Heme: a novel inducer of MCP-1 through HO-dependent and HO-independent mechanisms

Sharan K. R. Kanakiriya1, Anthony J. Croatt1, Jill J. Haggard1, Julie R. Ingelfinger2, Shiow-Shih Tang2, Jawed Alam3, and Karl A. Nath1

1 Division of Nephrology, Mayo Clinic/Foundation, Rochester, Minnesota 55905; 2 Pediatric Nephrology Unit, Massachusetts General Hospital, Boston, Massachusetts 02114; and 3 Department of Molecular Genetics, Ochsner Clinic/Foundation, New Orleans, Louisiana 70121


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study examined the effect of hemin on the expression of heme oxygenase-1 (HO-1) and monocyte chemoattractant protein-1 (MCP-1) in immortalized rat proximal tubular epithelial cells (IRPTCs). Hemin elicited a dose- and time-dependent induction of HO-1 and MCP-1 mRNA. HO activity contributed to MCP-1 mRNA expression at early time points (4-6 h) because inhibition of HO activity by zinc protoporphyrin (ZnPP) prevented hemin-induced expression of MCP-1 mRNA. Catalytically active intracellular iron was markedly increased in hemin-treated IRPTCs and contributed to the induction of HO-1 and MCP-1 mRNA because an iron chelator blocked hemin-induced upregulation of both genes, whereas a cell-permeant form of iron directly induced these genes. N-acetylcysteine completely blocked hemin-induced expression of HO-1 and MCP-1 mRNA, thereby providing added evidence for redox regulation of expression of these genes. The redox-sensitive transcription factor NF-kappa B was recruited in hemin-induced upregulation of MCP-1 because two different compounds that abrogate the activation of NF-kappa B (TPCK and BAY 11-7082) completely blocked hemin-induced upregulation of MCP-1 mRNA. In contrast to this HO-mediated induction of MCP-1 through redox-sensitive, iron-dependent, and NF-kappa B-involved pathways observed after 4-6 h, hemin also elicited a delayed induction of MCP-1 at 18 h through HO-independent pathways. We conclude that hemin is a potent inducer of MCP-1 in IRPTCs: HO-dependent, heme-degrading pathways lead to an early, robust, and self-remitting induction of MCP-1, whereas HO-independent mechanisms lead to a delayed expression of MCP-1.

heme oxygenase; monocyte chemoattractant protein-1; iron; oxidant stress


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

HEME OXYGENASE (HO) is the rate-limiting enzyme in the degradation of heme (1, 4, 26, 34, 45), and induction of its isozyme, HO-1, is recognized as a protective response against heme protein-mediated and other insults to diverse tissues (2, 25, 35, 43, 52, 63). While inducible by numerous stimuli (1, 4, 12, 22, 56), HO-1 is readily induced by heme proteins and their heme moiety (2, 5, 35). In pathological states, increased cellular content of heme may originate from two main sources (2, 9, 25, 29, 35, 56, 65). Heme may be released from intracellular heme proteins that are widespread in cells and include such members as hemoglobin, myoglobin, mitochondrial cytochromes, microsomal cytochromes, peroxide-scavenging enzymes, nitric oxide synthase, guanylate cyclase, and cyclooxygenases (9, 29, 56); such heme-containing proteins may be destabilized in the course of cell injury, such that heme is freed from its linkage with its respective protein moiety (9, 29, 56). Cellular content of heme in certain organs may also accrue from heme proteins originating from other tissues; for example, myoglobin released into the systemic circulation from injured skeletal muscle cells (as occurs in rhabdomyolysis), or hemoglobin released from lysed red blood cells (as occurs in hemolysis) can be readily incorporated by renal epithelial cells (2, 21, 25, 35, 65).

Augmentation in cellular content of heme can damage cells and their organelles through mechanisms that are, at least in part, prooxidant in nature (6, 8, 20, 36, 38, 40). Such prooxidant actions of heme are indicated, for example, by the capacity of heme to provoke cellular generation of hydrogen peroxide and peroxidation of membrane lipid; these effects may be ameliorated by antioxidants (6, 8, 20, 36, 38, 40). The induction of HO-1 in tissues exposed to heme provides a protective response, in part, by facilitating the degradation of heme and procuring antioxidant and other cytoprotective mechanisms (1, 4, 34, 45). The cellular mechanisms underlying the inductive effects of heme on HO-1 in tissues in general are poorly understood; notably, with regard to the kidney, there are no studies to date that explore the mechanisms underlying the inductive effect of heme on HO-1 in kidney-derived cells. The present study aimed to elucidate these mechanisms, focusing on the involvement of oxidant-related pathways.

Our laboratory (37, 41) has recently reported that in certain in vivo models of renal injury, upregulation of HO-1 is accompanied by induction of the chemokine monocyte chemoattractant protein-1 (MCP-1). MCP-1 is widely incriminated as a stimulus for mononuclear cellular infiltrate in diverse inflammatory conditions affecting the kidney and other organs (15, 50, 59, 62); additionally, special emphasis is assigned to MCP-1 in the evolution of atherosclerosis and in the pathogenesis of assorted vascular diseases (15, 23, 51). The basis for this upregulation of MCP-1 we have described in these in vivo models of renal injury has not been explored: such upregulation of MCP-1 may reflect an immediate early gene response to tissue injury (47); renal ischemia, which occurs invariantly in such models (46); the stimulatory effect of cytokines and other humoral factors elaborated in the course of such injury (65); and possibly, oxidative stress, which occurs in such states (3, 65). With regard to the last consideration, exposure to heme proteins occurs in these in vivo models, thereby raising the question of whether heme, possibly though oxidant pathways, induces renal expression of MCP-1.

The present study examined whether direct exposure of renal epithelial cells to heme elicits upregulation of MCP-1 in conjunction with HO-1, specifically determining whether such expression of MCP-1 is influenced by HO-1 induced in these cells. The latter possibility, that a dialogue exists between cellular expression of MCP-1 and HO-1 in heme-exposed cells, was considered because mcp-1 is an oxidant-inducible gene (42, 49), and ho-1 represents not only an oxidant-inducible gene but one that modulates cellular redox through its antioxidant [for example, bile pigments (19) and ferritin (5)] and prooxidant products [for example, iron (5)].


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents employed. Hemin (iron protoporphyrin chloride), zinc protoporphyrin (ZnPP), deferoxamine mesylate (DFO), N-acetylcysteine (NAC), TPCK, ferrous ammonium sulfate, and 8-hydroxyquinoline were obtained from Sigma (St. Louis, MO), as were all other chemicals employed unless otherwise stated; BAY 11-7082 was obtained from Calbiochem (San Diego, CA). Stock solutions of hemin and ZnPP were prepared in 0.05 M NaOH; TPCK was dissolved in DMSO; DFO was dissolved in cell culture media, whereas deionized water was used to prepare stock solutions of NAC, ferrous ammonium sulfate/8-hydroxyquinoline, and BAY 11-7082.

Cell culture. IRPTCs (immortalized rat proximal tubular cells, 93-p-2-1, developed and characterized as previously described) (58) were grown at 37°C in 95% air-5% CO2 in DMEM (Invitrogen, Grand Island, NY) containing low glucose (1 g/l), 20 mM HEPES, and 0.1 mM nonessential amino acids; the medium was supplemented with 5% FBS, 40 U/ml penicillin, and 40 µg/ml streptomycin. IRPTCs were studied as a confluent monolayer in all experiments. In all experiments, IRPTCs were incubated in the same medium as the one in which IRPTCs were grown, except that the medium was supplemented with 0.1% FBS instead of 5% FBS.

Dose-dependent and time-dependent effects of hemin on HO-1 and MCP-1 mRNA expression. The dose-dependent effect of hemin was examined by exposing IRPTCs to increasing concentrations of hemin (5, 10, and 20 µM) for 1 h in the medium described above supplemented with 0.1% FBS. The medium was then replaced by hemin-free medium, and, after 4 h of incubation, RNA was extracted for the assessment of HO-1 and MCP-1 mRNA expression. In protocols that examined the time-dependent effect of hemin on gene expression, IRPTCs were exposed to hemin (10 µM) for 1 h, after which the medium was replaced by hemin-free DMEM medium containing 0.1% FBS. After 2, 4, and 6 h of incubation, RNA was extracted for Northern analyses.

Studies examining mechanisms underlying hemin-induced gene expression in IRPTCs. In these protocols, IRPTCs were exposed to hemin (10 µM)-containing medium for 1 h, followed by incubation in hemin-free medium for an additional 4 h. Depending on the specific protocol, the hemin-containing medium also contained 10 µM ZnPP, 1 mM DFO, 1 mM NAC, 25 µM TPCK, 10 µM BAY 11-7082, or where relevant, the vehicle for these reagents. These reagents were added one-half hour before the addition of hemin and were maintained during the 1-h exposure to hemin. After this exposure to hemin and relevant reagent, the hemin-containing medium was replaced by hemin-free medium containing the respective reagents, as appropriate. After incubation for 4 h, RNA was extracted for the assessment of HO-1 and MCP-1 mRNA expression.

The effect of ZnPP on hemin-induced MCP-1 expression was examined at time points later than 4 h. In these studies, IRPTCs were exposed to hemin (10 µM) in the absence or presence of ZnPP (10 µM) for 1 h; the hemin-containing medium was replaced by hemin-free medium (containing ZnPP), and after 6, 10, 14, and 18 h of incubation, extraction of RNA was performed.

Additional protocols examined the effect of the cell-permeant form of iron, ferrous ammonium sulfate/8-hydroxyquinoline (7, 55), on gene expression. IRPTCs were exposed to ferrous ammonium sulfate (10 µM)/8-hydroxyquinoline (10 µM) in the presence or absence of DFO (1 mM). After 1 h of incubation, the ferrous ammonium sulfate/8-hydroxyquinoline-containing medium in the presence or absence of DFO was replaced by a ferrous ammonium sulfate/8-hydroxyquinoline-free medium, also containing, as appropriate, DFO (1 mM). After 4 h of incubation, RNA was extracted for the assessment of expression of HO-1 and MCP-1 mRNA.

RNA extraction and Northern analysis for HO-1 and MCP-1. To examine expression of HO-1 and MCP-1 mRNA, IRPTCs were washed with PBS, and RNA was extracted using the TRIzol method (Invitrogen, Carlsbad, CA). Ten micrograms of total RNA from each sample were separated on an agarose gel and transferred to a nylon membrane. Membranes were hybridized overnight with a 32P-labeled mouse HO-1 or rat MCP-1 cDNA probe. Autoradiograms were evaluated for loading and transfer by assessing the density of the 18S rRNA on an ethidium bromide-stained membrane, as previously described (13, 27).

Determination of HO activity. HO activity was measured by bilirubin generation in microsomes isolated from IRPTCs, as described previously (27). Cells were washed, scraped with a rubber policeman, and centrifuged at 1,000 g for 10 min at 4°C. The cell pellet was suspended in potassium phosphate buffer (100 mM, pH 7.4) and sonicated on ice before centrifugation at 12,000 g for 10 min at 4°C. The supernatant was centrifuged at 105,000 g for 60 min at 4°C. The pellet was suspended in potassium phosphate buffer (pH 7.4) containing 2 mM MgCl2 and designated as the microsomal fraction. An aliquot of the microsomal fraction was added to the reaction mixture (400 µl) containing rat liver cytosol (2 mg of cytosolic protein), 20 µM hemin, 2 mM glucose-6-phosphate, 0.2 units glucose-6-phosphate dehydrogenase, and 0.8 mM NADPH and incubated for 1 h at 37°C in the dark. The formed bilirubin was extracted with chloroform, and Delta OD 464-530 nm was measured (extinction coefficient, 40 mM/cm for bilirubin), where OD is optical density. HO activity was expressed as picomoles of bilirubin formed per hour per milligram protein.

Determination of catalytically active iron in IRPTCs. Catalytically active iron was measured in cellular lysates from IRPTCs using the bleomycin assay (17). Experimental media for incubations, wash buffer, and lysis buffer were treated with Chelex 100 to remove contaminating iron; reagents for the assay were prepared with Chelex-treated water in new plastic containers and subsequently treated with Chelex, except for the bleomycin, magnesium chloride, and iron standard.

In these studies, IRPTCs were exposed to hemin (10 µM) in the absence or presence of ZnPP (10 µM) for 1 h; the hemin-containing medium was then replaced by hemin-free medium in the absence or presence of ZnPP (10 µM). After 6 h, IRPTCs were washed with HBSS, lifted with a scraper into 5 ml of HBSS, and gently pelleted by centrifugation. The pelleted cells were resuspended in 0.5 ml of 25 mM HEPES buffer (pH 7.3), lysed in a sonicating water bath for 10 min, and centrifuged at 10,000 g for 10 min. The resulting supernatants were assayed for iron in an incubation mixture consisting of 0.1 ml calf thymus DNA (1 mg/ml), 20 µl bleomycin sulfate (1 U/ml), 20 µl MgCl2 (100 mM), 20 µl HEPES buffer (25 mM, pH 7.3), 20 µl sample, and 20 µl ascorbic acid (8 mM). This mixture was incubated for 2 h at 37°C with shaking, and the reaction was subsequently terminated with the addition of 0.1 ml of 0.2 M EDTA. Blank reactions for each sample were simultaneously incubated without bleomycin along with a calibration curve constructed using FeCl3. After the addition of 0.2 ml of thiobarbituric acid (1% in 0.5 N NaOH) and HCl (25% wt/vol), the samples were heated at 100°C for 15 min and cooled to room temperature. Chromagen formed was measured spectrophotometrically at 532 nm, standardized against the calibration curve, and expressed as nanomoles iron per milligram protein, the latter measured using the Lowry method.

Statistical analysis. Data are expressed as means ± SE. For comparisons involving two groups, Student's t-test was applied, whereas for comparisons involving more than two groups, ANOVA and the Student-Newman-Keuls test were applied. All results are considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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The exposure of IRPTCs to hemin for 4 h led to intense upregulation of HO-1 and MCP-1 mRNA in a dose-dependent manner (Fig. 1). This upregulation of MCP-1 and HO-1 mRNA was discernible as early as 2 h after the exposure to hemin for 1 h at a concentration of 10 µM (Fig. 2).


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Fig. 1.   Northern analysis demonstrating dose-dependent hemin-induced upregulation of heme oxygenase-1 (HO-1) and monocyte chemoattractant protein-1 (MCP-1) mRNA in immortalized rat proximal tubular epithelial cells (IRPTCs) assessed 4 h after exposure to hemin for 1 h. In this and subsequent figures, the equivalency of loading and transfer of RNA during the Northern analysis was assessed by the expression of 18S rRNA.



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Fig. 2.   Northern analysis demonstrating induction of HO-1 and MCP-1 mRNA in IRPTCs at 2, 4, and 6 h after exposure to 10 µM hemin for 1 h. C, studies undertaken in IRPTCs exposed to hemin-free medium; H, studies in IRPTCs exposed to hemin-containing medium.

To determine whether HO-1 is involved in the upregulation of MCP-1 mRNA, we studied the effect of ZnPP, the competitive inhibitor of HO activity. As shown in Fig. 3, ZnPP completely blocked the upregulation of MCP-1 mRNA induced by hemin without affecting hemin-induced upregulation of HO-1 mRNA. Along with these findings, we demonstrate that the induction of HO-1 mRNA by hemin is accompanied by a marked increase in HO activity and that ZnPP completely ablates cellular HO activity in either the absence or presence of hemin (Fig. 4). Thus the induction of MCP-1 mRNA by hemin is critically dependent on intact HO activity because inhibition of HO activity by ZnPP prevents such expression of MCP-1 mRNA.


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Fig. 3.   Northern analysis demonstrating the effect of 10 µM hemin, 10 µM zinc protoporphyrin (ZnPP), and hemin+ZnPP on expression of HO-1 and MCP-1 mRNA in IRPTCs assessed 4 h after exposure to hemin for 1 h.



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Fig. 4.   Measurement of HO activity in IRPTCs after exposure to 10 µM hemin, 10 µM ZnPP, and hemin+ZnPP assessed 6 h after exposure to hemin for 1 h (n = 5/group). * Significantly different from control (P < 0.05).

To examine mechanisms that may underlie such induction of HO-1 and MCP-1 mRNA, we considered the possibility that iron may be involved in such regulation: iron is a potent catalyst for oxidative stress (56), and both genes are inducible by oxidative stress (42, 49, 56); moreover, iron is released as heme is catabolized by HO activity (1, 4, 25). Indeed, we demonstrate large increments in cellular iron levels in hemin-treated cells and the marked attenuation in hemin-induced rise in cellular iron after concomitant treatment with ZnPP (Fig. 5); cellular levels of iron in hemin-exposed cells concomitantly treated with ZnPP were still significantly higher than levels in cells treated with ZnPP alone and cells studied under control conditions (Fig. 5).


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Fig. 5.   Measurement of catalytically active iron in IRPTCs after exposure to 10 µM hemin, 10 µM ZnPP, and hemin+ZnPP assessed 6 h after exposure to hemin for 1 h (n = 5/group). * Significantly different from all other conditions (P < 0.05). dagger Significantly different from all other conditions (P < 0.05). #Significantly different from all other conditions (P < 0.05).

We thus studied the effect of the iron chelator DFO on hemin-induced expression of MCP-1 and HO-1 mRNA. As shown in Fig. 6, the induction of MCP-1 and HO-1 mRNA by hemin were both reduced by DFO. To examine the capacity of iron per se to induce MCP-1 and HO-1 mRNA, we examined the effect of a cell-permeant form of iron. As demonstrated in Fig. 7, iron induced both MCP-1 and HO-1 mRNA, and this inductive effect of iron was blocked by the iron chelator DFO. Thus the upregulation of MCP-1 and HO-1 in response to hemin is dependent, at least in part, on increments in intracellular iron.


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Fig. 6.   Northern analysis demonstrating the effect of 10 µM hemin, 1 mM deferoxamine (DFO), and hemin+DFO on expression of HO-1 and MCP-1 mRNA in IRPTCs assessed 4 h after exposure to hemin for 1 h.



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Fig. 7.   Northern analysis demonstrating the effect of 10 µM ferrous ammonium sulfate/8-hydroxyquinoline (Fe), 1 mM DFO, and Fe+DFO on expression of HO-1 and MCP-1 mRNA in IRPTCs assessed 4 h after exposure to hemin for 1 h.

To determine whether alterations in cellular redox contribute to the upregulation of these genes, we studied the effect of the sulfhydryl-containing antioxidant N-acetylcysteine. As demonstrated in Fig. 8, N-acetylcysteine completely prevented the upregulation of MCP-1 and HO-1 mRNA.


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Fig. 8.   Northern analysis demonstrating the effect of 10 µM hemin, 1 mM N-acetylcysteine (NAC), and hemin+NAC on expression of HO-1 and MCP-1 mRNA in IRPTCs assessed 4 h after exposure to hemin for 1 h.

Because activation of the redox-sensitive transcription factor NF-kappa B (24) regulates expression of MCP-1 (15, 57, 61), we examined whether hemin-induced upregulation of MCP-1 can be interrupted by inhibiting activation of NF-kappa B. Evidence in support of this pathway was provided by two approaches. TPCK, a protease inhibitor that blocks activation of NF-kappa B (18, 64), completely prevented hemin-induced upregulation of MCP-1 (Fig. 9). Additional studies were undertaken with BAY 11-7082, a more specific inhibitor of NF-kappa B activation than TPCK; this compound inhibits the nuclear translocation of NF-kappa B by inhibiting the phosphorylation of Ikappa B (44). BAY 11-7082 completely prevented the upregulation of MCP-1 mRNA by hemin (Fig. 10). Thus inhibition of NF-kappa B-dependent pathways by two different approaches completely prevented hemin-induced upregulation of MCP-1. While TPCK blocked hemin-induced upregulation of HO-1 mRNA (Fig. 9), BAY 11-7082 only partially inhibited hemin-induced HO-1 mRNA accumulation and, by itself, BAY 11-7082 stimulated HO-1 expression in IRPTCs (Fig. 10).


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Fig. 9.   Northern analysis demonstrating the effect of 10 µM hemin, 25 µM TPCK, and hemin+TPCK on expression of HO-1 and MCP-1 mRNA in IRPTCs assessed 4 h after exposure to hemin for 1 h.



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Fig. 10.   Northern analysis demonstrating the effect of 10 µM hemin, 10 µM BAY 11-7082, and hemin+BAY 11-7082 on expression of HO-1 and MCP-1 mRNA in IRPTCs assessed 4 h after exposure to hemin for 1 h.

In our study of the inductive effect of hemin on MCP-1 expression, we also examined the time course of expression of MCP-1 mRNA in response to hemin in the absence and presence of ZnPP, the competitive inhibitor of HO activity. As shown in Fig. 11, the expression of MCP-1 mRNA after exposure to hemin in the absence of ZnPP peaked at 6 h and returned to basal levels by 18 h. In contrast, while there was no discernible expression of MCP-1 mRNA at 6 h elicited by hemin when ZnPP was concomitantly present, the expression of MCP-1 mRNA under such conditions thereafter increased. Standardized densitometric assessment confirmed these findings: expressed as a percentage of standardized densitometric expression of MCP-1 mRNA in the presence of hemin alone, standardized densitometric expression of MCP-1 mRNA in the presence of hemin and ZnPP increased from 10% at 6 h to 272% at 18 h.


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Fig. 11.   A: Northern analysis demonstrating the effect of 10 µM hemin, 10 µM ZnPP, and hemin+ZnPP on MCP-1 mRNA in IRPTCs at 6, 10, 14, and 18 h. B: standardized densitometric readings for MCP-1 mRNA expression in the presence of hemin and ZnPP as a percentage of standardized densitometric readings for MCP-1 mRNA expression in the presence of hemin alone at 6, 10, 14, and 18 h.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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We demonstrate that hemin is a vigorous inducer of HO-1 and MCP-1 via iron-mediated, redox-dependent mechanisms. First, at a time point at which hemin induced these genes (4-6 h), IRPTCs exhibited increased amounts of catalytically active iron, the latter representing a potent facilitator of oxidant stress. Second, the iron chelator DFO attenuated the induction of these genes by hemin, findings that indicate the fundamental involvement of catalytically active iron in the induction of these genes; moreover, direct evidence that increased cellular levels of catalytically active iron induced these genes was provided by studies in which marked upregulation of HO-1 and MCP-1 occurred in cells exposed to a cell-permeant form of iron, effects also abrogated by DFO. Third, the antioxidant sulfhydryl agent N-acetylcysteine prevented the induction of these genes by hemin. In the aggregate, these findings support an important role for iron-dependent, redox-involved pathways in hemin-induced expression of HO-1 and MCP-1. We point out that while the marked increase in MCP-1 mRNA levels in hemin-treated cells likely reflects increased transcriptional rates, additional studies that assess mRNA stability would be of interest so as to determine whether the latter mechanism contributes to increased mRNA levels; additionally, studies of MCP-1 protein levels would also be of interest so as to determine the extent to which alterations in gene expression are accompanied by analogous changes in MCP-1 protein levels in hemin-treated cells.

While the mechanisms underlying hemin-induced upregulation of HO-1 and MCP-1 exhibit several similarities, they differ in at least one important aspect: induction of MCP-1, but not of HO-1, is dependent on HO activity. This conclusion was derived from studies utilizing ZnPP, a widely employed, effective, and specific competitive inhibitor of HO activity. ZnPP completely prevented the expression of MCP-1 in hemin-exposed cells when studied at 4-6 h but had no effect on hemin-induced HO-1 mRNA levels. These findings lead us to conclude that the acute exposure of IRPTCs to hemin induces HO-1 and the attendant increase in HO activity, in turn, elicits the induction of MCP-1 mRNA. We wish to point out that ZnPP inhibits HO activity emanating not only from HO-1, but also from HO-2, the constitutive isoform; the third isoform of HO, HO-3, possesses trivial HO activity. Thus the results of studies which employ ZnPP as a competitive inhibitor of HO activity represent the combined inhibitory effect of ZnPP on HO enzyme activities originating from HO-1 (as this isoform is induced) as well as from HO-2 (the basally expressed, constitutive isoform). Because the catalytic activity of HO (from either HO-1 or HO-2) on hemin acutely elevates cellular iron content, we suggest that iron, released as hemin is degraded by HO, contributes to the induction of MCP-1. In support of this interpretation, we provide evidence that ZnPP totally blocked HO activity in cells (either in the control setting or after treatment with hemin) and that ZnPP attenuated the rise in catalytically active iron in hemin-treated cells.

ZnPP completely blocked HO activity, but ZnPP by itself stimulated HO-1 mRNA expression, albeit to a lesser extent than did hemin (Fig. 3). Induction of ho-1 gene/protein expression by analogs of heme that inhibit HO activity is well established (28, 48, 54). At least two mechanisms may account for this phenomenon: 1) heme may function as a structural cofactor for one or more proteins that participate in the HO-1 induction pathway, and ZnPP may be able to functionally substitute for the heme molecule; and 2) alternatively, the ho-1 gene may be negatively regulated as a consequence of product feedback inhibition. The inhibition of HO activity by ZnPP markedly reduces the availability of products of HO activity, thereby decreasing product feedback inhibition and, in turn, leading to increased ho-1 gene accumulation. It should also be pointed out that induction of HO-1 mRNA expression per se by ZnPP would not elicit upregulation of MCP-1 mRNA because the treatment of cells with ZnPP effectively blocks HO activity, and it is through HO activity that HO-1 exerts its cellular effects.

Because cleavage of heme and release of iron are not necessary for hemin-elicited induction of HO-1 mRNA, it is likely that hemin and iron can independently modulate HO-1 expression. Alternatively, or in conjunction, sources of iron other than hemin-iron may be responsible; for example, hemin, through its direct oxidative effects may mobilize iron from the "low-molecular-weight cellular iron pool" and other sources of iron (56), and such catalytically active iron may induce HO-1 mRNA. Indeed, in studies in which catalytically active iron was measured in IRPTCs exposed to hemin, levels of catalytically active iron in cells treated with hemin were reduced when ZnPP was concomitantly present; however, concentrations of catalytically active iron in hemin-treated cells in the presence of ZnPP were still significantly greater than levels in cells studied under control conditions, or in cells exposed to ZnPP alone.

To examine further the basis for hemin-induced upregulation of MCP-1, we considered the possibility that the oxidant-responsive transcription factor NF-kappa B (24) was involved. NF-kappa B binding sites are present in the promoter of the mcp-1 gene, and activation of NF-kappa B is regarded as an intermediary step in transcriptional control of expression of MCP-1 (15, 57, 61). To probe the involvement of NF-kappa B-dependent mechanisms, studies were undertaken with TPCK; TPCK is a protease inhibitor that blocks the degradation of Ikappa B and thereby prevents the nuclear translocation of NF-kappa B (18, 64). In hemin-exposed cells, TPCK completely blocked the induction of the MCP-1 gene. To complement this finding, we employed an additional approach that utilized the compound BAY 11-7082; this compound prevents the translocation of NF-kappa B to the nucleus by inhibiting the phosphorylation of Ikappa B (44). In our studies, BAY 11-7082 also completely blocked upregulation of MCP-1 in IRPTCs exposed to hemin. On the basis of these studies, we suggest that NF-kappa B-dependent pathways are involved in hemin-induced upregulation of MCP-1. Interestingly, BAY 11-7082 (a more specific inhibitor of activation of NF-kappa B than TPCK) only partially inhibited hemin-induced HO-1 mRNA accumulation and BAY 11-7082 by itself stimulated HO-1 expression in IRPTCs, thereby pointing to an additional mechanistic difference between the induction of the ho-1 and mcp-1 genes by hemin.

This marked expression of MCP-1 in hemin-treated cells observed at 2-6 h after exposure to hemin subsided by 10 h and completely abated by 14 and 18 h (Fig. 11). This temporal profile of expression of MCP-1 in hemin-treated cells was markedly altered when HO activity was inhibited by ZnPP. As shown in Fig. 11, while ZnPP prevented the expression of MCP-1 by hemin at 6 h, examination at later time points demonstrate an increasing level of expression of MCP-1 mRNA despite the continued presence of this inhibitor of HO activity; indeed, at 18 h, the level of expression of MCP-1 was increased almost threefold in the presence of ZnPP. Thus inhibition of HO altered the pattern of MCP-1 expression in IRPTCs exposed to hemin: while preventing the induction of MCP-1 observed at the early time point (4-6 h), such inhibition was associated with increased expression of MCP-1 at the later time point (18 h). From these findings, we suggest that induction of MCP-1 in IRPTCs in response to hemin occurs through HO-dependent and HO-independent pathways: HO-dependent heme-degrading pathways lead to an early, prominent, and self-remitting induction of MCP-1, whereas HO-independent mechanisms lead to a delayed expression of MCP-1.

As is well established, the induction of HO-1 by heme is coupled to secondary events that ultimately restore catalytically active iron to their basal levels (1, 5, 10, 14). For example, the induction of HO-1 entrains the synthesis of ferritin, the iron-binding protein that is the major intracellular repository for iron (5); induction of HO-1 is also linked to increased expression of iron-exporting proteins that facilitate the cellular egress of iron (10, 14). Thus to the extent that iron directly elicits the induction of MCP-1 in hemin-treated cells, the temporal regression in expression of this gene in hemin-treated cells (when HO activity is intact) likely reflects the reduction in cellular levels of iron due to increased availability of iron-binding and iron exporting-proteins.

The mechanisms that may underlie the delayed induction of MCP-1 in HO-inhibited cells merit comment. Heme can be degraded via HO-independent processes that involve nonenzymatic autooxidative reactions (1, 30-32, 56). For example, in pathophysiologically relevant concentrations, the interaction of hydrogen peroxide with ferrylheme leads to the degradation of heme, the liberation of iron, and the production of superoxide anion (30-32); superoxide anion can undergo dismutation to hydrogen peroxide, thereby providing a positive-feedback loop in the degradation of heme. Heme strongly stimulates cellular generation of hydrogen peroxide, as we have shown previously (36). Thus hemin itself may initiate a chain of oxidative events that culminate in the degradation of hemin through HO-independent, nonenzymatic processes. These nonenzymatic, autooxidative, heme-degrading processes lack the rapid, efficient, controlled, and coordinated features exhibited by the HO system. We speculate that such nonenzymatic autooxidative reactions, by promoting oxidative stress and/or increased availability of redox iron, may contribute to the delayed induction of MCP-1 we observed in hemin-treated cells concomitantly treated with ZnPP, the inhibitor of HO.

Our present findings also provide insights relevant to prior in vivo studies from this laboratory that demonstrate upregulation of HO-1 and MCP-1 in rat kidneys after exposure to heme proteins (37, 41) and studies in which HO-1+/+ and HO-1-/- mice were repetitively injected with hemoglobin at weekly intervals for 8 wk (41). In these latter in vivo studies, the kidneys in HO-1-/- mice, when examined 7 days after the last injection of hemoglobin, exhibit heightened inflammatory responses accompanied by accentuated expression of MCP-1 and NF-kappa B (41). These in vivo findings raised the possibility that the exacerbation of heme protein-induced inflammation in HO-1-deficient mice was due to the loss of an inhibitory effect of HO-1 on activation of NF-kappa B and NF-kappa B-dependent genes such as mcp-1. It should be pointed out that these in vivo studies did not examine the effect of the HO-1-deficient state on the nature of expression of MCP-1 acutely (that is, within hours) after the administration of heme proteins, a time frame explored in the present in vitro studies. Notwithstanding the differences in these studies, i.e., in vitro vs. in vivo, hemin compared with hemoglobin, MCP-1 expression within hours compared with MCP-1 expression after 1 wk, the findings from these in vitro studies make it unlikely that the marked upregulation of MCP-1 and exaggeration of inflammation in HO-1-/- mice subjected to heme proteins originate simply from the loss of a restraining effect of HO-1 directly at the level of expression of MCP-1; rather, the present in vitro findings underscore the fundamental propensity toward inflammation in an HO-1-deficient state in vivo and one that results from dysregulation at a level in the inflammatory cascade preceding that of a specific chemokine such as MCP-1.

That the induction of HO-1 is involved in the early induction of MCP-1 mRNA underscores an emerging perspective regarding the cell biology of HO, namely, the induction of HO-1 as a determinant of gene expression, in this case, mcp-1, in hemin-treated cells. While the functional significance of these findings is beyond the scope of the present studies, it is intriguing that this early expression of mcp-1, a gene conventionally regarded as a proinflammatory one, is dependent on the expression of ho-1, a gene increasingly recognized for its anti-inflammatory properties; and among the questions raised by these findings is whether this early, reversible upregulation of MCP-1 by hemin in cells with unrestrained HO activity is necessarily inflammatory or inimical to cellular vitality. In this regard, an analogy to TGF-beta 1 may be relevant: whereas the sustained upregulation of TGF-beta 1 provides a dominant pathway for chronic inflammation and fibrosis, TGF-beta 1, acutely and transiently upregulated, exerts anti-inflammatory and cytoprotective actions (11).

We conclude by suggesting that the induction of MCP-1 by hemin may be relevant to a number of inflammatory states in the kidney characterized by repetitive or unremitting exposure to heme proteins (21, 33, 37, 39, 41). This inductive effect of hemin on MCP-1 may also be germane to the proinflammatory effects, including thrombophlebitis, that attend the clinical use of heme-based compounds (16, 53). Finally, we raise the possibility that our findings may be relevant to atherosclerosis. Atherosclerosis is more likely to involve the vasculature at sites of turbulence, wherein red blood cells undergo mechanical trauma with the attendant insinuation of hemoglobin and heme in the walls of blood vessels (60). Because the sustained upregulation of MCP-1 is considered a critical chemokine in atherogenesis (15, 23, 51), we speculate that increased amounts of heme in the vasculature originating from these and other mechanisms may drive the expression of the proatherogenic chemokine MCP-1.


    ACKNOWLEDGEMENTS

We appreciate the secretarial expertise of Sharon Heppelmann in the preparation of this work.


    FOOTNOTES

These studies were funded by National Institutes of Health Grants R01-DK-47060 and HL-55552 (K. A. Nath), HL-48455 and DK-58950 (J. R. Ingelfinger), DK-50835 (S.-S. Tang), and DK-43135 (J. Alam).

Address for reprint requests and other correspondence: K. A. Nath, Mayo Clinic, 200 First St. SW, Guggenheim 542, Rochester, MN 55905 (E-mail: nath.karl{at}mayo.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajprenal.00298.2002

Received 16 August 2002; accepted in final form 13 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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