©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Macrophage Inflammatory Protein-1 mRNA by Oxidative Stress (*)

(Received for publication, November 15, 1995)

Michael Ming Shi John J. Godleski Joseph D. Paulauskis (§)

From the Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Accumulation of inflammatory cells within the lung has been implicated in oxidative injury. Recruitment of these cells to a tissue site is a complex process that depends in part upon the local expression of appropriate proinflammatory chemokines. Macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the CC subfamily of chemokines, has been shown to contribute to monocyte/macrophage and neutrophil chemotaxis and activation. Our previous work demonstrated that MIP-1alpha mRNA expression in macrophages is induced by bacterial endotoxin. The objective of this study was to test the hypothesis that an oxidative stress alone may trigger expression of MIP-1alpha mRNA in macrophages and to determine the mechanism leading to increased expression. A rat alveolar macrophage cell line (NR8383) was exposed to H(2)O(2) or menadione (2-methyl-1,4-naphthoquinone (MQ)), a quinone compound that undergoes redox cycling and generates reactive oxygen species continuously. Steady-state mRNA levels encoding MIP-1alpha were markedly increased (3-fold) in these cells after 1 h of exposure to 0.5 mM H(2)O(2), remained higher than control levels after 4 h, and decreased after 6 h. Similarly, MQ (25 or 50 µM) caused a significant increase of MIP-1alpha mRNA with a maximal induction after 4 h of exposure (5-fold). Both H(2)O(2) and MQ-induced up-regulation of MIP-1alpha mRNA was suppressed by co-treatment with N-acetylcysteine, a synthetic antioxidant. Co-treatment with actinomycin D reduced the MQ induction of MIP-1alpha mRNA to a greater extent than the H(2)O(2)-induced increase. Transcription of the MIP-1alpha gene was increased by exposure to both H(2)O(2) and MQ. H(2)O(2) treatment also induced a marked increase of the MIP-1alpha mRNA half-life, indicating post-transcriptional stabilization. These observations indicate that an oxidative stress can regulate MIP-1alpha mRNA expression by two distinct mechanisms: transcriptional activation of the MIP-1alpha gene and post-transcriptional stabilization of MIP-1alpha mRNA.


INTRODUCTION

Reactive oxygen species (ROS) (^1)have been implicated in the pathogenesis of several lung diseases including adult respiratory distress syndrome, asthma, chronic bronchitis, and lung fibrosis (reviewed by Halliwell and Gutterage(1989)). Increased production of ROS is believed responsible for tissue damage during pulmonary inflammation (Southern and Powis, 1988). Inflammatory cells such as neutrophils and macrophages play an important role in host defense but may also contribute to tissue injury through the release of tissuedamaging oxidants upon activation (reviewed by Sibille and Reynolds(1990)). In addition, activated inflammatory cells also produce mediators such as prostanoids and leukotrienes that can induce bronchoconstriction. Thus, leukocytes that are recruited into the lung and subsequently activated have profound effects on cells within the lung and may propagate inflammation.

Recruitment of inflammatory cells to a tissue site is a complex process that depends in part upon the local expression of appropriate chemoattractant proteins termed ``chemokines'' (reviewed by Oppenheim et al.(1991)). The chemokine superfamily can be subdivided into two subsets, which differ with respect to the relative positioning of the first two cysteines (CXC versus CC) at the N terminus. Macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the CC family of chemokines, contributes to monocyte/macrophage and neutrophil chemotaxis and activation. MIP-1alpha has been reported to be chemotactic for mononuclear phagocytes, neutrophils, eosinophils, basophils, and lymphocytes (Davatelis et al., 1988; Wolpe and Cerami, 1989; Taub et al., 1993; VanOtteren et al., 1994). MIP-1alpha also induces a respiratory burst in neutrophils and plays a role in the control of hemopoietic stem cell proliferation (Graham et al., 1990; Dunlop et al., 1992). MIP-1alpha mRNA has been reported to be constitutively expressed at low levels and up-regulated by inflammatory stimuli in pulmonary alveolar macrophages (Shi et al., 1995). Recombinant MIP-1alpha administered to the lungs of rats elicits a localized neutrophilic inflammatory response that can be neutralized with anti-MIP-1alpha antibody in vitro (Shanley et al., 1995). These features make this chemokine a very likely participant in initiation of inflammation by noxious stimuli.

A potential association specifically between oxidative stress and chemokine expression has been suggested (DeForge et al., 1992, 1993; Driscoll et al., 1993). Because of their role in pulmonary defense and their capacity for ROS production, alveolar macrophages are likely to be an important target for oxidative stress. Previous studies have shown that these cells are capable of expressing chemokines upon exposure to acute or chronic inflammatory stimuli (Huang et al., 1992a, 1992b; Farone et al., 1995; Shi et al., 1995). In this study we utilized a rat alveolar macrophage cell line to test the hypothesis that an oxidative stress alone can trigger expression of the chemokine MIP-1alpha and thus contribute to early inflammation.

Macrophages were exposed to both H(2)O(2) and MQ, and chemokine mRNA expression was measured in response to these oxidative stresses. In contrast to H(2)O(2)-induced oxidative stress, two major mechanisms are involved in quinone-induced cytotoxicity (reviewed by Brunmark and Cadenas(1989)). First, quinone is reduced to the hydroquinone or semiquinone radical by cellular reductase. The semiquinone radical undergoes rapid autoxidation with the generation of the parent quinone and concomitant formation of superoxide. The hydroquinone reacts rapidly with superoxide to form H(2)O(2) and the semiquinone (Fridovich 1981; Shi et al., 1994a). Secondly, most quinones, including MQ, also react with cellular nucleophiles, such as thiols and amines, and cause cellular damage (Chang et al., 1992; Shi et al., 1993). We now demonstrate that both H(2)O(2) and MQ induce MIP-1alpha mRNA expression in macrophages but through two distinct mechanisms. The up-regulation of MIP-1alpha mRNA by MQ is mediated through transcriptional activation of the MIP-1alpha gene. H(2)O(2) induces MIP-1alpha mRNA expression by induction of MIP-1alpha gene transcription as well as by increasing the stability of the mRNA transcript.


EXPERIMENTAL PROCEDURES

Cells and Culture Conditions

Tissue culture supplies and related materials were purchased from Sigma unless otherwise stated. The rat alveolar macrophage (RAM) cell line, NR8383, was generously provided by Dr. R. Helmke (Helmke et al., 1987). Cells were cultured in RPMI 1640 medium supplemented with 5% equine serum, 2 mML-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were grown in a humidified incubator at 37 °C with 5% CO(2).

Cell Treatment

Tissue culture plates were precoated with polyhydroxymethylacrylate to prevent cell adherence (Folkman and Moscona, 1987). Cells were plated at a density of 1 times 10^6 cells/ml in serum-free RPMI for 4 h prior to treatment. MQ was dissolved in dimethyl sulfoxide and subsequently diluted in RPMI medium. A 30% H(2)O(2) stock was diluted in RPMI immediately before treatment. When used, MQ was added to tissue culture plates at a final concentration of 25 or 50 µM for 1-6 h. Control cells were treated with the same concentrations of Me(2)SO as the MQ treatment group. Me(2)SO levels did not exceed 0.01%. No significant cytotoxicity was identified for any oxidant treatment used in this study as measured by trypan blue assays. When used, 1 mMN-acetylcysteine (NAC) was added to cells and preincubated for 1 h in serum-free RPMI before addition of MQ or H(2)O(2). In other studies, RAM cells were treated with MQ or H(2)O(2) along with 5 µg/ml actinomycin D or 5 µg/ml cycloheximide for 1 or 4 h to inhibit cellular RNA and protein synthesis, respectively.

RNA Extraction

Total cellular RNA was isolated from RAM cells using a modified guanidinum method (Chirgwin et al., 1979). Cells were lysed in 4 M guanidine thiocyanate/25 mM sodium citrate (pH 7.0)/0.5% N-lauroyl-sarcosine/0.1 M 2-mercaptoethanol. The mixture was layered on 5.7 M CsCl/0.1 M EDTA and centrifuged at 47,000 rpm (268,000 times g) for 4 h in a Beckman SW55 Ti rotor. Pelleted RNA was resuspended in diethyl pyrocarbonate-treated TE buffer (10 mM Tris/1 mM EDTA, pH 7.4).

Northern Analysis

Total RNA (10 µg/lane) was denatured in 50% formamide/7% formaldehyde, resolved in a 1% agarose/7% formaldehyde gel, and transferred and UV cross-linked to nylon membranes (MSI Inc., Westboro, MA). MIP-1alpha cDNA (Shi et al., 1995) was labeled with [alpha-P]dATP (6000 Ci/mmol, DuPont NEN) by random primer labeling (Life Technologies Inc.). Prehybridization (3 h) and hybridization with P-labeled MIP-1alpha cDNA (overnight) were carried out in 0.5 M NaPO(4)/1 mM EDTA/7% SDS/150 µg/ml tRNA (Church and Gilbert, 1984) at 65 °C. Blots were washed once in 0.1 times SSC (20 times SSC = 3 M NaCl, 0.3 M Na(3)citrate)/0.1% SDS at room temperature and twice at 52 °C before autoradiography and densitometry. The same blots were stripped of hybridized MIP-1alpha probe and rehybridized with a radiolabeled cDNA fragment of mouse beta-actin cDNA (ATCC, Rockville, MD) as an internal control.

mRNA Half-life Determination

Following treatment of RAM cells with 25 µM MQ for 4 h or 0.5 mM H(2)O(2) for 1 h as above, actinomycin D was added to the media to a final concentration of 5 µg/ml. Cells were sampled at times indicated through 6 h, and the levels of MIP-1alpha mRNA were quantified by Northern analysis. The integrated band values, as determined by densitometry, were normalized to beta-actin RNA.

Nuclear Run-on Transcription Assay

RAM cells were treated with 0 or 0.5 mM H(2)O(2) or 0 or 50 µM MQ as described above. Cells were then washed with ice-cold phosphate-buffered saline, and the nuclei were released by lysis in 0.5% Nonidet P-40 lysis buffer (Ausbel et al., 1990). Nuclei were collected by centrifugation and stored in 50 mM Tris, pH 8.3, 40% glycerol, 5 mM MgCl(2), 0.1 mM EDTA in liquid nitrogen. Upon thawing, nuclei were resuspended and incubated in reaction buffer (1times containing 100 mM Tris, pH 8.0, 300 mM (NH(4))(2)SO(4), 4 mM MgCl(2), 200 mM NaCl, 0.4 mM EDTA, 4 mM MnCl(2), 0.1 mM phenylmethylsulfonyl fluoride, 1.2 mM dithiothreitol), 1 mM each of ATP, GTP, and CTP, and 150 µCi of [alpha-P]UTP (800 Ci/mmol) (DuPont NEN) at 30 °C for 30 min, followed by 100 units DNase I treatment for 20 min at 30 °C. Proteinase K (100 µg) was then added, and the mixture was incubated overnight at 42 °C. RNA was then extracted using a RNA isolation kit (QIAGEN Inc., Chatsworth, CA), and labeled free nucleotides were removed by centrifugation through a Sephadex G-50 spin minicolumn (Pharmacia Biotech Inc.). Equal amounts of newly transcribed RNA were hybridized with 5 µg of denatured, linearized MIP-1alpha or glyceraldehyde-3-phosphate dehydrogenase cDNAs cross-linked to nitrocellulose membranes as described under ``Northern Analysis.'' A 0.8-kilobase pair rat MIP-1alpha cDNA in pCRII (Shi et al., 1995) and a 1.2-kilobase pair human glyceraldehyde-3-phosphate dehydrogenase cDNA in pBR322 (ATCC, Rockville, MD) were used in this assay.


RESULTS

Induction of MIP-1alpha mRNA by Oxidative Stress

RAM cells were exposed to H(2)O(2) up to 6 h, and steady-state mRNA levels encoding MIP-1alpha were measured by Northern analysis. Levels of MIP-1alpha mRNA were rapidly induced as early as 1 h following exposure to 0.5 mM H(2)O(2), remained higher than controls after 4 h of exposure, and returned to the control level by 6 h (Fig. 1). H(2)O(2) at 0.1 mM did not induce a change in MIP-1alpha mRNA levels.


Figure 1: Induction of MIP-1alpha mRNA levels by H(2)O(2) in a rat alveolar macrophage cell line, NR8383. Cells were incubated with 0, 0.1, or 0.5 mM H(2)O(2) in serum-free RPMI medium for 1, 4, or 6 h. Northern analysis was performed as described under ``Experimental Procedures'' using a MIP-1alpha cDNA as probe. Upper panels, autoradiogram of Northern blot hybridized with a radiolabeled MIP-1alpha cDNA. Lower panels, the same membrane hybridized with a mouse beta-actin cDNA to indicate relative amounts of hybridizable RNA per lane. The results are representative of two independent experiments.



RAM cells were also treated with a second source of reactive oxygen radicals, MQ, and MIP-1alpha mRNA levels were again quantified. MQ undergoes redox cycling and generates reactive oxygen species such as H(2)O(2) and O(2) continuously. MQ at both 25 and 50 µM also caused an induction of MIP-1alpha mRNA levels at 1 h, which persisted through 4 h of exposure with a maximum 5-fold increase over control levels (Fig. 2). After 6 h, MIP-1alpha mRNA levels had fallen back to near control levels at 25 µM MQ and were markedly reduced at 50 µM MQ (Fig. 2).


Figure 2: Induction of MIP-1alpha mRNA levels by MQ in rat alveolar macrophages. Cells were incubated with 0, 25, or 50 µM MQ in serum-free RPMI medium for 1, 4, or 6 h. Northern analysis was performed as described for Fig. 1. Upper panels, autoradiogram of Northern blot hybridized with radiolabeled MIP-1alpha cDNA. Lower panels, the same membrane hybridized with a mouse beta-actin cDNA to indicate relative amounts of hybridizable RNA per lane. The results are representative of two independent experiments.



N-Acetylcysteine Suppresses the Increase of MIP-1alpha mRNA by Oxidative Stress

NAC is a synthetic antioxidant that can replenish intracellular glutathione levels (Meister and Anderson, 1983). RAM cells were pretreated with 1 mM NAC for 1 h and then challenged with either MQ or H(2)O(2). NAC at this level completely eliminated the induction of MIP-1alpha mRNA induction by 25 and 50 µM MQ (Fig. 3). In a similar fashion, NAC treatment partially attenuated the induction of MIP-1alpha mRNA by 0.5 mM H(2)O(2) at both 1 and 4 h postexposure (Fig. 4).


Figure 3: Effect of NAC on MIP-1alpha mRNA levels in response to MQ in rat alveolar macrophages. Cells were incubated with 1 mM NAC for 1 h, followed by the addition of 0, 25, or 50 µM MQ and incubation for 4 h. Total cellular RNA was extracted, and Northern analysis was performed as described for Fig. 1. A, upper panel, autoradiogram of Northern blot hybridized with radiolabeled MIP-1alpha cDNA. Lower panel, the same membrane hybridized with a mouse beta-actin cDNA to indicate relative amounts of hybridizable RNA per lane. B, densitometric quantification of MIP-1alpha mRNA normalized to beta-actin RNA. The results are representative of two independent experiments.




Figure 4: Effect of NAC on MIP-1alpha mRNA levels in response to H(2)O(2) in rat alveolar macrophages. A, cells were incubated with 1 mM NAC for 1 h followed by the addition of 0.5 mM H(2)O(2) for 1 or 4 h. Total cellular RNA was extracted and Northern analysis was performed as described for Fig. 1. Upper panels, autoradiogram of Northern blot hybridized with a radiolabeled MIP-1alpha cDNA. Lower panels, the same membrane hybridized with a mouse beta-actin cDNA to indicate relative amounts of hybridizable RNA per lane. B, densitometric quantification of MIP-1alpha mRNA normalized to beta-actin RNA. The results are representative of two independent experiments.



Effect of Cycloheximide and Actinomycin D on the Elevation of MIP-1alpha mRNA by Oxidative Stress

Steady-state levels of mRNAs may be modulated by transcriptional or post-transcriptional mechanisms. To determine the route through which MQ and H(2)O(2) elevate MIP-1alpha mRNA concentrations, we applied commonly used translational and transcriptional inhibitor assays as described previously (Shi et al., 1994a, 1994b). RAM cells were exposed to 25 µM MQ in the presence of 5 µg/ml cycloheximide for 4 h to inhibit protein synthesis. Total cellular RNA was extracted and Northern analysis was performed. Cycloheximide alone caused a significant increase in MIP-1alpha mRNA levels, and did not appear to inhibit the induction of MIP-1alpha mRNA levels in response to MQ or H(2)O(2) (Fig. 5). These results only suggest that an increase of MIP-1alpha mRNA by MQ-induced oxidative stress may not be dependent on de novo protein synthesis.


Figure 5: Effect of cycloheximide on MIP-1alpha mRNA levels in response to MQ and H(2)O(2) in rat alveolar macrophages. Left panels, cells were co-incubated with 0 or 25 µM MQ and 0 or 5 µg/ml cycloheximide for 4 h. Right panels, cells were treated with 0 or 0.5 mM H(2)O(2) and 0 or 5 µg/ml cycloheximide for 4 h. Northern analysis was performed as described for Fig. 1. Upper panels, autoradiogram of Northern blot hybridized with radiolabeled-MIP-1alpha cDNA. Lower panels, the same membrane hybridized with a mouse beta-actin cDNA to indicate relative amounts of hybridizable RNA per lane. The results are representative of two independent experiments.



Experiments were also performed in the presence of actinomycin D to inhibit transcription. RAM cells were exposed to 25 µM MQ for 4 h or 0.5 mM H(2)O(2) for 1 h in the presence or the absence of 5 µg/ml actinomycin D. The elevation of MIP-1alpha mRNA by MQ was almost completely blocked by co-incubation with actinomycin D, suggesting that the increase by oxidative stress is transcriptionally regulated (Fig. 6, A and C). In contrast, actinomycin D only partially (60%) suppressed the increase of MIP-1alpha mRNA expression by H(2)O(2) (Fig. 6, B and D), suggesting that mRNA stability may also play an important role in the up-regulation of MIP-1alpha transcript levels.


Figure 6: Effect of actinomycin D (AD) on MIP-1alpha mRNA expression in response to MQ (A) and H(2)O(2) (B) in rat alveolar macrophages. Cells were treated with 0 or 25 µM MQ and 5 µg/ml actinomycin D for 4 h (A) or with 0 or 0.5 mM H(2)O(2) and 5 µg/ml actinomycin D for 1 h (B). Total cellular RNA was extracted, and Northern analysis was performed as described for Fig. 1. Upper panels, autoradiogram of Northern blot hybridized with radiolabeled MIP-1alpha cDNA. Lower panels, the same membrane hybridized with a mouse beta-actin cDNA to indicate relative amounts of hybridizable RNA per lane. C and D, densitometric quantification of MIP-1alpha mRNA normalized to beta-actin RNA in response to MQ and H(2)O(2), respectively. Intensity of control autoradiographic bands were defined as 1 in order to compare fold changes in MIP-1alpha mRNA in the absence or the presence of actinomycin D. The results are representative of two independent experiments.



Transcriptional Regulation of the MIP-1alpha Gene by an Oxidative Stress

Because transcriptional regulation of the MIP-1alpha gene by ROS was suggested by results obtained with actinomycin D, we performed nuclear run-on transcriptional rate assays to confirm the role of this mechanism in regulation of mRNA expression. RAM cells were treated with 0 or 0.5 mM H(2)O(2) or 0 or 50 µM MQ for 1 and 4 h. Cell nuclei were extracted, and transcription rates for the MIP-1alpha gene were determined as described under ``Experimental Procedures.'' At 1 h post-treatment, transcription of the MIP-1alpha gene markedly increased following treatment with both 0.5 mM H(2)O(2) and 50 µM MQ (Fig. 7). After 4 h, the transcriptional rate of the MIP-1alpha gene in MQ-treated cells was further increased; however, MIP-1alpha transcription in H(2)O(2)-treated cells had returned to near control levels. Hybridization to nonspecific, prokaryotic sequences (pBR322) was not evident. These results indicate that the observed induction of MIP-1alpha mRNA by both H(2)O(2) or MQ is at least in part caused by transcriptional activation of the gene.


Figure 7: Transcriptional rate of the MIP-1alpha gene in rat alveolar macrophages exposed to H(2)O(2) or MQ. Cells were exposed to 0 or 0.5 mM H(2)O(2) or 50 µM MQ for 1 and 4 h. Nuclei were then extracted, and nuclear run-on assays were performed as described under ``Experimental Procedures.'' Equivalent amounts of radiolabeled RNA prepared from nuclei isolated from untreated or H(2)O(2)- or MQ-treated cells were hybridized with denatured MIP-1alpha, glyceraldehyde-3-phosphate dehydrogenase (control) cDNA, or pBR322 plasmid DNA. The results are representative of two independent experiments.



Post-transcriptional Regulation of MIP-1alpha mRNA by H(2)O(2)

The MIP-1alpha mRNA 3`-untranslated region contains six copies of the reiterated AUUUA motifs (Shi et al., 1995) that are typically conserved in these regions of cytokines and growth factors mRNAs and are implicated in mRNA stability and translational control (Shaw and Kamen, 1986; Brewer, 1991). Our previous work suggested that the rapid up-regulation of MIP-1alpha mRNA by lipopolysaccharide treatment is the result of post-transcriptional regulation (Shi et al., 1995). The contribution of changes in MIP-1alpha mRNA stability to its increased expression in response to oxidative stress was evaluated by measuring MIP-1alpha mRNA half-life (t). In the presence of actinomycin D, MIP-1alpha mRNA from untreated macrophages decayed quickly with a t of approximately 1 h (Fig. 8). H(2)O(2) treatment significantly increased the half-life of MIP-1alpha mRNA, with a t greater than 6 h. MQ treatment did not change the t of MIP-1alpha mRNA in comparison with the control. These observations are in agreement with the results of the actinomycin D (Fig. 6, A and B) and nuclear run-on studies (Fig. 7), suggesting that the induction of MIP-1alpha mRNA by MQ results from transcriptional activation, whereas the elevation of MIP-1alpha mRNA by H(2)O(2) is the result of both transcriptional activation of the MIP-1alpha gene and post-transcriptional regulation of the mRNA.


Figure 8: Influence of H(2)O(2) and MQ on the half-life (t) of MIP-1alpha mRNA in rat alveolar macrophages. Cells were left untreated (Control) or treated with 0.5 mM H(2)O(2) for 1 h or 25 µM MQ for 4 h. Actinomycin D was then added to a final concentration of 5 µg/ml, and at the times indicated, total RNA was isolated and Northern analysis was performed as described in Fig. 1A, upper panels, autoradiogram of Northern blot hybridized with radiolabeled-MIP-1alpha cDNA. Lower panels, the same membrane hybridized with a mouse beta-actin cDNA to indicate relative amounts of hybridizable RNA per lane. B, densitometric quantification of the decay of MIP-1alpha mRNA normalized to beta-actin RNA. The MIP-1alpha mRNA t from control or MQ-treated cells was approximately 1 h, whereas H(2)O(2) treatment increased the t to greater than 6 h. The results are representative of two independent experiments.




DISCUSSION

Induction of MIP-1alpha mRNA in alveolar macrophages by H(2)O(2) or MQ strongly suggests a role for ROS in the regulation of chemokine gene expression. Although H(2)O(2) is a direct source of oxidative stress, MQ undergoes redox cycling, leading to a gradual increase in ROS levels (Chang et al., 1992; Shi et al., 1993). Both MQ- and H(2)O(2)-induced increases in MIP-1alpha mRNA were suppressed by the antioxidant NAC, further confirming that oxidative stress alone could influence MIP-1alpha expression. Although a number of other recent studies have explored the possible modulation of chemokine expression by ROS (DeForge et al., 1992, 1993), most used an endotoxin challenge as a stimulus for oxidant stress. The significance of the present study is that it directly links the ROS to increased MIP-1alpha mRNA expression in the absence of endotoxin.

Reactive oxygen species are now known to activate gene expression through modulation of the intracellular reduction-oxidation (redox) state of nuclear proteins. A number of site-specific DNA binding proteins have been identified whose redox state affects binding ability. Oxidative activation of a transcription regulatory protein was first identified in prokaryotes. cis-Acting elements in the 5`-flanking region of Escherichia coli and Salmonella typhimurium catalase and alkylhydroperoxide reductase genes are recognized by a trans-activator termed OxyR (Christman et al., 1989). Both reduced and oxidized OxyR binds the catalase promoter, but only the oxidized form activates transcription (Storz et al., 1990). In mammalian cells, ROS have also been implicated in the activation of the transcription factors c-Fos and c-Jun (Abate et al., 1990), NF-kappaB/Rel (Schreck et al., 1991). A cis-acting regulatory sequence, the antioxidant response element, in the 5`-flanking region of the rat glutathione transferase Ya subunit gene and rat NAD(P)H:quinone reductase, has also been identified to be responsive to a variety of redox cycling xenobiotics and H(2)O(2) (Rushmore et al., 1991; Favreau and Pickett, 1992). Constitutive antioxidant response element-binding factor(s) have been identified that are activated by ROS (Favreau and Picket, 1992). It has been reported that a neonatal rat lung protein forms specific complexes with catalase mRNA in a redox-sensitive manner. Neonatal rats exposed to hyperoxia show increased lung catalase mRNA stability associated with a larger proportion of catalase mRNA binding protein in oxidized form than from lungs of air-breathing neonatal rats (Clerch et al., 1991; Clerch and Massaro, 1992). These reports indicate that the intracellular redox state is crucial in the control of either active transcription factors or redox-sensitive proteins. Oxidant-induced conformational changes of regulatory proteins may influence a spectrum of genes by initiating transcription and/or stabilizing specific mRNAs.

We performed inhibitor studies to identify whether increases in MIP-1alpha mRNA levels by oxidative stress were dependent on new RNA synthesis or de novo protein synthesis. Cycloheximide alone was able to markedly stimulate the accumulation of MIP-1alpha mRNA (Fig. 5), possibly suggesting that MIP-1alpha gene may be controlled either directly or indirectly by a repressor protein with a short half-life. Co-incubation with actinomycin D almost completely eliminated the increase of MIP-1alpha mRNA by MQ (Fig. 6A) and partially by H(2)O(2), suggesting that the transcriptional regulation of the MIP-1alpha gene is involved. This suggestion was confirmed with nuclear run-on assays, which demonstrated increased transcriptional rates induced by MQ and H(2)O(2) (Fig. 7). Although the 5`-flanking region of rat MIP-1alpha is presently unavailable, its murine counterpart contains an NF-kappaB-like binding region (Grove and Plumb, 1993). NF-kappaB is a multi-subunit transcription factor that can rapidly activate the expression of genes involved in inflammation and the acute phase immune response (Baeuerle and Baltimore, 1988). As described above, NF-kappaB has been previously reported to be activated by oxidative stress, including H(2)O(2). MQ and H(2)O(2) may also induce transcription of MIP-1alpha through cis-acting element(s) like antioxidant response element. Whether the transcriptional induction of MIP-1alpha by MQ and H(2)O(2) is through an antioxidant response element-like element, an NF-kappaB binding region, or other 5` sequences deserves further study.

Stability of mRNA transcripts also plays an important role in the regulation of proinflammatory genes. Changes in turnover rate can affect steady-state levels over a relatively short period of time. As an example, the rapid, hyperoxia-induced elevation of lung catalase mRNA in neonatal rats is due to enhanced stability of its mRNA but not an increased rate of transcription (Clerch et al., 1991). In the present study, MIP-1alpha mRNA was elevated by H(2)O(2) and MQ in an exceedingly short period of time (1 h), suggesting that MIP-1alpha mRNA turnover might be influenced by oxidative stress. Also of note, the 3`-untranslated regions of the rat MIP-1alpha mRNA contains six copies of reiterated AU-rich motifs (Shi et al., 1995), implicated in mRNA stability and translational control (Shaw and Kamen, 1986; Brewer, 1991). A cytoplasmic protein termed adenosine-uridine binding factor has been shown to bind specifically to the AUUUA motifs of in vitro transcribed RNAs and form exceptionally stable complexes (Malter, 1989). Moreover, the binding of adenosine-uridine binding factor to RNA templates is also redox-sensitive (Malter and Hong, 1991). To distinguish between an increase in transcriptional rate and stabilization of pre-existing transcripts of MIP-1alpha, we performed an RNA decay assay. MQ-induced elevation of MIP-1alpha mRNA has a t of less than 1 h, which is similar to that of control cells. On the contrary, MIP-1alpha mRNA from H(2)O(2)-treated cells has a t greater than 6 h (Fig. 8). Thus, the induction of MIP-1alpha mRNA by MQ is primarily through transcriptional activation of the MIP-1alpha gene, whereas the effect of H(2)O(2) is through both transcriptional activation of the MIP-1alpha gene and the stabilization of MIP-1alpha mRNA. Post-transcriptional regulation of MIP-1alpha mRNA in macrophages has been previously reported following stimulation by bacterial endotoxin (Shi et al., 1995), which can also generate ROS (Adamson and Billings, 1990; Yoshikawa, 1990). It is noteworthy that two distinct mechanisms are involved in the regulation of MIP-1alpha gene expression; the one chosen is dependent on the specific stimulus. Enhanced stability of MIP-1alpha mRNA by H(2)O(2) may be due to an mRNA binding protein whose confirmation, synthesis, or degradation is changed during an oxidative stress.

In conclusion, the present study demonstrates that an H(2)O(2)- and MQ-generated oxidative stress rapidly elevates MIP-1alpha mRNA expression in macrophages. The up-regulation of MIP-1alpha by MQ was apparently mediated through transcriptional activation of the MIP-1alpha gene. H(2)O(2), on the other hand, induced MIP-1alpha mRNA expression by two mechanisms: transcriptional activation of the MIP-1alpha gene and stabilization of the MIP-1alpha mRNA.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL19170, HL07118, ES00002, ES05703, and ES05947. Portions of this work were presented at the 1995 Experimental Biology Meeting, Atlanta, Georgia. 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: Physiology Program, Dept. of Environmental Health, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. Fax: 617-432-3468.

(^1)
The abbreviations used are: ROS, reactive oxygen species; MIP, macrophage inflammatory protein; MQ, menadione; NAC, N-acetylcysteine; RAM, rat alveolar macrophage.


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