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Archives of Biochemistry and BiophysicsVol. 374, No. 2, February 15, pp. 325–333, 2000doi:10.1006/abbi.1999.1646, available online at http://www.idealibrary.com on

Proteasome Inhibition in Neuronal Cells Induces aProinflammatory Response Manifested by Upregulation ofCyclooxygenase-2, Its Accumulation as UbiquitinConjugates, and Production of the Prostaglandin PGE2

Patricia Rockwell,* Hongmei Yuan,* Ronald Magnusson,† and M. E. Figueiredo-Pereira*,1

*Department of Biological Sciences, Hunter College of CUNY, New York, New York 10021; and †Department ofPharmacology, Mount Sinai Medical School of CUNY, New York, New York 10028

Received August 20, 1999, and in revised form November 22, 1999

Inclusions containing ubiquitin-protein aggre-gates appear in neurons of patients with neurode-generative disorders such as Alzheimer’s diseaseand Parkinson’s disease. The relationship betweeninclusion production and cell viability is not under-stood. To address this issue, we investigated the re-sponse of an established mouse neuronal cell lineand of embryonic rat mesencephalic cultures to in-hibition of the ubiquitin/proteasome pathway. Twoproteasome inhibitors, a peptidyl aldehyde and anepoxy ketone, which cause accumulation of ubiqui-tinated proteins, were found to enhance expressionof stress-inducible genes, including HSP70i and thepolyubiquitin genes UbB and UbC. Under these con-ditions, mRNA and protein levels of the inducibleform of cyclooxygenase (COX-2) were upregulatedtogether with its product, PGE2, a proinflammatoryprostaglandin. Proteasomal inhibition also led tostabilization of COX-2 as ubiquitin conjugates, sug-gesting that the ubiquitin/proteasome pathway con-tributes to the regulation of COX-2 protein levels.Treatment with antioxidants known to inhibit NFkBand AP-1 transcriptional activation failed to abro-gate COX-2 upregulation. Instead, these inhibitorsexacerbated the stress response by potentiatingHSP70i levels while eliciting a decrease in PGE2 pro-

uction. These findings suggest that the accumula-ion of ubiquitinated proteins resulting from protea-ome inhibition in neuronal cells is associated withproinflammatory response that may be an impor-

ant contributor to neurodegeneration. © 2000 Academic

Press

1

To whom correspondence should be addressed. Fax: (212) 772-5227. E-mail: pereira@genectr.hunter.cuny.edu.

0003-9861/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

Key Words: proteasome inhibition; ubiquitinaggregates; inflammation; cyclooxygenase 2;neurodegeneration.

The ubiquitin/proteasome pathway rapidly degradesubiquitinated proteins. These generally do not accu-mulate in cells. However, ubiquitin-protein aggregatesare commonly detected in intraneuronal inclusions ofpatients with neurodegenerative disorders [reviewedin (1)]. The involvement of ubiquitin in neurologicaldisorders is generally accepted, but its role in the neu-rodegenerative pathway is not understood (2). Theubiquitin/proteasome pathway plays a major role inthe breakdown of abnormal proteins resulting fromstress conditions and in the degradation of short-livedregulatory proteins (3). Presumably, accumulation ofubiquitin-protein aggregates must result from a mal-function or overload of the ubiquitin/proteasome path-way or from structural changes on protein substratesthat halt their degradation. Failure to eliminate ubi-quitinated proteins disrupts homeostasis and thusmay contribute to cellular degeneration (4).

Recent epidemiological studies link nonsteroidal an-ti-inflammatory drugs (NSAID)2 with delays in theclinical expression of Alzheimer’s disease (AD) [re-viewed in (5)]. However, the mechanisms by whichNSAIDs affect the pathophysiological pathways lead-

2 Abbreviations used: AD, Alzheimer’s disease; COX-1 and COX-2,cyclooxygenase-1 and -2, respectively; HSP70i, inducible form of theheat shock protein 70; NSAIDs, nonsteroidal anti-inflammatorydrugs; PDTC, pyrrolidine dithiocarbamate; PGs, prostaglandins;

PSI, N-benzyloxycarbonyl-Ile-Glu(O-tert-butyl)-Ala-leucinal; Z-LL-CHO, N-benzyloxycarbonyl-Leu-leucinal.

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326 ROCKWELL ET AL.

ing to AD remain unclear. Some NSAIDs inhibit theactivity of cyclooxygenases, which are the rate-limitingenzymes in the synthesis of prostaglandins (PGs) fromtheir precursor arachidonic acid. Cyclooxygenases re-side in the lumen of the ER and are anchored to itsinner membrane layer by amphipathic helices, whichparticipate in the openings to the active sites (6). Whilethe constitutive isoform of cyclooxygenases (COX-1) isresponsible for synthesis of protective PGs, expressionof the inducible isoform (COX-2) leads to the produc-tion of PGs and reactive oxygen species that promotetissue damage (7).

Our studies investigate the response of neuronalcells to inhibition of the ubiquitin/proteasome path-way. Overall, they show that proteasome inhibition iscytotoxic and causes activation of two major pathways:(1) the stress pathway manifested by increased expres-sion of the stress-inducible genes HSP70, UbB, andUbC, and (2) the inflammatory pathway manifested byan increase in COX-2 expression and production of theproinflammatory prostaglandin PGE2. Moreover, theysuggest that COX-2 protein levels are modulated viadegradation by the ubiquitin/proteasome pathway. To-gether, our results suggest that the neurotoxicity as-sociated with malfunction of the ubiquitin/proteasomepathway and accumulation of ubiquitin-protein aggre-gates correlates with activation of the prostanoid syn-thesis pathway and possible generation of free radicalsby COX-2. Therefore, the ubiquitin/proteasome path-way may interact with the inflammatory pathway inthe cascade of events leading to neurodegeneration.

MATERIALS AND METHODS

Cell culture. HT4 cells were derived from a mouse neuroblastomacell line containing a recombinant temperature-sensitive mutant ofSV40 large T antigen (8). When grown at 39°C (nonpermissive tem-perature), HT4 cells differentiate with neuronal morphology, expressneuronal antigens, synthesize and secrete nerve growth factor, andexpress receptors for NGF (8) and for glutamate (9). Cells weremaintained as previously described (10). Cultures of rat embryonicmesencephalon were prepared and maintained as previously de-scribed (11).

Cell treatment. Cells were incubated at 37°C without (vehicleonly) or with increasing concentrations of the following proteaseinhibitors: (1) N-benzyloxycarbonyl-Ile-Glu(O-tert-butyl)-Ala-leuci-nal (PSI), a reversible inhibitor of mainly the chymotrypsin-likeactivity of the proteasome, which also affects calpain and cathepsinB at the higher concentrations tested (12), (2) N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO), a calpain/cathepsin B inhibitor, whichdoes not affect the proteasome (12), and (3) epoxomicin, an irrevers-ible inhibitor of mainly the chymotrypsin-like activity of the protea-some, which does not affect calpain/cathepsin B at the concentra-tions tested (13). The two peptidyl aldehydes were kindly provided byDr. Sherwin Wilk from Mount Sinai Medical School, New York, NY,and epoxomicin was obtained from Dr. Craig Crews from Yale Uni-versity, New Haven, CT. Protease inhibitor solutions were made indimethyl sulfoxide (DMSO) and added directly to the culture mediafor 24 h (Western blot, immunoprecipitation, Northern blot, RT-PCR,

and ELISA analyses) or also for 48 h (cytotoxicity studies). Inhibitorsof NFkB activation [pyrrolidine dithiocarbamate (PDTC) in DMSO]

and of AP-1 activation (curcumin in ethanol), both obtained fromSigma Chemical Co. (St. Louis, MO), were added to the culturemedia 10 min prior to treatment with PSI.

Preparation of cell extracts for Western blotting. Following theindicated treatments, cell extracts were prepared and subjected toSDS–PAGE as previously described (12). Identification of the ubiqui-tinated proteins, HSP70i, HSP90i, COX-1, and COX-2, was by West-ern blotting. The antigens were visualized by a horseradish peroxi-dase method (Bio-Rad Laboratories, Richmond, CA) utilizing thesubstrate 3,39,5,59-tetramethylbenzidine (Kirkegaard and PerryLaboratories, Gaithersburg, MD) or the ECL detection system (Am-ersham Corp., Arlington Heights, IL). Quantitative analysis of theimmunostaining was by image analysis with the ImagePC programfrom NIH as described previously (14).

Antibodies for Western blotting. Ubiquitin–protein conjugateswere detected with a rabbit polyclonal antibody (1:600) from DakoCorp. (Carpinteria, CA). Anti-HSP70i is a mouse monoclonal anti-body (1:3000) obtained from StressGen (Victoria, BC, Canada). Anti-HSP90i is a rabbit polyclonal antibody (1:2000) obtained from Affin-ity BioReagents (Neshanic, NJ). Anti-COX-1, a mouse monoclonalantibody (1:200), and anti-COX-2, a goat polyclonal antibody (1:300),

ere obtained from Santa Cruz Biotechnology (Santa Cruz, CA).Immunoprecipitation. Immunoprecipitation of COX-2 from total

ell extracts normalized to protein concentration was performed asreviously described (15). The immunocomplexes were resolved on% SDS gels, followed by Western blot analysis probed with thenti-COX-2 antibody, followed by stripping and reprobing with thenti-ubiquitin conjugate antibody.Northern blot analysis. Twenty micrograms of RNA per well was

lectrophoresed, blotted, and hybridized sequentially with HSP70ind b-actin cDNA probes and with the ubiquitin antisense oligonu-leotide probe, following a standard procedure (16). The mouseSP70i (HSP70-1) cDNA was from plasmid pH-7 (17), the b-actin

cDNA was from plasmid pA1 (18), and the ubiquitin probe (GAAT-GCAGACCGAATAATTCAGAG) from the mouse polyubiquitin Bgene (19) was synthesized by GenSet (La Jolla, CA). All lanes con-tained the same amount of RNA as judged by ethidium bromidestaining of the gel. Quantification was by densitometry.

RT-PCR analysis. RNA was isolated with the Totally RNA Kitfrom Ambion (Austin, TX). One microgram of RNA per sample wasreverse transcribed and the resultant cDNA was amplified withgene-specific primers for mouse UbA, UbB, UbC, COX-2, and anactin control using kits for two-step RT-PCR (Qiagen, CA). Primerswere designed from cDNA sequences obtained from GenBank underthe following Accession Numbers: M11690 for mouse UbA, X51703for mouse UbB, S40697 for mouse UbC, and M88242 for mouse

OX-2.PGE2 concentrations. Levels of PGE2 released into the media of

treated cells were measured by an enzyme-linked immunoassay(Cayman Chemicals, Ann Arbor, MI). For all experiments, cells wereseeded at a density of 4.4 3 103 cells/cm2 in six-well plates (35-mm-diameter wells). Amounts of PGE2 produced were expressed as %ontrol, which corresponded, in untreated HT4 cells, to 103.3 pg ofGE2/ml of media.Cell viability assay. Cell viability was assessed with a modifica-

tion of the method described in Mosmann (20) by the 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previouslydescribed (10).

Protein determination. Protein determination was by a bicincho-ninic acid assay kit (Pierce Chemical Co., Rockford, IL), using bovineserum albumin as a standard.

Statistical analysis. Statistical comparisons were performed

with the Tukey–Kramer multiple-comparison test (Instat 2.0,Graphpad Software, San Diego, CA).

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327PROTEASOME INHIBITION, UBIQUITIN INCLUSIONS, AND INFLAMMATION

RESULTS

Cytotoxic effect of prolonged inhibition of the chymo-trypsin-like activity of the proteasome. Cytotoxicitystudies were performed to investigate the long-termeffects of proteasome inhibition on HT4 cells. Treat-ment for 24 h with epoxomicin, the highly potent spe-cific inhibitor of the proteasome, reduced cell viabilitymoderately to a maximum of 43% in cells treated with10 mM of the drug (Fig. 1A). However, after a 48-hincubation with epoxomicin, cell viability was dramat-ically reduced to 10% of control in cells treated withconcentrations as low as 0.1 mM (Fig. 1A). Similarresults were obtained with PSI, as 48-h incubationswith 25 mM PSI resulted in 84% cell death (Fig. 1B).These data establish prolonged treatments (48 h) withproteasome inhibitors as detrimental incubation con-ditions for inducing cytotoxicity. Conversely, the dipep-tidyl aldehyde inhibitor of calpain/cathepsinB (Z-LL-CHO), which does not affect the proteasome activity(21), had no effect on HT4 cell survival even at aconcentration of 25 mM (Fig. 1B).

Proteasome inhibition increases the accumulation ofbiquitinated proteins. To address the effectivenessf proteasome inhibition mediated by PSI or epoxomi-in, Western blots of total lysates from control and4-h-treated cells were probed with a specific antibodyo detect ubiquitin-protein conjugates. These wereound to accumulate in cells treated with PSI concen-rations as low as 250 nM (Fig. 2A). Ubiquitin-conju-ate levels were increased 62-fold in cells exposed to 25

mM PSI and at least 3-fold in cells treated with ep-oxomicin concentrations as low as 100 nM (Fig. 2B).

FIG. 1. Effect of protease inhibitors on the viability of HT4 cellsssessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumromide assay. Data represent the mean and SE of eight determi-ations and are shown as percentage of cell viability measured witho inhibitors (control, 100%). Cell treatments were as follows: (h)poxomicin (24 h); (■) epoxomicin (48 h); (F) PSI (48 h); (E) Z-LL-

CHO (48 h).

Overnight incubations with Z-LL-CHO produced no

detectable accumulation of ubiquitinated proteins (Fig.2C) at the concentrations tested (2.5 nM to 25 mM).

Proteasome inhibition increases expression ofHSP70i and ubiquitin but not of HSP90i. It is widelyestablished that most cellular treatments resulting inaccumulation of abnormal or denatured proteins alsoinduce a stress response (22). Based on these findings,we sought to determine whether or not the accumula-tion of ubiquitinated proteins generated by proteasomeinhibition in HT4 cells occurred with a concomitantchange in the levels of stress-inducible proteins. West-ern blots of total lysates prepared from either PSI- orepoxomicin-treated HT4 cells were probed separatelywith antibodies that recognize the inducible form ofHSP70 (HSP70i) or HSP90 (HSP90i). Treatments withPSI (Fig. 2D) or epoxomicin (Fig. 2E), but not Z-LL-CHO (Fig. 2F), raised the concentrations of HSP70i tolevels that paralleled the increases in ubiquitinatedproteins shown in Figs. 2A and 2B. In cells treatedwith 25 mM PSI, the levels of HSP70i were 62-foldhigher than in those treated with 250 nM of the sameinhibitor. Incubations with 5 mM epoxomicin enhancedHSP70i levels by more than 18-fold compared to theuntreated control. Neither PSI nor Z-LL-CHO affectedthe expression of HSP90i (Figs. 2G and 2H, respec-tively).

To determine if proteasome inhibition inducedchanges in HSP70i and ubiquitin gene expression, to-

FIG. 2. Immunoblots showing ubiquitinated proteins (A–C),HSP70i (D–F), and HSP90i (G and H) in HT4 cell extracts (10 mg ofprotein/lane). Cells were incubated at 37°C for 24 h without (lanes 1)or with PSI (A, D, and G), epoxomicin (B and E), or Z-LL-CHO (C, F,and H). The following concentrations of inhibitors were tested inlanes 2–6: PSI (2.5 nM, 25 nM, 250 nM, 2.5 mM, and 25 mM),epoxomicin (0.1 mM, 0.5 mM, 1 mM, 5 mM, and 10 mM), and Z-LL-CHO (2.5 nM, 25 nM, 250 nM, 2.5 mM, and 25 mM). The blots

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328 ROCKWELL ET AL.

tal RNA preparations from control and PSI-treatedcells were subjected to Northern blot analyses. Theresults showed that HSP70i and ubiquitin mRNA lev-els in PSI-treated cells were increased in a concentra-tion-dependent manner (Fig. 3). In cells treated 24 hwith 25 mM PSI, HSP70i (left panel) and ubiquitin(center panel) mRNA levels were stimulated 20- and6-fold, respectively, relative to untreated controls,

FIG. 3. Northern blot hybridization of total RNA extracted fromcontrol (CON, vehicle only) and PSI (250 nM and 25 mM) treated HT4cells. The cells were treated for 24 h at 37°C with the inhibitor.Twenty micrograms of RNA per well was electrophoresed, blotted,and hybridized sequentially with HSP70i (left panel) and b-actincDNA probes (right panel) and with a ubiquitin antisense oligonu-cleotide probe (center panel). The two lanes from each condition arefrom duplicate samples.

FIG. 4. Semiquantitative RT-PCR detection of UbA (A), UbB (B), UNA from HT4 cells treated without (lanes 1) or with PSI (lanes 2–6f RNA were analyzed by semiquantitative RT-PCR. The results shohe left represent semiquantification of the respective bands by d

ifferent from control, with P , 0.05 or less for all values markedultiple-comparison test as described under Materials and Methods.

whereas those of b-actin mRNA levels (right panel)decreased 45% (Fig. 3).

PSI upregulates the expression of ubiquitin B and Cbut not of ubiquitin A. To determine which ubiquitingenes were upregulated in PSI-treated cells, RT-PCRanalyses were performed with total RNA using specificprimers for the three ubiquitin genes (UbA, UbB, and

bC) and actin as a control. These experiments in-olved analyses of HT4 cells incubated 24 h in thebsence and presence of 1–50 mM PSI. Amplification

products of the predicted fragment sizes and sequenceswere obtained for actin and each ubiquitin gene. Thespecific levels of gene expression obtained from eachtreatment are shown in Fig. 4 together with the plotsresulting from semiquantitative analyses of corre-sponding band intensities. Interestingly, RT-PCRanalysis of UbB mRNA yielded several bands that in-tensified with increasing PSI concentrations. Each ofthese bands corresponds to the predicted fragmentslocated within the mouse UbB gene, which codes forour ubiquitin units in mouse and humans. PSI con-entrations up to 25 mM (Fig. 4B) elicited a marked

increase in UbB mRNA levels to a maximum of 2-fold

(C), COX-2 (D), and actin (E) gene expression. After isolation of totalmM, 5 mM, 10 mM, 25 mM, and 50 mM, respectively), equal amountsare representative of three independent experiments. The plots on

itometry. The asterisk identifies the values that are significantly

bC, 1wnens

. Statistical comparison was performed with the Tukey–Kramer

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329PROTEASOME INHIBITION, UBIQUITIN INCLUSIONS, AND INFLAMMATION

of controls (P , 0.001) whereas UbC mRNA consis-tently showed increased levels that peaked to 1.5-foldof controls at 5 and 25 mM PSI (Fig. 4C). Inhibitorconcentrations of 50 mM resulted in inhibition of UbAand actin expression and no increases in UbB and UbClevels. However, the changes in UbA and actin mRNAlevels (Figs. 4A and 4E) were not statistically signifi-cant (P . 0.05).

Proteasome inhibition upregulates COX-2 gene andprotein expression and leads to accumulation of ubiqui-tinated COX-2. Given the cytotoxicity caused by pro-teasome inhibition, we then investigated the possiblerelationship between PSI treatment and a proinflam-matory response. RT-PCR analyses with gene-specificprimers revealed that PSI upregulated COX-2 expres-sion. COX-2 mRNA levels were strongly raised (P ,0.001), peaking with a 3.8-fold increase over controllevels in cells treated with 5 mM PSI (Fig. 4D). At 25mM PSI, a second peak of COX-2 upregulation wasobserved, yielding the maximum increase (4.4-fold) inmRNA levels. A decrease in COX-2 expression wasconsistently observed with 10 mM PSI and may reflecta transcriptional downregulation modulated by PGE2

levels as reported for porcine aortic smooth musclecells (23).

To determine if proteasome inhibition affects COX-2protein expression, Western blots of total lysates pre-pared from HT4 cells were probed with an anti-COX-2polyclonal antibody. In these studies, cell treatmentswere performed for 24 h with increasing concentrationsof PSI or epoxomicin. Anti-COX-2 immunoreactivitydetected a doublet of ;72/74 kDa and a high molecular

ass form at the top of the gel (Figs. 5A and 5B) in cellsreated with proteasome inhibitors but not in cellsreated with the calpain/cathepsin B inhibitor (Fig.C). Upregulation of cyclooxygenase by PSI was spe-

FIG. 5. Immunoblots showing COX-2 (A–C) and COX-1 (D and E)in HT4 cell extracts (20 mg of protein/lane). Cells were incubated at7°C for 24 h without (lanes 1) or with PSI (A and D), epoxomicin (Bnd E), or Z-LL-CHO (C). The following concentrations of inhibitorsere tested in lanes 2–6: PSI (1 mM, 5 mM, 10 mM, 25 mM, and 50

mM), epoxomicin (0.1 mM, 0.5 mM, 1 mM, 5 mM, and 10 mM), andZ-LL-CHO (1 mM, 5 mM, 10 mM, 25 mM, and 50 mM). The blotspresented are representative of one of at least three identical exper-iments for each condition tested. Ub-COX-2, ubiquitinated COX-2.

ific for COX-2. Protein expression of COX-1, which is

constitutive in HT4 cells, was not raised by treatmentwith increasing concentrations of either proteasomeinhibitor (Figs. 5D and 5E).

To further characterize the COX-2 immunoreactivehigh molecular mass form, total lysates prepared fromPSI-treated HT4 cells were subjected to immunopre-cipitation with the anti-COX-2 antibody. Western blotsof the immunoprecipitated proteins probed with theanti-COX-2 antibody revealed a pattern of reactivity(Fig. 6A) similar to that observed in Fig. 5A. Whenthese blots were stripped and reprobed with the anti-ubiquitin conjugate serum, the high molecular massCOX-2 immunoreactive form was also recognized bythe anti-ubiquitin conjugate antibody (Fig. 6B). Thesefindings demonstrate that proteasome inhibition re-sults in upregulation of COX-2 and its accumulation ashigh molecular mass ubiquitin conjugates.

Proteasome inhibition results in prostaglandin pro-duction. To evaluate changes in COX-2 functional ac-tivity, culture media from 24-h-treated cells were ana-lyzed for production of the proinflammatory prosta-glandin PGE2. These measurements revealed thatincreases in product formation accompanied rises inCOX-2 protein expression. Accordingly, Table I showsthat PSI and epoxomicin elicited a concentration-de-pendent increase in PGE2 production. For example, 25mM PSI enhanced PGE2 media concentrations by 8-foldover the control value. Treatment with the calpain/cathepsin B inhibitor did not alter PGE2 levels (notshown).

FIG. 6. Effect of PSI on COX-2 expression in HT4 cells incubated at37°C for 24 h without (lanes 1 and 3) or with 5 mM PSI (lanes 2 and4). The anti-COX-2 antibody was used to immunoprecipitate COX-2proteins from total cell extracts. Immunoblots of the precipitatedproteins were probed with anti-COX-2 antibody (A) and anti-ubiq-uitin conjugate antibody (B). The blots presented are representative

ot f one of at least three identical experiments for each conditionested. Ub-COX-2, ubiquitinated COX-2.

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330 ROCKWELL ET AL.

Proteasome inhibition induces accumulation of ubi-quitinated proteins, upregulation of HSP70i andCOX-2, and an increase in PGE2 levels in primarycultures of rat embryonic mesencephalon. Studies

ith primary cultures of rat embryonic mesencephalonere performed to establish that the PSI-mediatedffect was not restricted to the transformed HT4 neu-onal cell line utilized in this investigation. Like HT4ells, 24-h incubations of rat mesencephalic culturesith 10 mM PSI resulted in increased levels of ubiqui-

tinated proteins, HSP70i, COX-2, and PGE2 (Figs.7A–C and Table I, respectively). However, unlike HT4cells, the same responses were lowered by treatment

TABLE I

Effect of Proteasome Inhibition on Production of theProinflammatory Prostaglandin PGE2 by Mouse HT4

Neuronal Cells and Rat Embryonic PrimaryMesencephalic Cultures

PSI(mM)

% Control

Epoxomicin(mM)

% Control

HT4 cellsPrimarycultures HT4 cells

0 100 100 0 1001 208 — 0.01 2375 469 — 0.1 361

10 594 142 1 60125 802 91 5 52150 2319 — 10 403

Note. PGE2 concentrations are expressed relative to control condi-ions where cells were incubated in the absence of proteasome inhib-tors, which are assigned values of 100% 5 103.3 pg of PGE2/ml of

media for HT4 cells and 143.2 pg of PGE2/ml of media for the ratprimary cultures. The data represent means of at least two experi-ments for each condition.

FIG. 7. Immunoblots showing ubiquitinated proteins (A), HSP70i(B), and COX-2 (C) in rat embryonic mesencephalic cultures (15 mg of

rotein/lane). Cells were incubated at 37°C for 24 h without (lanes 1)r with the following concentrations of PSI: 10 mM (lanes 2) and 25

emM (lanes 3). The blots presented are representative of one of twoexperiments for each condition tested.

with 25 mM PSI. These findings are attributed to dif-ferent sensitivities of HT4 cells and mesencephalic cul-tures to PSI treatment.

Regulation of PSI-induced COX-2 gene expression.The COX-2 gene contains DNA-binding sites for NFkB,AP-1, AP-2, g-interferon activation site (GAS), C/EBP,and SP1 in its immediate promoter region (24, 25).However, a number of studies have provided evidencethat NFkB plays an important regulatory role inCOX-2 gene activation (26–28). To determine if NFkB

ediates COX-2 expression stimulated by proteasomenhibition, HT4 cells were treated with PSI in combi-ation with PDTC, an antioxidant that attenuatesFkB activity (29). The treated cells were then ana-

lyzed for changes in (1) COX-2 mRNA and proteinlevels, (2) accumulation of ubiquitinated proteins, and(3) PGE2 production. Pretreatment with 25 mM PDTCttenuated PSI-induced rises in COX-2 mRNA (Fig.A, lane 4) and protein levels (Fig. 8C, lane 5) by 23nd 11%, respectively, and PGE2 production by 55%

(Table II). In HT4 cells treated with 50 mM PDTC incombination with PSI, COX-2 protein expression and

FIG. 8. A: Semiquantitative RT-PCR detection of COX-2 gene ex-pression analyzed in HT4 cells. The bars on the right (B) representquantification of the respective bands by densitometry in one repre-sentative experiment. C–F: Immunoblots showing COX-2 (C and D)and HSP70i (E and F) in HT4 cell extracts (15 mg of protein/lane).The asterisk indicates a third band recognized by the COX-2 anti-body, which is present only in cells treated with PDTC and PSI. Theblots presented are representative of one of three experiments foreach condition tested. In these experiments, cells were incubated at37°C for 24 h as shown, with inhibitor concentrations expressed asmM.

PGE2 production were still increased over control lev-ls. These results demonstrate that upregulation of

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331PROTEASOME INHIBITION, UBIQUITIN INCLUSIONS, AND INFLAMMATION

COX-2 expression still occurs under conditions ex-pected to abrogate NFkB activation. Most likely, tran-scription factors other than NFkB contribute to theincrease in COX-2 expression, since this factor is pre-sumably inhibited by treatment with PSI in combina-tion with PDTC. In addition, increased levels of COX-2protein expression in PSI-treated cells may result fromprotein stabilization via a blockade of its degradationby the ubiquitin/proteasome pathway. The PSI-depen-dent COX-2 stabilization may be further exacerbatedin the presence of PDTC. Western blot analysis of cellstreated with a combination of PSI and either 25 or 50mM PDTC showed the presence of an additional COX-2immunoreactive band marked by an asterisk in Fig.8C, lanes 5 and 6, that is, most likely, the unglycosy-lated 65-kDa form of COX-2 (30). The appearance ofthis lower molecular mass band may reflect a PDTC-mediated interference in the mechanism responsiblefor conversion of the inactive unglycosylated 65-kDaform of COX-2 to the active N-glycosylated 72/74-kDaforms.

To determine whether AP-1 modulates PSI-inducedCOX-2 expression, HT4 cells were pretreated with cur-cumin, an anticancer agent known to inhibit activationof the transcription factor AP-1 and COX-2 expressionin human gastrointestinal epithelial cells (31) andNFkB in TNFa-activated Jurkat T lymphoma cells(32). Contrary to these findings, concentrations of 25mM curcumin failed to abrogate the rise in COX-2mRNA (Fig. 8A, lane 6) and protein levels (Fig. 8D,lane 5) triggered by PSI treatment. However, curcumininhibited PGE2 production by more than 75% (TableII). This dramatic decrease in PGE2 production proba-bly reflects the ability of curcumin to inhibit COX-2enzymatic activity via a mode of action that is unre-lated to its ability to inhibit AP-1 transcriptional acti-vation. Likewise, curcumin was shown to inhibit

TABLE II

Effect of the Antioxidants PDTC and Curcumin onPSI-Induced Production of the Proinflammatory

Prostaglandin PGE2 by Mouse HT4 Neuronal Cells

PSI(mM)

% Control

PDTC Curcumin

0 25 mM 50 mM 25 mM 50 mM

0 100 40 73 64 5110 594 259 152 135 85

Note. PGE2 concentrations are expressed relative to control condi-ions where cells were incubated in the absence of any drugs, whichre assigned values of 100% 5 103.3 pg of PGE2/ml of media for HT4

cells. The data represent means of at least two experiments for eachcondition.

COX-2 enzymatic activity in human gastrointestinal

epithelial cells (31). Neither PDTC nor curcumin af-fected the accumulation of ubiquitinated proteins in-duced by PSI treatment (not shown).

PDTC and curcumin exacerbate the PSI-inducedstress response. A previous study demonstrated thatPDTC inhibition of NFkB was linked to a stress re-sponse in primed endothelial cells (33). Consistent withthese findings, Western blot analysis revealed 2.4-foldincreases in HSP70i for HT4 cells treated with PSI and25 mM PDTC or curcumin (Figs. 8E and 8F, lanes 5)and 4.5-fold rises for combined treatments with PSIand 50 mM PDTC or curcumin (Figs. 8E and 8F, lanes6) relative to cells treated with PSI alone (Figs. 8E and8F, lanes 4). PDTC (50 mM) or curcumin (50 mM) alonealso elevated HSP70i levels (Figs. 8E and 8F, lanes 3).These data suggest that inhibition of the transcrip-tional activity of either NFkB or AP-1 affects expres-sion of genes important in the maintenance of cellularhomeostasis.

DISCUSSION

We have found that a 48 h exposure of mouse neu-ronal HT4 cells to proteasome inhibitors (PSI and ep-oxomicin) dramatically decreases cell viability whileepoxomicin incubations of 24 h are less toxic. However,24-h treatments with proteasome inhibitors still resultin accumulation of ubiquitinated proteins and of thestress-inducible protein HSP70i in the mouse neuronalHT4 cells and in rat primary mesencephalic cultures.

Our results are consistent with those of Goldbergand co-workers (34) and others (35) in providing evi-dence for activation of stress-inducible genes, includingHSP70i, in response to proteasome inhibition. In addi-tion, our studies show that mRNA levels of two otherstress-inducible genes, namely, UbB and UbC, werestrongly increased in HT4 cells treated with PSI. Inhumans, UbB and UbC code for four and nine copies ofthe ubiquitin gene, respectively (36). Upregulation ofthese polyubiquitin genes must be an important tran-scriptional stress response as a mechanism to preventubiquitin shortages when cells require increased ratesof ubiquitin/proteasome-dependent proteolysis. Ac-cordingly, studies with yeast showed that overexpres-sion of polyubiquitin genes conferred resistance to ox-idative stress in cells grown by respiration (37). Nev-ertheless, treatment of HT4 cells with 50 mM PSIresults in a loss of cell viability and an overall decreasein ubiquitin gene expression, with UbB being the mostaffected. Aberrant ubiquitin, resulting from a frame-shift mutation at the transcriptional level of the UbBgene, was detected in brain lesions of AD patients (38).Under these conditions, a shortage in wild-type ubiq-uitin may prevent ubiquitin/proteasome-dependentdegradation in affected cells and contribute to neuro-

nal death.

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332 ROCKWELL ET AL.

In addition to the stress response, proteasome inhi-bition triggered an inflammatory response manifestedas an increase in COX-2 mRNA and protein levels,together with an upregulation of one of its products,the proinflammatory prostaglandin PGE2. The highmolecular mass (;200 kDa) COX-2 immunoreactive

aterial, which accumulates in a PSI- or epoxomicin-oncentration-dependent fashion, corresponds toOX-2 conjugated to polyubiquitin chains of differentizes. These experiments are the first to reveal thatOX-2 is a substrate of the ubiquitin/proteasome path-ay. These results support the notion that elevated

evels of COX-2 protein induced by proteasome inhibi-ion in neuronal cells result from both an increase in itsene expression and a decrease in its proteolysis.Our results also show that the COX-2 protein de-

ected in PSI- or epoxomicin-treated cells appears as aoublet representing the 72/74-kDa bands reported asariable forms of N-glycosylated COX-2 (30). It is in-eresting to note that, in our studies, the 74-kDa bands less visible in the unstimulated control and intensi-es with increasing concentrations of either protea-ome inhibitor tested. These data indicate that lowevels of the 72-kDa COX-2 protein are normallyresent in neuronal cells and accumulation of its alter-ate N-glycosylated 74-kDa form occurs as a result ofroteasome inhibition. The role played by this mecha-ism will require future analysis.The transcriptional mechanisms responsible for PSI-

nduced activation of COX-2 were examined using var-ous approaches. The COX-2 promoter region containsinding sites for several transcriptional factors, includ-ng C/EBP, NFkB, AP-1, AP-2, GAS, and Sp1 (24, 25).

Of these, numerous reports have implicated NFkB asthe participatory factor in COX-2 induction under con-ditions of oxidative stress (28), hypoxia (27), and in-flammation (26). However, consistent with previousstudies (13, 39), we found that PSI and epoxomicinproduce little change in the protein levels of IkBa, anNFkB inhibitor that is degraded prior to NFkB activa-tion (results not shown). Moreover, PDTC, an inhibitorof NFkB transcriptional activation (33), decreases, butdoes not abrogate, PSI-induced COX-2 mRNA and pro-tein expression. To further investigate the regulatorymechanisms underlying COX-2 induction by protea-some inhibition, HT4 cells were treated with the anti-cancer agent curcumin, which inhibits AP-1-mediatedCOX-2 transcription in phorbol ester treated epithelialcells (31). In contrast to this result, we find that cur-cumin fails to diminish the PSI-induced COX-2 mRNAexpression. Evidence that curcumin inhibits mecha-nisms regulated by AP-1, NFkB, and protein kinase C(31, 32) rules out the possibility that these factors aresolely responsible for PSI-mediated COX-2 upregula-tion. Most likely, activation of distinct regulatory

mechanisms contributes to the PSI-mediated COX-2

1

expression in HT4 neuronal cells. A similar interpre-tation was used to explain the inhibitory mechanismsof aspirin (40) and caffeic acid phenethyl ester (41) inthe acute inflammation air-pouch model, where COX-2expression was shown to be prevented by targetingNFkB-independent pathways.

The increase in HSP70i expression observed in HT4cells treated with PSI, PDTC, or curcumin alone isconsistent with stress-related studies from other labo-ratories (33, 34, 42). Activation of the stress responseby PDTC and curcumin was attributed to inhibition ofNFkB activity (32, 33). Interestingly, potentiation ofthe stress response, manifested by a greater increasein HSP70i protein levels, was observed when PSI treat-ment was combined with either PDTC or curcumin.The stress response triggered by proteasome inhibitionmay be associated with deregulation of transcriptionalactivity. Whether accumulation of ubiquitinated pro-teins precedes or coincides with HSP70i and COX-2expression remains to be established, but the threephenomena occur prior to cell death. Delineation of thephysiological steps leading to these responses may de-fine novel therapeutic targets of intervention to pre-vent irreversible cell damage resulting from a proin-flammatory response.

ACKNOWLEDGMENTS

We thank Dr. S. Wilk of the Department of Pharmacology atMount Sinai School of Medicine, New York, NY, for PSI and Z-LL-CHO and Dr. C. Crews of the Department of Molecular, Cellular andDevelopmental Biology at Yale University, New Haven, CT, for ep-oxomicin. We are also grateful to Dr. C. Mytilineou of the Depart-ment of Neurology at Mount Sinai School of Medicine for the ratembryonic mesencephalic cultures and to Ms. Romia Bull for edito-rial comments. National Institutes of Health Grants NS34018 (toM.E.F.-P.) and RR03037 (Research Centers in Minority Institutions)supported this work.

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