(Received for publication, February 15, 1995; and in revised form, July 27, 1995)
From the
Heme-hemopexin or cobalt protoporphyrin (CoPP)-hemopexin (a
model ligand for hemopexin receptor occupancy) is shown to increase
transcription of the metallothionein-1 (MT-1) gene by activation of a
signaling pathway. Promoter deletion analysis followed by transient
transfection assays show that 110 base pairs (-153 to -43)
of 5`-flanking region of the murine MT-1 promoter are sufficient for
increasing transcription in response to heme-hemopexin or to
CoPP-hemopexin in mouse hepatoma cells. The protein kinase C inhibitor,
1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7),
prevented the increase in MT-1 transcription by heme-hemopexin,
CoPP-hemopexin, or phorbol 12-myristate 13-acetate, but the protein
kinase A inhibitor, HA1004, was without effect. N-Acetylcysteine (NAC) and glutathione, as well as superoxide
dismutase and catalase, inhibited both the increase in endogenous MT-1
mRNA and the activation of reporter gene activity by heme-hemopexin,
CoPP-hemopexin, and phorbol 12-myristate 13-acetate. In sum, these data
suggest that reactive oxygen intermediates are generated by
heme-hemopexin via events associated with receptor binding, including
protein kinase C activation. Induction of heme oxygenase-1 expression,
in contrast to MT-1, is significantly less sensitive to NAC. Deletion
and mutation analyses of the MT-1 proximal promoter revealed that the
sequence 5`-GTGACTATGC-3` (from -98 to -89 base pairs) is,
in part, responsible for the hemopexin-mediated regulation of MT-1
which is inhibited by H7. Regulation via this element is also induced
by HO
showing that it is an antioxidant
response element. Heme itself acts via more distal elements on the MT-1
promoter. In contrast to NAC and glutathione, diethyl dithiocarbamate
and pyrrolidine dithiocarbamate, which inactivate reactive oxygen
intermediates and chelate Zn(II), synergistically augment the induction
of MT-1 mRNA levels and reporter gene activity in response to
heme-hemopexin via the antioxidant response element by both
metal-responsive element-dependent and -independent mechanisms.
Hemopexin and transferrin are unique among known endocytic
transport systems since both the transport glycoprotein and its
receptor recycle(2, 3, 4, 5) . The
hepatic reclamation of heme ()by hemopexin (K
<1 pM) (6) sequesters heme from invading organisms (7) and
conserves heme-iron for reutilization, storage on ferritin(8) ,
or gene regulation(9, 10) . Hemopexin also acts as an
extracellular antioxidant(11, 12, 13) , as do
transferrin(14) , haptoglobin (15) , and
ceruloplasmin(16) , by coordinating and thus inactivating the
reactive heme-iron(12) .
Heme is a pleiotropic regulator of gene expression in Escherichia coli, yeast, and mammalian cells, although the mechanisms remain to be elucidated in each case. Work in this laboratory has established that the expression of heme oxygenase-1 (HO-1)(9) , the transferrin receptor(9) , and metallothionein-1 (MT-1) (10) is specifically and differentially regulated in response to heme-hemopexin in cultured mammalian cells. We have proposed (10) that the coordinate induction of MT-1 and HO-1 gene transcription by heme-hemopexin is an adaptive response of cells to maintain homeostasis by minimizing oxidative damage from heme and iron to survive under stress conditions.
Here, we address the mechanism of heme-hemopexin-mediated transcriptional activation of the MT-1 gene. MTs are small, cysteine-rich, metal (e.g. Cu(II), Zn(II), and Cd(II))-binding proteins. The mRNA levels of mouse metallothionein (MT-1) (10) have been shown to increase in response to heme-hemopexin. Notably, when heme or the heme analogs, tin-protoporphyrin (SnPP) and cobalt-protoporphyrin (CoPP), are bound to hemopexin, they are far more effective inducers of MT-1 gene transcription than when unbound(10, 17) . Interestingly, while the uptake of SnPP is facilitated by hemopexin, CoPP-hemopexin binds to the hemopexin receptor without intracellular transport of CoPP (17) yet is an effective regulator. This indicates that occupation of the hemopexin receptor itself produces intracellular events, postulated to be activation of a signaling pathway(17) , that enhances MT-1 gene transcription.
Protein kinase C (PKC) activation has been linked to the regulation of MT expression. First, phorbol 12-myristate 13-acetate (PMA), which activates PKC, induces MT in rats and cultured cells(18) . Second, the human MT-2A gene promoter has a functional AP-1 site (5`-TGAGTCA-3`) to which members of the AP-1 family of transcription factors, i.e. Jun/Fos, bind, and this DNA binding is induced by phorbol ester activation of PKC(19) . We also note that a putative AP-1 site (5`-TGAGTGA-3`) lies at -538 to -545 bp of the mouse MT-1 promoter. Since PMA also increases the rate of endocytosis of hemopexin (20) , the possibility of involvement of PKC in hemopexin-mediated MT-1 gene regulation is raised.
MT-1 is also readily induced in response to a
variety of stimuli including metals, glucocorticoids,
cytokines(22, 23, 24, 25) , and
ultraviolet radiation(26) . MTs have also been proposed (27, 28) to act as intracellular antioxidants by
sequestering reactive metals and inactivating hydroxyl radicals and
superoxide. Reactive oxygen intermediates (ROIs) ()have been
implicated in the regulation of MT gene expression since MT levels are
increased when cells are incubated with chemicals which undergo redox
cycling (e.g. paraquat) (29) or decrease glutathione
(GSH) concentration (e.g. diamide)(30) . ROIs are not
only produced as cytotoxic agents under pathological conditions, e.g. by granulocytes in inflammation, but are also generated
as side products of electron transfer reactions, both in mitochondria
and the endoplasmic reticulum, during normal cellular metabolism. Since
heme (iron-protoporphyrin IX) participates in oxygen-radical reactions
that can lead to the degradation of proteins, lipids, carbohydrates,
and DNA(1, 31, 32) , hemopexin-mediated heme
transport by increasing intracellular levels of heme and iron may
concomitantly raise ROI levels.
Several genes encoding proteins
which function to protect against oxidative stress, including the rat
GSH S-transferase Ya subunit and human quinone reductase
genes, contain within their respective promoters an antioxidant
response element (ARE, 5`-GTGACNNNGC-3`) which confers transcriptional
activation in response to -napthaquinone (33) or
H
O
(34, 35) . Interestingly,
there is a putative ARE sequence (5`-GTGACTATGC-3` from -98 to
-89 bp) in the mouse MT-1 promoter. The present study (
)was undertaken to define the regions of the MT-1 promoter
required for heme-hemopexin-mediated regulation and to assess the roles
of PKC, ROIs, and cis-acting elements, such as the putative
ARE, in this regulation.
Essentially identical results are found with
reporter gene constructs (Fig. 1, right-hand column)
transiently transfected into Hepa cells. H7, but not HA1004, prevented
the increases in -galactosidase reporter gene activity of
-750MT
Geo (Table 1) and -150MT
Geo (Fig. 1) in response to heme-hemopexin, CoPP-hemopexin, or PMA (Table 1). Thus, the responses monitored by
-galactosidase
activity of the reporter-gene constructs accurately reflect the
cellular responses of the endogenous MT-1 gene. Since CoPP-hemopexin
which binds to the receptor without tetrapyrrole transport is as
effective as heme-hemopexin, the results suggest that occupancy of the
receptor plays a role in transducing signals which result in PKC
activation as part of the signaling pathway which regulates MT-1 gene
transcription.
Figure 1:
Effects of
heme-hemopexin and CoPP-hemopexin on the transcriptional activities of
the MT-1 proximal promoter. The left panel shows a schematic
representation of the MT-1 fusion genes investigated, and the numbers
shown delineate the 5` end of the regulatory region covered by each
fusion gene. In the case of -750(110)MT
Geo, the 3` and
5` sites of the deletion are indicated. The right panel shows
the results of the transient transfection assays carried out as
described in the legend to Table 1. Twenty-four hours after
transfection heme-hemopexin (H-HPX), CoPP-hemopexin (10
µM), or PMA (50 ng/ml) were added. The
-galactosidase
activity of the fusion gene in cell extracts was measured 24 h later
and normalized to the CAT activity. Each data point represents the mean
value ± S.E. of four (pMT-lacF) or mean ± S.D.
of six or more (-750MT
Geo,
-750(
110)MT
Geo,
-150MT
Geo, -42MT
Geo, and MRE
Geo) independent transfections. N.D., not
determined. Two and 5 µM heme-hemopexin induces
-150MT
Geo reporter gene activity 76 ± 3 and 87
± 4% as effectively as 10 µM complex (data not
shown).
The induction of the -galactosidase
reporter gene by heme-hemopexin was abolished when the -153 to
-43 bp region was deleted (-750(
110)MT
Geo in Fig. 1). Moreover, the
-galactosidase activity of
-150MT
Geo, which contains up to -153 to -43 bp
of the promoter, is induced by heme-hemopexin, while
-42MT
Geo containing only the basal promoter does not respond (Fig. 1). Furthermore, the extent of induction of
-150MT
Geo by heme-hemopexin is similar to that seen with
either -750MT
Geo or pMTLacF (Fig. 1). Importantly,
CoPP-hemopexin also induces the reporter gene activity of
-750MT
Geo and -150MT
Geo, the former as
effectively as heme-hemopexin and the latter somewhat less effectively
than heme-hemopexin but similar to the levels seen with PMA (see
below). This shows that the effects of receptor-occupancy on regulation
are maintained in these constructs. H7 blocks the increase in
expression of -750MT
Geo by both heme and CoPP-hemopexin and
of -150MT
Geo by heme-hemopexin (Table 1).
The 110
bp of the shortened promoter in -150MTGeo contain the
putative ARE, four metal responsive elements (MRE a-d, which
share the heptad core TGCPuCNC), a major late transcription factor
consensus sequence, and an Sp1 site which overlaps with
MREc(43, 44, 45) . The five copies of MREd`,
the element which confers the highest response to Zn(II)(43) ,
in MRE
Geo did not restore induction by heme-hemopexin suggesting
that other or multiple elements (see below) are major factors in
regulation.
PMA, which activates the AP-1 family of transcription
factors, also induced expression of the fusion genes in
-750MTGeo and -150MT
Geo, although less
effectively than heme-hemopexin (Fig. 1). Thus, the putative
AP-1 site at -545 to -538 bp is not required for
transcriptional regulation by heme-hemopexin or PMA in transiently
transfected Hepa cells. PMA can activate via antioxidant response
elements as discussed below.
ROIs
themselves induce MT-1 gene expression in Hepa cells. Hydrogen peroxide
caused a 3-4-fold increase of MT mRNA levels within 3 h (Fig. 2A) and an increase in transcription of
-150MTGeo that was dose-dependent. Superoxide, generated
extracellularly by xanthine oxidase, increased transcription of
-150MT
Geo (Fig. 2B) in transient
transfection assays.
Figure 2:
Effects of hydrogen peroxide and a
superoxide generating system, xanthine oxidase, on MT-1 expression.
Hepa cells were incubated in serum-free, Hepes-buffered
Dulbecco's modified Eagle's medium containing either 100
µM hydrogen peroxide, in the presence or absence of 30
mM NAC as indicated, for 3 h. Total cellular RNA was isolated
as described under ``Materials and Methods.'' Panel A shows a dot blot analysis of MT-1 mRNA in 5 and 10 µg of total
cellular RNA. Tubulin mRNA levels are also shown. Panel B shows the results of transient transfection with
-150MTGeo and induction of reporter gene activity after
incubation of Hepa cells with increasing concentrations of xanthine and
70 units of xanthine oxidase to generate superoxide. In control
experiments when cells were incubated with either xanthine or xanthine
oxidase alone, there was no detectable change in reporter gene activity
(data not shown). Panel C shows the effects of NAC on basal
and heme-hemopexin (H-HPX)-induced HO-1 mRNA levels in
response to heme-hemopexin and PMA.
N-acetyl-L-cysteine (NAC), a
precursor of GSH that scavenges
ROIs(46, 47, 48) , prevented the increase in
MT-1 mRNA levels in response to HO
(Fig. 2A, lane 3), to heme-hemopexin or
CoPP-hemopexin, or to PMA (Table 3). In some cases treatment with
NAC produces MT-1 mRNA levels lower than the controls, but this was not
seen when GSH was added extracellularly in transient transfection
experiments with -750MT
Geo (Table 3, discussed below).
As additional controls, the effects of NAC on tubulin and HO-1 mRNA
levels were investigated (Fig. 2C). The HO-1 gene
contains two AP-1 binding sites in an enhancer element required for
increased transcription by heme (49) and is also induced by
PMA(10) . Heme-hemopexin and PMA raised HO-1 mRNA levels
5- and 6-fold by 3 h, respectively (Fig. 2C, lanes 2 and 5), while NAC lowered the induced HO mRNA
levels by about 40% (Fig. 2C, lanes 3 and 6), but NAC did not affect basal levels of HO-1 (Fig. 2A, lane 8) or tubulin mRNA (data not
shown). Thus, basal and heme-hemopexin- or PMA-induced HO-1 expression
is significantly less sensitive to NAC than the MT-1 gene. A 12-h
exposure of Hepa cells to 30 mM NAC produces no discernable
toxic effects or abnormal morphology.
The extent of changes in
reporter gene activity in the transient transfection assays are
essentially equivalent to those in the endogenous MT-1 gene measured by
Northern blot analysis. The increases in -galactosidase activity
of -750MT
Geo in response to heme-hemopexin, CoPP-hemopexin,
and PMA were also abolished by both NAC and GSH (Table 3). Taken
together, the results of both Northern analyses and transient
transfections are consistent with NAC and GSH acting as ROI scavengers
and indicate that the maintenance of thiol levels by NAC or GSH
prevents induction of MT-1 gene transcription in response to
heme-hemopexin, CoPP-hemopexin, or PMA. Thus, the increase in ROIs
which occurs in response to heme-hemopexin causes oxidation of a
critical thiol residue and/or depletion of intracellular thiols leading
to altered MT-1 gene transcription. However, NAC and GSH also chelate
zinc, and thus zinc availability as well as the links between
redox-mediated release of zinc from MT itself (50) and MT-1
gene expression are addressed below.
Figure 3:
Identification of the cis-acting
elements in the mouse MT-1 proximal promoter required for
transcriptional activation by hemehemopexin and evidence that this is
the same element involved in the response to hydrogen peroxide. Panel A summarizes the data from transient transfection
studies using -150MTGeo. Hepa cells were incubated in the
presence and absence of heme-hemopexin (H-HPX) and increasing
concentrations of either PDC (left) or DDC (right),
as indicated, for 24 h. Panel B shows a schematic
representation of the MT-1 fusion genes investigated containing MRE and
putative ARE elements. Constructs which contain fragments of the
proximal promoter are defined by their location in the promoter. Panel C shows the results of the transient transfection assays
carried out as described in the legend to Table 1. Twenty-four
hours after transfection heme-hemopexin (H-HPX; 10
µM) in the presence or absence of PDC or H7 was added as
indicated, and the
-galactosidase activity of the fusion gene in
cell extracts was measured 24 h later and normalized to the CAT
activity. In additional experiments the cells were incubated with
hydrogen peroxide or, as controls, H7 or PDC alone. Each data point
represents the mean ± S.D. of four independent transfections
(ARE
MT
Geo and ARE
-MT
Geo) or from 6
to 10 independent transfections (-750MT
Geo,
-153(-67)MT
Geo, -124(-67)MT
Geo,
124(-43)MT
Geo, AREMT
Geo, ARE
MT
Geo).
The fusion gene containing only one copy of the MT-ARE was not induced
by heme-hemopexin in six independent transfection experiments.
Heme-hemopexin and hydrogen peroxide increased the expression of
-150MT
Geo 4.9 ± 0.9- and 2.2 ± 0.6-fold,
respectively. The stimulatory and inhibitory effects, respectively, of
PDC and H7 on the induction of -150MT
Geo by heme-hemopexin
are presented elsewhere in the manuscript. A 3-fold induction of
MRE
Geo by hydrogen peroxide was observed with 500 µM reagent.
We originally investigated the effects of DDC
and PDC because they are structurally related compounds and
DDC inhibits superoxide dismutase by chelating zinc. Their effects on
MT mRNA levels were interpreted as being due to an increase in
intracellular ROIs since the regulation by heme-hemopexin appeared to
be independent of the element, MREd` (see Fig. 1). However,
since NAC and GSH are inhibitory while DDC and PDC are stimulatory,
they cannot act by the same mechanism. While this work was in progress,
a role for MREs in the response to oxidative stress of the chick MT
gene was suggested(53, 55) , and a model for MT gene
regulation was proposed involving increased intracellular zinc and a
constitutively expressed transcription factor, MTF-1. This factor binds
to the MREs upon release from a rapidly turning over inhibitor protein,
MTI(54) , a system analogous to NF
B and I
B.
The
PDC-induced expression of MREGeo in baby hamster kidney cells
required very low levels (0.5 µM) of extracellular Zn(II),
and it was proposed that PDC transported Zn(II) into cells and caused
dissociation of the MTI
MTF-1 complex(54) . However, the
lack of induction of MRE
Geo by heme-hemopexin ( Fig. 1and Fig. 3, Panel C) demonstrates that MT-1 gene regulation
in response to this heme transport system is not due to either a direct
or indirect effect on the MTI
MTF-1 interaction causing
dissociation of MTF-1 followed by binding to the MRE. It also seems
unlikely from this result that Zn(II) uptake has been stimulated.
Nonetheless, the regulation of MT-1 expression by hemopexin via the ARE
does not exclude or comment on regulation by changes in intracellular
Zn(II) pools (see below).
As summarized in Fig. 3,
heme-hemopexin induces to a similar extent the expression of three
fusion genes, -124(-67)MTGeo,
-153(-67)MT
Geo and -124(-43)MT
Geo,
all of which contain the MT-ARE, either with 5`- and 3`-flanking
regions, with 5`-flanking MREs c and d or with 3`-flanking MREs a and
b, respectively. Their responses were, however, only
50% that of
the fusion gene -150MT
Geo containing the complete region and
none were induced to higher levels than ARE
MT
Geo.
CoPP-hemopexin also induces these fusion genes but slightly less
effectively than heme-hemopexin (data not shown). The combined results
of deletion analyses of this 110-bp region are consistent with a
mechanism whereby the increased transcription of the MT-1 gene by
heme-hemopexin requires the ARE. The presence of additional elements
including MREs c and d does not restore the transcriptional activity of
the intact 110-bp region.
Heme is a reactive
form of iron able to participate in oxygen radical reactions, but
hemopexin in the plasma acts as an extracellular antioxidant by
coordinating and inactivating the reactive heme-iron (12) .
ROIs could be generated by redox cycling of heme released from
hemopexin, which would require both a change in heme coordination by
hemopexin, possibly induced by receptor binding because hemopexin in
solution is an antioxidant, and a source of electrons. CoPP is bound to
hemopexin similarly to heme(17) , and cobalt can undergo redox
cycling, but less readily than iron or heme under physiological
conditions. However, CoPP is not extensively taken up by cells when
presented as a CoPP-hemopexin complex(17) . Nevertheless, since
NAC abolishes CoPP-hemopexin-mediated MT gene activation, occupation of
the hemopexin receptor per se is implicated in the pathway
that generates free radicals. Binding of hemopexin complexes to the
hemopexin receptor, as does binding of diferric transferrin to its
receptor, may activate the transmembrane NADH oxidase which catalyzes
electron transfer from NADH to molecular
oxygen(57, 58) . This enzyme produces superoxide and
participates in ferric iron reduction as an electron source. Hemopexin
binds both ferri- and ferro-protoporphyrin(59) , and several
parallels exist between the hemopexin and transferrin systems. An as
yet to be defined ``ROI-inducing effect'' of PKC is thought
to be needed to stimulate NF-B DNA binding which is induced by
signals involving ROIs(56) . PKC activation is associated with,
and may be the direct mechanism for, activation of the NAD(P)H
oxidoreductase of the respiratory burst in phagocyte and
leukocytes(61) . The NADH oxidase in hepatic plasma membranes
has several features which distinguish it from other NADH
oxidoreductase or the leukocyte NADPH oxidoreductase and mitochondrial
NADH oxidase(58) .
Our current working hypothesis is that ROIs, including superoxide and hydrogen peroxide, are generated upon receptor occupancy as a consequence of PKC and plasma membrane NADH oxidase activation. Furthermore, production of ROIs at a low level may be a metabolic signal which helps set in motion a series of events including HO-1 and MT-1 activation to prepare the cell for survival since the presence of extracellular heme-hemopexin indicates hemolysis and/or tissue trauma. The signaling pathway results in phosphorylation or oxidation of key sulfhydryl group(s) of specific proteins of the regulatory pathway for gene regulation including transcription factors and proteins with which they associate. Possible additional sources of ROIs include redox cycling of heme and possibly of heme-hemopexin, interactions between intracellular iron and ROIs, and thiyl radicals from oxidation of a sulfhydryl group on the hemopexin receptor subunit(18) .
A role for heme itself in stimulating MT-1
gene transcription is also evident from the results presented here with
free heme and heme analogs. However, since -750MTGeo, but
not -150MT
Geo, responds to free heme, (
)more
distal elements in the region between -600 and -150 bp
appear to be involved. Heme is rapidly catabolized after uptake, and
there is evidence that iron can be bound directly to MT (60) but with an affinity that makes it unlikely that iron
would displace zinc.
Thus, we propose that regulation of MT-1
expression by hemopexin takes place by receptor-mediated signals from
the plasma membrane which affect gene regulation following activation
of signaling pathways involving PKC and ROIs. The latter act in part
through the ARE, perhaps as an early defense mechanism of the cell. If
additional events also occur, such as redox-sensitive release of Zn(II)
from MT or increased Zn(II) uptake as in the acute phase
response(62) , a rapid synergistic increase in transcription
would take place, probably due to released MTF-1. The inhibitory
effects of NAC and GSH on hemopexin-mediated induction of MT-1
expression may be due to their ability to bind Zn(II), but it seems
more likely here that they act as ROI quenchers. Quenching may prevent
oxidation of a critical thiol on a transcription factor, phosphatase or
other protein or prevent GSSG formation from HO
and GSH. However, since heme-hemopexin does not activate
MRE
Geo, redox-mediated Zn(II) release from MT is not caused by
hemopexin.
It seems likely, but is not yet proven, that member(s) of the AP-1 family of transcription factors recognize the AP-1-like element within the MT-ARE. However, as elegantly shown by Nguyen et al.(63) , an AP-1 site resembles an ARE in responding to xenobiotics if the terminal 3`-GC is present. The MT-ARE contains an internal sequence similar to an AP-1 binding site in the SV40 promoter(64) . The AP-1 family are leucine zipper proteins known to act synergistically with zinc finger proteins like MTF-1. The synergistic increases by PDC of hemopexin-mediated MT induction provide an example of a process whereby a variety of stimuli at the cell surface activate MT transcription in part via the MT-ARE and MREs.