Heat shock-mediated regulation of MKP-1

Hector R. Wong,1 Katherine E. Dunsmore,1 Kristen Page,1 and Thomas P. Shanley2

1Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center, and Cincinnati Children's Research Foundation, and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio; and 2Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan

Submitted 23 March 2005 ; accepted in final form 10 June 2005


    ABSTRACT
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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Heat shock modulates cellular proinflammatory responses, and we have been interested in elucidating the mechanisms that govern this modulation. The dual specific phosphatase, MAP kinase phosphatase-1 (MKP-1), is an important modulator of cellular inflammatory responses, and we recently reported that heat shock increases expression of MKP-1. Herein we sought to elucidate the mechanisms by which heat shock modulates MKP-1 gene expression. Subjecting RAW264.7 macrophages to heat shock increased MKP-1 gene expression in a time-dependent manner. Transfection with a wild-type murine MKP-1 promoter luciferase reporter plasmid demonstrated that heat shock activates the MKP-1 promoter. When the reporter plasmid was transfected into heat shock factor-1 (HSF-1)-null fibroblasts, the MKP-1 promoter was activated in response to heat shock in a manner similar to that of wild-type fibroblasts with intact HSF-1. Site-directed mutagenesis of two potential heat shock elements in the MKP-1 promoter demonstrated that both sites are required for basal promoter activity. mRNA stability assays demonstrated that heat shock increased MKP-1 mRNA stability compared with cells maintained at 37°C. Inhibition of p38 MAP kinase activity inhibited heat shock-mediated expression of MKP-1. These data demonstrate that heat shock regulates MKP-1 gene expression at both the transcriptional and posttranscriptional levels. Transcriptional mechanisms are HSF-1 independent but are dependent on putative heat shock elements in the MKP-1 promoter. Posttranscriptional mechanisms involve increased stability of MKP-1 mRNA that is partially dependent on p38 MAP kinase activity. These data demonstrate another potential mechanism by which heat shock can modulate inflammation-related signal transduction.

endotoxin; phosphatase; inflammation; heat shock factor; p38


HEAT SHOCK CONFERS PROTECTION against inflammation-associated cell and tissue injury. For example, heat shock has been demonstrated to confer protection against sepsis (3, 31), acute lung injury (30, 34), ischemia-reperfusion injury (4), and endotoxin-mediated apoptosis (32). One mechanism of protection involves modulation of cellular proinflammatory responses (19). For example, heat shock has been demonstrated to inhibit activation of the NF-{kappa}B pathway by mechanisms involving inhibition of I{kappa}B kinase activation (7, 14, 24, 27, 33, 36). In addition, the inhibitory effects of heat shock on the NF-{kappa}B pathway are partially dependent on intracellular phosphatase activity (7). On the basis of these observations, we have been interested in further elucidating the mechanisms by which heat shock modulates inflammation-associated signal transduction.

Phosphatases modulate signal transduction by their ability to dephosphorylate intracellular signaling proteins (26). MAP kinase phosphatase-1 (MKP-1) belongs to a family of dual-specific phosphatases, given their ability to dephosphorylate both threonine and tyrosine residues. MKP-1 is further distinguished by its nuclear localization. MKP-1 seems particularly important in signal transduction pathways involved in inflammation in that it can dephosphorylate key members of the MAP kinase pathway (2, 11). In this capacity, MKP-1 can be thought of as having an anti-inflammatory/counterregulatory role because dephosphorylation leads to decreased MAP kinase activity (13, 28). Indeed, we recently demonstrated that MKP-1-dependent regulation of p38 MAP kinase plays a central mechanistic role in the induction of endotoxin tolerance (22).

Recently, we reported that heat shock can independently increase expression of the MKP-1 gene in mononuclear cells (25). Many of the classic heat shock proteins are regulated at the transcriptional level and are dependent on the transcription factor, heat shock factor-1 (HSF-1), and heat shock elements (HSE) in the promoter regions of heat shock protein genes (6, 34). In addition, heat shock has also been demonstrated to increase mRNA stability of some genes (16, 20). On the basis of these background data, we sought to elucidate the mechanisms by which heat shock regulates MKP-1 gene expression, with a focus on HSF-1 and MKP-1 mRNA stability.


    EXPERIMENTAL PROCEDURES
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Cell culture and heat shock. Most of the experiments involved RAW264.7 murine macrophages (American Type Culture Collection, Rockville, MD). Cells were maintained in a 95% room air-5% CO2 incubator at 37°C using RPMI supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (GIBCO-BRL, Grand Island, NY). Selected experiments involved embryonic fibroblasts from wild-type (HSF-1+/+) and HSF-1-null mutant mice (HSF-1–/–) (21, 35). Fibroblasts were maintained in DMEM containing 10% fetal bovine serum, 55 µM {beta}-mercaptoethanol, 0.1 mM MEM nonessential amino acid solution, 2 mM L-glutamine, and 10 ml/l of antibiotic-antimycotic solution containing 10,000 U/ml penicillin G, 10,000 µg/ml streptomycin sulfate, and 25 µg/ml amphotericin B (GIBCO-BRL). Heat shock was induced by incubating cells in a 95% room air-5% CO2 incubator permanently set at 43°C. Cells were exposed to 43°C for 1 h and subsequently returned to a 95% room air-5% CO2 incubator set at 37°C.

Northern blot analysis. Total RNA was isolated using the TRIzol reagent (GIBCO-BRL). RNA concentrations were determined using spectrophotometry (260 nm), and 15 µg of RNA for each sample underwent electrophoresis in gels containing 1% agarose and 3% formaldehyde. RNA integrity was confirmed visually by ethidium bromide staining and brief UV light illumination. RNAs were transferred to nylon membranes (Micron Separations, Westboro, MA) and UV auto-crosslinked (UV Stratalinker 1800; Stratagene, La Jolla, CA). Membranes were prehybridized for 4 h at 42°C and subsequently hybridized overnight with a radiolabeled murine MKP-1 cDNA probe (9). The cDNA probe was labeled with {alpha}-[32P]dCTP (specific activity 3,000 Ci/mM, New England Nuclear Research Product) by random priming (Pharmacia, Piscataway, NJ). Membranes were subsequently washed twice with 2x SSC/0.1% SDS at 53°C, developed using a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA), and analyzed using ImageQuant Software (Molecular Dynamics).

mRNA stability assays. One group of cells was subjected to heat shock for 1 h. At the end of the heat shock, the cells were treated with actinomycin D (1 µg/ml) to induce transcriptional arrest and harvested for total RNA isolation at 0.5-h intervals. Control cells were maintained at 37°C, treated with actinomycin D, and harvested for total RNA isolation at 0.5-h intervals. Total RNA samples were then subjected to Northern blot analysis as described above.

Nuclear protein extraction. All nuclear extraction procedures were performed on ice with ice-cold reagents. Cells were washed twice with PBS and harvested by being scraped into 1 ml of PBS and pelleted at 6,000 rpm for 5 min. The pellet was washed twice with PBS, resuspended in one packed cell volume of lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, 0.2% vol/vol Nonidet P-40, 1 mM DTT, and 0.1 mM PMSF), and incubated for 5 min. After centrifugation at 6,000 rpm, one cell pellet volume of extraction buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 0.1 M EDTA, 1.5 mM MgCl2, 25% vol/vol glycerol, 1 mM DTT, and 0.5 mM PMSF) was added to the nuclear pellet and incubated on ice for 15 min with occasional vortexing. The nuclear proteins were isolated by centrifugation at 14,000 rpm for 15 min. Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA) and stored at –70°C until used for Western blot analysis.

Western blot analysis. Nuclear protein extracts were boiled in equal volumes of loading buffer (125 mM Tris·HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol) and 50 µg of protein loaded per lane on an 8–16% Tris-glycine gradient gel (Novex, San Diego, CA). Proteins were separated electrophoretically and transferred to nitrocellulose membranes with the use of the Novex X-cell Mini-Gel system. For immunoblot analysis, membranes were blocked with 10% nonfat dried milk in Tris-buffered saline (TBS) for 1 h. Primary MKP-1 antibody (polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA) was applied at 1:200 dilution for 2 h. After being washed two times in TBS containing 0.05% Tween 20 (TTBS), secondary antibody (peroxidase-conjugated goat anti-rabbit IgG; Calbiochem, La Jolla, CA) was applied at 1:10,000 for 1 h. The blots were washed in TTBS two times over 30 min, incubated in commercial enhanced chemiluminescence reagents (ECL, Amersham, Little Chalfont, UK), and exposed to photographic film.

Luciferase reporter plasmids. A luciferase reporter plasmid under the control of the wild-type murine MKP-1 promoter was used to quantify MKP-1 promoter activity in response to heat shock (10). With the use of this wild-type plasmid as a template, three mutant plasmids were constructed using site-directed mutagenesis (Stratagene). Mutations consisted of base substitutions within two potential HSEs identified in the murine MKP-1 promoter region at –517 and –558 bp, respectively (10, 23). Table 1 depicts the wild-type promoter regions containing the potential HSEs and their respective base substitution mutations. The plasmid designated "mutant 1" contained the designated base substitutions in the potential HSE located at –517 bp. The plasmid designated "mutant 2" contained the designated base substitutions in the potential HSE located at –558 bp. The plasmid designated "mutant 3" contained the designated base substitutions in both potential HSEs. The base substitutions in each of the three mutant plasmids were confirmed by standard sequencing protocols (University of Cincinnati DNA Core Facility).


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Table 1. Potential HSEs in murine MKP-1 promoter and their respective base substitution mutations

 
Transient transfection and luciferase assays. The wild-type reporter plasmid and the three mutant reporter plasmids described above were used individually for transfection-related experiments. Cells were transfected in duplicate, in six-well plates, at a density of 300,000 cells per well by incubation with FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN) and serum-free DMEM overnight. Four hours after heat shock, cells were washed with PBS once and cellular proteins were then extracted and analyzed for luciferase activity according to the manufacturer's instructions (Promega) with the use of a luminometer (AutoLumat LB953, Berthold). Luciferase activity was corrected for total cellular protein and reported as relative induction over respective control cells (cells that were transfected and treated with medium alone).

p38 MAP kinase activity and inhibition. p38 MAP kinase activity was measured using a solid-phase sandwich ELISA that incorporates a capture antibody specific for total p38 MAP kinase and a detection antibody specific for activated (phosphorylated) p38 MAP kinase (Oncogene Research Products, San Diego, CA). p38 MAP kinase activity was inhibited by treating cells with 5 µM p38 MAP kinase inhibitor SB-203580 (Calbiochem). The p38 MAP kinase inhibitor was added 1 h before heat shock.


    RESULTS
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 EXPERIMENTAL PROCEDURES
 RESULTS
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Heat shock increases MKP-1 gene expression. We (25) previously reported that heat shock increases MKP-1 expression in a human mononuclear cell line and in primary murine peritoneal macrophages. To determine whether this effect is also operative in other mononuclear cells, we subjected RAW264.7 murine macrophages to heat shock and measured MKP-1 gene expression. Northern blot analyses demonstrated that heat shock increased steady-state MKP-1 mRNA expression in a time-dependent manner (Fig. 1A). Western blot analyses in which we used nuclear protein extracts demonstrated that heat shock also increased MKP-1 peptide expression in a time-dependent manner (Fig. 1B). Collectively, these data confirm that the MKP-1 gene is expressed in response to heat shock in RAW264.7 murine macrophages and form the basis for the subsequent experiments described below.



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Fig. 1. A: representative Northern blot analysis demonstrating that heat shock increases steady-state MAP kinase phosphatase-1 (MKP-1) mRNA expression in a time-dependent manner. RAW264.7 murine macrophages were subjected to heat shock (43°C for 1 h), and total RNA was harvested at various time points after heat shock as indicated. Equal loading of total RNA was confirmed by ethidium bromide staining of 18S rRNA. B: representative Western blot analysis demonstrating that heat shock increases MKP-1 peptide expression in a time-dependent manner. RAW264.7 murine macrophages were subjected to heat shock (43°C for 1 h), and nuclear proteins were harvested at various time points after heat shock as indicated. Both gels represent 1 of 4 experiments with similar results.

 
Heat shock increases activation of the MKP-1 promoter. To assess whether heat shock activates the MKP-1 promoter, we transiently transfected RAW264.7 macrophages with a wild-type murine MKP-1 promoter luciferase reporter plasmid and measured luciferase activity in cells subjected to heat shock. Exposure to heat shock significantly increased luciferase activity in transfected cells compared with transfected cells maintained at 37°C (Fig. 2). These data demonstrate that heat shock activates the MKP-1 promoter.



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Fig. 2. Luciferase assay demonstrating that heat shock increases MKP-1 promoter activity. RAW264.7 murine macrophages were transiently transfected with a wild-type murine MKP-1 promoter luciferase reporter plasmid. Transfected cells were subjected to heat shock (43°C for 1 h), and total cellular proteins were harvested for luciferase assay 4 h after heat shock. Luciferase activity in cells exposed to heat shock is expressed as relative induction over control cells (transfected and maintained at 37°C). Data represents means ± SE of 5 individual experiments, with each experiment carried out in triplicate. *P < 0.05 vs. control.

 
Role of HSF-1 and HSEs in heat shock-mediated activation of the MKP-1 promoter. HSF-1 regulates the promoter activity of classic heat shock proteins, such as heat shock protein 70, via HSEs (21, 34, 35). To determine the role of HSF-1 in heat shock-mediated activation of the MKP-1 promoter, we transiently transfected HSF-1–/– and HSF-1+/+ murine fibroblasts with the murine MKP-1 promoter luciferase reporter plasmid described above and measured luciferase activity in response to heat shock. Exposure to heat shock increased luciferase activity in transfected HSF-1+/+ fibroblasts compared with transfected HSF-1+/+ fibroblasts maintained at 37°C (Fig. 3A). When transfected HSF-1–/– fibroblasts were subjected to heat shock, the relative induction of luciferase activity was similar to that of transfected HSF-1+/+ fibroblasts subjected to heat shock (Fig. 3A). These data indicate that heat shock-mediated activation of the MKP-1 promoter is independent of HSF-1.



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Fig. 3. A: Luciferase assay demonstrating that heat shock increases MKP-1 promoter activity in the absence of HSF-1. Embryonic murine fibroblasts from wild-type (HSF-1+/+) and HSF-1-null mice (HSF-1–/–) were transiently transfected with a wild-type murine MKP-1 promoter luciferase reporter plasmid. Transfected cells were subjected to heat shock (43°C for 1 h), and total cellular proteins were harvested for luciferase assay 4 h after heat shock. Luciferase activity in cells exposed to heat shock is expressed as relative induction over respective control cells (transfected and maintained at 37°C). B: luciferase assay demonstrating the role of two potential HSEs in the murine MKP-1 promoter on heat shock-mediated MKP-1 promoter activation. Three mutant (mut.) MKP-1 promoter luciferase reporter plasmids were generated as described in EXPERIMENTAL PROCEDURES. RAW264.7 murine macrophages were transiently transfected with either the wild-type MKP-1 promoter luciferase reporter plasmid or 1 of the 3 mutant plasmids. Transfected cells were subjected to heat shock and assayed as described above. Luciferase activity in cells transfected with the mutant plasmids and in cells exposed to heat shock is expressed as relative induction over control cells (transfected with the wild-type plasmid and maintained at 37°C). For both figures, data represent the means ± SE of 5 individual experiments, with each experiment carried out in triplicate. *P < 0.05 vs. respective control.

 
Examination of the murine MKP-1 promoter revealed the presence of two potential HSEs at positions –517 and –558 bp, respectively (10, 23). To determine the role of these two potential HSEs in heat shock-mediated activation of the MKP-1 promoter, we generated three mutant plasmids as described in EXPERIMENTAL PROCEDURES and Table 1. In cells transfected with mutant 1, basal luciferase activity was significantly decreased compared with cells transfected with the wild-type MKP-1 promoter luciferase plasmid (Fig. 3B). Heat shock-mediated luciferase activity was also decreased in cells transfected with mutant 1, compared with cells transfected with the wild-type MKP-1 promoter luciferase plasmid. Relative to the basal luciferase activity of cells transfected with mutant 1, however, cells transfected with mutant 1 and subjected to heat shock retained the ability to increase luciferase activity. Similar patterns of luciferase activity were observed in cells transfected with either mutant 2 or mutant 3 (Fig. 3B). These data indicate that the two potential HSEs in the murine MKP-1 promoter are necessary for basal promoter activity.

To rule out the possibility that the induction of luciferase activity described in Fig. 3, A and B, is due to a nonspecific effect (i.e., heat shock-mediated stabilization of luciferase mRNA or protein), we transiently transfected RAW264.7 macrophages with an NF-{kappa}B-dependent luciferase reporter that does not contain an HSE (1). In cells transfected with this NF-{kappa}B-dependent plasmid, heat shock did not induce luciferase activity (0.2 ± 0.1-fold induction relative to control) indicating that the data described in Fig. 3, A and B, are not due to artifact.

Heat shock increases MKP-1 mRNA stability. Because heat shock has been demonstrated to increase the mRNA stability of some genes (16, 20), we next determined the effect of heat shock on MKP-1 mRNA stability using a standard approach of actinomycin D-mediated transcriptional arrest. In control RAW264.7 macrophages maintained at 37°C and treated with actinomycin D, steady-state mRNA levels declined rapidly (Fig. 4). The calculated MKP-1 mRNA half-life (n = 4) was 63 ± 9 min in control cells. In RAW264.7 macrophages exposed to heat shock and treated with actinomycin D, steady-state mRNA levels remained substantially increased compared with that of control cells (Fig. 4). The calculated MKP-1 mRNA half-life (n = 4) was 211 ± 70 min in cells exposed to heat shock. These data demonstrate that heat shock increases MKP-1 mRNA stability.



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Fig. 4. Representative Northern blot analysis demonstrating the effect of heat shock on MKP-1 mRNA stability in RAW264.7 murine macrophages. Control cells were maintained at 37°C and treated with actinomycin D (1 µg/ml). Cells treated with heat shock were incubated at 43°C for 1 h and then returned to a 37°C incubator and treated with actinomycin D. Both groups of cells were harvested for total RNA extraction at 0.5-h intervals as indicated. 18S rRNA levels are depicted based on the ethidium bromide staining of the respective gel.

 
Role of p38 in heat shock-mediated expression of MKP-1. p38 MAP kinase is known to play a role in posttranscriptional (mRNA stability) gene regulation (5). Accordingly, we determined the role of p38 MAP kinase in heat shock-mediated expression of MKP-1. First, we measured p38 MAP kinase activation in response to heat shock and confirmed that heat shock activates p38 MAP kinase in RAW264.7 macrophages (Fig. 5A). Next, we inhibited p38 MAP kinase activity using the pharmacological inhibitor SB-203580 and measured MKP-1 expression by Western blot analysis. Inhibition of p38 MAP kinase activity had no effect on basal levels of MKP-1 (Fig. 5B). In cells exposed to heat shock, however, inhibition of p38 MAP kinase partially inhibited heat shock-mediated MKP-1 expression. Finally, we inhibited p38 MAP kinase activity and measured MKP-1 mRNA stability in cells treated with actinomycin D. In cells subjected to heat shock and simultaneous inhibition of p38 MAP kinase activity, the half-life of MKP-1 mRNA was similar to that of control cells maintained at 37°C (Fig. 5C). These data indicate that p38 MAP kinase activity is involved in heat shock-mediated expression of MKP-1 via increased stability of MKP-1 mRNA.



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Fig. 5. A: p38 MAP kinase assay (solid-phase sandwich ELISA) demonstrating the effect of heat shock on p38 MAP kinase activity. RAW264.7 murine macrophages were subjected to heat shock (43°C, for 1 h) and cellular proteins were harvested 1 h after heat shock for ELISA. p38 MAP kinase activity is expressed as relative induction over control cells maintained at 37°C. Data are normalized for total cellular protein and represent the means ± SE of 4 individual experiments with each experiment carried out in triplicate. *P < 0.05 vs. control. B: representative Western blot analysis demonstrating the effect of p38 MAP kinase inhibition on heat shock-mediated expression of MKP-1 peptide. RAW264.7 murine macrophages were subjected to heat shock (43°C for 1 h), and nuclear proteins were harvested 1 h after heat shock. One group of cells was treated with a p38 MAP kinase inhibitor (SB-203580, 5 µM) 1 h before heat shock as indicated. Gel represents 1 of 3 experiments with similar results. C: representative Northern blot analysis demonstrating the effect of p38 MAP kinase inhibition on heat shock-mediated increased stability of MKP-1 mRNA. Control cells were maintained at 37°C and treated with actinomycin D (1 µg/ml). Cells treated with heat shock were incubated at 43°C for 1 h and then returned to a 37°C incubator and treated with actinomycin D. One group of cells subjected to heat shock was also treated with a p38 MAP kinase inhibitor (SB-203580, 5 µM) 1 h before heat shock as indicated. Cells were harvested at 0.5-h intervals as indicated. 18S rRNA levels are depicted based on the ethidium bromide staining of the respective gel.

 

    DISCUSSION
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There are ~1,000 phosphatases encoded in the human genome (26). Many of the phosphatases are constitutively present, whereas others are dependent on de novo gene expression for activation. MKP-1 belongs to a family of dual-specific phosphatases by having the ability to dephosphorylate both threonine and tyrosine residues. This class of phosphatases also appears to be particularly important for regulation of the MAP kinases (2, 11, 29). MKP-1 activity is primarily dependent on de novo gene expression, and MKP-1 is further distinguished by its nuclear localization.

It is now well established that preconditioning with heat shock confers protection against inflammation-associated tissue and organ injury (31, 34). Accordingly, we have been interested in elucidating the mechanisms by which heat shock modulates inflammation-associated signal transduction, particularly in cells of the monocyte/macrophage line. The bulk of this work has focused on the I{kappa}B/NF-{kappa}B pathway and has clearly demonstrated that heat shock inhibits NF-{kappa}B activation both in vitro and in vivo (19). Interestingly, recent work (7) demonstrated that the inhibitory effect of heat shock on the NF-{kappa}B pathway is partially dependent on intracellular phosphatase activity. In keeping with this line of investigation, we recently demonstrated that heat shock induces de novo expression of MKP-1 in a human mononuclear cell line and in primary murine peritoneal macrophages (25). We also demonstrated that heat shock-mediated induction of MKP-1 was associated with inhibition of endotoxin-dependent activation of p38, activation of ERK, and induction of TNF-{alpha} expression (25). The central role of MKP-1 in this inhibitory process was further established by the demonstration that peritoneal macrophages from MKP-1-deficient mice were less sensitive to the inhibitory effects of heat shock on endotoxin-dependent signaling compared with wild-type macrophages with intact MKP-1 (25).

In the present study, we have confirmed that heat shock induces MKP-1 expression in cells of the monocyte/macrophage line. In combination with previous reports (12, 25), it now seems well established that MKP-1 is a heat shock-responsive gene. This is also consistent with previous reports demonstrating that MKP-1 expression can also be increased by other cellular stressors such as hypoxia (15), ultraviolet light (18), oxidative stress (12), and glucocorticoid stimulation (10). The most well known heat shock-responsive genes are the heat shock proteins (6, 34). Expression of many heat shock proteins, particularly heat shock protein 70, is primarily regulated at the level of transcription. Heat shock protein transcription involves interactions between the transcription factor HSF-1 and HSEs in the promoter regions of heat shock proteins (6, 34). Consistent with this concept, MKP-1 gene expression in response to heat shock also appears to be regulated at the level of transcription. This assertion is supported by the demonstration that heat shock-induced luciferase activity in cells transfected with a wild-type MKP-1 promoter luciferase reporter plasmid.

Cells and animals deficient in HSF-1 are known to have a drastically reduced capacity to induce heat shock protein expression in response to thermal stress (21, 35). Surprisingly, heat shock-mediated MKP-1 promoter activation seems to be independent of HSF-1. This assertion is supported by the demonstration that heat shock-induced luciferase activity in transfected HSF-1–/– fibroblasts to a degree similar that seen in wild-type fibroblasts with intact HSF-1. These data suggest that a transcription factor, or transcription factors, other than HSF-1 are responsible for activation of the MKP-1 promoter in response to heat shock.

HSEs are defined by tandem repeats of the pentamer sequence nGAAn ("n" denoting less strongly conserved nucleotides) arranged in alternating orientations (6, 34). At least two such units are necessary for high-affinity binding of HSF-1, and the units can be arranged either head to head (5'-nGAAnnTTCn-3') or tail to tail (5'-nTTCnnGAAnn-3'). Examination of the murine MKP-1 promoter region identified two potential HSEs (10, 23). The potential HSE located at –517 bp contains two sequential pentamers that are completely intact with regard to sequence and lack of intervening bases (Table 1). The potential HSE located a –558 bp has four sequential pentamers, two of which are not fully intact, and there is one intervening base (Table 1). Both elements appear to be required for basal MKP-1 promoter activity. This assertion is supported by the demonstration that transfection with mutant MKP-1 promoter luciferase reporter plasmids containing base substitutions in these potential HSEs decreased basal luciferase activity.

The degree of wild-type promoter activity seen in transfected cells subjected to heat shock was not fully consistent with the degree of MKP-1 gene expression seen in response to heat shock. This suggested to us that heat shock-mediated MKP-1 gene expression may also be dependent on posttranscriptional mechanisms. Standard mRNA stability assays demonstrated that heat shock increased MKP-1 mRNA stability by almost fourfold. Because even small increases in mRNA half-lives can lead to substantial increases of steady-state mRNA levels (8), we conclude that the level of MKP-1 gene expression that is induced in response to heat shock results from the combined effects of a modest increase in transcription, plus a substantial increase in MKP-1 mRNA stability.

p38 MAP kinase is thought to play a role in regulating mRNA stability, particularly that of inflammation-associated genes (5). Our inhibitor-related data indicate that MKP-1 mRNA stability, and thus MKP-1 gene expression, are partially dependent on p38 MAP kinase activity. These data complement previous data from our own laboratory (22, 25) and others (13, 28) indicating that MKP-1 can also inactivate p38 MAP kinase activity via dephosphorylation. Collectively, these data indicate that there is significant cross-talk between MKP-1 and p38 MAP kinase that regulates signal transduction mechanisms involved in both pro- and anti-inflammatory processes.

The mechanisms by which heat shock modulates inflammation-associated signal transduction are complex. Clearly, there is a high degree of regulation at the level of the I{kappa}B/NF-{kappa}B pathway (19). Evolving evidence implies that phosphatases also play an important role in modulating inflammation-associated signal transduction in the context of heat shock and that MKP-1 seems to play a central role in this process (7, 25). In support of this assertion is the recent demonstration by Lee and colleagues (17) that heat shock leads to MKP-1 expression in a respiratory epithelial cell line and that protein-protein interaction between heat shock protein 70 and MKP-1 is associated with increased phosphorylation of MKP-1. Further support is provided by the data presented herein demonstrating that heat shock regulates MKP-1 gene expression by a combination of transcriptional and posttranscriptional mechanisms. The transcriptional mechanism is independent of HSF-1 but seems to require putative heat shock elements in the MKP-1 promoter. The posttranscriptional mechanisms involve increased stability of MKP-1 mRNA that is partially dependent on p38 MAP kinase activity. Heat shock-mediated regulation of MKP-1 represents another potential mechanism by which the heat shock response can modulate inflammation-associated signal transduction.


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This study was supported by National Institute of General Medical Sciences Grants R01 GM-061723 (to H. R. Wong) and R01 GM-066839 (to T. P. Shanley).


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. R. Wong, Div. of Critical Care Medicine, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229 (e-mail: wonghr{at}cchmc.org)

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.


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