in reduced neointima formation. In addition, inhibition of EGFR blocked BI-induced Src, Jak2, and STAT3 phosphorylation. Similarly, interference with Src activation suppressed BI-induced Jak2 and STAT3 phosphorylation. Furthermore, adenovirus-mediated expression of dnJak2 also blocked BI-induced STAT3 phosphorylation. Consistent with the effects of 15(S)-HETE on the activation of EGFR-Src-Jak2-STAT3 signaling in VSMCs in vitro, adenovirus-mediated expression of 15-lipoxygenase 1 (15-Lox1) enhanced BI-induced EGFR, Src, Jak2, and STAT3 phosphorylation leading to enhanced MCP-1 expression in vivo. Blockade of Src or Jak2 suppressed BI-induced 15-Lox1-enhanced STAT3 phosphorylation, MCP-1 expression, and neointima formation. In addition, whereas dominant negative Src blocked BI-induced 15-Lox1-enhanced Jak2 phosphorylation, dnJak2 had no effect on Src phosphorylation. Together, these observations demonstrate for the first time that the 15-Lox1-15(S)-HETE axis activates EGFR via redox-sensitive manner, which in turn mediates Src-Jak2-STAT3-dependent MCP-1 expression leading to vascular wall remodeling.

In the present study, we show that the eicosanoid compound, 20-hydroxyeicosatetraenoic acid (20-HETE), an important arachidonic acid metabolite, activates mouse TRPC6 in a stable, overexpressing HEK293 cell line, Hek-t6.11. Application of 20-HETE rapidly induced an inward, non-selective current in whole-cell recordings, which was inhibited by N-methyl-d-glucamine, 1.8 mm Ca2+, and 100 microM Gd3+ but remained unaffected by flufenamate and indomethacin. The current-voltage relationship obtained at low concentrations of 20-HETE (1-10 microM) demonstrated slight inward rectification, whereas the highest concentration of 20-HETE tested (30 microM) showed outward rectification, as shown previously for these channels using 100 microM 1-oleoyl-2-acetyl-sn-glycerol. Dose-response curves indicate that 20-HETE activated TRPC6 channels with an EC50 = 0.8 microM. Single channel analysis using inside-out patches revealed that 20-HETE increased open probability of mouse TRPC6 channels approximately 3-fold, and this was in a membrane-delimited fashion. Interestingly, 20-HETE did not provoke changes in intracellular Ca2+ concentrations. Thus, we have identified an arachidonic acid metabolite, 20-HETE, as a novel activator for a TRP family member, TRPC6.

To understand the role of eicosanoids in angiogenesis, we have studied the effect of lipoxygenase metabolites of arachidonic acid on human microvascular endothelial cell (HMVEC) DNA synthesis. Among the various lipoxygenase metabolites of arachidonic acid tested, 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE) induced DNA synthesis in HMVEC. 5(S)-HETE also stimulated Jak-2, STAT-1, and STAT-3 tyrosine phosphorylation and STAT-3-DNA binding activity. Tyrphostin AG490, a specific inhibitor of Jak-2, significantly reduced tyrosine phosphorylation and DNA binding activity of STAT-3 and DNA synthesis induced by 5(S)-HETE. In addition, 5(S)-HETE stimulated phosphatidylinositol 3-kinase (PI3-kinase) activity and phosphorylation of its downstream targets Akt, p70S6K, and 4E-BP1 and their effector molecules ribosomal protein S6 and eIF4E. LY294002 and rapamycin, potent inhibitors of PI3-kinase and mTOR, respectively, also blocked the DNA synthesis induced by 5(S)-HETE. Interestingly, AG490 attenuated 5(S)-HETE-induced PI3-kinase activity and phosphorylation of Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. 5(S)-HETE induced the expression of basic fibroblast growth factor 2 (bFGF-2) in a Jak-2- and PI3-kinase-dependent manner. In addition, a neutralizing anti-bFGF-2 antibody completely blocked 5(S)-HETE-induced DNA synthesis in HMVEC. Together these results suggest that 5(S)-HETE stimulates HMVEC growth via Jak-2- and PI3-kinase-dependent induction of expression of bFGF-2. These findings also reveal a cross-talk between Jak-2 and PI3-kinase in response to 5(S)-HETE in HMVEC.

Human prostate tumors have elevated levels of 15-lipoxygenase-1 (15-LOX-1) and data suggest that 15-LOX-1 may play a role in the development of prostate cancer. In contrast, 15-LOX-2 expression is higher in normal rather than in tumor prostate tissue and appears to suppress cancer development. We recently reported that 13-(S)-HODE, the 15-LOX-1 metabolite, up-regulates the MAP kinase signaling pathway and subsequently down-regulates PPARgamma in human colorectal carcinoma cells. To determine whether this mechanism is applicable to prostate cancer and what the effects of 15-LOX-2 are, we investigated the effect of 15-LOX-1, 15-LOX-2, and their metabolites on epidermal growth factor (EGF)- and insulin-like growth factor (IGF)-1 signaling in prostate carcinoma cells. In PC3 cells, 13-(S)-HODE, a 15-LOX-1 metabolite, up-regulated MAP kinase while in contrast 15-(S)-HETE, a 15-LOX-2 metabolite, down-regulated MAP kinase. As a result, 13-(S)-HODE increased PPARgamma phosphorylation while a subsequent decrease in PPARgamma phosphorylation was observed with 15-(S)-HETE. Thus, 15-LOX metabolites have opposing effects on the regulation of the MAP kinase signaling pathway and a downstream target of MAP kinase signaling like PPARgamma. In addition to the EGF signaling pathway, the IGF signaling pathway appears to be linked to prostate cancer. 13-(S)-HODE and 15-(S)-HETE up-regulate or down-regulate, respectively, both the MAPK and Akt pathways after activation with IGF-1. Thus, the effect of these lipid metabolites is not solely restricted to EGF signaling and not solely restricted to MAPK signaling. These results provide a plausible mechanism to explain the apparent opposing effects 15-LOX-1 and 15-LOX-2 play in prostate cancer.

Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a > or =20 microm concentration of the fatty acid. We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on PKC, [(3)H]phorbol dibutyrate (PDB), increased binding of [(3)H]PDB within 15 s of exposure to > or =10-30 nm AA. Other unsaturated fatty acids, but not a saturated fatty acid, likewise stimulated PDB binding. These responses, similar to those caused by chemotactic factors, resulted from a rise in the number of diacylglycerol receptors that were plasma membrane-associated and therefore accessible to PDB. Unlike chemotactic factors, however, AA was fully active on cells overloaded with Ca(2+) chelators. The major metabolites of AA made by PMN, leukotriene B(4) and 5-hydroxyicosatetraenoate, did not mimic AA, and an AA antimetabolite did not block responses to AA. AA also induced PMN to translocate cytosolic PKCalpha, beta(II), and delta to membranes. This response paralleled PDB binding with respect to dose requirements, time, Ca(2+)-independence, resistance to an AA antimetabolite, and induction by another unsaturated fatty acid but not by a saturated fatty acid. Finally, HEK 293 cells transfected with vectors encoding PKCbeta(I) or PKCdelta fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKCbeta translocation from cytosol to plasma membrane at > or =0.5 microm, and EGFP-PKCdelta translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nm. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca(2+) or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.

The lipoxygenase-derived eicosanoids leukotrienes and lipoxins are well defined regulators of hemeodynamics and leukocyte recruitment in inflammatory conditions. Here, we describe a novel bioaction of lipoxin A(4) (LXA(4)), namely inhibition of leukotriene D(4) (LTD(4))-induced human renal mesangial cell proliferation, and investigate the signal transduction mechanisms involved. LXA(4) blocked LTD(4)-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity in parallel to inhibition of LTD(4)-induced mesangial cell proliferation. Screening of a human mesangial cell cDNA library revealed expression of the recently described cys-leukotriene(1)/LTD(4) receptor. LTD(4)-induced mesangial cell proliferation required both extracellular-related signal regulated kinase (erk) and PI 3-kinase activation and may involve platelet-derived growth factor receptor transactivation. LTD(4)-stimulated the MAP kinases erk and p38 via a pertussis toxin (PTX)-sensitive pathway dependent on PI 3-kinase and protein kinase C activation. On screening a cDNA library, mesangial cells were found to express the previously described LXA(4) receptor. In contrast to LTD(4), LXA(4) showed differential activation of erk and p38. LXA(4) activation of erk was insensitive to PTX and PI 3-kinase inhibition, whereas LXA(4) activation of p38 was sensitive to PTX and could be blocked by the LTD(4) receptor antagonist SKF 104353. These data suggest that LXA(4) stimulation of the MAP kinase superfamily involves two distinct receptors: one shared with LTD(4) and coupled to a PTX-sensitive G protein (G(i)) and a second coupled via an alternative G protein, such as G(q) or G(12), to erk activation. These data expand on the spectrum of LXA(4) bioactions within an inflammatory milieu.

In mammals, leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes hydrolysis of leukotriene A(4) into the proinflammatory leukotriene B(4) and also possesses an arginyl aminopeptidase activity. We have cloned, expressed, and characterized a protein from Saccharomyces cerevisiae that is 42% identical to human leukotriene A(4) hydrolase. The purified protein is an anion-activated leucyl aminopeptidase, as assessed by p-nitroanilide substrates, and does not hydrolyze leukotriene A(4) into detectable amounts of leukotriene B(4). However, the S. cerevisiae enzyme can utilize leukotriene A(4) as substrate to produce a compound identified as 5S,6S-dihydroxy-7,9-trans-11, 14-cis-eicosatetraenoic acid. Both catalytic activities are inhibited by 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (thioamine), a competitive inhibitor of human leukotriene A(4) hydrolase. Furthermore, the peptide cleaving activity of the S. cerevisiae enzyme was stimulated approximately 10-fold by leukotriene A(4) with kinetics indicating the presence of a lipid binding site. Nonenzymatic hydrolysis products of leukotriene A(4), leukotriene B(4), arachidonic acid, or phosphatidylcholine were without effect. Moreover, leukotriene A(4) could displace the inhibitor thioamine and restore maximal aminopeptidase activity, indicating that the leukotriene A(4) binding site is located at the active center of the enzyme. Hence, the S. cerevisiae leukotriene A(4) hydrolase is a bifunctional enzyme and appears to be an early ancestor to mammalian leukotriene A(4) hydrolases.

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 metabolite of arachidonic acid, is a potent vasoconstrictor, and has been implicated in the myogenic activation of renal and cerebral arteries. We examined the role of protein kinase C (PKC) in the signal transduction pathway by which 20-HETE induces vasoconstriction and inhibition of whole-cell K+ current in cat cerebral vascular smooth muscle. 20-HETE induced a concentration-dependent constriction in isolated pressurized cat middle cerebral arteries (-29 +/- 8% at 1 microM). However, in the presence of an N-myristoylated PKC pseudosubstrate inhibitor peptide (MyrPsiPKC-I(19-27)), 20-HETE induced a concentration-dependent vasodilation (26 +/- 4% at 1 microM). In whole-cell voltage clamp studies, application of 20-HETE inhibited whole-cell K+ current recorded in cat cerebral vascular smooth muscle cells, an effect that was attenuated by MyrPsiPKC-I(19-27). Further evidence for the role of PKC activation in response to 20-HETE is the finding that 20-HETE increased the phosphorylation of myristoylated, alanine-rich PKC substrate in cultured cat cerebral vascular smooth muscle cells in a concentration- and PKC-dependent manner. These data provide evidence that PKC is an integral part of the signal transduction pathway by which 20-HETE elicits vasoconstriction of cerebral arteries and inhibition of whole-cell K+ current in cat cerebral vascular smooth muscle.

Lipoxins (LX) are bioactive eicosanoids that activate human monocytes and inhibit neutrophils. LXA4 is rapidly converted by monocytes to inactive products, and to resist metabolism, synthetic analogs of LXA4 were designed. Here, we examined the bioactivity of several LXA4 analogs in monocytes and found, for chemotaxis, 15(R/S)-methyl-LXA4 and 15-epi-LXA4 were equal in activity, and 16-phenoxy-LXA4 was more potent than native LXA4. Both 15(R/S)-methyl-LXA4 and 16-phenoxy-LXA4 were approximately 1 log molar more potent than LXA4 in stimulating THP-1 cell adherence (EC50 approximately 1 x 10(-10) M). Dimethylamide derivatives of the LXA4 analogs also possessed agonist rather than antagonist properties for monocytes. Neither LXA4 nor 16-phenoxy-LXA4 affected monocyte-mediated cytotoxicity. We cloned an LXA4 receptor from THP-1 cells identical to that found in PMN. Evidence of receptor-mediated function of LXA4 and the stable analogs in monocytes included desensitization of intracellular calcium mobilization to a second challenge by equimolar concentrations of these analogs, but not to LTB4. Increases in [Ca2+]i by LXA4 and the analogs were specifically inhibited by an antipeptide antibody to the LXA4 receptor; and both LXA4- and analog-induced adherence and increments in Ca2+ were sensitive to pertussis toxin. Together, these results indicate that the LXA4 stable analogs are potent monocyte chemoattractants and are more potent than native LXA4 in stimulating THP-1 cell adherence, at subnanomolar concentrations. Moreover, they provide additional evidence that the LXA4 stable analogs retain selective bioactivity in monocytes and are valuable instruments for examining the functions and modes of action of LXA4.

Addition of submicromolar concentrations of arachidonic acid (AA) to human neutrophils induced a 2-fold increase in the activity of a cytosolic phospholipase A2 (PLA2) when measured using sonicated vesicles of 1-stearoyl-2-[14C]arachidonoylphosphatidylcholine as substrate. A similar increase in cytosolic PLA2 activity was induced by stimulation of neutrophils with leukotriene B4 (LTB4), 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (5-HETE). LTB4 was the most potent of the agonists, showing maximal effect at 1 nM. Inhibition of 5-lipoxygenase with either eicosatetraynoic acid or zileuton prevented the AA-induced increase in PLA2 activity but had no effect on the response induced by LTB4. Furthermore, pretreatment of neutrophils with a LTB4-receptor antagonist, LY 255283, blocked the AA- and LTB4-induced activation of PLA2 but did not influence the action of 5-HETE. Treatment of neutrophils with pancreatic PLA2 also induced an increase in the activity of the cytosolic PLA2; this response was inhibited by both eicosatetraynoic acid or LY 255283. The increases in PLA2 activity in response to stimulation correlated with a shift in electrophoretic mobility of the 85-kDa PLA2, as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA2 likely underlies its increase in catalytic activity. Although stimulation of neutrophils with individual lipoxygenase metabolites did not induce significant mobilization of endogenous AA, they greatly enhanced the N-formylmethionyl-leucyl-phenylalanine-induced mobilization of AA as determined by mass spectrometry analysis. Our findings support a positive-feedback model in which stimulus-induced release of AA or exocytosis of secretory PLA2 modulate the activity of the cytosolic 85-kDa PLA2 by initiating the formation of LTB4. The nascent LTB4 is then released to act on the LTB4 receptor and thereby promote further activation of the 85-kDa PLA2. Since 5-HETE and LTB4 are known to prime the synthesis of platelet-activating factor, the findings suggest that 85-kDa PLA2 plays a role in platelet-activating factor synthesis.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate gene transcription in response to peroxisome proliferators and fatty acids. PPARs also play an important role in the regulation of adipocyte differentiation. It is unclear, however, what naturally occurring compounds activate each of the PPAR subtypes. To address this issue, a screening assay was established using heterologous fusions of the bacterial tetracycline repressor to several members of the peroxisome proliferator-activated receptor (PPAR) family. This assay was employed to compare the activation of PPAR family members by known PPAR activators including peroxisome proliferators and fatty acids. Interestingly, the activation of PPARs by fatty acids was partially inhibited by the cyclooxygenase inhibitor indomethacin, which prevents prostaglandin synthesis. Indeed, prostaglandins PGA1 and 2, PGD1 and 2, and PGJ2-activated PPARs, while a number of other prostaglandins had no effect. We also screened a variety of hydroxyeicosatetraenoic acids (HETEs) for the ability to activate PPARs. 8(S)-HETE, but not other (S)-HETEs, was a strong activator of PPAR alpha. Remarkably, PPAR activation by 8(S)-HETE was stereoselective. In addition, 8(S)-HETE was able to induce differentiation of 3T3-L1 preadipocytes. These results indicate that PPARs are differentially activated by naturally occurring eicosanoids and related molecules.

Human eosinophils produce upon treatment with 5-oxo-eicosatetraenoic acid or (5S,15S)-dihydroxyeicosatetraenoic acid a potent eosinophil-chemotactic eicosanoid, 5-oxo-15-hydroxy-(6E,8Z,11Z,13E)-eicosatetraenoi c acid (5-oxo-15-HETE). 5-Oxo-15-HETE induces human eosinophil (Eo) chemotaxis at nanomolar concentrations with an efficacy in vitro comparable to that seen for platelet activating factor. Comparison of Eo chemotactic activities of several structurally related eicosanoids with different substituents and/or double bound geometry led to the conclusion that maximal potency and efficacy of eosinophil-chemotactic and chemokinetic activity is present in 5-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE). The presence of a hydroxyl group at position C-15 is not necessary for potent chemotactic activity, whereas a geometric isomer having trans instead of cis double bond at C-atom 8, as well as esterified 5-oxo-ETE usually show a 5-10-fold lower potency. 5-Oxo-eicosanoids elicit a dose-dependent transient rise of intracellular Ca2+ levels in human Eos, however, in contrast to some other Eo chemotaxins do not induce degranulation. Cross-desensitization of Ca2+ mobilization and Eo chemotaxis revealed that the geometric isomers of 5-oxo-eicosanoids, 5(S)-HETE, and (5S,15S)-di-HETE cross-deactivate Eo responses to each other, whereas other, unrelated stimuli did not interfere with these lipids indicating that 5-oxo-eicosanoids activate Eos via a separate receptor.

Arachidonic acid and its 15-lipoxygenase metabolite (15S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid ((15S)-HETE) modulates the beta-adrenergic response of cultured rat neonatal cardiomyocytes as indicated by an increase in the beating rate following stimulation of the cells with suboptimal isoproterenol concentrations. The effect of 15-HETE was enantioselective for the S-isomer and could be detected at concentrations as low as 10(-12) M. (11S)-HETE and (5S,15S)-dihydroxy-(6E,8Z,11Z,13E)-eicosatetraen oic acid did also exhibit this effect, whereas other mono-, di-, and trihydroxyeicosanoids as well as the 15-lipoxygenase products of 11,14-eicosadienoic acid, of two eicosatrienoic acid isomers and of 5,8,11,14,17-eicosapentaenoic acid were ineffective. Immunohistochemical studies indicated the expression of a 15-lipoxygenase in cardiomyocytes and in resident heart mast cells. Induction of the beta-adrenergic supersensitivity is paralleled by a selective incorporation of (15S)-HETE into the cellular phosphatidylinositol pool. In contrast, (12S)-HETE, which did not induce beta-adrenergic supersensitivity, was incorporated preferentially into phosphatidylcholine and phosphatidylethanolamine. Calphostin C, an inhibitor of protein kinase C, blocked both the induction of supersensitivity by (15S)-HETE and its incorporation into phosphatidylinositol. These data suggest that in cardiomyocytes 15-lipoxygenase metabolites specifically induces a signal transduction cascade leading to a supersensitivity of the cells toward beta-adrenergic agonists, which involves the phosphatidylinositol cycle and a protein kinase C.

We examined how 5-hydroxyicosatetraenoate (5-HETE) activates human neutrophils (PMN). 5-HETE stimulates PMN to mobilize Ca2+ but has little effect on degranulation or superoxide anion production. It nonetheless stereospecifically induced these responses in cells primed with tumor necrosis factor-alpha and likewise induced PMN plasma membranes to bind 35S-labeled guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and phosphohydrolyze [gamma-32P]GTP. Pertussis toxin blocked GTP gamma S binding responses. Scatchard analyses of GTP gamma S binding data indicated that 5-HETE raised the Ka of high affinity GTP gamma S binding sites without altering these sites' numbers or the parameters of low affinity GTP gamma S binding. Since N-formyl-Met-Leu-Phe, platelet-activating factor, and leukotriene (LT) B4 have these same bioactions, receptors for the latter agents might mediate responses to 5-HETE. However, 5-HETE desensitized degranulation responses to itself but not to the receptor agonists, the receptor agonists desensitized to themselves but not 5-HETE, and a LTB4 antagonist inhibited LTB4 but not 5-HETE in all assays. Finally, PMN and their membranes took up [3H] 5-HETE at 4 or 37 degrees C but, at both temperatures, also acylated the radiolabel into glycerolipids. Acylation nullified assessment of 5-HETE binding and questions reports that measure the cell binding, but not metabolism, of various HETEs. Our studies thus indicate 5-HETE acts by a down-regulatable, G protein-linked mechanism and represent the best available evidence that 5-HETE does not operate through, for example, LTB4 receptors.

Bone resorption requires cooperation between osteoclasts and mononuclear accessory cells by mechanisms which have not been elucidated. Since multinucleated cells in giant cell tumors of bone have many phenotypic and functional characteristics of normal osteoclasts, we have examined the interaction between the bone-resorbing multinucleated cells and the distinct mononuclear stromal cells from these tumors. We have found that these mononuclear cells produce an activity which stimulates both giant cells from giant cell tumors and rodent osteoclasts to resorb bone in vitro. We have identified the activity and found that it represents several products of the 5-lipoxygenase pathway of arachidonic acid metabolism, namely 5-hydroxyeicosatetraenoic acid and the leukotrienes. These data indicate that 5-lipoxygenase metabolites stimulate isolated osteoclasts to resorb bone in vitro and may represent a mechanism by which mononuclear stromal cells in human giant cell tumors communicate with the giant cells. In addition, these results may explain a possible mechanism for communication between accessory cells and osteoclasts involved in normal bone resorption.

Murine prostaglandin endoperoxide (PGH) synthase-1 and PGH synthase-2 expressed in cos-1 cells were found to be differentially sensitive to inhibition by common nonsteroidal anti-inflammatory drugs (NSAIDs). Aspirin completely inhibited bis-oxygenation of arachidonate by PGH synthase-1; in contrast, aspirin-treated PGH synthase-2 metabolized arachidonate primarily to 15-hydroxyeicosatetraenoic acid (15-HETE) instead of PGH2. ID50 values were determined for a panel of common NSAIDs by measuring instantaneous inhibition of cyclooxygenase activity using an oxygen electrode. Among common NSAIDs tested, indomethacin, sulindac sulfide, and piroxicam preferentially inhibited PGH synthase-1; ibuprofen, flurbiprofen, and meclofenamate inhibited both enzymes with comparable potencies; and 6-methoxy-2-naphthylacetic acid preferentially inhibited PGH synthase-2. These results demonstrate that the two PGH synthases are pharmacologically distinct and indicate that it may be possible to develop isozyme-specific cyclooxygenase inhibitors useful both for anti-inflammatory therapy and for delineating between the biological roles of the PGH synthase isozymes.

The mechanisms of stimulation of the inactive 5-lipoxygenase in mast/basophil PT-18 cells by microM 15-hydroxyeicosatetraenoic acid (15-HETE) was investigated. Treatment of PT-18 cells with pM 15-[3H]HETE at 4 degrees for 3 h resulted in the cell association of 10% of the ligand: two-thirds was incorporated into cellular lipids and a third was bound to specific 15-HETE cellular binding sites. Binding data analysis indicated a single class of 15-HETE binding sites with a Kd of 162 nM and a Bmax of 7.1 x 10(5) sites/cell. Unlabeled 15-HETE, 12-HETE, and 5,15-diHETE inhibited the binding of 15-[3H]HETE to cells, whereas LTB4 and PGF2 alpha were relatively ineffective. 2.4 microM 15-HETE (unlabeled) prevented 50% 15-[3H]HETE incorporation. Examination of the effects of 15-HETE methyl ester, 12-HETE, 5,15-diHETE, and pertussis toxin on both the 15-HETE-induced 5-lipoxygenase activation and 15-HETE cell association processes indicated a preponderant correlation of this activation process with specific 15-HETE binding rather than 15-HETE incorporation into phospholipids. In addition, 5,15-diHETE itself stimulated the inactive 5-lipoxygenase and eight times more [3H]diHETE was bound to cells than became incorporated into cellular lipids. The results support the involvement of low affinity 15-HETE receptors, rather than 15-HETE incorporation into cellular lipids, in the 15-HETE-induced stimulation of the 5-lipoxygenase in PT-18 cells.

Noncyclooxygenase metabolites of arachidonic acid may be potent modulators of the mitogenic response of renal mesangial cells to the mitogenic vasoactive peptide arginine vasopressin (AVP). Since Ca2+ is a critical second messenger in the response of mesangial cells to AVP, and Ca2+ has been implicated in the regulation of growth, we determined whether noncyclooxygenase metabolites altered the phospholipase C-Ca2+ signalling cascade which is activated by AVP. Pretreatment of mesangial cells for 10 min with lipoxygenase and cytochrome P450 monooxygenase inhibitors, nordihydroguaiaretic acid (NDGA, 10(-5) M) or SKF-525A (2.5 x 10(-5) M), but not the cyclooxygenase inhibitor indomethacin (2 x 10(-5) M), reduced the magnitude of the AVP (10(-8) and 10(-7) M)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) without affecting inositol trisphosphate production. With 10(-8) M AVP, [Ca2+]i increased to 250 +/- 47 nM in NDGA-treated cells versus 401 +/- 59 nM in control cells (p less than 0.01). [Ca2+]i, measured 2 min after exposure to AVP, was also lower with NDGA (152 +/- 21 nM) when compared with AVP alone (220 +/- 22 nM, p less than 0.01). 14,15-epoxyeicosatrienoic acid (EET) (10(-8) M), which had no effect on inositol trisphosphate production, completely reversed the NDGA-induced inhibition of the [Ca2+]i transient, whereas 5-hydroperoxyeicosatetraenoic acid (HPETE) (5 x 10(-7) M) did not. Pretreatment with higher concentrations of 14,15-EET (10(-7)-10(-6) M) markedly potentiated the AVP-induced increase in [Ca2+]i. NDGA-induced inhibition of the AVP-generated [Ca2+]i transient was also observed when cells were incubated in low Ca2+ media ([Ca2+] less than 5 x 10(-8) M), suggesting that NDGA pretreatment impaired intracellular release of Ca2+. Since NDGA had no direct effect on inositol 1,4,5-trisphosphate-induced Ca2+ release, we postulated that NDGA blocked production of a metabolite that releases Ca2+ from intracellular stores. 14,15-EET and 15-HPETE, but not 15-hydroxyeicosatetraenoic acid (each at 3 x 10(-7) M), raised [Ca2+]i when added directly to cells in low Ca2+ media. In permeabilized cells 14,15-EET and 15-HPETE (10(-7) M) potently released Ca2+ from intracellular stores. In summary, noncyclooxygenase metabolites of arachidonic acid, and in particular P450 metabolites, are potent endogenous amplifiers of the AVP-induced [Ca2+]i signal by mechanisms not directly involving phospholipase C activation. This effect is mediated, at least in part, by enhanced release of Ca2+ from intracellular storage sites by an inositol 1,4,5-trisphosphate-independent mechanism.

Neutrophils treated with optimal amounts of tumor promoters that activate protein kinase C (e.g. mezerein) release large quantities of superoxide (O2-) and exhibit an intense phosphorylation of two proteins with molecular masses of approximately 47 and 49 kDa. These cells can also be stimulated synergistically to release a comparable amount of O2-. This involves treatment with a suboptimal amount of a tumor promoter and an agent capable of elevating cellular Ca2+. Neutrophils treated in the former fashion exhibit a redistribution of the activity of protein kinase C from a soluble to a particulate fraction that is stable in the presence of Ca2+ chelators, whereas cells stimulated synergistically do not do so to an appreciable extent (Badwey, J. A., Robinson, J. M., Horn, W., Soberman, R. J., Karnovsky, M. J., and Karnovsky, M. L. (1988) J. Biol. Chem. 263, 2779-2786). In this paper, we report that neutrophils stimulated synergistically do exhibit a significant incorporation of 32P into the 47-kDa protein, but with little labeling of the 49-kDa species. This labeling of the 47-kDa protein was greater than the sum of those observed with each agent added separately but was less than that observed in cells stimulated with optimal amounts of tumor promoters alone. An inhibitor of protein kinase C (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) blocked O2- release and the phosphorylation of the 47-kDa protein under all conditions of stimulation mentioned, whereas an inhibitor of cyclic nucleotide-dependent kinases had no effect on these phenomena. Thus, labeling of the 47-kDa protein can occur in the absence of a "tight" translocation of protein kinase C to membrane and was always observed during synergy. The data support a role for protein kinase C and the 47-kDa phosphoprotein in the synergistic stimulation of neutrophils.

We recently demonstrated that 20-hydroxyeicosatetraenoic acid (20-HETE) constricts rat aortic rings. The contractile response was partially dependent on the presence of endothelium and was abolished by pretreatment of the rings with either indomethacin or the endoperoxide/thromboxane receptor antagonist, SQ29548. Addition of GSH or SnCl2 to the organ bath diminished the contractile response of 20-HETE, whereas preincubation of the rings with a thromboxane synthase inhibitor did not affect the 20-HETE induced contractions. Short time incubation (2 min) of 20-HETE with ram seminal vesicle microsomes in the presence of p-hydroxymercurybenzoate yielded metabolites which migrated similarly on thin layer chromatography to the known arachidonate endoperoxides prostaglandin (PG) G2 and PGH2 and possess vasoconstrictory properties. The vasoconstriction was dose-dependent with a half-life of approximately 6.3 +/- 0.6 min. Addition of SQ29548 to the aortic ring bath 1 min after metabolite elicited vasoconstriction produced immediate relaxation. Furthermore, pretreatment of the rings with SQ29548 totally abolished the contraction. SnCl2 reduction of the metabolites produced in incubation of rat seminal vesicles with 20-HETE and p-hydroxymercurybenzoate resulted in a single radioactive peak which was further identified by gas chromatography/mass spectrometry as 20-hydroxy-PGF2 alpha. The inhibitory effect of SQ29548, the appearance of labile metabolites with a half-life of approximately 6 min and the production of 20-hydroxy-PGF2 alpha by SnCl2 reduction clearly indicate that the vasoconstrictor metabolites of 20-HETE are the labile endoperoxides of 20-HETE, 20-hydroxy-PGG2, and 20-hydroxy-PGH2.

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.

Linoleate metabolism via the cyclooxygenase pathway enhances the proliferation of mammary epithelial cells in serum-free culture in the presence of epidermal growth factor and insulin (Bandyopadhyay, G.K., Imagawa, W., Wallace, D., and Nandi, S. (1987) J. Biol. Chem. 262, 2750-2756). Prostaglandin E2 (PGE2) can fully substitute for linoleic acid provided endogenous hydroxyeicosatetraenoic acids (HETEs, lipoxygenase metabolites) are available. The PGE2 effect is partial if lipoxygenase activity is inhibited by nordihydroguaiaretic acid. Any combination of two HETEs out of three tested (5-, 12-, and 15-HETEs) stimulates growth synergistically with PGE2; and together (i.e. PGE2 + HETEs), they completely substitute for linoleate. In the absence of PGE2, maximal stimulation cannot be attained with HETEs. Exogenous 5-HETE, compared with 12- or 15-HETE, is preferentially incorporated by the mammary epithelial cells, and about 25-30% of it is retained esterified in phospholipids. The cellular level of nonesterified, free HETE is low. Radioimmunoassay revealed that the concentrations of 12- and 15-HETEs in the culture media (with or without added linoleate) were always higher than that of 5-HETE. Both intra- and extracellular free HETEs are rapidly metabolized by the cells. Since these cells are capable of producing eicosanoids from linoleate, periodic supplementation of the cultures with linoleate allows maintenance of higher HETE and PGE2 levels. Thus, it appears that not only are HETEs short-lived in the cell cultures, but cells handle 5-HETE differently than 12- and 15-HETEs. Whatever may be the pathways of interaction, synergism between HETEs and PGE2 seems to explain how linoleate stimulates the growth of mammary epithelial cells in the presence of epidermal growth factor and insulin.

Arachidonic acid metabolism in ionophore A23187-activated human polymorphonuclear leukocytes (PMNs) proceeds predominantly via the 5-lipoxygenase pathway in comparison to metabolism by the 15-lipoxygenase route. Products of both lipoxygenase pathways appear to be involved in the mediation of inflammatory reactions. Pretreatment of polymorphonuclear leukocytes with micromolar amounts of the platelet-derived 12-lipoxygenase product 12-hydroxy-5,8,10,14- eicosatetraenoic acid (12-HETE) prior to the addition of A23187 and [14C]arachidonic acid resulted in the unexpected dose-dependent stimulation of the 15-lipoxygenase pathway, as evidenced by the formation of [14C]15-HETE. A concomitant inhibition of the 5-lipoxygenase pathway was also observed. The structural identity of 15-HETE was confirmed by retention times on straight-phase and reverse-phase high pressure liquid chromatography in comparison with an authentic standard, radioimmunoassay, and chemical derivatization. When other isomeric HETEs were tested, the order of stimulatory potencies was 15-HETE greater than 12-HETE greater than 5-HETE. When arachidonic acid metabolism via the 5-lipoxygenase route was inhibited by nordihydroguaiaretic acid, previously ineffective concentrations of exogenous 12-HETE were now able to stimulate the polymorphonuclear leukocyte 15-lipoxygenase. Thus, blockade of the 5-lipoxygenase pathway appeared to be a prerequisite for the activation of the 15-lipoxygenase. The HETE-induced activation of the 15-lipoxygenase occurred within 1-2 min, was a reversible process, and was enhanced in the presence of A23187. In nine donors tested, up to 14-fold stimulation of [14C]15-HETE production was observed. Our results indicate that endogenous HETEs can have a dual role in the post-phospholipase regulation of arachidonic acid metabolism since they can act as physiological stimulators of the 15-lipoxygenase as well as inhibitors of the 5-lipoxygenase.

Fluid phase interactions between arterial endothelial cells (EC) and smooth muscle cells (SMC) have been studied in vitro to assess the regulation of lipid metabolism in SMC (Hajjar, D. P., Falcone, D. J., Amberson, J. B., and Hefton, J. M. (1985) J. Lipid Res. 26, 1212-1223; Davies, P. F., Truskey, G. A., Warren, H. B., O'Connor, S. E., and Eisenhaure, B. H. (1985) J. Cell Biol. 101, 871-879). To identify EC-derived agonists which may modulate cholesterol metabolism in co-cultured SMC, we assessed the role of EC-derived eicosanoids and platelet-derived growth factor (PDGF) in the regulation of cholesteryl ester (CE) hydrolysis in SMC. The major eicosanoids synthesized by EC include PGI2 and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and, to a lesser extent, prostaglandin E2. Exogenously added PGI2 and 12-HETE stimulated CE hydrolytic activity in SMC by 49 and 35%, respectively, when co-cultured with aspirin-treated EC. Aspirin-treated EC when co-cultured with SMC did not stimulate CE hydrolytic activity in SMC, as was the case with non-aspirin-treated EC, suggesting a role of eicosanoids in the regulation of cholesterol metabolism. Other humoral agents derived from EC such as PDGFc stimulated CE hydrolytic activity almost 2-fold in SMC cultured alone or co-cultured with EC. Aspirin-treated EC, incubated with 10 ng/ml PDGF, did not stimulate CE hydrolytic activity in co-cultured SMC. These results suggest that growth factor-promoting activity may enhance CE hydrolysis via the PGI2-cyclic AMP-CE hydrolysis cascade. This hypothesis supports our observations that PDGF stimulates PGI2 production in SMC. Elevated PGI2, in turn, can stimulate CE hydrolysis in these cells. Our findings suggest that the regulation of cholesterol metabolism in SMC can involve, at least in part, growth factors and EC-derived eicosanoids. These may play a central role in the regulation of hemostasis and the inflammatory response.

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine. This induction of meiotic divisions can be triggered also by four fatty acids: 5,8,11-20:3; 5,8,11,14-20:4 (arachidonic acid); 6,9,12,15-20:4; 5,8,11,14,17-20:5, all other fatty acids being completely inactive. This maturation triggered by eicosanoids occurs in the micromolar range and is facilitated by the presence of calcium. A variety of arachidonic acid derivatives (esters, epoxides, etc.) and metabolites (cyclooxygenase and lipoxygenase products) has been tested; the biological activity is restricted to 8-hydroxyeicosatetraenoic acid (8-HETE), other mono- and poly-HETEs being completely inactive. Maturation triggered by 8-HETE occurs around 10 nM and is insensitive to the presence of calcium. 8-HETE methyl ester and 8-hydroperoxyeicosatetraenoic acid are able to induce maturation at higher concentrations. Both (8S) and (8R) stereoisomers have been tested; the biological activity is strictly restricted to the (8R) isomer. 8-HETE triggers a complete maturation, i.e. maturation-promoting factor appearance, germinal vesicle breakdown, emission of the polar bodies, and formation of a female pronucleus. (8R)-HETE, but not (8S)-HETE, triggers the typical decrease in cyclic AMP concentration induced by 1-methyladenine and the burst of protein phosphorylation associated with maturation. Starfish oocytes oxidize exogenous arachidonic acid into 8-HETE and other HETEs. 8-HETE was identified, after high pressure liquid chromatography purification, by gas chromatography mass spectrometry. Furthermore, it was found that the starfish oocytes only produce the (8R)-HETE isomer. This highly stereospecific induction of oocyte maturation by (8R)-HETE suggests that this fatty acid, or a very closely related fatty acid, may play a role in the transduction of the 1-methyladenine message at the plasma membrane level.

When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, two biologically active metabolites of arachidonic acid were formed. The structure of one of the metabolites, compound C, was previously reported to be 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid and was found to be a potent inhibitor of the Na+/K+-ATPase in the cornea. The second metabolite, compound D, was found to be a potent vasodilator as well as having the property of stimulating protein influx into the aqueous humor of the eye. Following purification of compound D by thin layer chromatography and high pressure liquid chromatography, it was found to lack a UV chromophore in contrast to the previously reported cytochrome P-450-dependent metabolite. Mass spectrometric analysis using positive and negative ionization modes was carried out on derivatized compound D that had been synthesized from a mixture of labeled [( 5,6,8,9,11,12,14,15-2H8]) and unlabeled arachidonic acid incubated with corneal microsomes. The novel arachidonate metabolite had abundant fragment ions consistent with compound D being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon 12 of the eicosanoid backbone; only seven deuterium atoms from [2H8]arachidonate were retained in the structure. Oxidative ozonolysis yielded a product indicating that the double bonds in metabolite D resided between carbons at positions 8 and 9 and positions 14 and 15 of the 20-carbon chain. Compound D was therefore characterized as 12-hydroxy-5,8,14-eicosatrienoic acid. Model compounds were synthesized from dimethyl malate with the hydroxy at the 12 position with both the R and S absolute configuration and with all double bonds of the cis configuration. Only the 12(R) isomer was found to be a potent vasodilator and to increase aqueous humor protein concentration, suggesting that the biologically active compound D was 12(R)-hydroxy-5,8,14-(Z,Z,Z)-eicosatrienoic acid. As this compound possesses proinflammatory properties, it may play a role in the wound-healing processes of corneal injury.