Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma.

BACKGROUND: Leukotrienes (LTs) are potent pro-inflammatory mediators involved in asthma. Exosomes, nanosized vesicles released from various cells, can stimulate or down-regulate immune responses, depending on the state and nature of the originating cell. We have recently shown an altered exosome profile in bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis, but their role in asthma is unknown. Our aims were to investigate whether exosomes from BALF have LT biosynthetic capacity and to explore phenotypic and functional characteristics of BALF exosomes in asthma. METHODS: Bronchoalveolar lavage fluid exosomes were collected from healthy individuals (n = 13) and patients with mild allergic asthma to birch pollen (n = 12) before and after birch allergen provocation. Exosomes were characterized by flow cytometry and Western blot. Their capacity to induce IL-8 and LT production in the human bronchial epithelial cell (BEC) line 16HB14o- was measured by ELISA and reverse-phase HPLC, respectively. RESULTS: Compared to BALF exosomes from healthy individuals, BALF exosomes from asthmatics displayed higher levels of exosome-associated markers, such as the tetraspanins CD63 and CD81 and the scavenger receptor CD36. No major differences were observed between BALF exosomes from before and after allergen provocation. Furthermore, we show that BALF exosomes contain enzymes for LT biosynthesis. The effect of exosomes to promote LTC(4) and IL-8 release in BEC was significantly increased for exosomes from asthmatics, and the CysLT(1) receptor antagonist Montelukast reduced exosome-induced IL-8 secretion. CONCLUSIONS: Bronchoalveolar lavage fluid exosomes from asthmatic and healthy individuals exhibit distinct phenotypes and functions. BALF exosomes from asthmatics might contribute to subclinical inflammation by increasing cytokine and LTC(4) generation in airway epithelium.

EGFR signaling upregulates expression of microsomal prostaglandin E synthase-1 in cancer cells leading to enhanced tumorigenicity.

In this report we describe the contribution of prostaglandin E(2) (PGE(2)) derived from the inducible microsomal PGE-synthase type-1 (mPGES-1) to the epidermal growth factor receptor (EGFR) oncogenic drive in tumor epithelial cells and in tumor-bearing mice. EGFR stimulation upregulated expression of mPGES-1 in HT-29, A431 and A549 cancer cells. Egr-1, a transcription factor induced by EGF, mediated this response. The Egr-1 rise provoked the overexpression of mPGES-1 messenger and protein, and enhanced PGE(2) formation. These changes were suppressed either by silencing Egr-1, or by upstream blockade of EGFR or ERK1/2 signals. Further, in a clonogenic assay on tumor cells, EGF induced a florid tumorigenic phenotype, which regressed when mPGES-1 was silenced or knocked down. EGF-induced mPGES-1 overexpression in epithelial cell reduced E-cadherin expression, whereas enhancing that of vimentin, suggesting an incipient mesenchymal phenotype. Additionally, inhibiting the EGFR in mice bearing the A431 tumor, the mPGES-1 expression and the tumor growth, exhibited a parallel decline. In conclusion, these findings provide novel evidence that a tight cooperation between the EGF/EGFR and mPGES-1 leads to a significant tumorigenic gain in epithelial cells, and provide clues for controlling the vicious association.

Microsomal prostaglandin E synthase-1 and 5-lipoxygenase: potential drug targets in cancer.

There is strong evidence for a role of prostaglandin (PG)E(2) in cancer cell proliferation and tumour development. In PGE(2) biosynthesis, cyclooxygenases (COX-1/2) convert arachidonic acid to PGH(2), which can be isomerized to PGE(2) by PGE synthases, including microsomal PGE synthase-1 (MPGES-1). Data describing genetic deletions of MPGES-1 are reviewed. The results suggest that MPGES-1 is an alternative therapeutic target for cancer cells and tumours that express this enzyme. Several compounds that target COX-2 or MPGES-1 also inhibit 5-lipoxygenase. This may be advantageous for treatment of some forms of cancer.

p38 MAP kinase mediates stress-induced leukotriene synthesis in a human B-lymphocyte cell line.

5-Lipoxygenase (5-LO), which catalyzes the first two steps in leukotriene biosynthesis, is a target for pharmacological treatment of inflammatory disorders. Previous studies have shown that B-lymphocytes express 5-LO. Here we demonstrate that several stimuli of cell stress such as osmotic shock (sorbitol, NaCl), oxidative stress (hydrogen peroxide, diamide), chemical stress sodium arsenite, and inflammatory cytokines enhanced cellular 5-LO activity in a B cell line (BL41-E95-A), when added simultaneously with ionophore plus arachidonate. It is interesting that sorbitol alone was sufficient for 5-LO product formation in the presence of exogenous arachidonic acid. These stimuli also activated p38 mitogen-activated protein (MAP) kinase and downstream MAP kinase-activated protein kinases in BL41-E95-A cells, which could phosphorylate 5-LO or heat shock protein 27 in vitro. The p38 MAP kinase inhibitor SB203580 abolished stress-induced leukotriene synthesis in B cells, without inhibition of 5-LO catalytic activity in cell-free systems. Our results indicate that p38 MAP kinase activation by cell stress is required for efficient leukotriene synthesis in B-lymphocytes.

Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction.

EPR investigation of the active site of recombinant human 5-lipoxygenase: inhibition by selenide.

Lipoxygenases are a group of non-heme iron dioxygenases which catalyze the formation of lipid hydroperoxides from unsaturated fatty acids. 5-Lipoxygenase (5LO) is of particular interest for formation of leukotrienes and lipoxins, implicated in inflammatory processes. In this study, electron paramagnetic resonance (EPR) spectroscopy was used to investigate the active site iron of purified recombinant human 5-lipoxygenase (5LO), and to explore the action of selenide on 5LO. After oxidation by lipid hydroperoxides, 5LO exhibited axial EPR spectra typified by a signal at g = 6.2. However, removal of the lipid hydroperoxides, their metabolites, and the solvent ethanol from the samples resulted in a shift to more rhombic EPR spectra (g = 5.17 and g = 9.0). Thus, many features of 5LO and soybean lipoxygenase-1 EPR spectra were similar, indicating similar flexible iron ligand arrangements in these lipoxygenases. Selenide (1.5 microM) showed a strong inhibitory effect on the enzyme activity of 5LO. In EPR, selenide abolished the signal at g = 6.2, typical for enzymatically active 5LO. Lipid hydroperoxide added to selenide-treated 5LO could not reinstate the signal at g = 6.2, indicating an irreversible change of the coordination of the active site iron.

Phorbol ester up-regulates capacities for nuclear translocation and phosphorylation of 5-lipoxygenase in Mono Mac 6 cells and human polymorphonuclear leukocytes.

The leukotrienes are inflammatory mediators derived from arachidonic acid. It was demonstrated that the priming of leukocytes with phorbol-12-myristate-13-acetate (PMA) leads to the increased formation of 5-lipoxygenase (5-LO) products in parallel with the increased association of 5-LO with the nucleus and the activation of kinases that can phosphorylate 5-LO in vitro. Stimulation of the monocytic cell line Mono Mac 6 with calcium ionophore gave low 5-LO product formation and no detectable redistribution of 5-LO. However, after priming of Mono Mac 6 cells with phorbol esters, ionophore led to the association of 45% to 75% of cellular 5-LO with the nuclear membrane, to 5-LO kinase activation, to enhanced release of arachidonate, and to substantial leukotriene synthesis. Similar results were obtained for human polymorphonuclear leukocytes stimulated with low-dose ionophore. In addition, for each cell type, PMA priming up-regulated leukotriene biosynthesis in the presence of exogenous arachidonic acid. A protein kinase inhibitor, calphostin C, reduced the association of 5-LO with the nucleus and 5-LO kinase activity, and the formation of 5-LO products was inhibited. These results suggest that PMA up-regulates leukotriene biosynthesis not only by increasing the release of endogenous arachidonate, but also by increasing the capacity for 5-LO phosphorylation and for the translocation of 5-LO to the nucleus in leukocytes.

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 interacts with coactosin-like protein.

We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonstrate a direct interaction between 5LO and CLP. 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates. In reciprocal experiments, 5LO was detected in CLP immunoprecipitates. Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca(2+)-independent manner. Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding. In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments. However, no F-actin-CLP.5LO ternary complex was observed. In contrast, 5LO appeared to compete with F-actin for the binding of CLP. Moreover, 5LO was found to interfere with actin polymerization. Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics.

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)

Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences.

5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO.

The N-terminal domain of 5-lipoxygenase binds calcium and mediates calcium stimulation of enzyme activity.

Human 5-lipoxygenase (5-LO) is a key enzyme in the conversion of arachidonic acid into leukotrienes and lipoxins, mediators and modulators of inflammation. In this study, we localized a stimulatory Ca(2+)-binding site to the N-terminal region of the enzyme. Thus, in a (45)Ca(2+) overlay assay, the N-terminal 128 amino acids of recombinant human 5-LO (fused to glutathione S-transferase) bound radioactive calcium to about the same extent as intact 5-LO. The glutathione S-transferase fusion protein of the C-terminal part of 5-LO (amino acids 120-673) showed much weaker binding. A model of a putative 5-LO N-terminal domain was calculated based on the structure of rabbit reticulocyte 15-LO. This model resembles beta-sandwich C2 domains of other Ca(2+)-binding proteins. Comparison of our model with the C2 domain of cytosolic phospholipase A(2) suggested a number of amino acids, located in the loops that connect the beta-strands, as potential Ca(2+) ligands. Indeed, mutations particularly in loop 2 (N43A, D44A, and E46A) led to decreased Ca(2+) binding and a requirement for higher Ca(2+) concentrations to stimulate enzyme activity. Our data indicate that an N-terminal beta-sandwich of 5-LO functions as a C2 domain in the calcium regulation of enzyme activity.

5-lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases.

5-lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes, a group of inflammatory mediators derived from arachidonic acid (AA). Here we describe that activation of p38 mitogen-activated protein kinase in human polymorphonuclear leukocytes and in Mono Mac 6 cells leads to activation of downstream kinases, which can subsequently phosphorylate 5-LO in vitro. Different agents activated the 5-LO kinase activities, including stimuli for cellular leukotriene biosynthesis (A23187, thapsigargin, N-formyl-leucyl-phenylalanine), compounds that up-regulate the capacity for leukotriene biosynthesis (phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor), and well known p38 stimuli as sodium arsenite and sorbitol. For all stimuli, 5-LO kinase activation was counteracted by SB203580 (3 microM or less), an inhibitor of p38 kinase. At least two p38-dependent 5-LO kinase activities were found. Based on migration properties in in-gel kinase assays and immunoreactivity, one of these was identified as mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2). The other appeared to be MAPKAP kinase 3; however, it could not be excluded that also other p38-dependent kinases contributed. When polymorphonuclear leukocytes were incubated with sodium arsenite (strong activator of 5-LO kinases), platelet-activating factor and exogenous AA, there was a 4-fold increase in 5-LO activity as compared with incubations with only platelet-activating factor and AA. This indicates that 5-LO phosphorylation can be one factor determining cellular 5-LO activity.

Mg2+ activates 5-lipoxygenase in vitro: dependency on concentrations of phosphatidylcholine and arachidonic acid.

Mg2+ gave dose-dependent activation of 5-lipoxygenase (5LO) in vitro. As for Ca2+, the activation depended on the presence of phosphatidylcholine (PC) vesicles, and the activation response was different at various combinations of arachidonate and PC. Stimulation of 5LO activity was observed with Mg2+ concentrations of 0.1-1 mM, similar to the concentration range of free Mg2+ in mammalian cells. However, to observe a clear increase in 5LO hydrophobicity, a higher concentration of Mg2+ (4 mM) was required, and at this concentration also 5LO activation was optimal. Combinations of Mg2+ with ATP (containing free Mg2+ and MgATP2- complex) gave better activation of 5LO than either agent alone. This effect of Mg2+ (and ATP) could be of interest in relation to basal 5LO activity in cells not subjected to a particular stimulus.

5-lipoxygenase binds calcium.

5-Lipoxygenase (5LO) catalyzes the first two steps in the biosynthesis of leukotrienes and lipoxins and has therefore become an important target for pharmacological treatment of inflammatory disorders. Binding of calcium to 5LO was shown using several different approaches. Human recombinant enzyme was expressed in E. coli and purified. Association of Ca2+ to 5LO was demonstrated by a calcium-induced mobility shift in gel electrophoresis, by calcium overlay, by gel filtration in the presence of calcium, and by equilibrium dialysis. The two latter methods also showed that calcium binds reversibly to 5LO. Equilibrium dialysis gave a Kd close to 6 microM; the stoichiometry of maximum calcium binding seemed to average around two Ca2+ per 5LO. Similar results were obtained when 5LO was inactivated during equilibrium dialysis, indicating that the calcium binding site(s) is (are) different from the active site. By Triton X-114 partitioning, it was confirmed that calcium increases the hydrophobicity of 5LO.

Interaction of 5-lipoxygenase with cellular proteins.

5-Lipoxygenase (5LO) plays a pivotal role in cellular leukotriene synthesis. To identify proteins interacting with human 5LO, we used a two-hybrid approach to screen a human lung cDNA library. From a total of 1.5 x 10(7) yeast transformants, nine independent clones representing three different proteins were isolated and found to specifically interact with 5LO. Four 1.7- to 1.8-kb clones represented a 16-kDa protein named coactosin-like protein for its significant homology with coactosin, a protein found to be associated with actin in Dictyostelium discoideum. Coactosin-like protein thus may provide a link between 5LO and the cytoskeleton. Two other yeast clones of 1.5 kb encoded transforming growth factor (TGF) type beta receptor-I-associated protein 1 partial cDNA. TGF type beta receptor-I-associated protein 1 recently has been reported to associate with the activated form of the TGF beta receptor I and may be involved in the TGF beta-induced up-regulation of 5LO expression and activity observed in HL-60 and Mono Mac 6 cells. Finally, three identical 2.1-kb clones contained the partial cDNA of a human protein with high homology to a hypothetical helicase K12H4. 8 from Caenorhabditis elegans and consequently was named DeltaK12H4. 8 homologue. Analysis of the predicted amino acid sequence revealed the presence of a RNase III motif and a double-stranded RNA binding domain, indicative of a protein of nuclear origin. The identification of these 5LO-interacting proteins provides additional approaches to studies of the cellular functions of 5LO.

Egr-1 and Sp1 interact functionally with the 5-lipoxygenase promoter and its naturally occurring mutants.

5-Lipoxygenase (5-LO), an enzyme essential for the formation of leukotrienes, is functionally modulated by a number of mechanisms, including transcriptional controls. The 5-LO promoter has a unique G+C-rich sequence, located between 176 and 147 base pairs upstream of the ATG translation start site, which contains five tandem Sp1 (a zinc-finger transcription factor) consensus binding sites overlapping five tandem early growth response protein 1 (Egr-1), a zinc-finger transcription factor, consensus binding sites. A family of naturally occurring mutations has been identified that consists of additions or deletions of these binding sites. The role of these overlapping Sp1/Egr-1 sites in the regulation of 5-LO transcription and the effects of these mutations on transcriptional regulatory mechanisms are unknown. We now show that Sp1 and Egr-1 bind specifically to the G+C-rich promoter sequence using in vitro deoxyribonuclease I footprinting. Both Sp1 and Egr-1 activate 5-LO promoter-reporter constructs in a minimally active drosophila SL2 cotransfection system, and the G+C-rich sequence is involved in this process. Moreover, studies comparing mutant promoter function indicate that both Sp1 and Egr-1 trans-activation are proportional to the number of Sp1/Egr-1 consensus binding sites within the G+C-rich sequence. It is possible that basal and inducible 5-LO gene transcriptions are mediated by an interplay of Sp1, Egr-1, and other transcription factors within the G+C-rich promoter region, and the naturally occurring mutations alter transcription by modifying their trans-activation potential.

Calcitriol and transforming growth factor-beta upregulate 5-lipoxygenase mRNA expression by increasing gene transcription and mRNA maturation.

Differentiation of the human monocytic cell line Mono Mac 6 with calcitriol plus transforming growth factor-beta (TGFbeta) strongly induces 5-lipoxygenase (5-LO) mRNA and protein expression. The mechanism of 5-LO mRNA induction by these agents was investigated. Analysis of mature 5-LO mRNA by reverse-transcriptase-PCR gave a 42-fold induction which was not due to alterations in 5-LO half life and which was only in part due to an induction of gene transcription. There was an up to fivefold increase in 5-LO primary transcripts by TGFbeta and calcitriol, which could be inhibited by cycloheximide. No significant effects on 5-LO transcription were observed with TGFbeta or calcitriol alone. However, treatment of the cells with either calcitriol or TGFbeta and addition of the corresponding second inducer lead to an about fourfold induction of primary transcript levels. Addition of cycloheximide together with the second inducer inhibited only the TGFbeta but not the calcitriol effects, which indicated that there is a direct stimulation of 5-LO transcription by calcitriol in the presence of TGFbeta-induced proteins. In order to investigate the effects of TGFbeta/calcitriol on 5-LO transcript, elongation and maturation, the relative changes in immature and mature 5-LO RNA species were analyzed by reverse-transcription-PCR. Analysis of exons 1-5 indicated an about threefold induction of 5-LO transcripts by calcitriol/TGFbeta, respectively. However, when exons 6-14 were determined, more pronounced increments were found (3.6-12-fold). Selective analysis of polyadenylated and spliced 5-LO mRNA species gave a 42-fold induction. The effects of both TGFbeta and calcitriol on transcript elongation and maturation were inhibited by cycloheximide. Our results show that induction of 5-LO mRNA by calcitriol and TGFbeta is due to a modest increase in 5-LO gene transcription and to the stimulation of transcript elongation and maturation.