(Received for publication, July 31, 1995; and in revised form, October 19, 1995)
From the
Glutathione transferase P (GST-P) is specifically induced in rat liver and kidney by lead cation. The increase of GST-P mRNA after lead administration is blocked by actinomycin D, suggesting that GST-P production by lead is regulated at the transcriptional level. To further determine which part of the flanking region of the GST-P gene has the lead-responsive cis-element in vivo, we utilized transgenic rats with five different constructs having GST-P and/or chloramphenicol acetyltransferase coding sequence. We studied the effect of lead on these transgenic rats and on transfected NRK (normal rat kidney) cells and found that GST-P induction by lead is indeed regulated at the transcriptional level and that the GST-P enhancer I (GPEI) enhancer is an essential cis-element required for the activation of the GST-P gene by lead. GPEI consists of two AP-1 (c-Jun/c-Fos heterodimer) site-like sequences that are palindromically arranged and can bind AP-1. c-jun mRNA in the liver increased after lead administration and GST-P, and c-Jun had patchy expression in the same hepatocytes 24 h after lead exposure. These results suggest that activation of the GST-P gene by lead is mediated in major part by enhancer GPEI and that AP-1 may be involved at least partially. GPEI has been shown to have essential sequence information for the trans-activation of the GST-P gene during chemical hepatocarcinogenesis of the rat (Morimura, S., Suzuki, T., Hochi, S., Yuki, A., Nomura, K., Kitagawa, T., Nagatsu, I., Imagawa, M., and Muramatsu, M.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2065-2068; Suzuki, T., Imagawa, M., Hirabayashi, M., Yuki, A., Hisatake, K., Nomura, K., Kitagawa, T., and Muramatsu, M. (1995) Cancer Res. 55, 2651-2655). The present study establishes that the same enhancer element does operate in the activation of the GST-P gene by lead regardless of the trans-activators involved.
Glutathione transferase P (GST-P) ()is an enzyme that
catalyzes the glutathione conjugation of electrophilic
xenobiotics(1) . This enzyme is known to be dramatically
increased during chemical hepatocarcinogenesis of the
rat(2, 3) . This is apparently an induction at the
transcriptional level(3, 4) , but is somewhat
different from usual induction in that it is not reversed by withdrawal
of the carcinogen but becomes constitutive in the precancerous liver
cells. It has also been reported that GST-P is induced by lead nitrate (5, 6, 7, 8) and lead
acetate(9) , although other metals can hardly affect its
production(10) . Glutathione is supposed to serve as a first
line of defense against heavy metal cytotoxicity prior to induction of
metallothionein(11) . Rats given lead acetate induce
metallothionein-like lead-bound protein and zinc-metallothionein. The
binding of lead to metallothionein-like lead-bound protein seems not so
tight but metallothionein-like lead-bound protein bound lead accounts
for about 60% of the lead in the rat liver cytosol at
maximum(12, 13) . Zinc-metallothionein is supposed to
sequester lead and donate zinc to other zinc-dependent
enzyme(11) . Therefore GST-P may play an important role in
cooperation with metallothionein-like lead-bound protein and
zinc-metallothioneins in the detoxification of lead.
To understand the mechanisms of tumor-specific expression of this gene during chemical hepatocarcinogenesis, we have cloned the GST-P gene (4, 14) and identified at -2.5 kb of the 5`-flanking region a strong enhancer, termed GPEI, whose core consisting of two AP-1 site-like sequences (1-base mismatch for each) arranged in a palindrome(14, 15, 16, 17) . By using transgenic rats we have recently demonstrated that the GST-P gene is activated by some transactivator(s) during chemical hepatocarcinogenesis and that the activation requires 5`-flanking region of GST-P gene containing GPEI(18) . We have also shown that GPEI itself is the necessary cis-element for GST-P gene expression during this process(19) .
Question arises as to whether GST-P expression at lead exposure is regulated at the transcriptional level and whether a common regulatory mechanism of GST-P gene expression is operative between the precancerous liver cells and lead-treated liver cells. To answer these questions, we have utilized transgenic rats having various transgene constructs. We have also transfected NRK fibroblast cells with a series of ECAT deletion mutant genes and determined CAT activity in order to narrow down the lead-responsive DNA region of the GST-P gene. The results indicate that GST-P induction by lead is regulated at the transcriptional level and that the essential cis-element for GST-P gene activation by lead is also the enhancer GPEI. Furthermore, to clarify the role of c-Jun in the activation of GST-P gene by lead, we studied the expression of c-Jun and GST-P in the liver cells at acute lead exposure using immunohistochemistry.
The data show that GPEI is an essential element for the activation of the GST-P gene by lead and that trans-acting factor AP-1 is likely to be involved at least in part in the transcriptional activation of GST-P gene by lead through GPEI sequence.
Untreated 10-week-old male Wistar rats were used as a control. Lead nitrate or lead acetate (Wako, Osaka, Japan) was dissolved in distilled water at a concentration of 100 µM just prior to use and injected intraperitoneally at a dose of 100 µmol/kg. Rats were given the same lead salt every 24 h three times and were sacrificed 24 h after the last lead salt administration. In some experiments, a single dose of lead acetate (100 µmol/kg) was administered and rats were sacrificed at 0, 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 12 h, 24 h, and 48 h later. In other experiments, actinomycin D (Serva, Heidelberg) was dissolved in 50% propylene glycol and administered intraperitoneally (0.8 mg/kg body weight) 4 h before single dose of lead nitrate (100 µmol/kg)(21) , and the rats were killed 12 h thereafter.
Figure 1: Western blot analysis that shows the increase of GST-P in the liver of control rats by lead nitrate. Lead salt was injected intraperitoneally three times at 24-h intervals and livers removed 24 h after the last administration. 100 µmol/kg/injection of lead nitrate treatment increased the amount of GST-P most effectively. 10 µg of protein were subjected to Western blots with anti-GST-P antibody. Lane 1, control; lane 2, 100 µmol/kg/injection of lead nitrate; lane 3, 200 µmol/kg/injection of lead nitrate.
Figure 2: A, Northern blot analysis that shows the changes of the GST-P, c-jun, junB, junD, and c-fos mRNA contents in the liver of control rats by single intraperitoneal lead acetate administration at a dose of 100 µmol/kg. Time after lead administration is indicated as ` (min) and h. Ten µg of total RNA was applied for each lane. Blotted filter was hybridized with GST-P, c-jun, junB, junD, c-fos, and GAPDH cDNA, successively. GAPDH mRNA contents are almost constant throughout the course of the study, which also indicates that almost equal amounts of undegraded RNA are loaded. B, Northern blot analyses of GST-P and c-jun mRNA expression after lead administration in another independent experiment different from that is shown in A. Note that the expression patterns shown here are essentially the same.
To see whether lead-induced GST-P mRNA accumulation was regulated at the transcriptional level, we administered actinomycin D to stop mRNA synthesis (21) before lead injection. The GST-P mRNA of rat liver increased 2.5-fold 12 h after lead nitrate treatment (Fig. 3, lane 1 versus lane 2), whereas actinomycin D completely blocked the increase (lane 3). In the kidney, GST-P mRNA increased 1.8-fold by lead nitrate (lane 4 versus lane 5), which was also blocked completely by actinomycin D (lane 6). The filter was dehybridized and rehybridized with GAPDH cDNA to confirm that RNA was not degraded by actinomycin D treatment and almost equal amount of RNA was loaded on the filter. These experiments have suggested that lead stimulates GST-P production at the transcriptional level.
Figure 3: Northern blot analysis that shows GST-P mRNA accumulation induced by a single lead nitrate administration in the liver and kidney of control rats was blocked by actinomycin D. Actinomycin D (0.8 mg/kg) was injected intraperitoneally 4 h prior to lead administration (100 µmol/kg), and the rats were killed 12 h thereafter. Ten µg of total RNA obtained from the control or experimental liver was applied for each lane. Lanes 1 and 4, control; lanes 2 and 5, lead nitrate-treated; lanes 3 and 6, actinomycin D and then lead nitrate-treated. The same filter was dehybridized and rehybridized with GAPDH cDNA to show that undegraded total RNAs were loaded. Expression of GAPDH was always higher in the kidney than in the liver.
Figure 4:
Transfection followed by CAT analysis
shows the requirement of the GPEI-containing region of GST-P gene for
lead induction in the NRK cells. Experimental cells were cultured with
10 nM lead nitrate. A, structure of the constructs
used for the transfection experiments. B, results of CAT
assay. Effect of lead nitrate on ECAT deletion mutants transfected into
NRK fibroblast cells was indicated. The amount of samples for CAT assay
was adjusted by the -galactosidase activity derived from
co-transfected pRSV
GAL. The stimulation index is the ratio of
acetylation for the lead-treated experimental versus untreated
control NRK cells. The mean values of the three independent experiments
are shown. CAT stimulation indexes were 2.7, 1.0, 1.1, 0.5, 1.1, and
0.7, in ECAT, 1CAT, 2CAT, 3CAT, 4CAT, and 5CAT,
respectively.
Because the expression of the GST-P mRNA was higher in the kidney than in the liver (see Fig. 3), we next examined the effect of lead on the ECAT gene transfected into NRK fibroblast cells (Fig. 4B). The results were shown by the mean values of three independent experiments. Basal ECAT activity was low, but lead could enhance the activity of the ECAT gene 2.7-fold of the control in these cells. Lead nitrate could not activate 1CAT, 2CAT, 3CAT, 4CAT, nor 5CAT deletion mutant genes, from which the GPEI had been deleted. Lead nitrate slightly, but reproducibly, reduced the activity of pSV2CAT transfected into NRK fibroblast cells (0.9-fold), and this might be due to the general toxicity of lead nitrate to the cells.
The above results show that the lead-responsive cis-element of the GST-P gene is located between -2.9 and -2.2 kb upstream from the transcription start site of GST-P gene. In this restricted region, there is a strong enhancer GPEI that we have reported previously (14, 15, 16, 17) .
Figure 5:
The GPEI-containing region is essential
for the in vivo induction of GST-P gene by lead. A,
constructs of ECAT and 1CAT used for the transgenic rats. B,
CAT assay with regards to the effect of lead acetate on the CAT
transgene in the liver and kidney of ECAT and 1CAT rats.
One-hundred-µg protein equivalent reactions were put on the thin
layer chromatography. A, liver samples reacted with acetyl-CoA
and [C]chloramphenicol for 1 h; B,
liver samples reacted for 16 h; C, kidney samples reacted for
1 h. CAT activity of the ECAT transgenic rat was always higher in the
kidney than in the liver.
To
pinpoint, in vivo, the enhancer region that is essential for
GST-P gene expression by lead, we used two more types of transgenic
rats. Fig. 6A illustrates the constructs. The construct
-56CAT has the minimum GST-P promoter connected to CAT coding
region. The construct
-56CAT GPEI has the genomic 122-bp GPEI (Fig. 6B) subcloned into the
-56CAT. We intentionally
placed the GPEI element at the 3` end of CAT coding sequence, i.e. 2.0 kb downstream from the transcription initiation site, because
we previously noted with transfected primary hepatocytes that the GPEI
enhancer had a constitutive enhancing activity when it was located
adjacent to the promoter(38) . We were afraid that CAT might be
constitutively expressed in the liver of transgenic rat, if the GPEI is
located too close to the promoter. We used each one line of
-56CAT
and
-56CAT GPEI rat. Southern blot showed that 5 and 21 copies of
the transgene were integrated in tandem arrays into the chromosome of
-56CAT and
-56CAT GPEI rats, respectively(36) . CAT
activity did not change significantly by lead in the liver of these
transgenic rats (data not shown). However, CAT activity of the kidney
samples from
-56CAT GPEI rats was enhanced 2-fold with lead
acetate (Fig. 6C). By contrast, CAT activity of the
kidney samples from
-56CAT rat was not stimulated at all. The
results show that lead stimulates the GST-P expression through the
122-bp GPEI sequence. Our previous data had suggested that 17-bp GPEI
core consisting of two AP-1 site-like sequences (see Fig. 7B) alone could confer a similar enhancing
activity to 122 bp of genomic GPEI in a cultured cell transfection
system(16, 17) . To determine whether lead
responsiveness of the GST-P gene depends upon the GPEI core sequence or
not, we have further made nCAT transgenic rats having only the 17-bp
GPEI core sequence inserted into
-56CAT at the 3`-end (Fig. 7A) and tested the effect of lead acetate on the
transgene. Six copies of the transgene were introduced into the
chromosome of nCAT rat (line 4) in a tandem manner(36) . The
CAT activity in the kidney was 2-fold enhanced by lead acetate
administration (Fig. 7C), but the CAT activity in the
liver did not change significantly (data not shown). The results
clearly indicate that GPEI core sequence is sufficient for the GST-P
gene expression in the kidney by lead under favorable conditions.
Although we could test the effect of lead only on each one line of the
transgenic rat having
-56CATGPEI or nCAT, we do not think that
this activation of transgene by lead was fortuitous due to mere
positional effect, for both of the transgenes were activated only after
lead administration.
Figure 6:
The 122-bp GPEI region is sufficient for
induction of GST-P by lead in vivo. A, constructs of
-56CAT and
-56CAT GPEI used for the transgenic rats.
-56CAT has minimum GST-P promoter (GC box and TATA box) but not
AP-1 site near the promoter. Genomic 122 bp of GPE was subcloned into
-56CAT and designated as
-56CAT GPEI. B, structure
of genomic GPEI. GPEI has two AP-1 site-like sequences (indicated by arrows) arranged palindromically 3 bases in between. Each site
has a 1-base mismatch in comparison with the authentic AP-1 site
(TGAC/GTCA). C, CAT assay with regard to the effect of lead
acetate on the CAT transgene in the kidney of
-56CAT and
-56CAT GPEI transgenic rats. One-hundred-µg protein equivalent
samples were used. Samples were reacted with acetyl-CoA and
[
C]chloramphenicol for 16
h.
Figure 7:
GPEI core
can act as a lead-responsive enhancer in vivo. A,
constructs of nCAT used for the transgenic rats. Synthetic GPEI core
sequence with 8-bp flanking was subcloned into -56CAT. B,
structure of GPEI core. GPEI core consists of 17 bp. AP-1 site-like
sequences are indicated by arrows. CAT assay of the effect of
lead acetate on the CAT transgene in the kidney of nCAT rats.
One-hundred-µg protein equivalent samples were used. Samples were
reacted with acetyl-CoA and [
C]chloramphenicol
for 16 h.
Figure 8: Immunohistochemical analysis using serial sections revealed that c-Jun and GST-P were expressed in the same hepatocytes located in the hepatic lobules 48 h after lead acetate injection. Livers of control and experimental rats (single intraperitoneal lead acetate administration at a dose of 100 µmol/kg) were obtained and used for the immunohistochemistry with antibody against GST-P or c-Jun. A, control rat liver immunostained by anti-GST-P antibody. B, control rat liver immunostained by anti-c-Jun antibody. C, lead-treated liver immunostained by anti-GST-P antibody. D, lead-treated liver immunostained by anti-c-Jun antibody.
Taken together, we conclude that GPEI has an essential sequence information for the activation of the GST-P gene by lead and that AP-1 may be involved at least partially in the induction of GST-P by lead.
We have demonstrated by means of transgenic rats that induction of the GST-P gene, a well known tumor marker for chemical hepatocarcinogenesis, by lead is regulated at the transcriptional level by means of the enhancer GPEI.
Although GST-P has been known to be induced by lead nitrate(5, 6, 7, 8) , notwithstanding the relative insensitivity to other substances, including heavy metals(10) , and most of the previous studies were performed with this compound, we confirmed that lead acetate could induce GST-P in the liver and kidney as good as or even better than lead nitrate did as reported by others(9) . This was probably because acetate ion was less toxic to cells covering the peritoneal cavity than nitrate ion, and this was confirmed by the morphological observation.
Two categorically different mechanisms may be considered for the activation of GST-P gene by lead. First, the GST-P gene may be activated, because the chromatin structure of a certain chromosomal locus is specifically altered and activated by lead cation. Second, some trans-acting factor(s) that is induced or activated by lead cation may bind to cis-elements of GST-P gene and thus activate GST-P gene. The present study showing that GST-P gene introduced into transgenic rats is activated in locus-independent manner supports the latter hypothesis. The GST-P gene is thus activated by some trans-activator(s) at lead exposure just as during chemical hepatocarcinogenesis(18) .
Actinomycin D blocked the
lead-responsive increase of GST-P mRNA in rat liver and kidney,
suggesting that the activation occurred at the transcriptional level.
This idea is clearly confirmed by the fact that CAT transgenes, ECAT,
-56CATGPEI, and nCAT, but not the
-56CAT, were activated by
lead in vivo. Others reported that transcription rate of the
GST-P gene is enhanced by lead using run-on assay(8) . Thus,
our transgenic system was found to be useful for investigating in
vivo transcription rate of the gene.
The next crucial question is what kind of trans-acting factor(s) will bind to the GPEI and activate GST-P gene after lead exposure. One of the candidates of the trans-activator would be AP-1, since c-jun mRNA elevation does precede GST-P mRNA accumulation, GPEI that is essential for the GST-P gene activation has two AP-1 binding site-like sequences, and GST-P protein is expressed in the hepatocytes having c-Jun 48 h after lead treatment. The data suggest that AP-1 may play an important role in the transient expression of GST-P after lead exposure.
The question as to whether activation of the GST-P gene by lead has any similarity to the activation during hepatocarcinogenesis is of special interest in view of the apparent difference in the mode of induction; the former is a transient induction, and the latter is a semi-constitutive change of gene expression during cell transformation. We note that the expression pattern of c-jun mRNA after lead exposure is complex. It has three peaks of expression after intraperitoneal injection of lead. N-Nitrosodiethylamine, which is given as an initiator for chemical hepatocarcinogenesis of Solt-Farber procedure(39) , also causes c-jun mRNA increase(40) . The pattern of c-jun mRNA expression after N-nitrosodiethylamine injection, however, was quite different from that after lead treatment. The amount of c-jun mRNA was kept unchanged at a high level from 2 h to 24 h after N-nitrosodiethylamine administration and GST-P mRNA content had its peak at 12 h(40) . The difference in the expression pattern of c-jun mRNA might have some relationship to the difference between semiconstitutive and transient GST-P activation during carcinogenesis and lead exposure. The expression pattern of junB and junD mRNA resembled but was slightly different from that of c-jun, indicating that these Jun family proteins might partially contribute to the regulation of GST-P expression by attenuating the activity of AP-1. Involvement of other trans-activators such as Maf family proteins (41, 42, 43, 44) that bind to the AP-1 site-like sequence cannot be ruled out and is now under study.
Activation of CAT enzyme activity by GPEI alone in -56CAT GPEI
rat or GPEI core alone in nCAT rat was significantly lower than that
seen in ECAT rat, an observation different from the transient
transfection into cell cultures(16) . This is probably due to
the flanking sequences that might affect the activity of the integrated
gene GPEI in the chromosomal context. The larger ECAT may be free from
various effects of the integration site, but the smaller GPEI or its
core may be subject to the effects of the adjacent sequences that are
different from one integration site to another. It is known that a
larger flanking region is often required for physiological regulatory
phenomenon when analyzed with transgenic animals in
general(45) . Although introduced GST-P genes were activated by
lead in a somewhat locus-independent manner in GST-P transgenic rats,
the degree of activation was different between liver and kidney. It is
reported that both c-Jun and c-Fos are rich in kidney, while they are
trace-positive or absent in liver(43, 46) . Thus, the
difference of GST-P activation between organs would be caused by the
difference in the distribution of trans-activators.
Identification and cloning of the transcription factor(s) other than c-Jun that bind to GPEI and activate or repress the GST-P gene expression are required for further understanding of the regulation of GST-P gene by lead.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) N00039 [GenBank](GenBank(TM)) and J02690 [GenBank](EMBL).