Heat Shock Protein 70 Suppresses Astroglial-inducible Nitric-oxideSynthase Expression by Decreasing NFkB Activation*
(Received for publication, March 20, 1996, and in revised form, April 24, 1996)
Douglas L. Feinstein‡§, Elena Galea‡, Dennis A. Aquino¶, Gloria C. Lii, Hui Xu‡,and Donald J. Reis‡
From the ‡Division of Neurobiology, Cornell University Medical College, New York, New York 10021, the ¶Department ofNeurology, Albert Einstein College of Medicine, Bronx, New York 10461, and the iRadiation and Hyperthermia BiologyLaboratory, Memorial Sloan Kettering Cancer Center, New York, New York 10021
In brain glial cells, expression of calcium independentnitric-oxide synthase (NOS-2) is induced following stim-ulation with bacterial endotoxin (lipopolysaccharide(LPS)) and/or pro-inflammatory cytokines. We have in-vestigated the effects of heat shock (HS), which can re-duce inflammatory responses in several cell types, onthe induction of glial NOS-2 expression. Preincubationof cells for 20–60 min at 43 °C decreased subsequentlevels of NOS-2 induction, with a maximal 80% reductionafter 60 min of HS. Following HS, cells were refractoryto NOS inducers for up to 4 h, after which time little orno suppression was observed. HS reduced cytosolicNOS-2 enzymatic activity (3-fold), steady state mRNAlevels (2–3-fold), and gene promoter activity (by 50%). HSalso reduced LPS-induced nuclear accumulation oftranscription factor NFkB p65 subunit, suggesting per-turbation of NFkB activation.A role for HS protein (HSP) 70 in NOS-2 suppression
by HS is supported by the demonstration that 1) trans-fectionwith humanHSP70 cDNA partially replicatedHSeffects; 2) antisense, but not sense, oligonucleotides di-rected against rat HSP70 partially blocked HS effects;and 3) rat fibroblasts stably expressing human HSP70did not express NOS-2 in response to LPS plus cytokines.As with heat-shocked cells, HSP70-expressing cells alsoexhibited decreased NFkB p65 subunit nuclear accumu-lation. These results demonstrate that in glial cells, aswell as other cell types, NOS-2 induction can be modu-lated by the HS response, mediated at least in part byHSP70 expression.
In brain, inflammatory responses of astroglial cells occurduring disease, infection, and ischemia. This response includesrelease of pro-inflammatory cytokines, such as interleukin 1-b(IL-1b)1 and tumor necrosis factor-a (TNF-a), as well as syn-
thesis and release of nitric oxide (NO). In astrocytes, NO isbiosynthesized by the calcium independent isoform of NO syn-thase (NOS-2) which is normally not present but whose expres-sion is activated by a variety of inflammatory stimuli (1, 2). Invitro studies using primary rat (1, 3, 6), mouse (4), and human(5) astrocytes and glioma cell lines (3, 6, 7) have demonstratedthat stimulation with bacterial endotoxin lipopolysaccharide(LPS) or with a combination of cytokines which nominallyincludes IL-1b leads to de novo expression of NOS-2. In vivo,astroglial NOS-2 expression has been described in demyelinat-ing diseases including experimental allergic encephalomyelitisin rodents (8) and human multiple sclerosis brain samples (9,45), following excitatory stimulation by kainate acid (10) andfollowing transient focal ischemia (11). In some cases, use ofthe selective NOS-2 inhibitor aminoguanidine can diminish theextent of damage (12), providing evidence that astroglial-de-rived NO contributes to pathological damage. Methods to re-duce and/or prevent astroglial NOS-2 induction will thereforebe of value in efforts to reduce inflammatory-related neurolog-ical damage.Several methods have been described for regulation of
NOS-2 expression in vitro (2, 13). Pretreatment of cells withanti-inflammatory agents such as dexamethasone or glucocor-ticoids prevents NOS-2 expression in macrophages (14) andastrocytes (6); while anti-inflammatory cytokines includingIL-4, IL-10, and transforming growth factor-b1 can reduce glialand macrophage NOS-2 induction (6, 16). NOS-2 induction ispotently blocked by specific inhibitors of protein-tyrosine ki-nases, whose activation represents an early, necessary step inthe inflammatory activation of cells (17–19). A third means ofglial NOS-2 regulation conceivably restricted to neural cells isvia endogenous neurotransmitters. Thus activation of b-adre-nergic receptors by norepinephrine reduced astroglial, but notRAW 264.7 macrophage NOS-2, expression (20). Other neuro-transmitters, including ATP and glutamate, can reduce astro-glial NOS-2 expression (21), whereas angiotensin II can reduceNOS-2 expression in C6 glioma (22) and smooth muscle cells(23). Finally, NOS-2 induction requires activation of transcrip-tion factor NFkB (24), and prevention of that activation pre-vents macrophage (24, 25), smooth muscle (26), and astroglial2
NOS-2 expression. The ability of NO itself to down-regulateNOS-2 expression (15, 27) has also been attributed to inhibitionof NFkB activation (28, 29).An additional mechanism for preventing or reducing inflam-
mation and associated damage is the heat shock (HS) response,present in virtually all species from bacteria to human. The HSresponse is elicited by a variety of stimuli, including thermal,chemical, and physical stress, and it is thought that the HS
* This work was supported by a grant from the National MultipleSclerosis Society (D.L.F.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence should be addressed: Division of Neuro-
biology, Cornell University Medical College, 411 East 69th St., NewYork, NY 10021. Tel.: 212-570-2900; Fax: 212-988-3672; E-mail:[email protected].
1 The abbreviations used are: IL-1b, interleukin 1b; TNF-a, tumornecrosis factor a; IFN-g, interferon g; HSP, heat shock protein; NOS-2,inducible form of NOS; NOS, nitric-oxide synthase; CM, cytokine mix-ture; LPS, lipopolysaccharide; GR, glucocorticoid receptor; ODN, oligo-nucleotide; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calfserum; CAT, chloramphenicol acetyltransferase; PCR, polymerasechain reaction; G3PDH, glyceraldehyde-3-phosphate dehydrogenase;PBS, phosphate-buffered saline; TBS, Tris-buffered saline; PAGE, poly-acrylamide gel electrophoresis. 2 D. L. Feinstein, unpublished observations.
response confers resistance against subsequent and more le-thal stress (30). In addition to producing a general down-regu-lation of cellular RNA and protein synthesis, the HS responsealso causes rapid expression of HS proteins (HSPs) which mayprotect cells by facilitating renaturation of partially denaturedproteins. In addition, HSPs can also restrict inflammatory re-sponses themselves. Thus, HSP expression can protect cellsfrom inflammatory damage occurring during zymosan-inducedsynovitis (31), acute pulmonary inflammation (32), cardiac is-chemia (33, 34), and endotoxemia (35, 36). Overexpression ofHSP70 also protects WEHI tumor cells against TNF-a cytotox-icity (37), rat pancreatic islet b-cells against IL-1b effects (38),inhibits LPS-induced monokine synthesis in macrophages (39),and blocks TNF-a production in retina (54). The mechanism(s)underlying these protective effects are not known, but onepossibility is that HSP expression can suppress NOS-2 induc-tion, since NO may contribute to many of these events.The effects of HS on astroglial immune responses have not
been examined. However, HSP expression can be induced inthese cells in vitro (40, 41) as well as in vivo (42), and HSPexpression can be protective in several neuropathologies (30).We have therefore tested the possibility that the HS responsecan reduce and/or prevent astroglial NOS-2 expression. A re-cent report indicates that the HS response can reduce NOS-2induction in rat pulmonary smooth muscle cells (67). We nowshow that HS reduces NOS-2 induction caused by LPS orcytokines in astrocytes and other cell types, and that this effectis mediated by expression of HSPs, notably HSP70. Finally, wepresent evidence suggesting that HS suppresses NOS-2 induc-tion by interfering with NFkB activation, suggesting a novelrole for HSPs in the modulation of inflammatory responses.
EXPERIMENTAL PROCEDURES
Materials—Cell culture reagents (DMEM, antibiotics), NOS cofac-tors (FMN, FADH, tetrahydrobiopterin), n-butyryl coenzyme A, andLPS (Salmonella typhimurium) were from Sigma. Fetal calf serum(FCS) was from Atlanta Biological (Norcross, GA). Recombinant humanTNF-a and IL-1b (both 107 units/mg) were purchased from Genzyme(Cambridge, MA). Recombinant rat IFN-g-g (4 3 106 units/mg), Lipo-fectin, geneticin, and Optimem were from Life Technologies, Inc. Taqpolymerase was from Promega Biotech Inc. Anti-HSP70 monoclonalantibody SPA810 was from Stressgen (Victoria, Canada), and anti-NFkB p65 polyclonal SC-109 was from Santa Cruz Biotechnology(Santa Cruz, CA). Peroxidase-conjugated goat secondary antibodieswere from Vector Laboratories (Burlingame, CA). [32P]dATP (3000 Ci/mmol), L-[14C]arginine (.300 Ci/mmol), [35S]methionine (.1000 Ci/mmol), [14C]chloramphenicol (54 mCi/mmol), and Enhanced Chemilu-minescence (ECL) detection kits were from Amersham. Syntheticphosphodiester oligonucleotides (ODNs) were purchased fromDNAgency (Malvern, PA). Plasmid pCAT-NOS-2 attached to chloram-phenicol transferase (CAT) reporter gene was provided by Qiao-wen Xie(Cornell University Medical College, New York). Plasmids pTK-HSP70and pTK-CAT were provided by Ruben Mestril (University of Califor-nia, San Diego, CA). C6-N cells were a gift of Chris Naus (University ofOntario, London, Canada). RAW 264.7 macrophage cell line was gen-erously provided by Carl Nathan (Cornell University Medical College,New York).Cell Culture—Primary astrocytes were prepared from cerebral cor-
tices of postnatal day 1 Sprague-Dawley rats as described (1). At con-fluency, the cultures were shaken overnight to remove adhering micro-glial cells and used within 2–3 days for experiments. These culturesconsist of greater than 95% astrocytes and between 1 and 3% microglialcells (1). C6-N cells were passaged once per week and maintained inDMEM with 10% FCS. The Rat-1 cell line and transfectants (43, 44)were grown in DMEM containing 10% FCS, and 400 mg/ml geneticinwas added to the transfectant cells when passaged. RAW 264.7 macro-phages were grown as C6 cells.NOS-2 Induction Protocol—The growth medium was removed from
confluent, or near-confluent cells, the cells were washed once in serum-free media, and then NOS-2 inducers were added in fresh serum-freemedia. In some experiments, 1% FCS was present with the inducers tosupply exogenous LPS receptors (CD14) which confer LPS responsive-
ness onto CD14 lacking cell types including glial cells (46). For astro-cytes, LPS-dependent induction was done with 1 mg/ml LPS; for C6cells, with LPS plus 20 units/ml IFN-g. For both cell types, cytokineinduction was done in the presence of a three-cytokine mixture (“CM”):IFN-g (20 units/ml), IL-1b (2 ng/ml), and TNF-a (10 ng/ml). For Rat-1cells and transfectants, NOS-2 was induced in 1% FCS by the combi-nation of LPS plus CM. Incubations were carried out up to 48 h.NOS-2 Activity Measurements—NOS-2 activity was indirectly meas-
ured by accumulation of NO2 in the cell culture media, from 12 to 48 hafter addition of inducers. An aliquot of the media (100 ml) was mixedwith 50 ml of Griess reagent (47), incubated 5 min at room temperature,and the absorbance at 546 nm was determined in a microplate reader.Solutions of NaNO2 diluted in DMEM served as standards.NOS-2 activity was measured directly in cytosolic lysates as the
conversion of L-[14C]arginine to L-[14C]citrulline (1). Assays were donein the presence of excess cofactors (FMN, FADH, tetrahydrobiopterin),20 mM cold arginine, and 2 mM EGTA to chelate free calcium.RNA Analysis—Total cytoplasmic RNA was prepared from cells by
the Nonidet P-40 lysis procedure (48). Levels of NOS-2 mRNA weredetermined by a competitive reverse transcriptase-PCR assay (49). Theprimers used for NOS-2 detection were 1704F (59-CTGCATGGAACAG-TATAAGGCAAAC-39), corresponding to bases 1704–1728; and 1933R(59-CAGACAGTTTCTGGTCGATGTCATGA-39), complementary tobases 1908–1933 of rat inducible NOS cDNA sequence (49). The mRNAlevels of constitutively expressed G3PDH were determined in parallelaliquots to control for differences in cDNA synthesis efficiency. PCRconditions were 35 cycles of denaturation at 93 °C for 35 s, annealing at63 °C for 45 s, and polymerization at 72 °C for 45 s, followed by 10 minat 72 °C. Amplifications were done in the presence of known amounts ofinternal deletion constructs which use the same primers as the cDNAsand [32P]dATP (100,000 dpm/50 ml of assay). PCR products were sepa-rated by agarose gel electrophoresis, bands were excised, and incorpo-rated radioactivity was determined by scintillation counting. cDNAlevels were calculated by comparison with synthesis of internal stand-ards as described (49).Heat Shock Procedure—Cells were incubated for the indicated times
in a cell culture incubator equilibrated to 43 6 0.5 °C, 95% humidity,and 5% CO2. Following incubation, the cells were returned to a 37 °Cincubator and allowed to recover for desired times before inducers wereadded or cells were harvested for analysis.Heat Shock Protein Analysis—Following HS, cells were allowed to
recover for 30 min, after which the medium was replaced with methi-onine-free DMEM containing 50 mCi/ml [35S]methionine. After an ad-ditional 2-h incubation at 37 °C, whole cell lysates were prepared bylysis in 8 M urea. Aliquots containing equivalent amounts of 35S wereelectrophoresed through denaturing 10% polyacrylamide gels, the gelswere dried, and incorporated [35S]methionine was visualized by expo-sure to Kodak XAR film overnight.Stable Transfection of C6 Cells—C6 cells were plated in 60-mm
dishes and, at 30–40% confluency, were transfected with plasmidpCAT-NOS-2 (50) by CaPO4-mediated co-precipitation. One to two mg ofpCAT-NOS-2 was co-precipitated with 0.25 to 0.5 mg of pBK-CMV(Stratagene) which contains a neomycin resistance gene, added to cellsin DMEM containing 10% FCS, and incubated for 4 h. The cells werethen incubated with 15% dimethyl sulfoxide for 3 min and washed,complete medium (DMEM containing 10% FCS) was added back, andcells were allowed to recover. Beginning 3 days later, the medium wasreplaced once per week with complete medium containing 1.2 mg/mlgeneticin. Stable transfectants (C6-1200T) were maintained by passag-ing in complete media containing 1.2 mg/ml geneticin. For experiments,transfectants were passaged into 6- or 12-well plates in the absence ofgeneticin and used within 3 to 4 days.CAT Assays—After an 18- to 24-h incubation in the presence of
NOS-2 inducers, an aliquot of the cell culture media was analyzed fornitrite levels. The cells were then washed in PBS, and 500 ml of lysisbuffer (Promega) was added. The cells were collected and centrifugedfor 3 min at 10,000 3 g to remove membranes. The resulting cell extractwas heated at 60 °C to inactivate endogenous CAT enzymes. A 10–50-mlaliquot of cell lysate (containing 20–100 mg of protein) was incubated at37 °C for 1 h in the presence of 12 mM [14C]chloramphenicol and 0.2mg/ml n-butyryl-CoA. The reaction was halted by extraction with 300 mlof mixed xylenes, the organic phase was back-extracted once with 100ml of 250 mM Tris-Cl, pH 8.0, and the amount of radiolabeled productrecovered in the organic phase was determined by liquid scintillationcounting. For TLC analysis, ethyl acetate was used instead of xylene,the organic phase was dried down, and the material (resuspended in 30ml of ethyl acetate) was separated by TLC in silica plates using chloro-form:methanol (9:1) solvent. After radiography, corresponding areas
were scraped from the plate and counted. Essentially identical resultswere obtained with the two methods of quantitation. In all cases, theamount of cell extract used and time of incubation were controlled toensure that product formation remained within the linear range of theassay.Transient Transfections—C6 cells were transiently transfected by
CaPO4 co-precipitation with plasmid pTK-HSP70 in which expressionof the human HSP70 cDNA clone pH2.3 (51) is under control of theherpes simplex virus thymidine kinase promoter and two copies of theSV40 enhancer. The control vector contained bacterial CAT gene inplace of HSP70. Two days after transfection, the cells were analyzed forNOS-2 induction by addition of LPS plus IFN-g.C6 cells were transiently transfected using Lipofectin and synthetic
phosphodiester oligonucleotides (ODNs). The ODNs (sense: 59-ATGGCC AAG AAA ACA-39; antisense: 59-TGT TTT CTT GGC CAT-39)flank the starting ATG codon of rat HSP70 (52) and differ in 6 of 15bases from the constitutively expressed HSC73 (66). ODNs (25 mg/ml)plus Lipofectin (25 mg/ml) were incubated together at room temperaturefor 15 min in Optimem, then diluted 5-fold with Optimem before use.Subconfluent C6 cells were washed twice with Optimem, then 400 ml ofdiluted LipofectinzODN complex were added. Transfections were car-ried out for 8 h, then cells were washed, and fresh DMEM containing10% FCS was added back. After an additional 16 h, the cells wereincubated at 43 °C for 0 or 40 min, allowed to recover at 37 °C for 30min, and then fresh medium containing LPS plus IFN-g was added toinduce NOS-2 expression, which was measured 18 to 28 h later byaccumulation of NO2 in the culture media.Preparation of Cell Extracts—Whole cell extracts were prepared for
immunoblot analysis by homogenization in 8 M urea, aliquots weremixed with an equal volume of 2 3 SDS gel sample buffer (124 mM
Tris-Cl, pH 6.8, 0.2% SDS, 10% b-mercaptoethanol, 10 mM EDTA, 50%glycerol) and boiled for 5 min. Cytosolic and nuclear extracts wereobtained using a Nonidet P-40 lysis procedure (53). Cells were washedin cold PBS, collected by centrifugation (1,000 3 g for 5 min), and thenresuspended in 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride.After a 15-min incubation on ice, Nonidet P-40 was added to a finalconcentration of 0.6%, the incubation continued for 15 min, and nucleiwere collected by centrifugation at 10,000 3 g for 15 min. The cytosolicfraction was mixed with SDS sample buffer and boiled. The nuclearpellet was washed once in lysis buffer without Nonidet P-40, then lysedin 8 M urea, and prepared with SDS sample buffer for immunoblotanalysis.Immunochemical Procedures—Protein samples (50–100 mg) were
mixed with an equal volume of 2 3 SDS sample buffer, boiled for 5 min,and then separated through 8 or 10% PAGE-SDS gels. After electro-phoresis, proteins were transferred to nylon membranes by semi-dryelectrophoretic transfer. The membranes were blocked in 5% dry milk(1 h), rinsed, and incubated with primary antibodies (1:2000 anti-HSP70; 1:2000 rabbit anti NFkB p65) in Tris-buffered saline (TBS)overnight at 4 °C. Primary antibody was removed, membranes werewashed 4 times in TBS, and 0.1 mg/ml peroxidase-labeled goat second-ary antibodies was added for 1 h. Following 4 washes in TBS, bandswere visualized by ECL and exposure to x-ray film.For immunostaining, cells were plated directly onto poly-L-lysine-
coated glass slides and grown for 2–3 days in complete media. Atdesired times after addition of LPS plus CM, the cells were washedtwice in ice-cold PBS, fixed 1 h at room temperature in 4% paraform-aldehyde, washed twice in TBS, permeabilized with 0.3% Triton X-100in TBS, and then incubated with anti NFkB p65 antibody overnight at4 °C. The cells were washed 4 times in TBS, and signals were visualizedusing the peroxidase-anti-peroxidase method with an ABC staining kit(Vector Laboratories) and diaminobenzidine as chromagen. The per-centage of cells which stained positively for nuclear p65 was determinedby counting at least 4 different fields of 30–50 cells each, and consid-ering only those cells with strong, clear nuclear staining as positive.Data Analysis—All experiments were done at least three times and
expressed as means 6 S.E. Statistical significance was assessed byone-way analysis of variance followed by Fisher’s post hoc tests, and pvalues ,0.05 were considered significant.
RESULTS
Heat Shock Reduces NOS-2 Expression—Incubation of ratastrocytes or C6 glioma cells with pro-inflammatory agents(LPS or a cytokine mixture, CM, consisting of IFN-g, IL-1b, andTNF-a) leads to de novo expression of NOS-2 mRNA, protein,and activity, and the extent of this induction can be assessed by
NO2 accumulation in the cell culture media (1, 3, 6, 7, 49). Todetermine if HS could influence NOS-2 induction and/or activ-ity, we incubated C6 cells or astrocytes for various times at43 °C, after which NOS-2 inducers were added, and NO2 levelswere determined 18–24 h later (Fig. 1). HS up to 20 min did notaffect NOS-2 induction. However, after a 45-min HS, subse-quent C6 cell NOS-2 induction was decreased to 63 6 9% (n 55) when induced by CM and to 41 6 8% (n 5 7) when inducedby LPS of non-heat-shocked cell induction. Essentially identi-cal results were obtained with primary astrocyte cultures, inwhich a 45-min HS resulted in 75 6 7% (CM, n 5 9) and 54 69% (LPS, n 5 9) induction compared to controls, and maximalsuppression occurred after a 60-min HS which reduced activityto 27 6 3% of control values for either inducer. HS of glial cellsfor up to 60 min did not promote release of intracellular lactatedehydrogenase (data not shown). Longer incubation times at43 °C and other HS temperatures were not examined.To determine if HS effects were long-lasting, cells were heat-
shocked, then allowed to recover for various times at 37 °Cbefore the addition of NOS-2 inducers (Fig. 2). The extent ofNOS-2 suppression decreased with increasing recovery time.Maximal suppression was observed if inducers were addedwithin 15 min of HS, and no significant reduction occurred ifinducers were added 4 h (or longer, not shown) after HS.Essentially identical kinetics of suppression were obtainedwith both astrocytes and C6 cells, and no significant differenceswere observed between the use of LPS or CM to induce NOS-2expression. These results indicate that the HS-induced effectsare transient. Furthermore, HS also suppressed NOS-2 expres-sion if carried out within 4 h after addition of the inducers,whereas HS at 8 h after addition of inducers was without effect(Fig. 2, filled squares). These findings suggest that for at least4 h after addition of LPS and/or cytokines, the pathways lead-ing to NOS-2 expression are sensitive to HS effects.To test the possibility that HS effects on NO2 accumulation
were due to reduced intracellular substrate and/or cofactoravailability, we directly measured NOS-2 activity in cytosoliclysates prepared from control or heat-shocked astrocytes thathad been incubated for 20 h with LPS (added 20 min after HS).When incubated in buffer containing excess cofactors and 20mM L-arginine, the heat-shocked lysates displayed 28 6 4%activity compared to control cells (31 6 5 versus 107 6 9 pmolof L-citrulline formed per 20 min per 200 mg of protein, p , 0.05,n 5 6). These results demonstrate that decreased NO2 accu-mulation correlated with a decrease in levels of active cytosolicNOS-2 protein, and that decreases in necessary cofactors orL-arginine do not account for HS effects.
FIG. 1. Effect of heat shock duration on NOS-2 expression. C6cells were incubated at 43 °C for the indicated times, placed at 37 °C for45 min, and then fresh medium containing NOS-2 inducers (either LPSplus IFN-g, or cytokine mixture, CM) was added. Accumulated NO2levels in the culture media were determined 18–24 h later using Griessreagent. Data shown are means 6 S.E. of 5–7 independent determina-tions and are relative NO2 accumulations of heat-shocked versus controlcells. *, p , 0.05 versus control cells.
Decreased NOS-2 protein levels could be due to changes inNOS-2 mRNA levels and/or protein translation. To determine ifmRNA levels were decreased, we measured NOS-2 mRNA lev-els in control and heat-shocked astrocytes 4 h after addition ofLPS (Fig. 3), a time at which NOS-2 mRNA levels are near-maximal (49). HS decreased NOS-2 mRNA levels approxi-mately 3-fold compared to control cells, a reduction comparableto that observed in NO2 accumulation. HS had little or no effecton levels of G3PDH mRNA, suggesting a selective suppressionof newly induced gene expression.To determine if decreased NOS-2 promoter activity could
contribute to HS effects on NOS-2 mRNA, we tested HS effectson C6-1200T cells which stably express the mouse NOS-2 pro-moter (50) attached to the bacterial CAT reporter gene (Fig. 4).Since the majority of the NOS-2 coding region and entire 39-untranslated region are absent from the CAT construct, anyeffects of HS on NOS-2 mRNA stability mediated by theseregions will be eliminated. The levels of CAT activity in cyto-solic lysates of heat-shocked cells were significantly decreasedcompared to control cells (43 6 2% of control, n 5 6–9, p ,0.005), and the extent of this reduction was comparable to,although somewhat less than, the decrease observed in NO2
accumulation measured in culture media from the same cells(23 6 1% of control, p , 0.005). These results indicate that HSblocks steps necessary for NOS-2 promoter activation, al-though additional effects on mRNA stability may also contrib-ute to the reduction in NOS-2 mRNA levels.To determine if HS effects were cell-specific, we examined
NOS-2 expression in the mouse macrophage RAW 264.7 cellline (13). RAW cells were heat-shocked for 40 min at 43 °C,allowed to recover for 10, 30, or 60 min, and then 1 mg/ml LPSwas added to induce NOS-2 expression assessed 20 h later bymeasurement of accumulated NO2 in the media. For all recov-ery times, HS slightly diminished NOS-2 expression (89 6 5%,91 6 2%, and 82 6 5% of control values at 10, 30, and 60 min,respectively, n 5 6 for each time point); however, only at 60 minwas this decrease statistically different from control values.This demonstrates that HS can suppress NOS-2 induction inother cell types (and see below), although the extent and kinet-ics of suppression may depend upon the particular cell typetested.HSP70 Mediates Heat Shock Effects on NOS-2 Expression—
The HS response includes both a general down-regulation of
cellular activities, including protein translation and mRNAtranscription, as well as selective induction of HSP expression(30). To determine which HSPs could mediate HS effects inglial cells, we monitored the pattern of [35S]methionine incor-poration in astrocytes and C6 cells following HS (Fig. 5). Inastrocytes, HS stimulated the synthesis of two major proteinproducts, the major band corresponding to HSP70, and asmaller protein corresponding to HSP32 (this band was notdetected with an antibody to HSP25, data not shown). HS of C6cells also stimulated synthesis of these two HSPs, as well as of
FIG. 2. Effect of time interval between heat shock and additionof NOS-2 inducers. C6 cells were heat-shocked for 45 min at 43 °C andthen allowed to recover at 37 °C for the indicated lengths of time beforeLPS plus IFN-g (open circles) or cytokines (filled circles) were added.Alternatively, LPS and IFN-g were added to C6 cells, and HS (45 minat 43 °C) was carried out at the indicated times (filled squares). NOS-2activity was assessed 18–24 h after inducers were added. The datashown are means 6 S.E. of 6–9 independent determinations and arerelative NO2 accumulation of heat-shocked versus control cells. *, p ,0.05 versus non-heat-shocked cells. §, p , 0.005 versus non-heat-shocked cells.
FIG. 3. Effect of heat shock on NOS-2 mRNA levels. Astrocyteswere heat-shocked for 0 or 45 min at 43 °C, allowed to recover for 40min at 37 °C, and then fresh medium containing LPS (1 mg/ml) wasadded. After 4 h, total cytoplasmic RNA was isolated, an aliquot wasconverted to cDNA, and NOS-2 and G3PDH mRNA levels were meas-ured by competitive PCR analysis (49). A, representative agarose gelshowing PCR amplification of NOS-2 and G3PDH in the presence ofindicated amounts of internal standards. B, log-log plot of ratio of[32P]dATP incorporated into cDNA to internal standard versus fg ofinternal standard added to PCR. The point at which log [ratio] 5 0corresponds to initial amounts of cDNA present, and in this experimentcorresponds to 1.6 fg (E, control) and 0.6 fg (●, heated) NOS-2 productpresent in 200 ng of template cDNA. The data shown are representativeof 2 separate experiments.
FIG. 4. Effect of heat shock on NOS-2 promoter activity. C6–1200T cells were heat-shocked for 45 min at 43 °C, allowed to recoverfor 40 min at 37 °C, and then fresh medium containing LPS plus CMwas added. After a 20-h incubation, NOS-2 activity was assessed bymeasurement of NO2 in the culture media, and NOS-2 promoter activ-ity was determined by cytosolic CAT activity. The data shown are themeans 6 S.E. of 6–9 independent determinations, and are the relativeactivities of heat-shocked versus control samples. **, p , 0.005 versuscontrol samples. Control NOS-2 activity was 80 6 5 nmol of NO2 per20 h per mg of protein; control CAT activity was 740 6 25 pmol ofacetylated chloramphenicol per h per 25 mg of cytosolic protein.
proteins corresponding to HSP60, -90, and -110. Since thepredominant labeled product in both cell types was HSP70, wefocused attention on the possible role of this protein in medi-ating suppressive effects of HS on NOS-2 induction. Immuno-blot analysis (Fig. 6) of whole cell extracts revealed that follow-ing HS of astrocytes, HSP70 accumulation began within 30min, reached maximal levels between 4 and 8 h, and was stillpresent at 24 h. Low levels of HSP70 were detectable in thenon-heat-shocked cells. Similar kinetics of appearance wereobserved for HSP70 expression in C6 cells.To determine if HSP70 could mediate HS effects, we heat-
shocked C6 cells which had first been transfected with sense orantisense oligonucleotides (ODNs) directed against rat HSP70mRNA (Fig. 7). As expected, HS reduced subsequent NOS-2activity to 45 6 3% of control values. Transient transfection (8h) with antisense, but not sense, ODNs partially reversed thisreduction, resulting in cells expressing 70 6 7% of controlNOS-2 activity levels (n 5 3, p , 0.05). Longer transfectiontimes were less effective than the 8 h used (not shown). Thatonly partial reversal of the HS effect was observed may suggestinvolvement of other HSPs, degradation of ODNs during thevarious incubation periods, and/or limited transfectionefficiencies.The above results suggested that HSP70 expression could
account, at least in part, for the ability of HS to reduce NOS-2expression. To confirm this possibility, we transfected C6 cells
with the human HSP70 cDNA (51) or with vector alone (Fig. 8).The cells were allowed to express for 2 days, then were treatedwith LPS and IFN-g. Cells transfected with vector alone exhib-ited significantly reduced NOS-2 expression (53 6 2% of controlcells, n 5 3, p , 0.05) possibly due to induction of endogenousrat HSPs, as previously noted (43, 44). However, transfectionwith the HSP70 containing plasmid led to a 2-fold furtherdecrease in NOS-2 induction (27 6 2% of control cells, n 5 3,p , 0.05 versus vector alone) suggesting that expression ofhuman HSP70 protein in rat glial cells can also reduce NOS-2induction.The results obtained from transient transfection experi-
ments may be complicated by activation of endogenous HSPsby the transfection procedures. To further confirm that HSP70could replicate HS effects, we examined NOS-2 expression (Fig.9) in transfected Rat-1 cells which stably express full-lengthhuman HSP70 (43, 44). Rat-1 cell lines, in contrast to astro-cytes or C6 cells, required a mixture of LPS plus CM as well asthe presence of 1% FCS to obtain maximal levels of NOS-2induction (data not shown). NOS-2 expression in parentalRat-1 cells amounted to roughly 15% of that obtained with glialcells (NO2 accumulation was 11 6 3 nmol of per 24 h per mg ofprotein, n 5 10). As found for glial cells, prior HS treatment ofRat-1 cells reduced subsequent NOS-2 induction (to 70% of
FIG. 5.HSP induction in astrocytes and C6 cells. Glial cells wereheat-shocked at 43 °C for 40 min, allowed to recover for 30 min at 37 °C,and then the medium was replaced with methionine-free DMEM and 25mCi/ml [35S]methionine. After a further 2-h incubation at 37 °C, wholecell lysates were prepared in 8 M urea, subjected to SDS-PAGE electro-phoresis, and newly synthesized proteins were visualized by overnightexposure of the dried gel to x-ray film. The positions of molecular weightmarkers (left side) and approximate molecular weights of induced HSPs(right side) are indicated. Similar results were obtained in a second setof experiments.
FIG. 6. Time course of HSP70 appearance in heat-shocked as-trocytes. Astrocytes were heat-shocked for 40 min at 43 °C, then al-lowed to recover at 37 °C for the indicated times before whole celllysates were prepared. Equal aliquots of protein (50 mg) were separatedby SDS-PAGE and analyzed for HSP70 levels by immunoblot analysis.The gel shown is representative of three separate experiments.
FIG. 7. Antisense oligonucleotides to HSP70 reduce heat shockeffects on NOS-2 expression. C6 cells were transfected with 5 mg/mlsense or antisense ODNs directed against rat HSP70 for 8 h, allowed torecover for 16 h in complete media, then heat-shocked for 0 or 40 min at43 °C before LPS plus IFN-g was added to induce NOS-2 expression.NOS-2 activity was assessed by NO2 accumulation in the culture media(18–28 h). Data shown are means 6 S.E. from three independentdeterminations and are relative NOS-2 activities compared to non-transfected, non-heat-shocked cells. *, p , 0.05 versus nontransfectedheat-shocked cells.
FIG. 8. Transfection with human HSP70 reduces NOS-2 ex-pression. C6 cells were transfected with 4 mg of plasmid p2.3 (contain-ing human HSP70) or vector only. Two days later, LPS plus IFN-g wereadded to induce NOS-2 expression, assessed 22 h later by measurementof NO2 levels in the culture media. data are means 6 S.E. of threeindependent determinations, and similar results were obtained in asecond set of experiments. *, p , 0.005 versus vector-transfected cells.
control values). Neomycin selection alone caused a significantup-regulation of NOS-2 expression (MV-6 cell activity was 20 66 nmol of NO2 per 24 h per mg of protein, n 5 7) possiblyrelated to the decreased division rate of transfectants com-pared to Rat-1 cells (data not shown). HS of MV-6 cells reducedNOS-2 induction to 63 6 7% of control values (p , 0.05 versuscontrol cells, n 5 3). In contrast to MV-6 cells, two independ-ently isolated lines of Rat-1 cells stably expressing humanHSP70 protein exhibited markedly reduced NOS-2 expression(in absence of HS) compared to either parental Rat-1 cells orMV-6 cells (M-21 cells, 1.7 6 1, n 5 8; M-25 cells, 3.5 6 1, n 54; nanomoles of NO2 per 24 h per mg of protein, p , 0.05 forboth versus either Rat-1 or MV-6 cells). The expression ofHSP70 in unheated M-21 and M-25 cells, and not in MV-6 cells,was confirmed by immunoblot analysis (43, 44, and data notshown). These observations demonstrate that constitutive ex-pression of HSP70 can also prevent NOS-2 induction.To determine if HSP70 expression, achieved in the absence of
thermal stress, could also reduce NOS-2 mRNA levels, wemeasured NOS-2 mRNA in MV-6 and M-21 cells 4 h afteraddition of LPS plus CM. In MV-6 cells, LPS plus CM elevatedNOS-2 mRNA levels 63-fold over background levels (3,800 ver-sus 60 fg of NOS-2 cDNA per mg of RNA, n 5 2), whereas inM-21 cells NOS-2 mRNA was increased less than 3-fold (20versus 8 fg of NOS-2 cDNA per mg of RNA). In neither cell typewere G3PDH mRNA levels altered by LPS plus CM treatment.Thus, HSP70 expression alone is sufficient to block NOS-2mRNA accumulation.Heat Shock Reduces NFkB Translocation—The above results
suggest that heat shock or HSP70 reduces NOS-2 expression byblocking NOS-2 promoter activation. Since activation of tran-scription factor NFkB is necessary for NOS-2 induction (24–26), we tested if HS or HSP70 expression perturbed NFkBsubunit p65 activation as assessed by nuclear accumulation(Fig. 10). In control C6 cells, incubation with LPS plus CMcaused nuclear uptake of p65, beginning at 20 min (not shown),maximal at 60 min (77% of cells showed clear nuclear staining),and diminished, but still present, at 90 min (28% positive). Inheat-shocked C6 cells, nuclear uptake of p65 commenced atapproximately the same time as control cells (not shown), at alltimes examined was reduced compared to control cells (at 60min 38% positive), and was almost absent at 90 min (,15%positive). Similar results were obtained when comparing con-
trol to heat-shocked astrocyte cultures (not shown). In Rat-1cells, LPS plus CM induced comparable levels of nuclear p65staining in both MV-6 and M-21 cells when examined from 0 to60 min after inducer addition (approximately 67% positive at30 min in both cell types). However, at 90 min, MV-6 cellscontinued to have strong nuclear staining (38% positive), butM-21 cells exhibited little or no staining (,5% positive). Thepresence of the p65 protein in M-21 cells rules out the possi-bility that lack of inducible NOS-2 expression in these cells isdue to absence of p65.To confirm that nuclear p65 levels were reduced in heat-
shocked cells, as well as verify the identity of the nuclearantigen detected, we subjected cytosolic and nuclear extractsfrom control and heat-shocked astrocytes to immunoblot anal-ysis (Fig. 11). In the absence of LPS, the NFkB p65 subunit wasdetected in the cytosolic, but not the nuclear fraction of bothcontrol and heat-shocked astrocytes (lane 2). In control cells, a30-min incubation with LPS caused appearance of p65 in thenuclear fraction, as well as a corresponding loss from the cyto-plasm (lane 1). In contrast, nuclear p65 levels were greatlyreduced in heat-shocked astrocytes, although still present inthe cytosol (lane 3). Together, these results support the conclu-sion that HS and HSP70 expression reduces NFkB p65 nuclearaccumulation following stimulation with LPS and/or cytokines.
DISCUSSION
In this paper we demonstrate that inflammatory activationof the NOS-2 gene by either LPS or cytokines can be modulated
FIG. 9. NOS-2 expression in Rat-1 fibroblast transfectants.Rat-1 cells, stably transfected with human HSP70 (M-21, M-25 cells) orvector only (MV-6 cells), were incubated for 24 to 48 h with LPS plusCM in the presence of 1% FCS to induce NOS-2 expression, assessed24–48 h later by NO2 accumulation. In some cases, cells were heat-shocked at 43 °C for 40 min prior to addition of NOS-2 inducers (HS).Data shown are means 6 S.E. of between 3 and 10 independent deter-minations and are nanomoles of NO2 accumulated per 24 h per mg ofprotein. *, p , 0.05 versus non-heat-shocked cells; **, p , 0.005 versusnon-heat-shocked MV-6 or Rat-1 cells.
FIG. 10. Heat shock and HSP70 expression reduce NFkB sub-unit p65 nuclear accumulation. C6 cells (control or heat-shocked for40 min at 43 °C), MV-6, and M-21 cells were incubated with LPS plusCM in the presence of 1% FCS for the indicated times, fixed, and thenprocessed for immunostaining for the presence of NFkB p65 subunit.The results shown are representative of two separate experiments.Magnification is 3 200.
by the HS response. In glial cells, HS reduced NO2 accumula-tion, cytosolic L-citrulline formation, steady state NOS-2mRNA levels, and NOS-2 promoter activity. That the effects ofHS are mediated, at least in part, by HSP70 expression, andnot due to the general down-regulation of transcriptional andtranslation processes that accompany HS, is supported by ourfindings that: 1) HSP70 is synthesized in these cells followingHS; 2) antisense ODNs directed against rat HSP70 partiallyblocked HS effects; and 3) in glial cells and in Rat-1 fibroblasts,overexpression of HSP70, achieved in the absence of thermalstress, also reduced NOS-2 expression. HS also decreasedNOS-2 expression in mouse RAW 264.7 cells and Rat-1 fibro-blasts indicating that HSP70 regulation of NOS-2 expression iscommon to several cell types, although the magnitude of sup-pression varied between the three cell types examined. Thesefindings suggest that HSPs, in addition to providing protectiveeffects against protein denaturation, can also regulate the ini-tiation of inflammatory events themselves.HS reduced NOS-2 expression by blocking transcription of
this gene, a conclusion supported by the observations that HSreduced steady state NOS-2 mRNA levels, promoter activity(as assessed by induction of CAT activity), and nuclear accu-mulation of the NFkB p65 subunit, a key step in NFkB activa-tion (55–57) and necessary for NOS-2 gene expression (24–26).Nuclear uptake of p65 was also perturbed in HSP70 expressingM-21 cells; however, decreased nuclear levels were only ob-served at 90 min after addition of NOS-2 inducers, in contrastto results with heat shock which diminished nuclear p65 levelsat all times examined. The mechanisms by which HS caninterfere with the activation of NFkB are not yet known. How-ever, one possibility is that HSP70, which also translocates tothe nucleus (59), impedes NFkB nuclear translocation by com-peting for access to nuclear pore complexes through whichNFkB is transported (58). Alternatively, HSP70 could impedeNFkB activation by direct interaction with one (or more) of theNFkB constituents. The association of inhibitory IkB withNFkB p50 and p65 subunits occurs via interaction of IkBankyrin domains with nuclear localization sites present in thep50 and p65 proteins (57). Mutational analysis has confirmedthe presence of a nuclear localization site region in humanHSP70 (59), which raises the possibility that HSP70 can spe-cifically interact with ankyrin domains present in IkB. Such aninteraction could conceivably hinder IkB phosphorylation and
subsequent dissociation of NFkB. However, whether HSP70physically interacts with IkB remains to be determined.A precedent for ascribing a role for HSP70 in regulating
nuclear uptake of NFkB exists in the regulation of steroidreceptor nuclear translocation (60, 61, 65). The glucocorticoidreceptor (GR) resides in the cytosol as a large heteromericcomplex containing two molecules of HSP90, and, upon hor-mone binding, dissociation of HSP90 allows the GR to moveinto the nucleus. Moreover, HSP70 is also a part of the GRcomplex in transfected Chinese hamster ovary cells (62), rathepatocytes (63), and in recombinant human GR (64). Thus, theability to regulate protein uptake into the nucleus may be acommon feature of several members of the HSP family.Whereas our results suggest that both HS and HSP70 dimin-
ish p65 nuclear uptake, others have failed to detect effects ofHS or HSP70 on NFkB activation (37, 54). One factor whichmay contribute to this discrepancy is the time at which NFkBmeasurements are made. Thus, in our cells clear differences innuclear p65 levels were observed between control and heat-shocked C6 cells at all times examined, while in Rat-1 cellsdifferences were observed only at the 90-min time point. Asecond consideration is that conclusions that HSP70 does notaffect transcriptional activation, based solely upon DNA shiftassays (37, 54), may be complicated by the fact that transcrip-tionally inactive NFkB complexes lacking p65, for example p50homodimers, can also bind to kB sites and result in decreasedelectrophoretic mobilities.Another difference between our observations and others con-
cerns the activation of the NOS-2 promoter. Our results dem-onstrate that expression of the bacterial CAT gene, under con-trol of the NOS-2 promoter, is also reduced by HS (Fig. 4),suggesting that HS blocks transcriptional activity at theNOS-2 promoter. This conclusion is strengthened by our obser-vations that HS reduced NFkB p65 nuclear uptake, which isnecessary for NOS-2 promoter activation. It has recently beenreported (67) that in pulmonary smooth muscle cells the HSresponse, achieved by incubation with sodium arsenite, abol-ished the increase in NOS-2 mRNA levels induced by IL-1b,with no reduction in NOS-2 promoter activity. It was concludedthat arsenite-induced HS response either decreased NOS-2mRNA stability, or that the reporter gene construct used waslacking HS-sensitive regions. Since the portion of the NOS-2promoter used in our studies is identical to the one used byWong et al. (67), it is likely that HS-sensitive elements arepresent in this region. It is conceivable that arsenite inductionof HS response, unlike thermal stress-induced HSP70 expres-sion, results in a pattern of HSP expression that does notimpede NFkB activation. Alternatively, the use of stable celllines (here) versus transient lines (Wong et al. (67)) could ac-count for the contradictory results. Finally, it should be notedthat interpretation of the experiments of Wong et al. (67), maybe complicated by their observations that addition of arsenitealone (in the absence of IL-1b) was, in some experiments, alsoan effective inducer of NOS-2 promoter activity.Comparison of the duration of the HS-mediated inhibition
(Fig. 2) to the time course of HSP70 protein expression (Fig. 6)revealed that, in glial cells, the maximal suppressive effects ofHS were obtained when NOS-2 inducers were added immedi-ately or soon after HS, times at which HSP70 was not yetpresent. A similar discordance was observed for HS suppres-sion of LPS-induced TNF-a mRNA increase (39), and thoseauthors proposed that cellular events preceding HSP70 induc-tion were responsible for inhibition of LPS effects. However, wefavor an alternate explanation, namely that following additionof LPS and/or cytokines, the cascade of events leading to NOS-2induction requires a period of time such that HSP70 is present
FIG. 11. Heat shock reduces nuclear NFkB p65 subunit levels.Astrocytes were heat-shocked for 0 or 40 min at 43 °C, allowed torecover at 37 °C for 30 min, LPS was added, and nuclear and cytosolicextracts were prepared 30 min later. Equal amounts of protein (50 mg)were subjected to SDS-PAGE, and transferred proteins were analyzedby immunoblot for the presence of the p65 subunit. Similar results wereobtained in two other experiments.
at the same time an HSP70-sensitive step occurs. Consistentwith this possibility are our results (Fig. 2) demonstrating thatHS potently blocked NOS-2 induction even when carried out upto 4 h after the addition of LPS plus IFN-g. Although theidentity of the HSP70 sensitive step is not yet known, a likelycandidate is nuclear uptake of p65, which does not commencefor at least 10 to 20 min following addition of NOS-2 inducers.In this case, the initial nuclear uptake of p65 may not beimpeded immediately after HS; however, HSP70 expressionduring the next several hours could interfere with sustainedp65 uptake and thereby lead to significantly reduced finallevels.On the other hand, 4 h after HS, when HSP70 levels were at
or near maximal, NOS-2 induction was no longer impeded. Onefactor that may help reconcile these observations is if thesubcellular localization of the HSP70 protein is important topromoting suppressive effects. It is known that at early timesfollowing HS, HSP70 protein accumulates mostly in the nu-cleus while several hours later localization is mainly in thecytosol (56). Similar kinetics of nuclear localization are ob-served in heat-shocked glial cells.2 Thus, the window of sup-pression in heat-shocked cells may reflect a restricted periodduring which time HSP70 is in the correct subcellular locationto exert suppressive effects.Based upon our results and the above discussions, we pro-
pose the following model to explain the effects of HS on NOS-2expression: Following HS, HSP70 expression begins within 30min and continues to accumulate for the next several hours.During this time, stimulation with NOS-2 inducers initiatesNFkB activation, resulting in gradual release of NFkB fromIkB, and nuclear uptake commences within 20–30 min afterstimulation. At this time, HSP70 levels are still low, so initialnuclear uptake is probably not impeded. However, within thenext 30–60 min, HSP70 levels are sufficient to reduce NFkBnuclear uptake. The mechanism by which HSP70 blocks NFkBuptake are not yet known. However, the knowledge thatHSP70 also enters the nucleus suggests that simple competi-tion for nuclear pore complexes may be occurring. Alterna-tively, or in addition, HSP70 could bind to the NFkB complex asit dissociates from IkB, a state which may share features of apartially denatured protein. The decreased NFkB nuclear up-take results in decreased maximal levels attained as well as ashorter duration of nuclear NFkB content. As a consequence,we expect that binding of NFkB to the NOS-2 promoter isgreatly reduced, thereby preventing efficient transcriptionalactivity and NOS-2 expression. Although the precise molecularmechanisms involved require further elaboration, this modelprovides a working basis for further studies of HSP70 effects onNFkB activation.The expression of NOS-2 during brain pathologies has been
suggested to contribute to the damage occurring during ische-mia, demyelinating diseases, including multiple sclerosis, fol-lowing excitotoxic damage, and during viral infection. In mostof these pathologies, there is induction of HSPs, considered tobe an internal response of neurons to protect themselves fromfurther damage. Protective effects of HS and/or HSP70 expres-sion in cardiac ischemia (33, 34, 52), sepsis (35, 36), and otherinflammatory diseases (32, 36–38) have also been ascribed tothe ability of HSPs to prevent irreversible protein denatur-ation. However, reports that HS, or HSP70, can block cytokinesynthesis (39, 54), phospholipase A2 activation (37), and NOS-2induction (67), together with the findings presented here, leadto the conclusion that prevention of inflammatory responsesmay contribute to the protective actions of HSPs. We thereforepropose that expression of HSPs, and particularly HSP70, pro-
vides a novel mechanism by which cells can restrict inflamma-tory reactions.
Acknowledgment—We thank Liubov Lyandvert for continued excel-lence in the preparation and maintenance of cell cultures.
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