What are cyclooxygenases and lipoxygenases doing in the driver's seat of carcinogenesis?

Substantial evidence supports a functional role for cyclooxygenase- and lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Genetic intervention studies firmly established cause-effect relations for cyclooxygenase-2, but cyclooxygenase-1 may also be involved. In addition, pharmacologic cyclooxygenase inhibition was found to suppress carcinogenesis in both experimental mouse models and several cancers in humans. Arachidonic acid-derived eicosanoid or linoleic acid-derived hydro[peroxy]fatty acid signaling are likely to be involved impacting fundamental biologic phenomena as diverse as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, long chain unsaturated fatty acid oxidation reactions indicate antipodal functions of distinct lipoxygenase isoforms in carcinogenesis, i.e., the 5- and platelet-type 12-lipoxygenase exhibit procarcinogenic activities, while 15-lipoxygenase-1 and 15-lipoxygenase-2 may suppress carcinogenesis.

IL-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression.

Dendritic cell (DC) differentiation from human CD34(+) hematopoietic progenitor cells (HPCs) can be triggered in vitro by a combination of cytokines consisting of stem cell factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha. The immune response regulatory cytokines, IL-4 and IL-13, promote DC maturation from HPCs, induce monocyte-DC transdifferentiation, and selectively up-regulate 15-lipoxygenase 1 (15-LO-1) in blood monocytes. To gain more insight into cytokine-regulated eicosanoid production in DCs we studied the effects of IL-4/IL-13 on LO expression during DC differentiation. In the absence of IL-4, DCs that had been generated from CD34(+) HPCs in response to stem cell factor/granulocyte-macrophage colonystimulating factor/tumor necrosis factor alpha expressed high levels of 5-LO and 5-LO activating protein. However, a small subpopulation of eosinophil peroxidase(+) (EOS-PX) cells significantly expressed 15-LO-1. Addition of IL-4 to differentiating DCs led to a marked and selective down-regulation of 5-LO but not of 5-LO activating protein in DCs and in EOS-PX(+) cells and, when added at the onset of DC differentiation, also prevented 5-LO up-regulation. Similar effects were observed during IL-4- or IL-13-dependent monocyte-DC transdifferentiation. Down-regulation of 5-LO was accompanied by up-regulation of 15-LO-1, yielding 15-LO-1(+) 5-LO-deficient DCs. However, transforming growth factor beta1 counteracted the IL-4-dependent inhibition of 5-LO but only minimally affected 15-LO-1 up-regulation. Thus, transforming growth factor beta1 plus IL-4 yielded large mature DCs that coexpress both LOs. Localization of 5-LO in the nucleus and of 15-LO-1 in the cytosol was maintained at all cytokine combinations in all DC phenotypes and in EOS-PX(+) cells. In the absence of IL-4, major eicosanoids of CD34(+)-derived DCs were 5S-hydroxyeicosatetraenoic acid (5S-HETE) and leukotriene B(4), whereas the major eicosanoids of IL-4-treated DCs were 15S-HETE and 5S-15S-diHETE. These actions of IL-4/IL-13 reveal a paradigm of eicosanoid formation consisting of the inhibition of one and the stimulation of another LO in a single leukocyte lineage.

5-lipoxygenase expression in dendritic cells generated from CD34(+) hematopoietic progenitors and in lymphoid organs.

The 5-lipoxygenase (5-LO) pathway in human CD34(+) hematopoietic progenitor cells, which were induced to differentiate into dendritic cells (DCs) by cytokines in vitro and in DCs of lymphoid tissues in situ, was examined. Extracts prepared from HPCs contained low levels of 5-LO or 5-LO-activating protein. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor-alpha (TNF-alpha) promoted DC differentiation and induced a strong rise in 5-LO and FLAP expression. Fluorescence-activated cell sorter (FACS) analyses identified a major DC population coexpressing human leukocyte antigen (HLA)-DR/CD80 and monocytic or Langerhans cell markers. Transforming growth factor-beta1 (TGF-beta-1), added to support DC maturation, strongly promoted the appearance of CD1a(+)/Lag(+) Langerhans-type cells as well as mature CD83(+) DCs. TGF-beta-1 further increased 5-LO and FLAP expression, recruited additional cells into the 5-LO(+) DC population, and promoted production of 5-hydroxyeicosatetraenoic acid and leukotriene B(4) in response to calcium (Ca(++)) ionophore A23187. These in vitro findings were corroborated by 5-LO expression in distinct DC phenotypes in vivo. Scattered 5-LO and FLAP in situ hybridization signals were recorded in cells of paracortical T-lymphocyte-rich areas and germinal centers (GCs) of lymph nodes (LNs) and tonsil and in cells of mucosae overlying the Waldeyer tonsillar ring. 5-LO protein localized to both CD1a(+) immature DCs and to CD83(+) mature interdigitating DCs of T-lymphocyte-rich areas of LNs and tonsil. As DCs have the unique ability to initiate naive lymphocyte activation, our data support the hypothesis that leukotrienes act at proximal steps of adaptive immune responses. (Blood. 2000;96:3857-3865)

5-Lipoxygenase expression in Langerhans cells of normal human epidermis.

We studied expression of the 5-lipoxygenase (5-LO) pathway in normal human skin. In situ hybridization revealed a 5-LO mRNA-containing epidermal cell (EC) population that was predominantly located in the midportion of the spinous layer, in outer hair root sheaths, and in the epithelial compartment of sebaceous glands. Examination of skin specimens by immunohistochemistry and of primary ECs by flow cytometry mapped the 5-LO protein exclusively to Langerhans cells (LCs). The LC 5-LO protein was largely found in the nuclear matrix, in nuclear envelopes, and perinuclear regions as indicated by in situ confocal laser scan microscopy. Reverse transcription-PCR and immunoblot analyses of purified primary EC populations further indicated that LCs are major 5-LO expressing cells. Enriched primary LCs were also found to contain 5-LO activating protein (FLAP), leukotriene (LT) C4 synthase, and LTA4 hydrolase. By contrast, 5-LO, FLAP, and LTC4 synthase were undetectable or largely reduced, but LTA4 hydrolase transcripts and protein were identified in ECs depleted of LCs. These data show that naive LCs are major, and possibly the sole, 5-LO pathway expressing cells in the normal human epidermis.

Differential regulation of cyclo-oxygenase-2 and 5-lipoxygenase-activating protein (FLAP) expression by glucocorticoids in monocytic cells.

1. The objective of the present study was to determine the effects of dexamethasone on key constituents of prostaglandin and leukotriene biosynthesis, cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP). The human monocytic cell line THP-1 was used as a model system. mRNA and protein levels of COX-2 and FLAP were determined by Northern and Western blot analyses, respectively. 2. Low levels of COX-2 and FLAP mRNA were expressed in undifferentiated THP-1 cells, but were induced upon differentiation of the cells along the monocytic pathway by treatment with phorbol ester (TPA, 5 nM). Maximal expression was observed after two days. 3. Coincubation of the undifferentiated cells with dexamethasone (10(-9) - 10(-6) M) and phorbol ester prevented induction of COX-2 mRNA, but did not affect the induction of FLAP mRNA. 4. Dexamethasone downregulated COX-2 mRNA and protein in differentiated, monocyte-like THP-1 cells. In contrast, FLAP mRNA and protein were upregulated by dexamethasone in differentiated THP-1 cells. After 24 h, FLAP mRNA levels were increased more than 2 fold. Dexamethasone did not change 5-lipoxygenase mRNA expression. 5. Release of prostaglandin E2 (PGE2) and peptidoleukotrienes was determined in cell culture supernatants of differentiated THP-1 cells by ELISA. Calcium ionophore-dependent PGE2 synthesis was associated with COX-2 expression, whereas COX-1 and COX-2 seemed to participate in arachidonic acid-dependent PGE2 synthesis. Very low levels of peptidoleukotrienes were released from differentiated THP-1 cells upon incubation with ionophore. Treatment with dexamethasone did not significantly affect leukotriene release. 6. These data provide evidence that prostaglandin synthesis is consistently downregulated by glucocorticoids. However, the glucocorticoid-mediated induction of FLAP may provide a mechanism to maintain leukotriene biosynthesis through more efficient transfer of arachidonic acid to the 5-lipoxygenase reaction, in spite of inhibitory effects on other enzymes of the biosynthetic pathway.

Expression of 5-lipoxygenase in differentiating human skin keratinocytes.

We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.

Quantitative lipoxygenase product profiling by gas chromatography negative-ion chemical ionization mass spectrometry.

An assay for the quantitative determination of the hydroxylation profile of long-chain fatty acids is described for gas chromatography negative-ion chemical ionization mass spectrometry and stable isotope dilution using [carboxyl-18O2]-labeled internal standards. The assay has been applied to the study of fatty acids isolated from body fluids, tissue, and cultured cells. Examples for the analyses of biological systems expressing 5-, 8-, 12-, or 15-lipoxygenase activity are given and the most important sources of analytical errors are addressed. Increased specificity compared to analysis by negative-ion chemical ionization, at the cost of sensitivity, can be achieved by the use of positive-ion electron impact ionization for the investigation of hydrogenated pentafluorobenzylester/trimethylsilylether derivatives. The method described provides complete, specific, and quantitative profiles of hydroxylated fatty acids originally present in biological samples or generated in vitro by incubation with polyunsaturated fatty acid substrates such as linoleic or arachidonic acid.

The arachidonic acid cascade, eicosanoids, and signal transduction.

Eicosanoid biosynthesis in animal cells either results from agonist-stimulated phospholipase activation (endogenous pathway) or from lipoprotein receptor-mediated uptake and lysosomal lipid hydrolase-dependent release of AA (exogenous pathway) (see Fig. 1 for schematic representation). LDL stimulates eicosanoid formation through delivery of substrate AA to enzymes of oxidative AA metabolism. The classical LDL receptor is a control point of the effects of LDL AA on eicosanoid formation in different tissues: LDL AA metabolism occurs in several cell types of mesenchymal and epithelial origin and generates the formation of distinct eicosanoid patterns in each case. The LDL AA pathway does appear to couple directly to the PGH synthase reaction, whereas it does not couple directly to the 5-lipoxygenase reaction. We expect that a more complete characterization of the LDL unsaturated fatty acid pathway in different tissue will yield additional information on the biochemistry of lipoproteins, AA, and eicosanoids.

Lipoprotein-dependent unsaturated fatty acid transport and metabolism in cultured cells.

Our results can be summarized as follows. LDL stimulates eicosanoid formation through delivery of substrate AA to enzymes of oxidative AA metabolism. The classical LDL receptor controls the effect of LDL AA on eicosanoid formation. LDL AA metabolism occurs in several cell types of mesenchymal and epithelial origin and generates the formation of distinct eicosanoid patterns in a tissue-specific way. LDL inhibits the PGH synthase, and the LDL-dependent inhibition of the enzyme resembles the inhibition by unesterified AA. The LDL AA pathway does appear to couple directly to the PGH synthase reaction, but it does not appear to couple directly to the 5-lipoxygenase reaction. We expect that a more complete characterization of the LDL unsaturated fatty acid pathway in different tissues will yield additional information on the biochemistry of both lipoproteins and AA metabolism.

Selective eicosanoid formation during HL-60 macrophage differentiation. Regulation of thromboxane synthase.

Earlier studies on HL-60 cells induced to differentiate into macrophages by phorbol esters have shown a selective stimulation of thromboxane (Tx) formation from endoperoxide prostaglandin (PG) H2, indicating that Tx synthesis is regulated at the level of Tx synthase (TxS), one of the peripheral enzymes of the PGH-synthase pathway. We now report on the regulation of TxS during HL-60 macrophage differentiation using monoclonal anti-TxS serum and comparing turnover rates of TxS and its biological activity with those of other enzymes of arachidonic acid metabolism. Western-blot analysis, enzyme-linked immunosorbent assay, immunohistochemical staining and [35S]methionine-labeling experiments suggested a phorbol-ester-dependent early induction of synthesis of TxS. [35S]Methionine incorporation into TxS was stimulated within 4 h after initiation of differentiation and was associated with a major rise in the TxS catalytical activity. Pulse-chase experiments showed a half life for the TxS protein of 16.4 h in both control and phorbol-ester-treated cells. The biological half life of TxS was 10.5 h, as determined by PGH2 incorporation into TxB2 after cycloheximide treatment. In contrast, the biological half lives of PGH synthase, prostacyclin synthase and 5-lipoxygenase were significantly shorter and were 3, 2.5 and 2.5 h, respectively. These results reveal that Tx synthesis in macrophages is mediated by at least two distinct mechanisms; a protein-kinase-C-dependent induction of de novo synthesis of TxS and the selective resistance of the enzyme against the activity of protein kinase C.

Differential low density lipoprotein receptor-dependent formation of eicosanoids in human blood-derived monocytes.

We studied the ability of low density lipoproteins (LDLs) to provide arachidonic acid (AA) for eicosanoid biosynthesis in human blood-derived monocytes. When incubated in the presence of reconstituted LDL that contained cholesteryl [1-14C]arachidonate (recLDL-[14C]AA-CE), resting monocytes formed three labeled products of the prostaglandin (PG) H synthase pathway: 6-keto-PGF1 alpha, thromboxane B2, and PGE2. The amounts of these eicosanoids in response to recLDL-[14C]AA-CE were comparable to or exceeded those that were produced in response to the addition of 10 microM unesterified [1-14C]AA. By contrast, resting monocytes formed only small amounts of products of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4 from either recLDL-[14C]AA-CE or [14C]AA, indicating preferential utilization of AA in the PGH synthase reaction. However, they converted LDL-derived [14C]AA efficiently into LTB4 and LTC4, when they were first incubated with recLDL-[14C]AA-CE and subsequently stimulated with the chemotactic peptide N-formylmethionylleucylphenylalanine or the Ca2+ ionophore A23187. The classical LDL receptor pathway mediated the synthesis of all of the above eicosanoids from LDL but not from unesterified AA. These results demonstrate that the LDL receptor pathway preferentially promotes the synthesis of PGH synthase products in resting human blood-derived monocytes and that an additional mechanism is required to promote effective synthesis of 5-lipoxygenase pathway products from AA that originates in LDL cholesteryl esters.

LDL receptor-dependent polyunsaturated fatty acid transport and metabolism.

It is widely assumed that eicosanoid biosynthesis is initiated by an increase in the intracellular concentration of unesterified arachidonic acid (AA) as a consequence of the activation of cellular phospholipases and/or inhibition of AA reacylation reactions. Here, we describe a mechanism of eicosanoid formation that is entirely dependent on low density lipoprotein (LDL) receptor-mediated delivery of AA to eicosanoid producing target cells. This LDL AA pathway introduces a new regulatory component into the provision of unsaturated fatty acids to mammalian cells.

[Molecular mechanisms in atherogenesis]. / Molekulare Mechanismen der Atherogenese.

Disturbances of the lipid metabolism play a key role in atherogenesis, cholesterol, however, is not stored passively in the arterial wall, but on the basis of complex, partly still unknown humoral and cellular processes. The proliferative lesion of the arteriosclerotic vascular wall is characterized by injured endothelial cells, differentiating macrophages, immunocompetent T-lymphocytes and proliferating smooth muscle cells. Molecular biological investigations on cells of arteriosclerotic plaques among others clarified growth factors, cytokines, factors of angiogenesis and growth inhibitors in form and significance. Risk factors damage the endothelium and thus cause the production of these mediators. The adhesion proteins and the proto-oncogene c-sis play a further role. The latter might produce a connection between arteriosclerosis and cancer research.

Production of leukotrienes in gonadotropin-releasing hormone-stimulated pituitary cells: potential role in luteinizing hormone release.

Gonadotropin-releasing hormone (GnRH) stimulated the formation of two major metabolites of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4, as well as luteinizing hormone (LH) release in primary cultures of rat anterior pituitary cells. Several lines of evidence suggested the presence of a GnRH-dependent pituitary endocrine system in which LTs act as second messengers for LH release: (i) GnRH-dependent LT formation was observed within 1 min and immediately preceded GnRH-induced LH release, whereas exogenous LTs stimulated LH release at low concentrations; (ii) the dose responses of GnRH-induced LT production and LH release were similar and both effects required the presence of extracellular Ca2+ ions; (iii) GnRH-induced LH release was blocked by up to 45% following the administration of several LT receptor antagonists; (iv) LTE4 action on LH secretion was entirely abolished by LT receptor antagonists; and (v) an activator of protein kinase C acted synergistically with LTE4 to induce LH release. The major source of LT formation in the pituitary cell cultures appeared to be the gonadotrophs, as shown by GnRH receptor desensitization experiments. The results demonstrate the presence of a GnRH-activatable 5-lipoxygenase pathway in anterior pituitary cells and provide strong support for the hypothesis that LTs play a role in LH release in the GnRH signaling pathway.

A new role for the low density lipoprotein receptor.

It is well established that the low density lipoprotein (LDL) pathway functions to maintain a constant concentration of cellular cholesterol, but LDL effects that are unrelated to cholesterol metabolism have not been studied in great detail. In the present investigation we demonstrate that the LDL receptor pathway regulates cellular levels of free arachidonic acid (AA) and hence prostaglandin (PG) synthesis. We used platelet-derived growth factor (PDGF)-stimulated fibroblasts as a model system to investigate mechanism of LDL-dependent PG synthesis. PDGF-stimulated but not quiescent cells formed radiolabelled prostacyclin (PGI2) and PGE2 upon incubation with LDL that had been reconstituted with cholesteryl-(1-14C)-arachidonate (rec-LDL), while fibroblasts from patients that are afflicted with the LDL receptor negative phenotype of familial hypercholesterolaemia (FH) failed to synthesize significant amounts of PGs. Furthermore cells that had been preincubated with chloroquine or an anti LDL receptor antibody, that prevents binding of LDL to its receptor, did not produce significant amounts of PGs upon incubation with rec-LDL. Moreover incubation of PDGF-stimulated cells with LDL or AA led to a time and concentration-dependent inactivation of PGH synthase, the rate limiting enzyme of PG synthesis. When taken together our results establish a new role of the classical LDL receptor pathway of Brown and Goldstein by demonstrating that LDL provides AA to fibroblasts for eicosanoid formation and that LDL has a profound inhibitory effect on the key enzyme of PG synthesis, the PGH synthase.

The LDL receptor pathway delivers arachidonic acid for eicosanoid formation in cells stimulated by platelet-derived growth factor.

Animal cells can convert 20-carbon polyunsaturated fatty acids into prostaglandins (PGs) and leukotrienes. These locally produced mediators of inflammatory and immunological reactions act in an autocrine or paracrine fashion. Arachidonic acid (AA), the precursor of most PGs and leukotrienes, is present in the form of lipid esters within plasma lipoproteins and cannot be synthesised de novo by animal cells. Therefore, AA or its plant-derived precursor, linoleic acid, must be provided to cells if PGs or leukotrienes are to be formed. Because several classes of lipoproteins, including low-density lipoproteins (LDL), very-low-density lipoproteins, and chylomicron remnants, are taken up by means of the LDL receptor, and because LDL and very-low-density lipoproteins, but not high-density lipoproteins, stimulate PG synthesis, we have suggested previously that PG formation is directly linked to the LDL pathway. Using fibroblasts with the receptor-negative phenotype of familial hypercholesterolaemia and anti-LDL receptor antibodies, we show here that LDL deliver AA for PG production and that an LDL receptor-dependent feedback mechanism inhibits the activity of PGH synthase, the rate-limiting enzyme of PG synthesis. These results indicate that the LDL pathway has a regulatory role in PG synthesis, in addition to its well-known role in the maintenance of cellular cholesterol homeostasis.