(Received for publication, May 1, 1995; and in revised form, June 15, 1995)
From the
We recently reported that -prostaglandin (PG)
J
caused various cells to synthesize heme oxygenase, HO-1
(Koizumi, T., Negishi, M., and Ichikawa, A.(1992) Prostaglandins 43, 121-131). Here we examined the molecular mechanism
underlying the
-PGJ
-induced HO-1
synthesis.
-PGJ
markedly stimulated the
promoter activity of the 5`-flanking region of the rat HO-1 gene from
-810 to +101 in rat basophilic leukemia cells. From
functional analysis of various deletion mutant genes we found that the
-PGJ
-responsive element was localized in
a region from -690 to -660, containing an E-box motif,
which was essential for the
-PGJ
-stimulated promoter activity. When
the region containing the
-PGJ
-responsive
element was combined with a heterologous promoter, SV40 promoter, in
the sense and antisense direction, the element showed an enhancer
activity in response to
-PGJ
. Gel
mobility shift assays demonstrated that
-PGJ
specifically stimulated the binding of two nuclear proteins to
the E-box motif of this region. These results indicate that
-PGJ
induces the expression of the rat
HO-1 gene through nuclear protein binding to a specific element having
an E-box motif.
Eicosanoids are oxygenated metabolites of arachidonic acid, and
are regarded as modulators of cellular functions in various
physiological and pathological processes(1) . Eicosanoids are
divided into two groups, conventional eicosanoids and
cyclopentenone-type prostaglandins (PGs) ()according to
their mechanisms of action. Conventional eicosanoids, such as PGE
and PGD
, act on a cell surface receptor to exert
their actions, and the molecular structures of their receptors have
been revealed recently(2) . Cyclopentenone PGs, such as
-PGJ
and PGA
, have no cell
surface receptor, but are actively transported into cells and
accumulated in nuclei, where they act as potent inducers of cell growth
inhibition and cell differentiation(3) . The actions of
cyclopentenone PGs are attributed to the synthesis of the various
proteins induced by them, such as heat shock proteins
(HSPs)(4, 5) ,
-glutamylcysteine
synthetase(6) , collagen(7) , gadd 153(8) , and
heme oxygenase(9) . In contrast to conventional eicosanoids,
the molecular characterization of cyclopentenone PG actions has been
hardly carried out.
Heme oxygenase is one of the most prominent
proteins induced by -PGJ
, and it is a key
enzyme in heme catabolism, oxidatively clearing heme to yield
biliverdin, iron, and carbon monoxide(10) . The biological
functions of this enzyme are the production of biliverdin as a
physiological antioxidant and the conservation of the
iron(11) . Furthermore, carbon monoxide produced on the
enzymatic degradation of heme has been suggested to function as a
neural messenger(12) . Two isozymes of heme oxygenase, HO-1 and
HO-2, have been identified(13) . HO-2 is constitutively
expressed, while HO-1 is drastically induced in response to a variety
of stresses, including heavy metals, heat shock, and UV
irradiation(14) . We previously found that
-PGJ
preferentially induced the synthesis
of HO-1 in various cells involved in the reticuloendothelial system, in
which active degradation of heme by HO-1 takes place during
inflammation(9, 15) . In order to elucidate the
mechanism underlying
-PGJ
-induced protein
synthesis, we examined the effect of
-PGJ
on the promoter activity of the HO-1 gene. We report here that
-PGJ
induces the expression of the rat
HO-1 gene through nuclear protein binding to a specific
-PGJ
-responsive element, located 660 base
pairs upstream from the transcription initiation site.
Rat basophilic leukemia
(RBL)-2H3 cells were obtained from the Japanese Cancer Research
Resources Bank (Tokyo, Japan). The cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum, 4 mM glutamine, 0.2 mg/ml streptomycin,
and 100 units/ml penicillin under humidified air containing 5% CO at 37 °C.
Successive deletion plasmids as to the 5`-flanking region, RHO600 (-600 to +101), RHO320 (-320 to +101), RHO270 (-270 to +101), RHO-0 (+1 to +101), and RHO810-600 (-810 to -600), were also generated by PCR using the respective forward primers with a BamHI site. To prepare a deletion plasmid without region (-600 to -270), two DNA fragments (-810 to -600, and -270 to +101) were prepared by PCR and tandemly constructed into pUC00CAT. For the functional analysis using a heterologous promoter, the 5`-flanking region from -810 to -600 was inserted into pCAT promoter vector (Promega), which carries the SV40 promoter upstream from the CAT gene, and the DNA fragment from -810 to -600 was located upstream from the SV40 promoter, in the sense (pCAT-HOs) and antisense direction (pCAT-HOa).
Reactions were started by the addition of
the test agents. After incubation for the indicated times, cellular
extracts were prepared by four cycles of freezing and thawing. The CAT
assay was performed as described previously(20) . Cellular
extracts containing equal amounts of protein were treated at 65 °C
for 10 min to inactivate deacetylase, and then incubated for 4 h at 37
°C with [C]1-deoxychloramphenicol (50 nCi)
and 0.1 mg/ml acetyl-CoA. The reaction mixtures were extracted with
ethyl acetate, and then separated on a silica TLC plate (F1500,
Schleicher & Schnell). After development of the TLC plate, the
radioactivity was quantitated with a Fuji BAS 2000 imaging analyzer
(Fuji Film, Co., Tokyo). CAT activity was normalized as to
-galactosidase activity due to co-transfected Rous sarcoma
virus-
-galactosidase, which was assayed as described
previously(20) .
Nuclear extracts were prepared by
detergent lysis method of Block et al.(21) . The
nuclear extracts (2 µg) were incubated with 2 µg of poly(dI-dC)
and 1 ng of a P-labeled probe (10,000 cpm) for 30 min at
30 °C in 25 mM Hepes-NaOH (pH 7.9), containing 0.5 mM EDTA, 50 mM KCl, 10% glycerol, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride.
The reaction mixtures were electrophoresed on native 4% polyacrylamide
gels at 4 °C at 150 V for 2 h in 50 mM Tris-HCl (pH 8.5),
containing 380 mM glycine and 2 mM EDTA. The gels
were dried on Whatman 3MM paper and then autoradiographed with x-ray
film (Fuji RX).
Figure 1:
HO-1 mRNA induction by
-PGJ
in RBL-2H3 cells. A, time
course. After RBL-2H3 cells (3
10
cells) had been
treated with 10 µM
-PGJ
for
the indicated times, total cellular RNA was extracted from the cells. B, concentration dependence. After cells had been treated with
the indicated concentrations of
-PGJ
for
3 h, total cellular RNA was extracted. Total cellular RNA (10 µg)
was subjected to Northern blot analysis, as described under
``Experimental Procedures.'' The hybridized bands for HO-1
and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were
indicated by the arrows. The HO-1 (
) and
glyceraldehyde-3-phosphate dehydrogenase (
) mRNA values are
expressed as fold of the untreated cell values, and are representative
of three independent experiments that yielded similar
results.
To identify the cis-regulatory element for
-PGJ
-induced expression of the HO-1 gene,
we isolated the 5`-flanking region of the HO-1 gene by PCR
amplification, which included the 810 base pairs upstream of the
transcription initiation site, containing two known cis-acting
elements, the heat shock element (HSE) and the metal-responsive element
(MRE). To determine whether or not
-PGJ
stimulates the promoter activity of the 5`-flanking region, we
examined the effect of
-PGJ
on the
transient expression of the bacterial CAT reporter gene harboring the
5`-flanking region (-810 to +101) in RBL-2H3 cells. As shown
in Fig. 2, RHO810 containing the 5`-flanking region gave low but
detectable CAT activity, and
-PGJ
markedly stimulated it by about 5-fold, indicating that the
region contains an element responsible for
-PGJ
. Fig. 3shows the time course
and concentration dependence of the effect of
-PGJ
on the promoter activity of RHO810.
-PGJ
stimulated the promoter activity in
a time-dependent manner, the time course being consistent with those of
the
-PGJ
-induced HO-1 protein synthesis
and HO-1 activity(9) .
-PGJ
concentration-dependently stimulated it, the maximum being
reached at 10 µM (Fig. 3B). This
concentration dependence was consistent with that of the mRNA induction
by
-PGJ
(Fig. 1B). We
next examined the specificity of stimulation of the promoter activity
for various PGs. As shown in Fig. 4A,
-PGJ
and
-PGA
markedly stimulated the activity, but the other PGs did not
significantly stimulate it. HO-1 synthesis is known to be induced by
other stimuli, such as heat shock, thiol-reactive agents, arsenite and
diethylmaleate, and hemin, which is the substrate of heme oxygenase and
a known strong inducer of HO-1 (23, 24, 25) .
Thus, we examined the effects of these agents on the promoter activity
of RHO810. As shown in Fig. 4B, heat shock strongly
stimulated the promoter activity, while hemin did not stimulate it at
all. Arsenite and diethylmaleate only slightly stimulated it.
Figure 2:
Effect of -PGJ
on the promoter activity of the 5`-flanking region of the HO-1
gene. After cells (4
10
cells) had been transiently
transfected with the vehicle (1), pUC00CAT (2 and 3), and RHO810 (4 and 5), they were treated
for 9 h with (3 and 5) or without (1, 2, and 4) 10 µM
-PGJ
. The CAT assay was performed for
cellular extracts, as described under ``Experimental
Procedures.'' Arrows indicate: O, origin; C, 1-deoxychloramphenicol; AC,
3-acetyl-1-deoxychloramphenicol. The percentages of the acetylated
chloramphenicol of RHO810-transfected cells treated with (5)
and without (4)
-PGJ
were 1.15
and 0.230, respectively. The results are representative of three
independent experiments that yielded similar
results.
Figure 3:
Time course and concentration dependence
of the -PGJ
-stimulated promoter activity
of RHO810. After cells (4
10
cells) had been
transiently transfected with RHO810, they were treated with 10
µM
-PGJ
for the indicated
times (A), or with the indicated concentrations of
-PGJ
for 9 h (B). The CAT assay
was performed for cellular extracts, as described under
``Experimental Procedures.'' The values are expressed as fold
of the untreated cell value, and are the means for three independent
experiments, which varied by less than 10%. The values for the
acetylated chloramphenicol of untreated cells were 0.253 ±
0.034% (A) and 0.315 ± 0.023% (B).
Figure 4:
Effects of various PGs and stimulants on
the promoter activity of RHO810. After cells (4 10
cells) had been transiently transfected with RHO810, they were
treated with 10 µM of the indicated PGs for 9 h (A), or with 10 µM
-PGJ
, 20 µM arsenite, 150
µM diethylmaleate (DEM), or 150 µM hemin for 9 h, or heat shock (43 °C) for 1 h followed by an
8-h incubation at 37 °C (B). The CAT assay was performed
for cellular extracts, as described under ``Experimental
Procedures.'' The values are expressed as fold of the untreated
cell value, and are the means for three independent experiments, which
varied by less than 10%. The values for the acetylated chloramphenicol
of untreated cells were 0.316 ± 0.042% (A) and 0.215
± 0.054% (B).
Figure 5:
Functional analysis of various deletion
mutant genes of RHO810. After cells (4 10
cells)
had been transiently transfected with RHO810, RHO600, RHO320, RHO270,
RHO-0, RHO810-600, or RHO810 without region (-600 to
-270), (dRHO810), they were treated with (
) or without
(
) 10 µM
-PGJ
for 9
h. The CAT assay was performed for cellular extracts, as described
under ``Experimental Procedures.'' The values are expressed
as fold of the untreated cell value of RHO810, and are the means for
three independent experiments, which varied by less than 10%. The value
for the acetylated chloramphenicol of the untreated cells was 0.295
± 0.043%.
Figure 6:
Functional analysis of the
-PGJ
-responsive element using SV40
promoter. After cells (4
10
cells) had been
transiently transfected with pCAT, pCAT-HOs, or pCAT-HOa, they were
treated with (
) or without (
) 10 µM
-PGJ
for 9 h. The CAT assay was performed
for cellular extracts, as described under ``Experimental
Procedures.'' The values are expressed as fold of the untreated
cell value of pCAT, and are the means for three independent
experiments, which varied by less than 10%. The value for the
acetylated chloramphenicol of the untreated cells was 0.223 ±
0.038%.
Figure 7:
Point mutation of the E-box motif in the
region from -690 to -660. A, gel mobility shift
assay. After cells had been treated with (J) or without (C) 10 µM -PGJ
for
2 h, nuclear extracts were prepared. The gel mobility shift assay was
performed using the
P-labeled DNA fragment (-690 to
-660) in the absence (1 and 2) or presence of a
100-fold excess of the unlabeled antisense single-stranded DNA
(-690 to -660) (3), sense single-stranded DNA
(-690 to -660) (4), double-stranded DNA fragment
(-690 to -660) (5), or the E-box
motif-point-mutated double-stranded DNA fragment (6), as
described under ``Experimental Procedures.'' I and II represent specific protein-DNA complexes. B, UV
cross-linking. The gel mobility shift assay was performed using the
nuclear extract from
-PGJ
-treated cells
and the
P-labeled DNA fragment (-690 to -660).
The wet gel was UV-irradiated and the protein-DNA complexes, I and II,
were separately electroeluted. They were subjected to SDS-12.5% PAGE,
followed by autoradiography. C, CAT assay. After cells (4
10
cells) had been transiently transfected with
RHO810 or RHO810 point mutated at the E-box motif (mRHO810), they were
treated with (J) or without (C) 10 µM
-PGJ
for 9 h. The CAT assay was
performed for cellular extracts, as described under ``Experimental
Procedures.'' The values are expressed as percentages of
acetylated chloramphenicol, and are the means for three independent
experiments, which varied by less than 10%.
We further examined the effect of -PGJ
on the promoter activity of RHO810 point-mutated at the E-box
motif. As shown in Fig. 7C, this point mutation
completely abolished the
-PGJ
-induced
stimulation of the promoter activity of RHO810. These results indicate
that the region (-690 to -660) contains the
-PGJ
-responsive element and that the
E-box motif in this element is essential for this stimulation of the
promoter activity.
We demonstrated here that -PGJ
drastically induced HO-1 mRNA through a
-PGJ
-specific cis-regulatory element,
containing a E-box motif, in RBL-2H3 cells.
-PGJ
induces the syntheses of a variety
of proteins(3) . Among the
-PGJ
-induced protein syntheses, the
mechanism for the synthesis of HSP has been well characterized. A wide
range of external stress stimuli, including heat shock, heavy metals,
amino acid analogues, and oxidizing agents, drastically induce the
expression of the HSP gene through activation of HSF, which binds to
HSE located in the 5`-flanking sequence of the HSP gene(29) .
-PGJ
induces the expression of the HSP
gene, and this induction is also mediated by HSF activation, as well as
the above mentioned stimuli(26, 27) . We here showed
that
-PGJ
stimulated the promoter
activity of the 5`-flanking region of the HO-1 gene and then induced
HO-1 mRNA in RBL-2H3 cells ( Fig. 1and Fig. 2). Although
the 5`-flanking region contains HSE, HSE is not necessary for the
-PGJ
-stimulated promoter activity (Fig. 5), but this stimulation requires the specific region
(-690 to -660) containing the E-box motif (Fig. 7),
indicating that
-PGJ
induces the gene
expression through a specific element other than HSE. On the other
hand, heat shock stimulated the promoter activity of RHO810, but this
stimulation was completely abolished on removal of HSE, indicating that
heat shock induces this gene expression through HSE. Furthermore, other
stimuli, such as hemin and arsenite, did not stimulate the promoter
activity of RHO810 or showed only very low stimulation (Fig. 4B). These findings demonstrate that this element
specifically responds to
-PGJ
activation.
Thus, the element could be referred to as the
-PGJ
-responsive element, and this is the
first example of a cis-regulatory element showing a
-PGJ
-specific response. Among various
PGs, the stimulation of the promoter activity of HO-1 is specific for
-PGJ
and
-PGA
(Fig. 4A). The stimulation was not observed with
A type cyclopentenone PGs as well as conventional PGs. Generally, the
biological actions of PGA
and PGA
are much
weaker than those of
-PGJ
and
-PGA
(3) . A characteristic of
cyclopentenone PGs, such as
-PGJ
and
PGA
, is that they contain
,
-unsaturated ketones,
which are very susceptible to nucleophilic addition reactions with
thiols, and are essential for the actions of the
PGs(30, 31) . PGA
or PGA
forms
a monoconjugate with a thiol (32, 33) , but
-PGJ
or
-PGA
can form a bisconjugate with two thiols(30) .
Furthermore, the binding of PGA
to synthetic
polymer-supported thiols as the model of thiol-containing proteins is
reversible, but that of
-PGJ
or
-PGA
is irreversible(34) . An
irreversible bisconjugate with thiol groups of proteins may be required
for the stimulation of the HO-1 promoter activity, or the stimulation
by PGA
or PGA
might be marginal in the
detection of the stimulation of the activity of CAT.
The 5`-flanking
region of the HO-1 gene contains a number of DNA sequences of potential
regulatory elements. The 5`-flanking region of the rat HO-1 gene up to
position -600 contains several potential binding sites for
different transcription factors: a transcription factor, Sp1, a
positive regulator for the control of amino acid synthesis (GCN4), a
heat shock transcription factor, and a metal-dependent transcription
factor(18) . The proximal promoter region within 149 base pairs
of the upstream sequence of the mouse HO-1 gene contains several
sequence elements for AP-1, AP-4, C/EBP, and c-Myc:Max/USF, and is
required for basal promoter activity (35) . Several NFB
and AP-2-like binding sites have been found in the 5`-flanking region
of the human HO-1 gene up to position -500(36) . However,
the more upstream region of rat HO-1 (-810 to -600),
containing the
-PGJ
-responsive element,
has not yet been reported to contain potential binding sites for so far
known transcription factors. Thus, the
-PGJ
-responsive element is a novel
cis-regulatory element and plays an important role in HO-1 gene
expression. Recently, the heme oxygenase transcription factor, an
essential transcription factor, was shown to interact with the
cis-acting element (-51 to -35), located just upstream of
the TATA box of the rat HO-1 gene, and this binding is essential for
the basal expression of the HO-1 gene in rat glioma cells(37) .
In RBL-2H3 cells, the proximal 5`-flanking region (-270 to
+101) containing the heme oxygenase transcription factor binding
element and TATA box showed the basal promoter activity. The proximal
promoter region, containing the heme oxygenase transcription factor
binding element and TATA box, is essential for the basal promoter
activity. Whereas the region (-810 to -600) containing the
-PGJ
-responsive element itself did not
show the basal promoter activity, this region (-810 to
-600), when combined with the proximal 5`-flanking region
(-270 to +101), enhanced the promoter activity of the region
(-270 to +101) in a
-PGJ
-dependent manner (Fig. 5).
Furthermore, this region (-810 to -600) enhanced activity
of the exogenous promoter, SV40 promoter (Fig. 6). The
-PGJ
-responsive element thus appears to
act as an enhancer.
HO-1 mRNA was increased more than 30-fold by
-PGJ
(Fig. 1), although the
magnitude of stimulation of CAT activity was 5-fold (Fig. 3).
Such a difference may simply represent that the fusion genes lack the
additional element required for the maximal stimulation by
-PGJ
. Alternatively, this may be due to a
limitation of transient expression assays; namely, integration of the
fusion genes into the genomic DNA is required for the maximal
stimulation.
Using UV cross-linking, we identified two nuclear
proteins, the -PGJ
-responsive factors,
which specifically bind to the
-PGJ
-responsive element, their apparent
molecular weight being 80,000 and 24,000 as DNA complex form,
indicating that two different proteins bind to the element in response
to
-PGJ
(Fig. 7B). The
-PGJ
-responsive element contains an E-box
motif, and this motif is essential for the
-PGJ
-responsive factor binding to this
element and for the
-PGJ
-stimulated
promoter activity (Fig. 7, A and C). The
transcription factors which bind to the E-box motif contain a
helix-loop-helix domain and an adjacent basic amino acid region, and a
variety of transcription factors, such as MyoD, c-myc, and
Myf-5, belong to this family(38) . In RBL-2H3 cells, the
-PGJ
-responsive factors appear to belong
to a family of basic helix-loop-helix type transcription factors.
Mast cells synthesize and subsequently release PGD,
which is converted into
-PGJ
by serum
albumin during the process of inflammation(39) . The
-PGJ
produced stimulates the
transcription of the HO-1 gene in various cells, such as basophils,
fibroblasts, and vascular endothelial cells, which are actively
involved in the inflammatory response, and the reticuloendothelial
system is the site at which the active degradation of heme by HO-1 may
take place during inflammation. The induction of HO-1 by
-PGJ
may play an important role in the
fate of heme liberated during inflammation.
In summary, we here
identified the -PGJ
-specific
cis-regulatory element responsible for the expression of the rat HO-1
gene. This study will contribute not only to understanding of the HO-1
gene expression mechanism but will also facilitate elucidation of the
molecular mechanisms of cyclopentenone PG actions.