©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Nuclear Receptor for Melatonin Represses 5-Lipoxygenase Gene Expression in Human B Lymphocytes (*)

(Received for publication, December 23, 1994; and in revised form, January 30, 1995)

Dieter Steinhilber (1) Martina Brungs (1) Oliver Werz (1) Irmgard Wiesenberg (2) Carina Danielsson (3) Jean-Pierre Kahlen (3) Sepideh Nayeri (3) Magdalena Schräder (3) Carsten Carlberg (3)(§)

From the  (1)Pharmazeutisches Institut der Universität Tübingen, D-72076 Tübingen, Germany, (2)Pharma-Forschung, Ciba-Geigy AG, CH-4002 Basel, Switzerland, and (3)Clinique de Dermatologie, Hôpital Cantonal Universitaire, CH-1211 Genève 14, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The two subtypes of retinoid Z receptor (RZR alpha and beta) and the three splicing variants of retinoid orphan receptor (ROR alpha1, alpha2, and alpha3) form a subfamily within the superfamily of nuclear hormone receptors. Very recently we found that the pineal gland hormone melatonin is a natural ligand of RZRalpha and RZRbeta. Ligand-induced transcriptional control is therefore proposed to mediate physiological functions of melatonin in the brain where RZRbeta is expressed, but also in peripheral tissues, where RZRalpha was found. However, no natural RZR responding genes have been identified yet. Here, we report that a response element in the promoter of 5-lipoxygenase binds specifically RZRalpha and RORalpha1, but not RORalpha2 and alpha3. 5-Lipoxygenase is a key enzyme in the biosynthesis of leukotrienes, which are known to be allergic and inflammatory mediators. We could show that the activity of the whole 5-lipoxygenase promoter as well as of the RZR response element fused to the heterologous thymidine kinase promoter could be repressed by melatonin. The hormone down-regulated the expression of 5-lipoxygenase about 5-fold in B lymphocytes, which express RZRalpha. In contrast, 5-lipoxygenase mRNA levels were not affected in differentiated monocytic and granulocytic cell lines, which do not express RZRalpha. This indicates that 5-lipoxygenase is the first natural RZRalpha responding gene. Furthermore, our results open up a new perspective in understanding the involvement of melatonin in inflammatory and immunological reactions.


INTRODUCTION

Many aspects of vertebrate development, differentiation, and homeostasis are regulated by hormones. Steroids (glucocorticoid, mineralocorticoid, estrogen, progesterone, and androgen), 1,25-dihydroxyvitamin D(3), 3,5,3`-triiodothyronine, and retinoids control gene expression by binding with high affinity to their specific nuclear receptors(1, 2) . This superfamily of structurally related transcription factors also includes a still growing number of orphan receptors for which no ligand is known so far. The identification of specific ligands is therefore a crucial step toward understanding the physiological role of orphan receptors. A first example was the discovery of 9-cis-retinoic acid (3, 4) to be a specific ligand for retinoid X receptors(5) . Very recently we reported that the pineal gland hormone melatonin binds and activates the brain-specific nuclear receptor RZRbeta(^1)(6) . RZRbeta is a member of the RZR/ROR family of orphan receptors, which also comprises RZRalpha (7) and the three splicing variants of RORalpha1, alpha2, and alpha3(8) . Sequence comparison showed that RZRalpha is a further splicing variant of RORalpha, i.e. the DNA and ligand binding domains of all four receptors are identical and they vary only in their amino-terminal domain. We could also show that RZR/RORalpha is a nuclear receptor for melatonin(9) . RZRs have sequence homology with retinoid receptors but bind to their response elements as a monomer(10) . In contrast to the brain-specific RZRbeta, RZR/RORalpha is expressed in many peripheral tissues and cells, e.g. in the peripheral blood, liver, smooth muscle, and testes(7, 11, 12) . Due to their affinity to certain retinoid response elements, RZR/RORs were thought to play a role as dominant negative regulators of retinoid signaling(8, 10) . However, neither natural RZR responding genes nor genes that are directly regulated by melatonin are known to date.

5-Lipoxygenase catalyzes the transformation of arachidonic acid to leukotrienes, which are involved in host defense reactions and are considered to play a role in diseases like asthma, inflammatory bowel disease, and arthritis(13, 14) . The expression of 5-lipoxygenase is restricted to specific cell types and tissues. The enzyme is found in granulocytes, monocytes/macrophages, mast cells, and B-lymphocytes, but no 5-lipoxygenase mRNA or protein expression was detected in platelets, endothelial cells, T cells, and erythrocytes(15, 16) . It has been shown for the human myeloid cell lines HL-60 and Mono Mac 6 that cellular differentiation is accompanied with an increase in 5-lipoxygenase mRNA expression(17) . After dimethyl sulfoxide (Me(2)SO)/transforming growth factor (TGF) beta treatment of HL-60 cells and 1,25-dihydroxyvitamin D(3)/TGFbeta treatment of Mono Mac 6 cells, 5-lipoxygenase mRNA and protein expression, as well as 5-lipoxygenase activity, are comparable to normal human granulocytes and monocytes, respectively(18, 19) . Furthermore, it has been shown that human B-lymphocytes and continuously growing B lymphocytic cell lines constitutively express 5-lipoxygenase(15) .

Here, we describe the identification of a functional RZR response element in the promoter of the human 5-lipoxygenase gene. We could show that 5-lipoxygenase expression in RZRalpha-expressing B-lymphocytes was repressed by stimulation with melatonin, whereas in differentiated HL-60 and Mono Mac 6 cells, which do not have RZRalpha, 5-lipoxygenase mRNA levels were not affected. These results show that 5-lipoxygenase is the first identified natural RZR responding gene.


MATERIALS AND METHODS

DNA Constructs

For the RZR response element (see Fig. 1), a pair of oligonucleotides with adjacent XbaI sites was annealed and fused to the thymidine kinase (tk) promoter to drive the expression of the chloramphenicol acetyltransferase (CAT) reporter gene by subcloning into the XbaI site of pBLCAT2 (20) . The human 5-lipoxygenase promoter fragments (positions -1551 to +79 and -1476 to +79) were amplified by polymerase chain reaction (PCR) from human genomic DNA (Promega) using the primers HSLIP1.3 (5`-ATTTCTAGACTTCTATCGCTCCATGCATA-3`) and HSLIP-1 (5`-ATTTCTAGACTGAGGTAGATGTAGCTGTCA-3`) or HSLIP1.4 (5`-ATTTCTAGACCTAGAGGAAAACCTAGGAG-3`) and HSLIP-1, respectively, and subcloned into the XbaI site of pBLCAT3(20) .


Figure 1: Putative RZR response element. A gel shift analysis was performed using in vitro translated RORalpha1, RORalpha2, RORalpha3, and RZRalpha and the RZR response element probe that was identified in the promoter of human 5-lipoxygenase(24) . The specificities of the shifted bands were proved by comparison with unprogrammed lysate (firstlane).



In Vitro Translation and DNA Binding Assays

Linearized cDNAs for human RZRalpha, RORalpha1, RORalpha2, and RORalpha3 were used for in vitro transcription as recommended by the supplier (Promega). Five µg of each RNA were mixed with 175 µl of rabbit reticulocyte lysate, 100 units of RNasin, and 20 µM complete amino acid mixture (all from Promega) in a total volume of 250 µl and incubated at 30 °C for 180 min. The RZR response element probe was labeled by a fill-in reaction using [alpha-P]dCTP and the Klenow fragment of DNA polymerase I (Promega). Five µl of in vitro translated receptor was incubated for 20 min at room temperature in a total volume of 20 µl of binding buffer (10 mM Hepes, pH 7.9, 80 mM KCl, 1 mM dithiothreitol, 0.2 µg/µl poly(dI/dC), and 5% glycerol) with about 1 ng of labeled probe (50,000 cpm). Protein-DNA complexes were resolved on a 5% nondenaturing polyacrylamide gel (at room temperature) in 0.5 times TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.3).

Cell Culture

The human cell lines MCF-7 (breast carcinoma), HL-60 (promyelocytes), RPMI 1788 (B lymphocytes), and the B cell clone P16 were grown in RPMI (Life Technologies, Inc.) supplemented with 10% fetal calf serum (FCS). HL-60 cells were differentiated with Me(2)SO (1.5%) and TGFbeta (1 ng/ml) for 4 days. Mono Mac 6 cells (monocytes) (21) were grown as described (19) and differentiated with 1,25-dihydroxyvitamin D(3) (50 nM) and TGFbeta (1 ng/ml) for 4 days.

Transfection and CAT Assays

For transfection of human breast cancer MCF-7 cells, 2 times 10^5 cells/well were seeded into six-well plates and grown overnight in phenol red-free RPMI (Life Technologies) supplemented with 10% charcoal-treated FCS. Liposomes were formed by incubating 2 µg of the reporter plasmid and 1 µg of the reference plasmid pCH110 (Pharmacia Biotech Inc.) with 20 µg of DOTAP (Boehringer Mannheim) for 15 min at room temperature in a total volume of 100 µl. After dilution with 0.9 ml of phenol red-free RPMI, the liposomes were added to the cells. Phenol red-free RPMI (500 µl) supplemented with 30% charcoal-treated FCS and 100 nM melatonin (Fluka) or solvent were added 8 h after transfection. P16, RPMI 1788 cells, differentiated HL-60, and differentiated Mono Mac 6 cells have been transfected by electroporation at 280 V and 960 microfarads using 30 µg of the reporter plasmid (in some cases also 10 µg of pSG5-based RZRalpha expression vector; (10) ) and 15 µg of the reference plasmid pCH110 per 2 times 10^7 cells. Cells were then diluted to 10^6 cells/ml with phenol red-free RPMI supplemented with 10% charcoal-treated FCS and treated with 100 nM melatonin or solvent. All transfected cells were harvested 40 h after onset of the stimulation, and CAT assays were performed as described(22) . The CAT activities were normalized to beta-galactosidase activity; each condition was analyzed at least in triplicate, and data are shown as mean with standard deviation.

mRNA Quantification

Total cellular RNA was isolated from cells stimulated for 24 h with 100 nM melatonin or from non-stimulated cells and reverse-transcribed into cDNA, and PCR analysis was performed as described(23) . The efficiency of the reverse transcription was checked by detection of beta2 microglobulin or beta-actin mRNA with RT-PCR. 5-Lipoxygenase expression was analyzed using the primers LIP1 (CACTGACGACTACATCTACC) and LIP-1 (GTTCCACTCCATCCATCGAT) or LIP2 (ACCATTGAGCAGATCGTGGACACGC) and LIP-2 (GCAGTCCTGCTCTGTGTAGAATGGG). The RZR/RORalpha isoforms were quantified using the primer RORalpha1P (AAACATGGAGTCAGCTCCG) for RORalpha1, RORalpha2P (CTCCAAATACTCCATCAGTGTATCC) for RORalpha2, RORalpha3P (CAACTTGAGCACATAAACTGG) for RORalpha3, and RORalpha4P (TGTATTTTGTGATCGCAGAG) for RZRalpha, and the RORalphaCP (CATACAAGCTGTCTCTCTGC) as reverse primer for all four. The PCR products were quantified by scanning densitometry.


RESULTS

The members of the RZR/ROR family bind with high affinity as monomers to DNA(8, 10) . For both RZRalpha and beta, we found that the heptameric sequence TRGGTCA (R = A or G) appears to be optimal for receptor protein-DNA interaction(10) . This is consistent with the results of Giguère et al.(8) . In order to identify a natural RZR responding gene, we screened several promoter sequences from the EMBL data base for the sequence TRGGTCA and found a putative RZR response element between positions -1521 and -1510 of the human 5-lipoxygenase gene promoter (24) (Fig. 1). We used this sequence as a probe in gel shift experiments with in vitro translated RORalpha1, RORalpha2, RORalpha3, and RZRalpha (Fig. 1). Interestingly, we observed that only RORalpha1 and RZRalpha, not RORalpha2 and RORalpha3, bind to this response element. This supports the observation of Giguère et al.(8) that RORalpha1 and RORalpha2 have different preferences for sequences 5`-flanking to the core binding motif.

To test the functionality of the response element, we fused it to the tk promoter driving the CAT reporter gene. We transfected this construct, the parental reporter plasmid pBLCAT2 and two CAT reporter constructs containing 5-lipoxygenase promoter fragments into the human breast cancer cell line MCF-7 (Fig. 2). This cell line is our model system for analyzing nuclear signaling pathways, since it endogenously expresses various nuclear receptors(25, 26) including members of the RZR/ROR family (Fig. 3B). Although 5-lipoxygenase expression is mainly restricted to myeloid cells, its promoter is also active in other mammalian cell lines(24) . It was therefore not surprising that both 5-lipoxygenase promoter constructs show constitutive activity in MCF-7 cells (Fig. 2). However, only the activity of the longer promoter fragment (position -1551 to +79) containing the RZR response element, but not that of the shorter fragment (position -1476 to +79), was reduced by stimulation with melatonin. The activity of the heterologous RZR response element/tk promoter construct was also repressed by melatonin, whereas the basal activity obtained with the parental reporter plasmid pBLCAT2 containing only the tk promoter was not affected (Fig. 2).


Figure 2: Melatonin represses 5-lipoxygenase promoter activity. MCF-7 cells were transfected with CAT reporter constructs containing either 5-lipoxygenase promoter fragments or the RZR response element fused to the tk promoter and the parental CAT reporter plasmid pBLCAT2, as schematically depicted. The cells were stimulated for 40 h with 100 nM melatonin or solvent, as indicated. Relative CAT activity is shown; columns represent mean values of at least three independent experiments, and the bars indicate standard deviations.




Figure 3: Expression of 5-lipoxygenase and RZR/RORalpha isoforms in different human cell lines. Expression of 5-lipoxygenase (A) and the four RZR/RORalpha isoforms (B) determined by RT-PCR in RPMI 1788, P16, differentiated HL-60, differentiated Mono Mac 6, and MCF-7 cells. The cells have been stimulated for 24 h with either 100 nM melatonin or solvent (RPMI 1788, P16, and MCF-7 cells) or even for 48 h with 1 µM melatonin (HL-60 and Mono Mac 6 cells). PCR products were quantified using scanning densitometry; columns represent the mean of relative values of at least three independent experiments, and the bars indicate standard deviations.



We next asked whether 5-lipoxygenase promoter activity correlates with 5-lipoxygenase mRNA levels in cells naturally expressing 5-lipoxygenase. By RT-PCR we analyzed 5-lipoxygenase mRNA expression in five human cell lines in the presence or absence of melatonin: RPMI 1788 (B lymphocytes), P16 (B cell clone), Me(2)SO/TGFbeta-differentiated HL-60 (granulocytes), 1,25-dihydroxyvitamin D(3)/TGFbeta-differentiated Mono Mac 6 (monocytes), and MCF-7 (Fig. 3A). The four myeloid cell lines express 5-lipoxygenase, whereas MCF-7 cells do not. 5-Lipoxygenase expression was reduced about 4-5-fold in B lymphocytes, whereas in HL-60 and Mono Mac 6 cells mRNA levels were not significantly affected. In parallel we analyzed the expression of all four RZR/RORalpha isoforms (Fig. 3B). We observed that all five cell lines tested express RORalpha2 and RORalpha3, but no RORalpha1. However, a major difference is the level of RZRalpha mRNA expression. In HL-60 and Mono Mac 6 cells, we found only very faint amounts of RZRalpha mRNA, whereas it was expressed in MCF-7 cells and B lymphocytes.

We then transfected the four myeloid cell lines with the CAT reporter construct containing the longer 5-lipoxygenase promoter fragment (position -1551 to +79) (Fig. 4). Stimulation with melatonin significantly reduced promoter activity only in B lymphocytes, but not in HL-60 and Mono Mac 6 cells. This is consistent with the transcriptional activity of the 5-lipoxygenase gene (Fig. 3A). Interestingly, when HL-60 cells were co-transfected with a RZRalpha expression vector stimulation with melatonin resulted also in a reduction of 5-lipoxygenase promoter activity (Fig. 4). We therefore conclude that melatonin down-regulates 5-lipoxygenase expression only in cells that co-express a RZR/RORalpha isoform that binds to the 5-lipoxygenase response element, i.e. RORalpha1 or RZRalpha.


Figure 4: 5-lipoxygenase promoter activity in different human cell lines. RPMI 1788, P16, differentiated HL-60, and differentiated Mono Mac 6 cells were transfected with a CAT reporter construct containing the 5-lipoxygenase promoter (position -1551 to +79). In an additional experiment, HL-60 cells have also been co-transfected with a RZRalpha expression vector. The cells were stimulated for 40 h with 100 nM melatonin or solvent, as indicated. Relative CAT activity is shown; columns represent mean values of at least three independent experiments, and the bars indicate standard deviations.



Furthermore, we determined 5-lipoxygenase activity in leukocyte homogenates as described previously(23) . We observed that the addition of 1 µM melatonin had no effect on 5-lipoxygenase activity. Additionally, the incubation of human polymorphonuclear leukocytes with 0.3 µM melatonin for 17 h did not affect 5-lipoxygenase activity in cell homogenates or in intact cells stimulated with 10 µM ionophore. Thus, melatonin appears not to have an effect on the activation or the catalytic activity of 5-lipoxygenase in granulocytes.


DISCUSSION

Here, we report the identification of human 5-lipoxygenase as the first natural responding gene for the nuclear receptor RZRalpha. The expression of 5-lipoxygenase in myeloid cells is in correlation with the expression of RZRalpha in a comparison of 16 human tissue; the highest expression of RZRalpha was found in peripheral blood leukocytes (7) .

The best known function of the RZRalpha ligand, the pineal gland hormone melatonin, is its role as a transmitter of photoperiodic information and regulator of seasonal reproductive cycles. Melatonin is secreted during the hours of darkness, and it has been assumed to be primarily active in certain brain tissues(27) . However, melatonin is circulating and all peripheral tissues have access to the hormone. Melatonin has been shown to exhibit anti-stress, anti-aging, and oncostatic properties and to influence various immunological and endocrinological functions(28, 29, 30, 31) . Moreover, binding sites for melatonin with K(d) values in the picomolar range have been described in membrane fractions of different tissues(27, 32) , and a membrane receptor for melatonin with a K(d)-value of 0.063 nM has been cloned recently from frog(33) , sheep, and human(34) . The function of melatonin at the level of gene regulation is not well understood; however, the presence of a nuclear and a membrane-coupled receptor for melatonin predicts two types of signaling pathways. We have shown here that the 5-lipoxygenase gene responds to the nuclear melatonin signaling pathway, i.e. 5-lipoxygenase is also the first clearly defined melatonin responding gene. Interestingly, for nuclear melatonin signaling, there seem to be at least two different types of responding genes: those that contain in their promoter a response element recognized by RORalpha1 and RZRalpha, like the 5-lipoxygenase, and those that contain a response element specific for RORalpha2 and RORalpha3.

To our knowledge no reasonable evidence has been reported that melatonin is involved in host defense reactions. However, the down-regulation of 5-lipoxygenase expression in B lymphocytes by melatonin would suggest a clear link. The physiological conditions that lead to activation of the 5-lipoxygenase enzyme in intact B-lymphocytes are unknown, and the function of 5-lipoxygenase in these cells is still unclear. There is some evidence that 5-lipoxygenase may have additional functions aside from the release of leukotrienes. It has been shown that 5-lipoxygenase is located in the nucleus of certain cell types (35) . Recently, it was demonstrated that 5-lipoxygenase contains a binding site for the Src homology 3 domain of growth factor receptor-bound protein 2 and it was suggested that 5-lipoxygenase may be involved in tyrosine kinase signaling(36) . It is reasonable to assume that down-regulation of 5-lipoxygenase mRNA expression by melatonin results in a decrease in 5-lipoxygenase activity. Thus, it will be interesting to investigate the effects of melatonin in inflammatory models and on B lymphocyte functions.

The use of melatonin as a drug is probably limited by its side effects on the day-night rhythm. It is likely that some of these side effects are mediated by the membrane signaling pathway of melatonin. Therefore, the availability of specific RZR ligands should allow repression of 5-lipoxygenase without interfering with other physiological functions of melatonin.


FOOTNOTES

*
This work was supported by Swiss National Foundation Grant 3100-040314.94 (to C. C.) and Deutsche Forschungsgemeinschaft Grant 458/2-1 (to D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 41-22-37-29428; Fax: 41-22-37-29460.

(^1)
The abbreviations used are: RZR, retinoid Z receptor; ROR, RAR-related orphan receptor; TGF, transforming growth factor; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; FCS, fetal calf serum; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate.


ACKNOWLEDGEMENTS

We thank J.-H. Saurat for discussions, and M. Becker-André for RZRalpha expression vector, and V. Giguère for RORalpha1, RORalpha2, and RORalpha3 expression vector.


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