(Received for publication, December 23, 1994; and in revised form, January 30, 1995)
From the
The two subtypes of retinoid Z receptor (RZR and
)
and the three splicing variants of retinoid orphan receptor (ROR
1,
2, and
3) 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 RZR
and RZR
.
Ligand-induced transcriptional control is therefore proposed to mediate
physiological functions of melatonin in the brain where RZR
is
expressed, but also in peripheral tissues, where RZR
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 RZR
and ROR
1, but not
ROR
2 and
3. 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 RZR
. In contrast,
5-lipoxygenase mRNA levels were not affected in differentiated
monocytic and granulocytic cell lines, which do not express RZR
.
This indicates that 5-lipoxygenase is the first natural RZR
responding gene. Furthermore, our results open up a new perspective in
understanding the involvement of melatonin in inflammatory and
immunological reactions.
Many aspects of vertebrate development, differentiation, and
homeostasis are regulated by hormones. Steroids (glucocorticoid,
mineralocorticoid, estrogen, progesterone, and androgen),
1,25-dihydroxyvitamin D, 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 RZR
(
)(6) . RZR
is a member of the
RZR/ROR family of orphan receptors, which also comprises RZR
(7) and the three splicing variants of ROR
1,
2, and
3(8) . Sequence comparison showed that RZR
is a
further splicing variant of ROR
, 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/ROR
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 RZR
,
RZR/ROR
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
(MeSO)/transforming growth factor (TGF)
treatment of
HL-60 cells and 1,25-dihydroxyvitamin D
/TGF
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
RZR-expressing B-lymphocytes was repressed by stimulation with
melatonin, whereas in differentiated HL-60 and Mono Mac 6 cells, which
do not have RZR
, 5-lipoxygenase mRNA levels were not affected.
These results show that 5-lipoxygenase is the first identified natural
RZR responding gene.
Figure 1:
Putative RZR response
element. A gel shift analysis was performed using in vitro translated ROR1, ROR
2, ROR
3, and RZR
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).
The members of the RZR/ROR family bind with high affinity as
monomers to DNA(8, 10) . For both RZR and
,
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 ROR
1,
ROR
2, ROR
3, and RZR
(Fig. 1). Interestingly, we
observed that only ROR
1 and RZR
, not ROR
2 and ROR
3,
bind to this response element. This supports the observation of
Giguère et al.(8) that
ROR
1 and ROR
2 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/ROR isoforms in different human cell lines. Expression of
5-lipoxygenase (A) and the four RZR/ROR
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),
MeSO/TGF
-differentiated HL-60 (granulocytes),
1,25-dihydroxyvitamin D
/TGF
-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/ROR
isoforms (Fig. 3B). We observed that
all five cell lines tested express ROR
2 and ROR
3, but no
ROR
1. However, a major difference is the level of RZR
mRNA
expression. In HL-60 and Mono Mac 6 cells, we found only very faint
amounts of RZR
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 RZR 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/ROR
isoform that binds to the 5-lipoxygenase response
element, i.e. ROR
1 or RZR
.
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 RZR 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.
Here, we report the identification of human 5-lipoxygenase as
the first natural responding gene for the nuclear receptor RZR.
The expression of 5-lipoxygenase in myeloid cells is in correlation
with the expression of RZR
in a comparison of 16 human tissue; the
highest expression of RZR
was found in peripheral blood leukocytes (7) .
The best known function of the RZR 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
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
-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 ROR
1
and RZR
, like the 5-lipoxygenase, and those that contain a
response element specific for ROR
2 and ROR
3.
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.