Articles

Heat Shock Protein 27 Expression in Human Proximal Tubule Cells Exposedto Lethal and Sublethal Concentrations of CdCI2Seema Somji, Donald A. Sens, Scoff H. Garrett, Mary Ann Sens, and John H. Todd

Robert C. Byrd Health Sciences Center, Department of Pathology, West Virginia University, Morgantown, West Virginia, USA

The expression of hsp 27 mRNA and protein was determined in cultured human proximaltubule (HPT) cells exposed to lethal and sublethal concentrations of Cd2+ under both acute andextended conditions. Initial procedures demonstrated that HPT cells display the classic stressresponse following physical and chemical stess. Heat stres (42.5`C for 1 hr) caused an increasein both hsp 27 mRNA and protein as well as a shf in the protein to a more phosphorylatedstate. Results were similar when the edls were subjected to chemical s (exposure to 100 pMsodium arsenite for 4 hr). Acute exposure to 53 pM CdCI2 for 4 hr also resulted in an increase inhsp 27 mRNA and protein and a shift to the more phosphorylated protein isoform. ExtndedCd2+ exposure involved continuous treatment with +C.2 at both lethal and sublethal level overa 16-day ime coure. The results of this treatment showed that chronic exposure to Cd2* failedto increase either hsp 27 mRNA or protein expression in HPT cell, even at lethal C2 concen-trations. In fict, hisp 27 protein levels decreased as compared to contros at both lethal and sub-lethal eposure to C&. These findings imply that hp 27 expressionin hum an proximal tubulecells may have two distinct modes depending on the natur (acute vs. chronic) of the stress. Keyuorda cadmium, gene expression, heat shock, heavy metals, hp 27, proximal tubule, sodiumarsenite. Envin Heaut Perpe 107:545-552 (1999). [Online 2 June 1999]http://ehpnetl.niehs.nih.gov/docs/I999/107p545-552somji/absraatMmnl

The kidney, and in particular the proximaltubule, is critically affected by chronic expo-sure to the environmental pollutant Cd2+ inboth animals and humans (1,2). Nephro-toxicity results from a slow accumulation ofCdP in the proximal tubules of the kidney.The earliest markers of chronic Cd2+ nephro-toxicity are disorders of proximal tubuletransport characterized by low-molecular-weight proteinuria, increased high-molecular-weight protein excretion, and a variety ofother proximal tubule ion transport disorders(1,3-6). Selective and direct absorption ofCd2+ by proximal tubules has been demon-strated using microinjection techniques (7).In an effort to define the fundamental mech-anistic processes underlying human Cd2+_induced nephrotoxicity, we used a cell culturemodel of the human proximal tubule (HPT).The HPT cell culture model retains impor-tant features of proximal tubule cell differen-tiation (8-10). These retained features arestable with cell passage and include a consis-tent enzyme histochemical profile, sodium-dependent glucose transport, parathyroidhormone stimulation of cAMP, generation ofan apical-negative potential difference, andthe presence of gap junctions. When thesecells are exposed to sublethal concentrationsof Cd2+, they exhibit the transport and ultra-structural alterations expected from in vivoknowledge of Cd-induced nephrotoxicity(11-13). Also in agreement with in vivo find-ings is the fact that at lethal CdP concentra-tions, the cells undergo necrotic cell death(11). This model system is currently beingused to define the roles and interactions of

the stress response superfamily of proteinsin protection and recovery from Cd2+ expo-sure. Initial examinations centered on themetallothionein gene family because theseproteins are known for their ability to bindand sequester heavy metals (14-16). Thecurrent study focuses on the role of thestress response protein, hsp 27, as a possiblemediator of Cd2+-induced nephrotoxicity.This examination was motivated by recentstudies demonstrating that enhanced hsp 27expression has a role in the protection andrecovery of the proximal tubule cell duringand after brief periods of renal ischemia(17-19). This finding suggested that hsp 27expression may also have a role in the pro-tection of the renal proximal tubule fromthe cytotoxic effects of the environmentalpollutant, cadmium.

Hsp 27 is a member of a large superfam-ily of proteins with molecular weights rang-ing from 8 to 170 kD and collectivelyreferred to as the heat shock (hsp) or stressresponse proteins (20,21). In humans, hsp27 is encoded by a single active gene locatedon chromosome 9 (22). Cell lines that over-express hsp 27 protein exhibit an enhancedability to survive and recover from heat stress(23-29). Increasing hsp 27 expression bytransiently or stably transfecting cell linesconfers increased cellular resistance to a vari-ety of toxicants including doxorubicin,daunorubicin, actinomycin D, vincristine,colchicine, arsenite, hydrogen peroxide, andtumor necrosis factor (23-25,27,30). Hsp27 appears to exert its effects on cell survival,at least in part, through a chaperone action

that stabilizes microfilament dynamics. Hsp27 regulates actin dynamics, and hsp 27 over-expression prevents microfilament disruptionand enhances mitogen-stimulated actin poly-merization (23-25,31,32). Hsp 27 alsodemonstrates actin capping activity (25,31).Hsp 27 is phosphorylated at serine residues inresponse to heat shock or mitotic stimuli,suggesting a role in the regulation of signaltransduction pathways. Recent studies alsoindicate that hsp 27 is involved in the regula-tion of programmed cell death in several celllines (33-35).

Materials and MethodsCell culture. Stock cultures of HPT cellswere grown in 75-cm2 T-flasks using proce-dures previously described by this laboratory(8,X2. The growth medium was a serum-freeformulation consisting of a 1:1 mixture ofDulbecco's modified Eagles' medium andHam's F- 12 growth medium supplementedwith selenium (5 ng/mL), insulin (5 pg/mL),transferrin (5 pg/mL), hydrocortisone (36ng/mL), triiodothyronine (4 pg/mL), andepidermal growth factor (10 ng/mL). Thegrowth surface was treated with a collagenmatrix to promote cell attachment and sub-culture. The cells were fed fresh growthmedium every 3 days, and at confluence(normally 3-6 days post subculture) weresubcultured using trypsin-EDTA (0.05%,0.02%). For use in experimental protocols,the cells were subcultured in six-well plates ata 1:2 ratio and allowed to reach confluence(6 days after subculture) before initiation ofexperimental protocols. The cells were fedevery 3 days. HPT cells between passages 5and 7 were used in the present study. Thethree isolates of HPT cells were derived fromnormal cortical tissue obtained from kidneysremoved for renal cell carcinoma. The kid-neys were from a 72-year-old female, a 63-year-old male, and a 58-year-old female.

Cell viability. The effect of the varioustreatments on the viability of confluent cell

Address correspondence to J.H. Todd, Departmentof Pathology, West Virginia University, PO Box9203, Morgantown, WV 26506 USA. Telephone:(304) 293-3212. Fax: (304) 293-6249. E-mail:jtodd2@wvu.eduThis publication was made possible by grant

ES07687 from the National Institute of EnvironmentalHealth Sciences, NIH, and by the Department ofPathology Research and Education find.Received 30 November 1998; accepted 23

February 1999.

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monolayers was determined by countingcell nuclei of viable cells remaining attachedto the culture surface using the nuclear stainDAPI (4',6-diamidino-2-phenylindole) andKontron KS400 image analysis software(Zeiss, Thornwood, NY), as described pre-viously (15). At the indicated time points,wells containing the cell monolayers werefixed for 15 min in 70% ethanol, rehydrat-ed with phosphate-buffered saline (PBS),and stained with 10 pL DAPI (10 pg/mL indistilled water). Each well was examinedunder epifluorescent illumination at 40 xmagnification on a Zeiss Axiovert 35 (Zeiss)linked to the computer with an OptronicsDEI 470 CCD camera (Optronics, Goleta,CA). For each time point, a minimum of20 fields per well and three wells per datapoint were determined. Both nuclearcounts and total nuclear area were obtainedfrom the program and yielded equivalentresults.

Isolation of total RNA, RT-PCR, andNorthern analysis. Total RNA was isolatedaccording to the protocol supplied withTRI REAGENT (Molecular ResearchCenter, Inc., Cincinnati, OH) as describedpreviously by this laboratory (15). The con-centration and purity of the RNA sampleswere determined using spectrophotometerscan in the ultraviolet (UV) region andethidium bromide (EtBr) visualization of

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intact 18S and 28S RNA bands followingagarose gel electrophoresis.

For reverse transcriptase polymerasechain reaction (RT-PCR), 500 ng of totalRNA from cultured HPT cells was reversetranscribed by incubating for 20 mim at42°C in a 20pL reaction mixture containing2.5 ,uM random hexamers, 5 mM MgCl2,1X PCR buffer (10 mM Tris-Cl, pH 8.3, 50mM KCI), 4 mM dNTPs, 1 U/pL RNaseinhibitor, and 2.5 U/pL MuLV reverse tran-scriptase. The resulting cDNA was amplifiedin 100 pL reaction mixtures containing 2mM MgCl2, IX PCR buffer, 0.025 U/pLTaq polymerase, and 0.0 15 ,uM of therespective primers. The primer pair foramplification of hsp 27 cDNA consisted ofoligonucleotide sequences 5'CACGAGGA-GCGGCAGGACGAG3' and 5'CAGT-GGCGGCAGCAGGGGTGG3' (PCRprimer pair STM-500; StressGen, Victoria,British Columbia, Canada). Reaction vol-umes were incubated at 95°C for 2 min, fol-lowed by 30 cycles of 95°C for 30 sec/58°Cfor 30 sec. PCR products were elec-trophoresed in 2% agarose gels, stained withEtBr, and photographed over LV light. ADNA ladder (Bio-Rad, Hercules, CA) wasincluded in each gel to verify the size ofPCRproducts. For Northern analysis, 5.0 ,ug ofcellular RNA was separated on 1.2% agarosegels containing 0.003% (w/v) EtBr, 1 x

ExposConiroi . . ... . . . .. : ;t ..hsp 27|

GAPDHControl 1 hr 1 hr 2hr 4hr 8hr 12hr 16hr 24hr 36hr 48hr

Exposure Recovery

MOPS buffer [0.1 M 3-(N-morpholino)propanesulfonic acid, 40 mM sodiumacetate, 5 mM EDTA, pH 8.0], and 0.6 Mformaldehyde. After electrophoresis, RNAwas transferred to nylon membranes andcovalently attached by microwave treatmentfor 1.5 min. Hsp 27 cDNA produced byreverse transcription PCR of heat-shockedHeLa cell control RNA (provided byStressGen) was purified using a PCR prod-uct purification kit (QIAquick kit 28104;QIAGEN, Chatsworth, CA). Thirty-fivenanograms of purified PCR product was

labeled to > 1.2 x 109 cpm/pg with 50 pCix32PP-dCTP (3,000 Ci/mmol; AmershamCorp., Arlington Heights, IL) using a ran-dom primed synthesis kit (Promega Corp.,Madison, WI). Nylon membranes were pre-hybridized for 2 hr at 50°C in a solutioncontaining 50% (v/v) formamide, 0.25 MNaCl, 0.25 M sodium phosphate (pH 7.2),0.7% SS, 1 mM EDTA (pH 8.0), and 100pg/mL sonicated herring sperm DNA. Afterprehybridization, radiolabeled cDNA probewas hybridized to Northern transfers in thesame solution as prehybridization for 16 hrat 52°C. Membranes were washed twice in 2x standard saline citrate 0.1% sodium dode-cyl sulfate (SDS) for 15 min at 50°C, twicein 25 mM sodium phosphate (pH 7.2) 1mM EDTA 0.10% SDS for 15 min at 50°C,and twice in 25 mM sodium phosphate (pH

sure I Recoveryal 1 hr 1 hr 2hr 4hr 8hr 12hr 16hr 24hr 36hr 48hr

Exposure Recovery

|7 ; 000 iU <Q : fi;< i 0 hsp27

Exposure - GAPDH

Control 1 hr 1lhr 2hr 4 hr 8Shr l2hr 16 hr 24hr 36 hr 48 hr

Ixour Recovery

Figure 1. HPT cells exposed to heat shock (42.50C) for 1 hr then returned to 37°C for a 48-hr recovery period. Abbreviations: GAPDH, glyceraldehyde-3-phosphatedehydrogenase; hsp, heat shock protein; HPT, human proximal tubule; 100, integrated optical density; RT-PCR, reverse transcriptase polymerase chain reaction;SEM, standard error of the mean. (A) and (8) Northern analysis of hsp 27 and GAPDH mRNA. (A) Average (± SEM) 100 of bands representing hsp 27 mRNA forthree HPT cell isolates. (8) Northern blots for one HPT cell isolate. (C) and (6) RT-PCR analysis of hsp 27 and GAPDH mRNA. (C) Average (± SEM) relative 100 ofinverted bands representing hsp 27 mRNA for three HPT cell isolates. (D) Ethidium-bromide-stained agarose gels for PCR products representing hsp 27 andGAPDH mRNA for one HPT cell isolate. Individual 100 values were divided by the respective GAPDH lODs and normalized to control lODs.

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7.2) 1 mM EDTA 1.0% SDS for 15 min at50°C. Blots were wrapped in plastic andexposed overnight at -76°C for autoradiog-raphy. Radioactive probes were removedfrom membranes by immersion in boiling0.1% SDS. Stripped blots were reprobedwith radiolabeled cDNA complementary tohuman glyceraldehyde 3-phosphate dehy-drogenase mRNA (Clontech, Palo Alto,CA) for loading correction.

Western analysis. Cells were washedtwice with PBS and lysed directly in theflask by addition of400 pL (85°C) 1 x SDSbuffer (2% SDS, 100 mM dithiothreitol,and 50 mM Tris-HCI, pH 6.8). The celllysate was heated in a boiling water bath for10 min. DNA was sheared by repeated pas-sage through a 23-gauge needle. The sam-ples were centrifuged at 10,000g for 10 minat room temperature and the supernatanttransferred to a new tube. The concentra-tion of protein in the samples was deter-mined by the bicinchoninic acid proteinassay (Pierce Chemical Co., Rockford, IL).Equal amounts of total cellular protein wereseparated on 12% SDS-containing polyacry-lamide gels and electrophoretically trans-ferred to polyvinylidene difluoride (PVDF)membranes (Bio-Rad). Membranes wereblocked with 10% (w/v) nonfat milk in PBS,incubated with a mouse monoclonal anti-body specific for human hsp 27 (StressGen)diluted 1:2000 in PBS containing 1% (w/v)bovine serum albumin as carrier, followed byincubation with a goat antimouse, alkalinephosphatase conjugated secondary antibody

Exposure I Recovery

c°s oX No'Wo .ell'Rso 4s

hap 27

a

(Promega). Colorimetric detection used analkaline phosphatase Vectastain ABC-AP kit(Vector, Burlingame, CA).

Analysis of hsp 27 phosphoisoforms.Proteins were extracted from cell monolay-ers with 9.0 M urea, 1.0 mM phenyl-methyl sulfonyl fluoride, 10.0 mM NaF,2% ampholines, 5% f-mercaptoethanol,and 2% Triton X-100. The proteins werefocused on 4% polyacrylamide capillarytube gels containing 9 M urea, 1.5% 5/7Biolyte, and 0.5% 3/10 Biolyte ampholines(Bio-Rad). Capillary tube gels containingfocused proteins were placed at the top of12% polyacrylamide slab minigels, fol-lowed by separation of proteins in the sec-ond dimension. Resolved proteins wereelectrotransferred onto PVDF membranes(Bio-Rad). Hsp 27 phosphoisoforms weredetected using procedures identical tothose described for Western analysis.

Integrated optical density (IOD). IODvalues were determined using an imageanalysis work station configured withKontron KS 400 software. For IOD evalu-ations of EtBr-stained gels, inverted imageswere used. In the heat-shock, acute CdCI2,and sodium arsenite protocols, IOD valuesfor Northern blots and RT-PCR gels are{[IOD hsp 27 experimental/IOD hsp 27control]/[IOD glyceraldehyde-3-phosphatedehydrogenase (GAPDH) experimental/IOD GAPDH control]}. IOD values forWestern blots are IOD experimental/IODcontrol. In the 16-day CdCI2 exposure, theIOD for each Hsp 27 band was divided by

the IOD for GAPDH at the appropriatetime point and Cd concentration. Bandsrepresenting the three Cd2P concentrationswere then divided by the control values foreach time point.

ResultsHsp 27 expression in HPT cells exposed toheat shock. The classic method used toexamine the response of cultured mam-malian cells to physical stress is exposure toelevated temperature, usually 42-440C, fol-lowed by a recovery period at normal tem-perature. To determine the effect of heatshock on hsp 27 expression in HPT cells,confluent cells from three independent cellisolates were exposed to an elevated temper-ature of 42.5°C for 1 hr followed by a recov-ery period of 48 hr at 370C. Exposure toheat shock clearly resulted in an increase inthe amount of hsp 27 mRNA for all threeHPT cell isolates as determined byNorthern analysis (Figure IA, B). Hsp 27mRNA was increased at the end of the 1-hrheat shock period, continued to increase dur-ing the initial hour of the recovery period,and remained elevated for the next 12-16 hrbefore returning to control values 24 hr post-heat shock. Hsp 27 mRNA levels were alsoexamined on the same total RNA samplesusing an RT-PCR assay to determine ifresults would be equivalent to those foundwith Northern analysis (Figure IC, D). Thisis important because RT-PCR analysis con-sumes a much smaller amount of total RNAthan Northern analysis. The relative

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Figure 2. Western analysis (A, B) and two-dimensional gel electrophoresis (C, D) of heat shocked HPT cells during exposure and recovery periods. Abbreviations:a, unphosphorylated; b, one serine phosphorylated; c, two serines phosphorylated; d, three serines phosphorylated; hsp, heat shock protein; HPT, human proxi-mal tubule; IOD, integrated optical density. (A) Western blot for a single isolate. (B) Relative (normalized to control values) lODs of bands representing hsp 27 pro-tein at the various time points for three HPT cell isolates. (C) Blot showing increasing phosphorylation of hsp 27 protein represented by the a, b, c, and d phos-phoisoforms. (D) IOD values for each of the phosphoisoforms in three HPT cell isolates.

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amounts of hsp 27 mRNA were similar forboth Northern and RT-PCR assay methods.

The expression of hsp 27 protein wasalso determined at selected time points ofthe heat shock protocol by Western analy-sis (Figure 2A, B). Hsp 27 protein wasexpressed under control conditions and didnot increase during the initial 1-hr period at42.5°C. Hsp 27 protein levels increased inthe recovery period, reaching peak values by8-12 hr. The phosphorylation state of hsp27 protein was also determined as a func-tion of heat shock and recovery (Figure 2C,D). In the control condition, hsp 27 pro-tein was present in the unphosphorylatedand mono-phosphorylated state. There wasan increase in phosphorylation during the1-hr heat shock, as noted by a faint addi-tional immunoreactive spot representingthe di-phosphorylated state. There was noloss of cell viability during the treatmentand recovery periods (data not shown).

Hsp 27 expression in HPTcellsfollowingacute exposure to arsenite and CdClI Theclassic method to evaluate the response ofcultured cells to chemical stress is exposure tosodium arsenite followed by a recovery peri-od that involves the removal of the chemicalstress through a change of the culture medi-um and renewed incubation at 37°C. Thismethod was used to determine if acute expo-sure to Cd2+ induces hsp 27 expression inHPT cells. Confluent cells were exposed to53 pM Cd2+ for 4 hr, followed by a 48-hr

recovery period in Cd2+-free growth media.The Cd2+ concentration used was establishedin preliminary experiments to be an exposurelevel resulting in the death of 15-30% of thecells by the end of the recovery period. Theeffect of this level of exposure was confirmedby monitoring HPT cell viability over thetotal time course of exposure and recovery(Figure 3A). Exposure to 53 iM Cd2+ clearlyresulted in an increase in the amount of hsp27 mRNA, as determined by RT-PCRanalysis (Figure 3B, C). The increase in hsp27 mRNA was rapid, 5-10-fold over con-trol, occurred largely within the first 4 hr ofCd> exposure, and was not dependent on aCd2P-free recovery period. This elevated levelof hsp 27 mRNA was maintained 4-8 hrinto the recovery period and returned tocontrol values by 48 hr of recovery in Cd_free growth medium.

The level of hsp 27 protein was alsoincreased by acute Cd2+ exposure (Figure3D, E). Hsp 27 protein was maximally ele-vated following 1 hr of Cd2+ exposure,remained elevated 8-12 hr into the recoveryperiod, and returned to control values fol-lowing 24 hr of recovery in Cd2+-freegrowth medium. Exposure to 53 j1M Cd2+resulted in an immediate and prolongedshift of the hsp 27 isoform pattern to anenhanced phosphorylation state (Figure 3F).The shift in the phosphoisoform patternoccurred within the first hr of Cd2+ expo-sure and was retained in the recovery period.

For comparison, confluent HPT cellswere also exposed to 100 pM sodium arsen-ite for 4 hr, followed by a 48-hr recoveryperiod (Figure 4). Exposure to 100 pMsodium arsenite resulted in an increase inthe amount of hsp 27 mRNA (Figure 4B,C). The level of hsp 27 mRNA was relative-ly constant during the initial hours of sodi-um arsenite exposure and began to increaseafter 4 hr. Four hours into the recovery peri-od, there was a large increase in the level ofhsp 27 mRNA. The increased level of hsp27 mRNA was sustained for 12 hr of recov-ery and thereafter rapidly returned to con-trol values. Hsp 27 protein also increased asa consequence of sodium arsenite treatment(Figure 4D, E). Hsp 27 protein levelsincreased following 1 hr of sodium arsenitetreatment and remained elevated 24 hr intothe recovery period. The phosphorylationstate of hsp 27 protein was also evaluated forHPT cells exposed to sodium arsenite. Theunphosphorylated and mono-phosphorylat-ed forms of hsp 27 were evident in the con-trol condition. Sodium arsenite exposureresulted in a shift in the isoform pattern ofhsp 27 to a more phosphorylated state fol-lowing 4 hr of exposure (Figure 4F).

Hsp 27 expression in HPT cells follow-ing chronic exposure to lethal and sub-lethallevels of Cct+. Because the stress response istypically assessed under conditions of acuteagent exposure, an experiment was designedto determine if the hsp 27 stress response of

CCD

1 hr 2 hr 4 hr 4 hr 8 hr 12 hr 16hr 24 hr 36 hr 48 hr Control 1 hr 2 hr 4 hr 4 hr 8 hr 12 hr 16hr 24hr 36 hr4hr

Exposure | Recovery Exposure | Recovery

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Exposure | Recovery

h.p ...;h.i.-21.c

hapn7

GAPDH

F±i a h c d

ControlhD27 o o ;o ,c; s

hsp 27

Exposure Recovery

Figure 3. HPT coils exposed to CdCI2. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glycer-aldehyde-3-phosphate dehydrogenase; hsp, heat shock protein; HPT, human proximal tubule; IOD, inte-grated optical density; RT-PCR, reverse transcriptase polymerase chain reaction; SEM, standard error ofthe mean. (A) Computer-assisted cell counts for one HPT cell isolate exposed to 53 pM CdCI2 for 4 hr, fol-lowed by a 48-hr recovery period. DAPI-stained nuclei in 20 fields for each triplicate well were countedand results are expressed as percentage of control. (B) and (C) RT-PCR analysis of hsp 27 and GAPDHmRNA in HPT cells exposed to Cd2P. (B) Average (± SEM) IOD of bands, normalized to control, represent-ing hsp 27 mRNA for three HPT cell isolates. (C) Ethidium-bromide-stained agarose gels for PCR productsrepresenting hsp 27 and GAPDH mRNA for one HPT cell isolate. (D) and (E8 Western analysis of hsp 27protein following Cd2P exposure and recovery periods. (D) Average (± SEM) IOD of bands, normalized tocontrol values, representing hsp 27 protein at the various time points for three HPT cell isolates. (8)Western blot for a single isolate. (Iq Two-dimensional gel electrophoresis. Blot shows increasing phos-phorylation of hsp 27 protein represented by the a, b, c, and d phosphoisoforms.

Exposure 1 hr

2 hr

Recovery 4hr

12 hr

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HPT cells is different with extended expo-sure. To accomplish this, HPT cells werecontinuously exposed to Cd2P over a 16-daytime course. Three concentrations of Cd2Pwere used: 9 PM, which produces no celldeath over the 16-day time course; 27 pM,which produces cell death late in the 16-daytime course; and 45 ,M, which producescell death early in the 16-day time course(Figure 5). Hsp 27 mRNA and proteinexpression were determined after 1, 4, 7, 10,13, and 16 days of exposure. The level ofhsp 27 mRNA expression was constant overthe 16-day time course for control HPTcells (Figure 6B). The level of hsp 27mRNA expression was also not altered ascompared to control for any of the Cd2+treatment groups regardless of Cd2P dose orCd2+-induced cell lethality (Figure 6). Thelevel of hsp 27 protein expression was alsorelatively constant over the 16-day timecourse for control cells (Figure 7B). In con-trast, the level of hsp 27 protein wasreduced as compared to control at eachconcentration of Cd2+ (Figure 7). The pat-tern of hsp 27 phosphoisoforms was evalu-ated after 24 hr of exposure to each Cd2+concentration and was identical to controlcells (data not shown).

DiscussionThe first goal of the present study was todetermine if acute exposure to Cd2+ evokesthe hsp 27 stress response in cultured HPT

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cells. This was accomplished by mimickingthe classic protocols used to determine acell's response to physical or chemical stress:a short exposure to the agent, followed byagent removal and monitoring of theresponse during a recovery period. For theHPT cells, this involved treatment with 53pM Cd2+ for 4 hr, followed by removal ofthe metal through a change in growth medi-um, and subsequent monitoring of the hsp27 response during a 48-hr recovery period.The results demonstrated that Cd2+ treat-ment induces the hsp 27 stress response, asevidenced by an increase in both hsp 27mRNA and protein as well as by a shift inhsp 27 isoforms to a pattern of increasedphosphorylation. To confirm that the Cd2P-induced hsp 27 response in HPT cells wassimilar to the classic response induced byheat and sodium arsenite, these agents werealso used to induce hsp 27 expression. It wasdemonstrated that the hsp 27 response wassimilar for heat, sodium arsenite, and Cd2 .The only feature of the hsp 27 response inHPT cells that was different from other sys-tems was that a recovery phase was not nec-essary for induction of the hsp 27 response.In HPT cells the induction of hsp 27mRNA and protein occurred in the presenceof Cd2+ and did not require a recovery phase.This implies that acute exposure to lethalconcentrations of Cd2+ does not immediatelyinactivate any of the cellular componentsnecessary for transcription, translation, or

phosphorylation of hsp 27 in HPT cells.Otherwise, the hsp 27 response to acuteCd2+ exposure is what would be expectedbased on many studies in other cell systems.

Although studies in the renal system arelimited, a role for the hsp 27 response inprotection of the proximal tubule cell fromacute Cd2+ exposure can be inferred fromrecent studies on renal ischemia. In studiesusing the rodent model, evidence showsthat induction of the hsp 27 stress responsecan attenuate the effects of acute renalischemia (17-19). The expression andintracellular distribution of hsp 25 (therodent homologue to human hsp 27) wasevaluated in rat renal cortex following 45min of renal ischemia with subsequentreflow (17). Cortical hsp 25 was inducedwithin 2 hr of reflow, peak values werereached by 6 hr, and elevated levels weremaintained after 24 hr of reflow. The shiftin hsp 25 between the detergent soluble andinsoluble cytoskeletal fractions and thelocalization of hsp 25 within the proximaltubule cell as a function of ischemia andrecovery both suggested specific interactionsbetween hsp 25 and actin during the earlypostischemic reorganization of thecytoskeleton. The suggestion that hsp 25provides assistance in the reorganization ofthe actin cytoskeleton following renalischemia is consistent with one of theknown functions of hsp 27 and the alter-ations of the actin cytoskeleton that are

hplhap 27C20

Exposurjo1\Nos

Recovery

GAPDH

Exposure I Recovery

Exposure | Recovery

hs 2,7* <.o .. .. oohSpZl

Control thr 2hr 4hr 4hr 8hr 12hr 16hr 24hr

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XU a h c

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Exposure 4hr

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Figure 4. HPT cell isolates exposed to 100 pM sodium arsenite for 4 hr, followed by a 48-hr recovery period. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hsp, heat shock protein; HPT, human proximal tubule; IOD, integrated optical density; RT-PCR, reversetranscriptase polymerase chain reaction; SEM, standard error of the mean. (A) Computer-assisted cell counts for one HPT cell isolate. DAPI-stained nuclei in 20fields for each triplicate well were counted and results are expressed as percentage of control. (81 and (C) RT-PCR analysis of hsp 27 and GAPDH mRNA in HPTcells. (B) Average (± SEM) IOD of bands normalized to control values representing hsp 27 mRNA for three HPT cell isolates. (C) Ethidium-bromide-stained agarosegels of PCR products representing hsp 27 and GAPDH mRNA for one HPT cell isolate. (D) and (E) Western analysis of hsp 27 protein. (D) IOD for bands, normalizedto control values, representing hsp 27 protein at the various time points for three HPT cell isolates. (E) Western blot for a single isolate. (F) Two-dimensional gelelectrophoresis. Blot shows increasing phosphorylation of hsp 27 protein represented by the a, b, and c phosphoisoforms.

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Days 1 4

aM Cd 0 9 27 45 0 9 27 45

hsp 27

GAPDH

7 10 13 16

0 9 27 0 9 27 0 9 27 0 9 27

1 4 7 10 13 16

Days

Figure 5. HPT cell continuous exposure to CdCI2.Abbreviations: DAPI, 4',6-diamidino-2-phenylin-dole; HPT, human proximal tubule; IOD, integratedoptical density.Three HPT cell isolates wereexposed to 9, 27, and 45 pM CdCI2 for a period of16 days. Computer-assisted cell counts areshown for one HPT cell isolate. DAPI-stainednuclei in 20 fields for each triplicate well werecounted and results are expressed as percentageof control.

known to occur in ischemia. A series ofstudies demonstrated that the loss of proxi-mal tubule cell structure and transportfunction associated with renal ischemiainvolves alterations in the actin cytoskeleton[reviewed by Molitoris (36)]. The inductionof hsp 27 in HPT cells by acute Cd2+ expo-sure would likewise be expected to stabilizethe actin filament network and help pre-serve proximal tubule transport function.

Recent studies in other systems havedemonstrated that expression ofhsp 27 regu-lates apoptosis (33-35). In studies usingL929 cells that constitutively express the Fasreceptor, expression of human hsp 27inhibits apoptosis mediated by stimulation ofthe Fas receptor (34). In addition, hsp 27expression interfered with apoptotic celldeath mediated by staurosporine (33,34). Asimilar finding for a protective effect of thesmall hsp against apoptosis was also observedin U937 and Wehi-s cells exposed to actino-mycin D, camptothecin, and etoposide (35).HPT cells express Fas under normal growthconditions and increased Fas expressionoccurs after treatment with interferon-y (37).These observations suggest that the rapidinduction of hsp 27 protein and phosphory-lation early in the time course of acute expo-sure to Cd2+ may protect the proximaltubule cell against programmed cell death.

The dassic methods used to examine thestress response in cultured cells evaluate thepresence or absence of the response after a

short duration of agent exposure. Theresults discussed for hsp 27 expression inCd2+-exposed HPT cells can then be catego-rized as early events within the initial 4 hr ofCd2+ exposure. Although this illustrates thehsp 27 stress response following acute expo-

sure to Cd2 , it is likely that actual exposurein many instances is more prolonged andinvolves lower concentrations. An additional

3

FControl~ 2279jM

CD

1 4 7 10 13 16 1 10 6Days Days

Figure 6. RT-PCR analysis of HPT mRNA during 16-day exposure to three Cd2+ concentrations.Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hsp, heat shock protein; HPT,human proximal tubule; IOD, integrated optical density; RT-PCR, reverse transcriptase polymerase chainreaction; SEM, standard error of the mean. (A) Ethidium-bromide-stained agarose gels for PCR productsrepresenting hsp 27 and GAPDH mRNA for one HPT cell isolate. Average (± SEM) IOD of (inverted) bandsrepresenting (B) hsp 27 mRNA in controls and (C) cells exposed to 9, 27, and 45 pM CdCI2 for three HPTcell isolates over the 16-day time course. IOD values were divided by the respective GAPDH IOD valuesand normalized to lODs for control cells.*Only two viable samples.

Days 1 4 7 10 13 16iM Cd 0 9 27 45 0 9 27 0 9 27 0 9 27 0 9 0 9

hop 27

a

0CD

1U 10 1 1 4 7 10

Days Days

Figure 7. Western analysis of hsp 27 protein following 16-day exposure to CdCI2. Abbreviations: GAPDH,glyceraldehyde-3-phosphate dehydrogenase; hsp, heat shock protein; HPT, human proximal tubule; IOD,integrated optical density; SEM, standard error of the mean. (A) Western blots for a single isolate.Average (± SEM) relative IOD values for bands representing (B) hsp 27 protein in controls and (C) cellsexposed to 9, 27, and 45 pM CdCI2 for three HPT cell isolates. IOD values were divided by the respectiveGAPDH IOD values and normalized to lODs for control cells.*Only two viable samples.

goal of this study was to determine if theCd2P-induced early increase in hsp 27 levelwas sustained when HPT cells were exposedover a longer time course. Sustained Cd2+exposure was modeled by continuous treat-ment ofHPT cells with both lethal and sub-

lethal Cd2P levels over a 16-day time course

while monitoring cell viability and hsp 27mRNA and protein levels at days 1, 4, 7, 10,13, and 16. For hsp 27 mRNA, the level ofhsp 27 mRNA did not increase over controllevels at any point in the time course regard-less of sublethal (9 pM) or lethal (27 and 45PM) levels of Cd2P exposure. The finding

that hsp 27 mRNA was not elevated inHPT cells following 24 hr of exposure toany of the three Cd2+ concentrations was

surprising because the described acute expo-sure experiments showed a large elevation ofhsp 27 mRNA following 4 hr of exposure to53 pM Cd2 . Subsequent examination ofhsp 27 mRNA levels in HPT cells withinthe initial 24 hr of exposure demonstratedthat 9 PM Cd2+ caused no increase in hsp27 mRNA levels, 27 PM Cd2+ elicited a

maximal 2-fold increase in hsp 27 mRNAonly between 4 and 12 hr of exposure, and45 pM Cd2+ resulted in a 2- to 4-fold

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increase between 2 and 20 hr of exposure,with all dosage groups returning to controlvalues following 24 hr of exposure (38).There was no sustained elevation of hsp 27mRNA levels in HPT cells during extendedexposure to both lethal and sublethal con-centrations of Cd2P. Analysis of hsp 27 pro-tein levels over the extended time coursedemonstrated a marked decrease in hsp 27protein as compared to control cells. Thisdecrease in hsp 27 protein was evident atboth lethal and sublethal levels of Cd2+exposure, although the decrease was greatestat lethal levels of Cd2+ exposure. To ourknowledge, this is the only example of non-lethal levels of toxicant exposure thatreduces the constitutive level of hsp 27 pro-tein expression in the cell.

In a previous study using identical HPTcell lines, Cd2+ concentrations, and expo-sure time course, Cd2+ exposure also elicit-ed a sustained induction of metallothionein(MT) protein (15). Within the first 24 hrof exposure to 9 pM Cd2+, HPT cellsshowed a 10- to 20-fold increase in MTprotein levels and this increase continued tothe end of the time course where MT repre-sented 7 to 10% of total cell protein.Because of the high binding affinity ofMTfor Cd2+, it would be expected that Cd2+ isin the unsequestered state for only a briefperiod following the initial exposure of thecells before complexing with MT.Assuming that hsp 27 induction occurs inresponse to Cd2+ in the unsequestered state,then hsp 27 induction would only beexpected to occur immediately after Cd2+exposure-the brief interval before MTprotein induction and complexation withMT. This would provide a mechanism toexplain the current finding that hsp 27expression is limited to an early transientinduction in Cd2+-exposed HPT cells.Because the Cd- sequestering MT proteinlevel continues to increase with time, thisexplanation is also consistent with the find-ing that hsp 27 expression is not increasedlater in the time course. Although theinduction ofMT may explain the lack of asustained or long-term induction of hsp 27expression by Cd2+, it does not explain whythe levels of hsp 27 protein are decreased byprolonged exposure to lethal and sublethalconcentrations of Cd2+. Although there isno explanation for this finding at present, itdoes not appear to be due to a nonspecificoverall decrease in protein synthesis becauseMT protein is increased at the same timehsp 27 protein is decreased.

Two distinct roles can be proposed forhsp 27 expression when the HPT cell isexposed to Cd2+. Induction early in thetime course of Cd21 exposure may protectthe cell while the Cd2+-binding protein MT

is being synthesized. This would serve tostabilize the actin filament network of thecell in a fashion similar to that proposed tooccur during renal ischemia and subsequentreflow (17-19). Direct evidence that a tran-sient elevation of hsp 27 expression canprovide cellular resistance to Cd2+ toxicitycomes from recent studies with mouseembryonic stem cells transfected with senseor antisense hsp 27 cDNA (39). In thesestudies, the level of hsp 27 expression wasdirectly correlated with cellular resistance tothe toxicity of CdCl2, HgCl2, cis-platinum(11)-diamine dichloride, or sodium arsenitewithin a 12-hr exposure period. Protectionagainst programmed cell death provided bythe early induction of hsp 27 is speculativebased solely on hsp 27 involvement in pro-grammed cell death decision in other cellsystems (33-35). In contrast, the inabilityto sustain constitutive levels of hsp 27 pro-tein may potentially have deleterious effects,as was demonstrated when HPT cells weresubjected to a longer period of sustainedCd2+ exposure. Whereas the induction ofhsp 27 protein has been proposed to stabi-lize actin filament dynamics, the loss of hsp27 protein would be expected to render theactin filaments susceptible to damage. Thedemonstration that constitutive levels ofhsp 27 protein are not maintained in HPTcells during a chronic course of Cd2+ expo-sure suggests that the cytoskeleton might bea site particularly susceptible to damage incadmium-induced nephropathy.

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