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 HO(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.

MATERIALS AND METHODS

Hemopexin and Heme and Heme Analog Complexes of Hemopexin

Plasmids

Cell Culture and Transient Transfections

Isolation of Total Cellular RNA and Northern Blot Analysis

RESULTS AND DISCUSSION

Involvement of PKC in Hemopexin-mediated MT-1 Expression

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 -750MTGeo (Table 1) and -150MTGeo (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)MTGeo, 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 (-750MTGeo, -750(110)MTGeo, -150MTGeo, -42MTGeo, and MREGeo) independent transfections. N.D., not determined. Two and 5 µM heme-hemopexin induces -150MTGeo reporter gene activity 76 ± 3 and 87 ± 4% as effectively as 10 µM complex (data not shown).

Elements of the Mouse MT-1 Proximal Promoter Involved in Transcriptional Activation by Hemopexin

The induction of the -galactosidase reporter gene by heme-hemopexin was abolished when the -153 to -43 bp region was deleted (-750(110)MTGeo in Fig. 1). Moreover, the -galactosidase activity of -150MTGeo, which contains up to -153 to -43 bp of the promoter, is induced by heme-hemopexin, while -42MTGeo containing only the basal promoter does not respond (Fig. 1). Furthermore, the extent of induction of -150MTGeo by heme-hemopexin is similar to that seen with either -750MTGeo or pMTLacF (Fig. 1). Importantly, CoPP-hemopexin also induces the reporter gene activity of -750MTGeo and -150MTGeo, 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 -750MTGeo by both heme and CoPP-hemopexin and of -150MTGeo 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 MREGeo 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 -150MTGeo, 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.

Roles for ROIs in Hemopexin-mediated MT-1 Gene Regulation

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 -150MTGeo (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 -750MTGeo (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 -750MTGeo 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.

Role for ARE in Hemopexin-mediated MT-1 Gene Regulation

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 (AREMTGeo and ARE-MTGeo) or from 6 to 10 independent transfections (-750MTGeo, -153(-67)MTGeo, -124(-67)MTGeo, 124(-43)MTGeo, AREMTGeo, AREMTGeo). 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 -150MTGeo 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 -150MTGeo by heme-hemopexin are presented elsewhere in the manuscript. A 3-fold induction of MREGeo by hydrogen peroxide was observed with 500 µM reagent.

Involvement of cis-Acting Elements in Addition to the ARE in Hemopexin-mediated MT-1 Gene Regulation

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 NFB and IB.

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 MTIMTF-1 complex(54) . However, the lack of induction of MREGeo 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 MTIMTF-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)MTGeo and -124(-43)MTGeo, 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 -150MTGeo containing the complete region and none were induced to higher levels than AREMTGeo. 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.

Augmentation by PDC of MT-1 Gene Regulation by Heme-Hemopexin via Both MRE-dependent and MRE-independent Effects

()

Effect of H7 on Activation of the MT-ARE by Heme-Hemopexin

Conclusion

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 -150MTGeo, 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 MREGeo, 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.

FOOTNOTES

*

This research was supported by United States Public Health Service, National Institutes of Health Grant DK-37463. 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.: 816-235-2579; Fax: 816-235-5158; annsmith{at}cctr.umkc.edu.

()

The abbreviations used are: heme, iron-protoporphyrin IX; HO, heme oxygenase; MT, metallothionein; MT-1, metallothionein-1 isozyme form; ARE, antioxidant response element; MRE, metal-responsive element; H7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride; HA1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride; PMA, phorbol 12-myristate 13-acetate; CoPP, cobalt-protoporphyrin; SnPP, tin-protoporphyrin; GSH, glutathione; DDC, diethyl dithiocarbamate; PDC, pyrrolidine dithiocarbonate; NAC, N-acetylcysteine; ROI, reactive oxygen intermediate; PKC, protein kinase C; PKA, protein kinase A; CAT, chloramphenicol acetyltransferase; bp, base pair(s).

()

The term ROI encompasses singlet oxygen, hydrogen peroxide (HO), superoxide anion (O), and the hydroxyl radical (OH), all of which can be interconverted in reactions that depend in part on redox metals such as iron(1) .

()

Preliminary accounts of portions of this work were presented at the Scientific Education Partnership symposium on ``Molecular Mechanisms of Aging,'' Kansas City, MO, October 1992; at the American Society for Biochemistry and Molecular Biology meeting, San Diego, CA, May 1993 (FASEB J.7, A1235 (Abstr. 1054); and at the Keystone Symposium on Molecular and Cellular Biology, Keystone, CO, January 1993 (J. Cell. Biochem., Suppl. 17A, 124a (Abstr. B410).

()

Y. Ren and A. Smith, unpublished observation.

()

Y. Ren and A. Smith, unpublished results.

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