Heme: a novel inducer of MCP-1 through HO-dependent andHO-independent mechanism - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Sharan K. R.Kanakiriya, Anthony J.Croatt, Jill J.Haggard, Julie R.Ingelfinger, Shiow-ShihTang, JawedAlam,  Karl A.Nath作者单位：1 Division of Nephrology, Mayo Clinic/Foundation,Rochester, Minnesota 55905; Pediatric Nephrology Unit,Massachusetts General Hospital, Boston, Massachusetts 02114; and Department of Molecular Genetics, OchsnerClinic/Foundation, New Orleans, Louisiana 70121 * j5 Q& E7 m2 @& Z
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      【摘要】  D& V; G$ |+ y. [& |$ {
   This studyexamined the effect of hemin on the expression of heme oxygenase-1(HO-1) and monocyte chemoattractant protein-1 (MCP-1) in immortalizedrat proximal tubular epithelial cells (IRPTCs). Hemin elicited a dose-and time-dependent induction of HO-1 and MCP-1 mRNA. HO activitycontributed 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 activeintracellular iron was markedly increased in hemin-treated IRPTCs andcontributed to the induction of HO-1 and MCP-1 mRNA because an ironchelator blocked hemin-induced upregulation of both genes, whereas acell-permeant form of iron directly induced these genes. N -acetylcysteine completely blocked hemin-induced expressionof HO-1 and MCP-1 mRNA, thereby providing added evidence for redoxregulation of expression of these genes. The redox-sensitivetranscription factor NF- B was recruited in hemin-inducedupregulation of MCP-1 because two different compounds that abrogate theactivation of NF- B (TPCK and BAY 11-7082) completely blockedhemin-induced upregulation of MCP-1 mRNA. In contrast to thisHO-mediated induction of MCP-1 through redox-sensitive, iron-dependent,and NF- B-involved pathways observed after 4-6 h, hemin alsoelicited a delayed induction of MCP-1 at 18 h throughHO-independent pathways. We conclude that hemin is a potent inducer ofMCP-1 in IRPTCs: HO-dependent, heme-degrading pathways lead to anearly, robust, and self-remitting induction of MCP-1, whereasHO-independent mechanisms lead to a delayed expression of MCP-1.
      【关键词】 heme oxygenase monocyte chemoattractant protein iron oxidantstress
   INTRODUCTION
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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, isrecognized as a protective response against heme protein-mediated andother 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 cellularcontent of heme may originate from two main sources ( 2, 9, 25, 29, 35, 56, 65 ). Heme may be released from intracellular hemeproteins that are widespread in cells and include such members ashemoglobin, myoglobin, mitochondrial cytochromes, microsomalcytochromes, peroxide-scavenging enzymes, nitric oxide synthase,guanylate cyclase, and cyclooxygenases ( 9, 29, 56 ); suchheme-containing proteins may be destabilized in the course of cellinjury, such that heme is freed from its linkage with its respectiveprotein moiety ( 9, 29, 56 ). Cellular content of heme incertain organs may also accrue from heme proteins originating fromother tissues; for example, myoglobin released into the systemiccirculation from injured skeletal muscle cells (as occurs inrhabdomyolysis), or hemoglobin released from lysed red blood cells (asoccurs in hemolysis) can be readily incorporated by renal epithelialcells ( 2, 21, 25, 35, 65 ).- N' p: ?: \) d2 y+ F6 j

Augmentation in cellular content of heme can damage cells and theirorganelles through mechanisms that are, at least in part, prooxidant innature ( 6, 8, 20, 36, 38, 40 ). Such prooxidant actions ofheme are indicated, for example, by the capacity of heme to provokecellular generation of hydrogen peroxide and peroxidation of membranelipid; these effects may be ameliorated by antioxidants ( 6, 8, 20, 36, 38, 40 ). The induction of HO-1 in tissues exposed toheme provides a protective response, in part, by facilitating thedegradation of heme and procuring antioxidant and other cytoprotectivemechanisms ( 1, 4, 34, 45 ). The cellular mechanismsunderlying the inductive effects of heme on HO-1 in tissues in generalare poorly understood; notably, with regard to the kidney, there are nostudies to date that explore the mechanisms underlying the inductiveeffect of heme on HO-1 in kidney-derived cells. The present study aimedto elucidate these mechanisms, focusing on the involvement ofoxidant-related pathways., [8 f4 Y' m. V

Our laboratory ( 37, 41 ) has recently reported that incertain 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 formononuclear 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 ofatherosclerosis and in the pathogenesis of assorted vascular diseases( 15, 23, 51 ). The basis for this upregulation of MCP-1 wehave described in these in vivo models of renal injury has not beenexplored: such upregulation of MCP-1 may reflect an immediate earlygene response to tissue injury ( 47 ); renal ischemia, which occurs invariantly in such models( 46 ); the stimulatory effect of cytokines and otherhumoral factors elaborated in the course of such injury( 65 ); and possibly, oxidative stress, which occurs in suchstates ( 3, 65 ). With regard to the last consideration,exposure to heme proteins occurs in these in vivo models, therebyraising the question of whether heme, possibly though oxidant pathways,induces renal expression of MCP-1.- ?7 S" c% E! N

The present study examined whether direct exposure of renal epithelialcells to heme elicits upregulation of MCP-1 in conjunction with HO-1,specifically determining whether such expression of MCP-1 is influencedby HO-1 induced in these cells. The latter possibility, that a dialogueexists between cellular expression of MCP-1 and HO-1 in heme-exposedcells, was considered because mcp-1 is an oxidant-induciblegene ( 42, 49 ), and ho-1 represents not only anoxidant-inducible gene but one that modulates cellular redox throughits antioxidant [for example, bile pigments ( 19 ) andferritin ( 5 )] and prooxidant products [for example, iron ( 5 )].
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MATERIALS AND METHODS" }3 f, O! t. `8 v5 s

Reagents employed. Hemin (iron protoporphyrin chloride), zinc protoporphyrin (ZnPP),deferoxamine mesylate (DFO), N -acetylcysteine (NAC), TPCK, ferrous ammonium sulfate, and 8-hydroxyquinoline were obtained fromSigma (St. Louis, MO), as were all other chemicals employed unlessotherwise 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.) n$ p' m- u1 t& Z7 [/ p8 Q
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Cell culture. IRPTCs (immortalized rat proximal tubular cells, 93-p-2-1, developedand characterized as previously described) ( 58 ) were grownat 37°C in 95% air-5% CO 2 in DMEM (Invitrogen, GrandIsland, NY) containing low glucose (1 g/l), 20 mM HEPES, and 0.1 mMnonessential amino acids; the medium was supplemented with 5% FBS, 40 U/ml penicillin, and 40 µg/ml streptomycin. IRPTCs were studied as aconfluent monolayer in all experiments. In all experiments, IRPTCs wereincubated 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.2 s. C. k6 e" n) m! ^; r3 A

Dose-dependent and time-dependent effects of hemin on HO-1 andMCP-1 mRNA expression. The dose-dependent effect of hemin was examined by exposing IRPTCs toincreasing concentrations of hemin (5, 10, and 20 µM) for 1 h inthe medium described above supplemented with 0.1% FBS. The medium wasthen 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 geneexpression, IRPTCs were exposed to hemin (10 µM) for 1 h, afterwhich the medium was replaced by hemin-free DMEM medium containing0.1% FBS. After 2, 4, and 6 h of incubation, RNA was extractedfor Northern analyses.% K& s& z% f1 z, `: |% D; h
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Studies examining mechanisms underlying hemin-induced geneexpression in IRPTCs. In these protocols, IRPTCs were exposed to hemin (10 µM)-containingmedium for 1 h, followed by incubation in hemin-free medium for anadditional 4 h. Depending on the specific protocol, the hemin-containing medium also contained 10 µM ZnPP, 1 mM DFO, 1 mMNAC, 25 µM TPCK, 10 µM BAY 11-7082, or where relevant, the vehiclefor these reagents. These reagents were added one-half hour before theaddition of hemin and were maintained during the 1-h exposure to hemin.After this exposure to hemin and relevant reagent, the hemin-containingmedium was replaced by hemin-free medium containing the respectivereagents, as appropriate. After incubation for 4 h, RNA wasextracted for the assessment of HO-1 and MCP-1 mRNA expression.# I7 Q) Q( |. U* P; v0 V

The effect of ZnPP on hemin-induced MCP-1 expression was examined attime points later than 4 h. In these studies, IRPTCs were exposedto hemin (10 µM) in the absence or presence of ZnPP (10 µM) for1 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.6 s" ~" O1 f, _! f- x; x7 s

Additional protocols examined the effect of the cell-permeant form ofiron, 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 ferrousammonium sulfate/8-hydroxyquinoline-containing medium in the presenceor absence of DFO was replaced by a ferrous ammoniumsulfate/8-hydroxyquinoline-free medium, also containing, asappropriate, DFO (1 mM). After 4 h of incubation, RNA wasextracted for the assessment of expression of HO-1 and MCP-1 mRNA.
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RNA extraction and Northern analysis for HO-1 and MCP-1. To examine expression of HO-1 and MCP-1 mRNA, IRPTCs were washed withPBS, 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 32 P-labeledmouse HO-1 or rat MCP-1 cDNA probe. Autoradiograms were evaluated forloading and transfer by assessing the density of the 18S rRNA on anethidium bromide-stained membrane, as previously described ( 13, 27 ).+ m5 _/ O) h8 W/ d- t3 b

Determination of HO activity. HO activity was measured by bilirubin generation in microsomes isolatedfrom IRPTCs, as described previously ( 27 ). Cells werewashed, 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 beforecentrifugation at 12,000 g for 10 min at 4°C. Thesupernatant was centrifuged at 105,000 g for 60 min at4°C. The pellet was suspended in potassium phosphate buffer (pH 7.4)containing 2 mM MgCl 2 and designated as themicrosomal fraction. An aliquot of the microsomal fraction was added tothe reaction mixture (400 µl) containing rat liver cytosol (2 mg ofcytosolic protein), 20 µM hemin, 2 mM glucose-6-phosphate, 0.2 unitsglucose-6-phosphate dehydrogenase, and 0.8 mM NADPH and incubated for1 h at 37°C in the dark. The formed bilirubin was extracted withchloroform, and OD 464-530 nm was measured (extinctioncoefficient, 40 mM/cm for bilirubin), where OD is optical density. HOactivity was expressed as picomoles of bilirubin formed per hour permilligram protein.: d, f' B1 @) T1 ?
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Determination of catalytically active iron in IRPTCs. Catalytically active iron was measured in cellular lysates from IRPTCsusing the bleomycin assay ( 17 ). Experimental media forincubations, wash buffer, and lysis buffer were treated with Chelex 100 to remove contaminating iron; reagents for the assay were prepared withChelex-treated water in new plastic containers and subsequently treatedwith Chelex, except for the bleomycin, magnesium chloride, and iron standard.
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In these studies, IRPTCs were exposed to hemin (10 µM) in the absenceor presence of ZnPP (10 µM) for 1 h; the hemin-containing mediumwas then replaced by hemin-free medium in the absence or presence ofZnPP (10 µM). After 6 h, IRPTCs were washed with HBSS, liftedwith a scraper into 5 ml of HBSS, and gently pelleted by centrifugation. The pelleted cells were resuspended in 0.5 ml of 25 mMHEPES buffer (pH 7.3), lysed in a sonicating water bath for 10 min, andcentrifuged 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 MgCl 2 (100 mM), 20 µl HEPES buffer (25 mM,pH 7.3), 20 µl sample, and 20 µl ascorbic acid (8 mM). This mixturewas incubated for 2 h at 37°C with shaking, and the reaction wassubsequently terminated with the addition of 0.1 ml of 0.2 M EDTA.Blank reactions for each sample were simultaneously incubated withoutbleomycin along with a calibration curve constructed usingFeCl 3. 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 at100°C for 15 min and cooled to room temperature. Chromagen formed wasmeasured spectrophotometrically at 532 nm, standardized against thecalibration curve, and expressed as nanomoles iron per milligramprotein, the latter measured using the Lowry method.( V& y6 U, _0 f$ H# D8 Q

Statistical analysis. Data are expressed as means ± SE. For comparisons involving twogroups, Student's t -test was applied, whereas forcomparisons involving more than two groups, ANOVA and theStudent-Newman-Keuls test were applied. All results are consideredsignificant at P6 V# Q+ h' m6 ?- v3 s! j. a: B
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RESULTS! H5 K3 K1 F) R. `
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The exposure of IRPTCs to hemin for 4 h led to intenseupregulation of HO-1 and MCP-1 mRNA in a dose-dependent manner (Fig. 1 ). This upregulation of MCP-1 and HO-1mRNA was discernible as early as 2 h after the exposure to heminfor 1 h at a concentration of 10 µM (Fig. 2 ).
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Fig. 1. Northern analysis demonstrating dose-dependenthemin-induced upregulation of heme oxygenase-1 (HO-1) and monocytechemoattractant protein-1 (MCP-1) mRNA in immortalized rat proximaltubular epithelial cells (IRPTCs) assessed 4 h after exposure tohemin for 1 h. In this and subsequent figures, the equivalency ofloading and transfer of RNA during the Northern analysis was assessedby the expression of 18S rRNA.4 L; W1 H( I2 v1 r7 b

Fig. 2. Northern analysis demonstrating induction of HO-1 andMCP-1 mRNA in IRPTCs at 2, 4, and 6 h after exposure to 10 µMhemin for 1 h. C, studies undertaken in IRPTCs exposed tohemin-free medium; H, studies in IRPTCs exposed to hemin-containingmedium.2 R0 ]- z  X! a4 q4 O$ A/ |, H

To determine whether HO-1 is involved in the upregulation of MCP-1mRNA, we studied the effect of ZnPP, the competitive inhibitor of HOactivity. As shown in Fig. 3, ZnPPcompletely blocked the upregulation of MCP-1 mRNA induced by heminwithout affecting hemin-induced upregulation of HO-1 mRNA. Along withthese findings, we demonstrate that the induction of HO-1 mRNA by heminis accompanied by a marked increase in HO activity and that ZnPPcompletely ablates cellular HO activity in either the absence orpresence of hemin (Fig. 4 ). Thus theinduction of MCP-1 mRNA by hemin is critically dependent on intact HOactivity because inhibition of HO activity by ZnPP prevents suchexpression of MCP-1 mRNA.. c# J+ ?" }5 N& R# a( {
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Fig. 3. Northern analysis demonstrating the effect of 10 µMhemin, 10 µM zinc protoporphyrin (ZnPP), and hemin ZnPP on expressionof HO-1 and MCP-1 mRNA in IRPTCs assessed 4 h after exposure tohemin for 1 h.

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

To examine mechanisms that may underlie such induction of HO-1 andMCP-1 mRNA, we considered the possibility that iron may be involved insuch 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 iscatabolized by HO activity ( 1, 4, 25 ). Indeed, wedemonstrate large increments in cellular iron levels in hemin-treatedcells and the marked attenuation in hemin-induced rise in cellular iron after concomitant treatment with ZnPP (Fig. 5 ); cellular levels of iron inhemin-exposed cells concomitantly treated with ZnPP were stillsignificantly higher than levels in cells treated with ZnPP alone andcells studied under control conditions (Fig. 5 ).
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Fig. 5. Measurement of catalytically active iron in IRPTCs afterexposure 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 Significantly different from all otherconditions ( P P

We thus studied the effect of the iron chelator DFO on hemin-inducedexpression of MCP-1 and HO-1 mRNA. As shown in Fig. 6, the induction of MCP-1 and HO-1 mRNAby hemin were both reduced by DFO. To examine the capacity of iron perse to induce MCP-1 and HO-1 mRNA, we examined the effect of acell-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 isdependent, at least in part, on increments in intracellular iron.8 c7 E* v- t* P7 W' T8 `
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Fig. 6. Northern analysis demonstrating the effect of 10 µMhemin, 1 mM deferoxamine (DFO), and hemin DFO on expression of HO-1 andMCP-1 mRNA in IRPTCs assessed 4 h after exposure to hemin for1 h.# H4 T0 j2 x& J  G

Fig. 7. Northern analysis demonstrating the effect of 10 µMferrous ammonium sulfate/8-hydroxyquinoline (Fe), 1 mM DFO, andFe DFO on expression of HO-1 and MCP-1 mRNA in IRPTCs assessed 4 hafter exposure to hemin for 1 h.' S" C# ?6 h* W3 O2 @3 c& g4 ^
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To determine whether alterations in cellular redox contribute to theupregulation of these genes, we studied the effect of thesulfhydryl-containing antioxidant N -acetylcysteine. Asdemonstrated in Fig. 8, N -acetylcysteine completely prevented the upregulation ofMCP-1 and HO-1 mRNA.

Fig. 8. Northern analysis demonstrating the effect of 10 µMhemin, 1 mM N -acetylcysteine (NAC), and hemin NAC onexpression of HO-1 and MCP-1 mRNA in IRPTCs assessed 4 h afterexposure to hemin for 1 h.7 H, c" N( _! x3 S
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Because activation of the redox-sensitive transcription factor NF- B( 24 ) regulates expression of MCP-1 ( 15, 57, 61 ), we examined whether hemin-induced upregulation of MCP-1 canbe interrupted by inhibiting activation of NF- B. Evidence in support of this pathway was provided by two approaches. TPCK, a protease inhibitor that blocks activation of NF- B ( 18, 64 ),completely prevented hemin-induced upregulation of MCP-1 (Fig. 9 ). Additional studies were undertakenwith BAY 11-7082, a more specific inhibitor of NF- B activation thanTPCK; this compound inhibits the nuclear translocation of NF- B byinhibiting the phosphorylation of I B ( 44 ). BAY 11-7082 completely prevented the upregulation of MCP-1 mRNA by hemin (Fig. 10 ). Thus inhibition ofNF- B-dependent pathways by two different approaches completelyprevented hemin-induced upregulation of MCP-1. While TPCK blockedhemin-induced upregulation of HO-1 mRNA (Fig. 9 ), BAY 11-7082 onlypartially inhibited hemin-induced HO-1 mRNA accumulation and, byitself, BAY 11-7082 stimulated HO-1 expression in IRPTCs (Fig. 10 ).
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Fig. 9. Northern analysis demonstrating the effect of 10 µMhemin, 25 µM TPCK, and hemin TPCK on expression of HO-1 and MCP-1mRNA in IRPTCs assessed 4 h after exposure to hemin for 1 h./ a+ V! }% {0 {6 c1 S) X3 }+ Z

Fig. 10. Northern analysis demonstrating the effect of 10 µMhemin, 10 µM BAY 11-7082, and hemin BAY 11-7082 on expression of HO-1and MCP-1 mRNA in IRPTCs assessed 4 h after exposure to hemin for1 h., Q! A% v5 A4 f* S

In our study of the inductive effect of hemin on MCP-1 expression, wealso examined the time course of expression of MCP-1 mRNA in responseto hemin in the absence and presence of ZnPP, the competitive inhibitorof HO activity. As shown in Fig. 11, the expression of MCP-1 mRNA after exposure to hemin in the absence ofZnPP peaked at 6 h and returned to basal levels by 18 h. In contrast, while there was no discernible expression of MCP-1 mRNA at6 h elicited by hemin when ZnPP was concomitantly present, theexpression of MCP-1 mRNA under such conditions thereafter increased.Standardized densitometric assessment confirmed these findings:expressed as a percentage of standardized densitometric expression ofMCP-1 mRNA in the presence of hemin alone, standardized densitometricexpression of MCP-1 mRNA in the presence of hemin and ZnPP increasedfrom 10% at 6 h to 272% at 18 h.3 I4 Q4 z6 y. D
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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 densitometricreadings for MCP-1 mRNA expression in the presence of hemin and ZnPP asa percentage of standardized densitometric readings for MCP-1 mRNAexpression in the presence of hemin alone at 6, 10, 14, and 18 h.; ]" @2 K( X9 A" }& w+ P
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DISCUSSION
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We demonstrate that hemin is a vigorous inducer of HO-1 and MCP-1via iron-mediated, redox-dependent mechanisms. First, at a time pointat which hemin induced these genes (4-6 h), IRPTCs exhibitedincreased amounts of catalytically active iron, the latter representinga potent facilitator of oxidant stress. Second, the iron chelator DFOattenuated the induction of these genes by hemin, findings thatindicate the fundamental involvement of catalytically active iron inthe induction of these genes; moreover, direct evidence that increasedcellular levels of catalytically active iron induced these genes wasprovided by studies in which marked upregulation of HO-1 and MCP-1occurred in cells exposed to a cell-permeant form of iron, effects alsoabrogated by DFO. Third, the antioxidant sulfhydryl agent N -acetylcysteine prevented the induction of these genes byhemin. In the aggregate, these findings support an important role foriron-dependent, redox-involved pathways in hemin-induced expression ofHO-1 and MCP-1. We point out that while the marked increase in MCP-1mRNA levels in hemin-treated cells likely reflects increasedtranscriptional rates, additional studies that assess mRNA stabilitywould be of interest so as to determine whether the latter mechanismcontributes to increased mRNA levels; additionally, studies of MCP-1protein levels would also be of interest so as to determine the extentto which alterations in gene expression are accompanied by analogouschanges in MCP-1 protein levels in hemin-treated cells.

While the mechanisms underlying hemin-induced upregulation of HO-1 andMCP-1 exhibit several similarities, they differ in at least oneimportant aspect: induction of MCP-1, but not of HO-1, is dependent onHO activity. This conclusion was derived from studies utilizing ZnPP, awidely employed, effective, and specific competitive inhibitor of HOactivity. ZnPP completely prevented the expression of MCP-1 inhemin-exposed cells when studied at 4-6 h but had no effect onhemin-induced HO-1 mRNA levels. These findings lead us to conclude thatthe acute exposure of IRPTCs to hemin induces HO-1 and the attendantincrease in HO activity, in turn, elicits the induction of MCP-1 mRNA.We wish to point out that ZnPP inhibits HO activity emanating not onlyfrom HO-1, but also from HO-2, the constitutive isoform; the thirdisoform of HO, HO-3, possesses trivial HO activity. Thus the results ofstudies which employ ZnPP as a competitive inhibitor of HO activityrepresent the combined inhibitory effect of ZnPP on HO enzymeactivities originating from HO-1 (as this isoform is induced) as wellas from HO-2 (the basally expressed, constitutive isoform). Because thecatalytic activity of HO (from either HO-1 or HO-2) on hemin acutelyelevates cellular iron content, we suggest that iron, released as heminis degraded by HO, contributes to the induction of MCP-1. In support ofthis interpretation, we provide evidence that ZnPP totally blocked HOactivity in cells (either in the control setting or after treatmentwith hemin) and that ZnPP attenuated the rise in catalytically activeiron in hemin-treated cells.4 W( }3 ~, q# _6 \  w
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ZnPP completely blocked HO activity, but ZnPP by itself stimulated HO-1mRNA expression, albeit to a lesser extent than did hemin (Fig. 3 ).Induction of ho-1 gene/protein expression by analogs of hemethat 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 proteinsthat participate in the HO-1 induction pathway, and ZnPP may be able tofunctionally substitute for the heme molecule; and 2 )alternatively, the ho-1 gene may be negatively regulated asa consequence of product feedback inhibition. The inhibition of HOactivity by ZnPP markedly reduces the availability of products of HOactivity, thereby decreasing product feedback inhibition and, in turn,leading to increased ho-1 gene accumulation. It should alsobe pointed out that induction of HO-1 mRNA expression per se by ZnPPwould not elicit upregulation of MCP-1 mRNA because the treatment ofcells with ZnPP effectively blocks HO activity, and it is through HOactivity that HO-1 exerts its cellular effects.
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Because cleavage of heme and release of iron are not necessary forhemin-elicited induction of HO-1 mRNA, it is likely that hemin and ironcan independently modulate HO-1 expression. Alternatively, or inconjunction, sources of iron other than hemin-iron may be responsible;for example, hemin, through its direct oxidative effects may mobilizeiron from the "low-molecular-weight cellular iron pool" and othersources of iron ( 56 ), and such catalytically active ironmay induce HO-1 mRNA. Indeed, in studies in which catalytically activeiron was measured in IRPTCs exposed to hemin, levels of catalyticallyactive iron in cells treated with hemin were reduced when ZnPP wasconcomitantly present; however, concentrations of catalytically activeiron in hemin-treated cells in the presence of ZnPP were stillsignificantly greater than levels in cells studied under controlconditions, or in cells exposed to ZnPP alone.4 O% z: j3 M& U7 g

To examine further the basis for hemin-induced upregulation of MCP-1,we considered the possibility that the oxidant-responsive transcriptionfactor NF- B ( 24 ) was involved. NF- B binding sitesare present in the promoter of the mcp-1 gene, andactivation of NF- B is regarded as an intermediary step intranscriptional control of expression of MCP-1 ( 15, 57, 61 ). To probe the involvement of NF- B-dependent mechanisms,studies were undertaken with TPCK; TPCK is a protease inhibitor thatblocks the degradation of I B and thereby prevents the nucleartranslocation of NF- B ( 18, 64 ). In hemin-exposed cells,TPCK completely blocked the induction of the MCP-1 gene. To complementthis finding, we employed an additional approach that utilized thecompound BAY 11-7082; this compound prevents the translocation ofNF- B to the nucleus by inhibiting the phosphorylation of I B( 44 ). In our studies, BAY 11-7082 also completely blockedupregulation of MCP-1 in IRPTCs exposed to hemin. On the basis of thesestudies, we suggest that NF- B-dependent pathways are involved inhemin-induced upregulation of MCP-1. Interestingly, BAY 11-7082 (a morespecific inhibitor of activation of NF- B than TPCK) only partiallyinhibited hemin-induced HO-1 mRNA accumulation and BAY 11-7082 byitself stimulated HO-1 expression in IRPTCs, thereby pointing to anadditional mechanistic difference between the induction of the ho-1 and mcp-1 genes by hemin.8 _: X4 A1 }# |* n2 L
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This marked expression of MCP-1 in hemin-treated cells observed at2-6 h after exposure to hemin subsided by 10 h and completely abated by 14 and 18 h (Fig. 11 ). This temporal profile ofexpression of MCP-1 in hemin-treated cells was markedly altered when HOactivity was inhibited by ZnPP. As shown in Fig. 11, while ZnPPprevented the expression of MCP-1 by hemin at 6 h, examination atlater time points demonstrate an increasing level of expression ofMCP-1 mRNA despite the continued presence of this inhibitor of HOactivity; indeed, at 18 h, the level of expression of MCP-1 wasincreased almost threefold in the presence of ZnPP. Thus inhibition ofHO altered the pattern of MCP-1 expression in IRPTCs exposed to hemin: while preventing the induction of MCP-1 observed at the early timepoint (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 tohemin occurs through HO-dependent and HO-independent pathways: HO-dependent heme-degrading pathways lead to an early, prominent, andself-remitting induction of MCP-1, whereas HO-independent mechanismslead to a delayed expression of MCP-1.
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As is well established, the induction of HO-1 by heme is coupled tosecondary events that ultimately restore catalytically active iron totheir basal levels ( 1, 5, 10, 14 ). For example, theinduction of HO-1 entrains the synthesis of ferritin, the iron-bindingprotein 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 irondirectly elicits the induction of MCP-1 in hemin-treated cells, thetemporal 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 ironexporting-proteins.% r: L1 h4 T& O; W* K3 V* C

The mechanisms that may underlie the delayed induction of MCP-1 inHO-inhibited cells merit comment. Heme can be degraded viaHO-independent processes that involve nonenzymatic autooxidative reactions ( 1, 30-32, 56 ). For example, inpathophysiologically relevant concentrations, the interaction ofhydrogen peroxide with ferrylheme leads to the degradation of heme, theliberation of iron, and the production of superoxide anion( 30-32 ); superoxide anion can undergo dismutation tohydrogen peroxide, thereby providing a positive-feedback loop in thedegradation of heme. Heme strongly stimulates cellular generation ofhydrogen peroxide, as we have shown previously ( 36 ). Thushemin itself may initiate a chain of oxidative events that culminate inthe degradation of hemin through HO-independent, nonenzymaticprocesses. These nonenzymatic, autooxidative, heme-degrading processeslack the rapid, efficient, controlled, and coordinated featuresexhibited by the HO system. We speculate that such nonenzymaticautooxidative reactions, by promoting oxidative stress and/or increasedavailability of redox iron, may contribute to the delayed induction ofMCP-1 we observed in hemin-treated cells concomitantly treated withZnPP, the inhibitor of HO.- r2 T; W, c$ n0 \' \( T, ~6 g

Our present findings also provide insights relevant to prior in vivostudies from this laboratory that demonstrate upregulation of HO-1 andMCP-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 inHO-1 / mice, when examined 7 days after the last injection ofhemoglobin, exhibit heightened inflammatory responses accompanied byaccentuated expression of MCP-1 and NF- B ( 41 ). These invivo findings raised the possibility that the exacerbation of hemeprotein-induced inflammation in HO-1-deficient mice was due to the lossof an inhibitory effect of HO-1 on activation of NF- B andNF- B-dependent genes such as mcp-1. It should be pointedout that these in vivo studies did not examine the effect of theHO-1-deficient state on the nature of expression of MCP-1 acutely (thatis, within hours) after the administration of heme proteins, a timeframe 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 withMCP-1 expression after 1 wk, the findings from these in vitro studiesmake it unlikely that the marked upregulation of MCP-1 and exaggerationof inflammation in HO-1 / mice subjected to heme proteins originatesimply from the loss of a restraining effect of HO-1 directly at thelevel of expression of MCP-1; rather, the present in vitro findingsunderscore the fundamental propensity toward inflammation in anHO-1-deficient state in vivo and one that results from dysregulation ata level in the inflammatory cascade preceding that of a specificchemokine such as MCP-1.

That the induction of HO-1 is involved in the early induction of MCP-1mRNA underscores an emerging perspective regarding the cell biology ofHO, namely, the induction of HO-1 as a determinant of gene expression,in this case, mcp-1, in hemin-treated cells. While thefunctional significance of these findings is beyond the scope of thepresent 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 geneincreasingly recognized for its anti-inflammatory properties; and amongthe questions raised by these findings is whether this early,reversible upregulation of MCP-1 by hemin in cells with unrestrained HOactivity is necessarily inflammatory or inimical to cellular vitality. In this regard, an analogy to TGF- 1 may be relevant: whereas thesustained upregulation of TGF- 1 provides a dominant pathway forchronic inflammation and fibrosis, TGF- 1, acutely and transiently upregulated, exerts anti-inflammatory and cytoprotective actions ( 11 ).0 E* [: d7 l# ]* s" K" @( n2 M
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We conclude by suggesting that the induction of MCP-1 by hemin may berelevant to a number of inflammatory states in the kidney characterizedby repetitive or unremitting exposure to heme proteins ( 21, 33, 37, 39, 41 ). This inductive effect of hemin on MCP-1 may also begermane 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 berelevant to atherosclerosis. Atherosclerosis is more likely to involvethe vasculature at sites of turbulence, wherein red blood cells undergomechanical trauma with the attendant insinuation of hemoglobin and hemein the walls of blood vessels ( 60 ). Because the sustainedupregulation of MCP-1 is considered a critical chemokine inatherogenesis ( 15, 23, 51 ), we speculate that increased amounts of heme in the vasculature originating from these and othermechanisms may drive the expression of the proatherogenic chemokineMCP-1.
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ACKNOWLEDGEMENTS0 E0 d0 b' o6 u

We appreciate the secretarial expertise of Sharon Heppelmann in thepreparation of this work." m- I$ Q5 T/ n6 W. R4 H) E
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