17-β-estradiol induces heat shock proteins in brain arteries and potentiates ischemic heat shock...

17-�-Estradiol Induces Heat Shock Proteins in Brain Arteriesand Potentiates Ischemic Heat Shock Protein Induction in Glia

and Neurons

Aigang Lu, Rui-qiong Ran, Joseph Clark, Melinda Reilly, Alex Nee, and Frank R. Sharp

Department of Neurology and Neurosciences Program, Vontz Center for Molecular Studies, University of Cincinnati,Cincinnati, Ohio, U.S.A.

Summary: Estradiol reduces brain injury from many diseases,including stroke and trauma. To investigate the molecularmechanisms of this protection, the effects of 17-�-estradiol onheat shock protein (HSP) expression were studied in normalmale and female rats and in male gerbils after global ischemia.17-�-Estradiol was given intraperitoneally (46 or 460 ng/kg, or4.6 �g/kg) and Western blots performed for HSPs. 17-�-Estradiol increased hemeoxygenase-1, HSP25/27, and HSP70in the brain of male and female rats. Six hours after the ad-ministration of 17-�-estradiol, hemeoxygenase-1 increased 3.9-fold (460 ng/kg) and 5.4-fold (4.6 �g/kg), HSP25/27 increased2.1-fold (4.6 �g/kg), and Hsp70 increased 2.3-fold (460 ng/kg).Immunocytochemistry showed that hemeoxygenase-1,HSP25/27,and HSP70 induction was localized to cerebral ar-teries in male rats, possibly in vascular smooth muscle cells.17-�-Estradiol was injected intraperitoneally 20 minutes before

transient occlusion of both carotids in adult gerbils. Six hoursafter global cerebral ischemia, 17-�-estradiol (460 ng/kg) in-creased levels of hemeoxygenase-1 protein 2.4-fold comparedwith ischemia alone, and HSP25/27 levels increased 1.8-foldcompared with ischemia alone. Hemeoxygenase-1 was inducedin striatal oligodendrocytes and hippocampal neurons, andHSP25/27 levels increased in striatal astrocytes and hippocam-pal neurons. Finally, Western blot analysis confirmed that es-trogen induced heat shock factor-1, providing a possiblemechanism by which estrogen induces HSPs in brain and othertissues. The induction of HSPs may be an important mechanismfor estrogen protection against cerebral ischemia and othertypes of injury. Key Words: Estrogen—Heat shock—Heatshock proteins—Heat shock factor—Stroke—Ischemia—Blood vessels—Arteries—Smooth muscle cells.

Epidemiologic studies associate postmenopausal es-trogen use with a reduction in risk of Alzheimer andParkinson disease, and reduced death from stroke andcardiovascular disease (Garcia-Segura et al., 2001;Green and Simpkins, 2000; Hurn and Macrae, 2000).Estrogen protects in several neuronal culture model sys-tems, including serum deprivation, �-amyloid–inducedtoxicity, excitotoxicity, and oxygen-glucose deprivation.Estrogens attenuate neuronal death in rodent models ofcerebral ischemia, traumatic injury, and Parkinson dis-ease (Hurn and Macrae, 2000). Although estrogens areknown to exert several direct and indirect effects on neu-rons and the brain, the cellular and molecular mecha-

nisms of protection by this steroid are just beginning tobe elucidated (Garcia-Segura et al., 2001; Green andSimpkins, 2000; Hurn and Macrae, 2000). One mecha-nism of protection may be that native estrogens or ex-ogenous 17-�-estradiol act as direct vasodilators orthrough vasodilatory mediators during ischemia. Estro-gen-mediated improvement in cerebral blood flow hasbeen most clearly identified in models of global cerebralischemia (Hurn and Macrae, 2000).

Increased expression of heat shock protein (HSP) 70,27 (HSP27), and 32 (hemeoxygenase-1) in the brain hasbeen extensively documented in association with a vari-ety of insults, including ischemia, and may play a role incell survival and recovery after injury (Rajdev et al.,2000; Sharp et al., 1999; Xanthoudakis and Nicholson,2000). Estrogen has been shown to modulate HSPs in avariety of tissues. Estrogen increases heat-shock factorexpression in endometrium (Yang et al., 1995), and ac-tivates HSF-1 and induces HSP72 in isolated cardiacmyocytes (Knowlton and Sun, 2001). Estradiol increases

Received July 26, 2001; final revision received October 1, 2001;accepted October 2, 2001.

Supported by National Institutes of Health grants NS28167 andNS38743 (to F.R.S.).

Address correspondence and reprint requests to Dr. Frank R. Sharp,Department of Neurology, University of Cincinnati, Vontz Center forMolecular Studies, Room 2327, 3125 Eden Avenue, Cincinnati, OH45267, U.S.A.; e-mail: [email protected]

Journal of Cerebral Blood Flow & Metabolism22:183–195 © 2002 The International Society for Cerebral Blood Flow and MetabolismPublished by Lippincott Williams & Wilkins, Inc., Philadelphia

183

HSP90 and HSP70 mRNA in myometrium and endome-trium of nonpregnant ewes (Wu et al., 1996), and inducesHSP27 in human endometrial glandular epithelium (Pad-wick et al., 1994), endothelial cells (Piotrowicz et al.,1995), and in MCF7 human breast cancer cells (Porter etal., 2001).

There are only a few isolated reports that estrogenmight modulate HSP expression in the brain. Estrogenincreases HSP90 protein in ventromedial hypothalamus,heat shock cognate 73 (Hsc73) in the pituitary and ven-tromedial hypothalamus, and induces HSP72, HSP27,and HSP90 in astrocytes in the arcuate nucleus and in thewall surrounding the third ventricle (Olazabal et al.,1992; Krebs et al., 1999; Mydlarski et al., 1995). Thesignificance of the localized estrogen-related HSP induc-tion is not known, nor is it known whether this localizedinduction relates to the protection afforded by estrogen.

In this study, we postulated that estradiol would in-duce HSPs in the brain because of reports that estrogeninduces heat shock factors (HSFs) and binds HSP90(Yang et al., 1995; Knowlton and Sun, 2001), both ofwhich should induce HSPs. We found that 17-�-estradiolinduced hemeoxygenase-1, HSP25/27, and HSP70 inbrains of male and female rats, and that this inductionoccurred mainly in cerebral blood vessels in male rats.17-�-Estradiol also increased hemeoxygenase-1 andHSP25/27 brain expression 6 hours after global cerebralischemia in gerbils. Hemeoxygenase-1 was induced instriatal oligodendrocytes and hippocampal neurons, andHSP25 was induced in striatal astrocytes and hippocam-pal neurons. These results suggest that estrogen induc-tion of HSPs may be one of the important mechanismsby which estrogen protects against ischemia.

MATERIALS AND METHODS

Animal studiesMale and female Sprague-Dawley rats (n � 40, 200–240 g)

and adult male Mongolian gerbils (n � 12, 60–80 g) were usedin this study. All procedures were approved by the Universityof Cincinnati Animal Care Committee in accordance with theNational Institutes of Health Guide for Care and Use of Labo-ratory Animals. Water-soluble 17-�-estradiol (E4389; Sigma,St. Louis, MO, U.S.A.) was dissolved in 0.9% saline solutionand administered intraperitoneally at a dose of 46 or 460 ng/kgor 4.6 �g/kg in male and female rats. Controls were injectedwith 0.9% saline. Positive controls were injected with kainicacid (10 mg/kg, intraperitoneally). After 6 or 24 hours, ratswere anesthetized with isoflurane and euthanized for Westernblot analysis (two animals for each group); or animals wereanesthetized with ketamine (100 mg/kg) and xylazine (20mg/kg) and perfused with 4% paraformaldehyde in 0.1 mol/Lphosphate buffer (pH 7.4) for immunohistochemical analysis(two animals for each group).

Adult male Mongolian gerbils were anesthetized with 3%isoflurane in a mixture of 20% oxygen and 77% nitrogen. 17-�-Estradiol (460 ng/kg or 4.6 �g/kg) or saline (0.9%) vehiclewas injected intraperitoneally 20 minutes before ischemia. Toproduce ischemia, both common carotid arteries were exposed

and occluded with aneurysm clips for 5 minutes. The clips werethen removed to restore cerebral blood flow. The rectal tem-perature was maintained at 36.5 to 37.5°C with a heating blan-ket until the animals recovered from surgery. After recovery,the animals were monitored for 2 hours to prevent hypothermia.At 6 or 24 hours after ischemia, the gerbils were anesthetizedwith isoflurane and euthanized before the brains were removedfor Western blot analysis (two animals for each group), or wereanesthetized with ketamine (100 mg/kg) and xylazine (20mg/kg) and perfused with 4% paraformaldehyde before thebrains were processed for immunohistochemical analysis (twoanimals for each group).

Tissue preparation and Western blot analysisBrains were homogenized in ristocetin-induced platelet ag-

glutination buffer (9.1 mmol/L dibasic sodium phosphate, 1.7mmol/L monobasic sodium phosphate, 150 mmol/L sodiumchloride, 10 �l/mL Igepal (I-3021, Sigma) CA-630, 5 mg/mLsodium deoxycholate, 1 mg/mL sodium dodecyl sulfate, pH7.4) with 0.01% PMSF, 3% aprotinin, and 0.02% sodium or-thovanadate and centrifuged at 10,000 g for 10 minutes. Thesupernatant was removed and centrifuged again; the tissue wasmaintained at 4°C throughout. Protein concentrations were de-termined by the method of BCA (200 Protein Assay Kit)(PIERCE, Rockford, IL, U.S.A.), and gel samples prepared byadding sample buffer containing final concentrations of 50mmol/L Tris (pH 6.7), 2% sodium dodecyl sulfate, 2% �-mer-captoethanol, and the marker bromphenol blue. Samples wereboiled for 3 minutes, and 30 to 50 �g protein was loaded on10% sodium dodecyl sulfate-polyacrylamide gels. Proteinswere transferred to nitrocellulose membranes (Schleicher andSchuell, Keene, NH, U.S.A.) using standard electroblottingprocedures. Nitrocellulose membranes were blocked with 10%nonfat dry milk. Blocking solutions were prepared in a buffercontaining 100 mmol/L Tris-hydrochloride, 0.9% sodiumchloride, and 0.05% Tween 20 (Tris-buffered saline-Tween(TBST), pH7.6).

Blots were incubated overnight at 4°C with antibodiesagainst HSP70 (1:4000, 386032; Calbiochem, San Diego, CA,U.S.A.), hemeoxygenase-1 (1:1000, OSA-111; Stressgen, Vic-toria, BC, Canada), rodent HSP25 (1: 3500, SPA-801; Stress-gen), HSP90 (1:5000, SPA-771; Stressgen), HSP60 (1:5000,SPA 807; Stressgen), HSP47 (1:500, SPA 470; Stressgen) andHSF1 (1:10000, Stressgen SPA-901), respectively. The rodentHSP25 is probably identical to the human HSP27 HSP and isreferred to as HSP25/27 in this report. Immunoblots werewashed in several changes of TBST at room temperature andthen incubated with antimouse or antirabbit immunoglobulinlinked to horseradish peroxidase (Santa Cruz Biotechnology,Santa Cruz, CA, U.S.A.). Immunoreactivity was detected withenhanced chemiluminescence (Amersham Pharmacia Biotech,Piscataway, NJ, U.S.A.) according to the manufacturer’s rec-ommended conditions. Blots were quantified using densitomet-ric analyses with the MCID digital image analysis system(IMAGE Research, St. Catherines, Ontario, Canada). Westernblots were performed in duplicate for two separate animals foreach protein and each experimental condition.

ImmunohistochemistryAnimals were anesthetized with ketamine (100 mg/kg) and

xylazine (20 mg/kg) hydrochloride and perfused transcardiallywith 0.9% saline, followed by 4% paraformaldehyde in 0.1mol/L phosphate buffer (pH 7.4). The brains were removed,postfixed for 6 hours in 4% paraformaldehyde phosphatebuffer, and placed in 30% sucrose overnight. Fifty-micrometercoronal sections were cut on a sliding microtome and placed in

A. LU ET AL.184

J Cereb Blood Flow Metab, Vol. 22, No. 2, 2002

2% horse serum (or goat serum) and 0.2% Triton X-100/0.1%bovine serum albumin in phosphate buffer for 2 hours at roomtemperature, and then incubated for 2 hours at room tempera-ture with mouse anti-HSP70 monoclonal antibody (1:1000,SPA-810; Stressgen), rabbit anti-HSP25 polyclonal antibody(1:1000, SPA-801; Stressgen), or rabbit antihemeoxygenase-1polyclonal antibody (1:6000; gift from Ortiz De Montellano,University of California Davis) in 2% goat serum (or horseserum), 0.2% Triton X-100, and 0.1% bovine serum albumin inphosphate buffer. After three 10-minute phosphate bufferwashes, the sections were incubated for 2 hours at room tem-perature with a biotinylated horse antimouse second antibody(1:200; Vector Labs) or goat antirabbit second antibody (1:400;Vector Labs), diluted in 2% goat serum (or horse serum), 0.2%Triton X-100, and 0.1% bovine serum albumin in phosphatebuffer. After three 10-minute PB washes, the sections wereincubated with ABC reagent (Vector Labs) for 2 hours. Afterthree 10-minute phosphate buffer washes, the sections wereincubated with a solution of 0.6 mg/mL diaminobenzidine and0.05% hydrogen peroxide for 3 minutes. This incubation wasterminated by three 10-minute phosphate buffer washes. Sec-tions were mounted onto gelatin-coated slides and dried over-night before being covered with a cover slip. Representativesections from each animal were then photographed.

RESULTS

Hemeoxygenase-1 protein inductionThe administration of 17-�-estradiol induced

hemeoxygenase-1 at 6 hours. In female rats, 17-�-estradiol doses of 460 ng/kg (Fig. 1A, lane 7) and 4.6

�g/kg (Fig. 1A, lane 8) increased hemeoxygenase-1 pro-tein 3.9-fold and 5.4-fold, respectively, compared withthe 6-hour control (Fig. 1A, lane 2). There was no in-duction of hemeoxygenase-1 at 24 hours with any dose(Fig. 1A, lanes 3–5) compared with the 24-hour control(Fig. 1A, lane 1). In male rats, the highest 17-�-estradioldose of 4.6 �g/kg increased hemeoxygenase-1 protein3.7-fold at 6 hours (Fig. 1B, lane 8) compared with con-trols (Fig. 1B, lane 1). There was no induction ofhemeoxygenase-1 at 6 hours with lower doses (Fig. 1B,lanes 6,7) and no induction of hemeoxygenase-1 at 24hours after any dose (Fig. 1B, lanes 3–5). Six hours afterglobal ischemia in gerbils, 17-�-estradiol (460 ng/kg ad-ministered 20 minutes before ischemia) increasedhemeoxygenase-1 protein 2.4-fold (Fig. 1C, lane 2) com-pared with ischemia alone (Fig. 1C, lane 1). However, 24hours after global ischemia, 17-�-estradiol (460 ng/kg)decreased hemeoxygenase-1 protein nearly fourfold(Fig. 1C, lane 4) compared with ischemia alone (Fig. 1C,lane 3).

Immunohistochemical analysis of the normal adult ratbrain showed that 17-�-estradiol (4.6 �g/kg) inducedhemeoxygenase-1 in medium-sized vessels (most likelyarteries) in the cerebral cortex (Fig. 2D), striatum (Fig.2E), and hippocampus (Fig. 2F). There was no compa-rable staining of vessels in the control cortex (Fig. 2A),striatum (Fig. 2B), or hippocampus (Fig. 2C).

Six hours after brief global ischemia in gerbils, therewas little induction in the cortex (not shown) or striatum(Fig. 3A) in these ischemic animals. After the adminis-tration of 17-�-estradiol (4.6 �g/kg) and 6 hours afterglobal ischemia, hemeoxygenase-1 was induced in whitematter tracts in the striatum (Fig. 3D) compared withischemia alone (Figs. 3A). The staining in the white mat-ter tracts appeared to be in cells with small cell bodiesand fine processes, and probably represented oligoden-drocytes (data not shown). 17-�-Estradiol (4.6 �g/kg)also induced hemeoxygenase-1 in CA1 (Fig. 3E), CA2(Fig. 3F), and CA3 pyramidal neurons (data not shown),and to a lesser degree in dentate granule cell neurons ofthe hippocampus (data not shown) compared with ve-hicle-treated animals 6 hours after ischemia (Fig. 3B, C).The ischemia-induced staining in the lacunosom molecu-lare after ischemia alone was only slightly increased byestradiol (data not shown).

HSP25 induction17-�-Estradiol also induced HSP25 in the rat brain

(Figs. 4–6). Administration of 17-�-estradiol at doses of460ng/kg (Fig. 4A, lane 7) and 4.6 �g/kg (Fig. 4A, lane8) induced HSP25/27 approximately 1.5-fold and 2.1-fold, respectively, compared with the 6-hour control(Fig. 4A, lane 1). At 24 hours after the administration of460 ng/kg 17-�-estradiol, HSP25/27 levels increased2.1-fold (Fig. 4A, lane 4) compared with the 24-hour

FIG. 1. Western blots showing hemeoxygenase-1 protein fromfemale rat brain (A), male rat brain (B), and ischemic gerbil brain(C). Female rats in A were injected with 17-�-estradiol (lanes3–8) or vehicle (lanes 1, 2) and allowed to survive for 6 (lanes 2,6–8) or 24 hours (lanes 1, 3–5). 17-�-Estradiol was injected indoses of 46 (lanes 3, 6) or 460 ng/kg (lanes 4, 7) and 4.6 µg/kg(lanes 5,8), respectively. Male rats in B were treated identicallyexcept that lane 2 shows hemeoxygenase-1 protein induced 6hours after the administration of intraperitoneal kainic acid (10mg/kg). In C, male gerbils subjected to global ischemia 6 (lanes1, 2) or 24 hours previously (lanes 3, 4) were given saline vehicle(lanes 1, 3) or 17-�-estradiol (lanes 2, 4; 460 ng/kg) 20 minutesbefore ischemia. Lane 9 in A and B and lane 5 in C show thepurified hemeoxygenase-1 protein. Each lane represents asample from one animal. A replicate experiment showed similarfindings.

EFFECT OF 17-�-ESTRADIOL 185


control (Fig. 4A, lane 2). In male rats, 17-�-estradiolonly minimally increased HSP25/27 at the higher dosesof 460ng/kg and 4.6 �g/kg at 24 hours (Fig. 4B, lanes4,5) compared with 6 hours (Fig. 4B, lanes 7,8) and

vehicle control (Fig. 4A, lane 1). Six hours after globalischemia in the gerbil, 17-�-estradiol (460 ng/kg) in-creased HSP25/27 levels 1.8-fold (Fig. 4C, lane 2) com-pared with ischemia alone (Fig. 4C, lane 1), whereas

FIG. 2. Hemeoxygenase-1 immunostaining in the cortex (A and D), striatum (B and E), and hippocampus (C and F) in male rats 6 hoursafter injection of saline (A–C) or 4.6 µg/kg intraperitoneal 17-�-estradiol (D–F). Arrowheads show hemeoxygenase-1–stained vessels,probably arteries and arterioles, in estradiol-injected animals.

A. LU ET AL.186


FIG. 3. Hemeoxygenase-1 immunohistochemistry of ischemic gerbil brain (A–F) of the striatum (A and D), hippocampus CA1 (B and E)and hippocampus CA2 regions (C and F) 6 hours after ischemia. One group of animals received saline vehicle (A–C) and the otherreceived 4.6 µg/kg intraperitoneal 17-�-estradiol (D–F) 20 minutes before ischemia. Arrows in (D) point to hemeoxygenase-1 staining ofwhite matter bundles in animals given estradiol (D) compared with no white matter staining in animals given saline (A). In the hippo-campus, estradiol increased hemeoxygenase-1 staining in CA1 (E, arrow) and CA2 (F, arrow) with less staining in vehicle-injectedischemic controls (B and C).



there was little difference between estrogen-treated (Fig.4C, lane 4) and ischemia-alone (Fig. 4C, lane 3) groups24 hours after ischemia.

Immunocytochemical analysis showed similar, scat-tered HSP25/27 glial staining in brains of animals thatreceived saline (Figs. 5A to 5C) and 17-�-estradiol (Figs.5D to 5F). However, 6 hours after administration of 4.6�g/kg 17-�-estradiol, there was marked upregulation ofHSP25/27 in cerebral blood vessels (Figs. 5D to 5F)compared with saline-injected rats (Figs. 5A to 5C).Compared with vehicle-injected animals that had slightvascular staining (Figs. 5A to 5C), staining was localizedto medium-sized vessels (probably arteries) in the cortex(Fig. 5D), striatum (Fig. 5E), hippocampus (Fig. 5F), andthroughout other brain regions (data not shown). Sixhours after global ischemia in gerbils, the HSP25/27 pro-tein was induced in small arteries in many brain regions,including the striatum (Fig. 6A). There was also someHSP25/27 staining of neurons in hippocampal CA re-gions (Fig. 6B) and dentate gyrus (Fig. 6C). Six hoursafter global ischemia in gerbils pretreated with 17-�-estradiol (4.6 �g/kg), HSP25/27 was markedly inducedin striatum astrocytes (arrow heads, Fig. 6D). HSP25/27staining also increased, to some degree, in CA1 pyrami-dal neurons (Fig. 6E) and dentate gyrus granule cell neu-rons and hilar neurons (Fig. 6F) compared with ischemiaalone (Figs. 6 B and 6C).

Heat shock protein 70 inductionThe antibody used to detect HSP70 on Western blots

stained two bands (Fig. 7A and 7B, lane 7). The lowerband represents the constitutive Hsc70 protein thatshowed little change 6 (Figs. 7A and 7B, lanes 6–8) or24 hours (Figs. 7A,B, lanes 3–5) after administrationof varying doses of estrogen in female (Fig. 7A) ormale rats (Fig. 7B). The upper band represents the in-ducible HSP70 (Figs. 7A and 7B, lane 9). Six hours afterthe administration of 17-�-estradiol, the 460-ng/kg doseinduced HSP70 levels 2.3-fold in female rats (Fig. 7A,lane 7) compared with controls (Fig. 7A, lane 1); andthe 460-ng/kg (Fig. 7B, lane 7) and 4.6-�g/kg (Fig. 7B,lane 8) doses of estradiol induced HSP70 levels 3.2-foldand twofold, respectively, in male rats comparedwith controls (Fig. 7B, lane 1). The levels of HSP70decreased to control levels by 24 hours after estradiolin female (Fig. 7A, lanes 3–5) and male rats (Fig. 7B,lanes 3–5).

Immunocytochemical analysis revealed that 17-�-estradiol (4.6 �g/kg) induced HSP70 mainly in smallarteries in the cortex (Fig. 8D), striatum (Fig. 8E), hip-pocampus (Fig. 8F) compared with no vascular stainingin vehicle-injected controls (Figs. 8A-C). The immuno-cytochemistry is thought to show HSP70 induction be-cause only HSP70 and not Hsc70 was induced on theWestern blots.

Heat shock protein 90, 47, and 60Administration of 46 ng/kg, 460 ng/kg, and 4.6 �g/kg

17-�-estradiol did not affect HSP90 (Fig. 9A, lanes 6–8),HSP47 (Fig. 9B, lanes 6–8), or HSP60 (Fig. 9C, lanes6–8) 6 hours after administration compared with 6-hourcontrols (Figs. 9A to 9C, lane 1). Similarly, 17-� estra-diol did not affect HSP90 (Fig. 9A, lanes 3–5), HSP47(Fig. 9B, lanes 3–5), or HSP60 (Fig. 9C, lanes 3–5) 24hours after administration compared with 24-hour con-trols (Figs. 9A to 9C, lane 2).

It should be emphasized that the changes of hemeoxy-genase-1, HSP25/27, and HSP70 on Western blots ofbrains of estrogen-treated rats were generally modest.This finding appears to be due to dilution of the HSPinduction, mainly in vessels with the HSPs in other cel-lular compartments that are not changing. During theinterpretation of these HSP90, HSP47, and HSP60 data,it should be considered that changes in an underrepre-sented cell type will not be detected on the Western blots.

Heat shock factor-1 protein inductionThe last study explored one possible mechanism by

which estrogen might induce HSPs in the brain. We pos-tulated that if HSF had an estrogen response element inits promoter, then estrogen could induce HSF. Adminis-tration of 17-�-estradiol (460 ng/kg) to nonischemic

FIG. 4. Western blots showing heat shock protein (HSP) 25/27from the female (A) and male rat brain (B), and the ischemicgerbil brain (C). Female rats in A were injected with 17-�-estradiol (lanes 3–8) or vehicle (lanes 1, 2) and allowed to survivefor 6 (lanes 1, 6–8) or 24 hours (lanes 2, 3–5). 17-�-Estradiol wasinjected in doses of 46 (lanes 3, 6) or 460 ng/kg (lanes 4, 7) and4.6 µg/kg (lanes 5, 8), respectively. Male rats in B were treatedidentically except that lane 2 shows HSP25/27 induced 6 hoursafter intraperitoneal kainic acid (10 mg/kg). In C male gerbilssubjected to global ischemia 6 (lanes 1, 2) or 24 hours previously(lanes 3, 4) were given saline vehicle (lanes 1, 3) or 460 ng/kg17-�-estradiol (lanes 2, 4) 20 minutes before ischemia. Lane 9 inA and B and lane 5 in C show the purified HSP25/27. Each lanerepresents a sample from one animal. A replicate experimentshowed similar findings.

A. LU ET AL.188


male rats increased levels of HSF1 protein 1.6-fold (Fig.10A, lane 3) compared with controls (Fig. 10A, lane 1) at6 hours, and at 24 hours increased HSF1 protein levels1.5-fold (Fig. 10A, lane 4) compared with controls (Fig.

10A, lane 2). Six hours after global ischemia, 17-�-estradiol (460 ng/kg) increased HSF1 protein levels 1.6-fold (Fig. 10B, lane 2) compared with ischemia alone(Fig. 10B, lane 1). At 24 hours after global ischemia,

FIG. 5. Heat shock protein (HSP) 25/27 immunostaining in the cortex (A and D), striatum (B and E), and hippocampus (C and F) in malerats 6 hours after injection of saline (A–C) or 4.6 µg/kg intraperitoneal 17-�-estradiol (D–F). Arrowheads show HSP25/27-stained vesselsin the cortex (D), striatum (E), and hippocampus (F) that are especially prominent in estradiol-injected animals and that are probablyarteries and arterioles.



FIG. 6. Heat shock protein (HSP) 25/27 immunohistochemistry of the ischemic gerbil brain (A–F) of striatum (A and D), hippocampusCA1 (B and E), and dentate gyrus (C and F) 6 hours after ischemia. One group of animals received saline vehicle (A–C) and the otherreceived 4.6 µg/kg intraperitoneal 17-�-estradiol (D–F) 20 minutes before ischemia. Vehicle-treated ischemic animals showed somestaining in glia and scattered blood vessels in the striatum (A) and in other regions. Estradiol-treated ischemic animals showed similarfindings, except there was marked induction of HSP25/27 in astrocytes in the striatum (D, arrows). Moreover, estradiol-treated animalshad increased HSP25/27 staining in CA1 (E, arrow) and dentate gyrus of the hippocampus (F, arrow) compared with saline-injectedsubjects (B and C).

A. LU ET AL.190


17-�-estradiol increased levels of HSF1 protein 2.1-fold(Fig. 10B, lane 4) compared with ischemia alone (Fig.10B, lane 3).

DISCUSSION

The results of this study show that estradiol induceshemeoxygenase-1, HSP25/27, and HSP70 in the brain 6to 24 hours after acute administration. Estradiol induc-tion of these HSPs occurs mostly in arteries in the normalbrain and in arteries, glia, and neurons in the ischemicbrain. Estrogen induction of heat-shock factors providesat least one mechanism by which estrogen induces HSPsin the brain and in other tissues. Finally, it is proposedthat acute neuroprotection afforded by estrogen may bedue at least in part to the induction or facilitation of theHSP response.

The present study provides the first evidence that 17-�-estradiol induces hemeoxygenase-1 protein in the nor-mal and ischemic brain. Hemeoxygenase-1 was inducedin blood vessels of nonischemic rats and in oligodendro-cytes and hippocampal neurons of ischemic rats. Thehemeoxygenase-1 gene is induced by various stimuli.Oxidative stress activates mitogen-activated kinases, mi-togen-activated protein kinases that induces Nrf2, whichinduces hemeoxygenase-1 through an AP1 site (Elbirtand Bonkovsky, 1999; Immenschuh and Ramadori,2000). Because estrogen activates mitogen-activated pro-tein kinases (Green and Simpkins, 2000), it may inducehemeoxygenase-1 in part through this pathway. Nitricoxide is a potent hemeoxygenase-1 inducer (Bouton andDemple, 2000; Chen and Maines, 2000). Estradiol in-duces the association between HSP90 and endothelialnitric oxide synthase, the activation of endothelial nitric

oxide synthase, and the release of nitric oxide (Russell etal., 2000). The estrogen receptor is localized to the ca-veola, where it directly interacts with the p85 subunit ofPI3-kinase to activate the PI3-kinase-Akt endothelial ni-tric oxide synthase pathway (Mendelsohn, 2000). Cyclicadenosine monophosphate and cyclic guanosine mono-phosphate can activate protein kinase A and proteinkinase G and increase hemeoxygenase-1 transcription(Immenschuh and Ramadori, 2000). Estrogen increasescyclic adenosine monophosphate accumulation andphosphorylation of cyclic adenosine monophosphate re-sponse element binding protein (Green and Simpkins,2000). Therefore, estrogen likely induces hemeoxygen-ase-1 through multiple pathways, though the nitric oxidesynthase and cyclic guanosine monophosphate pathwaysmay be particularly important because estrogen induction of hemeoxygenase-1 appears to occur mainly insmooth muscle cells around cerebral arteries and arterioles.

Hemeoxygenase-1 is cytoprotective in various cellculture and animal models. Hemeoxygenase-1 gene in-duction is postulated to be an adaptive cellular defensemechanism against increases in intracellular heme (Leeet al., 1996; Yang et al., 1999). Hemeoxygenase-1 ex-pression in vascular endothelial cells protects againstoxidative stress-induced apoptosis (Foresti et al., 1999).Hemeoxygenase-1 and the products of heme catabolismattenuate the arterial response to injury and ensure vas-cular wall remodeling (Tulis et al., 2001). Neurons thatover express hemeoxygenase-1 are resistant to glutamateand hydrogen peroxide-mediated cell death (Chen et al.,2000). Hemeoxygenase-1 overexpression in transgenicmice protects the brain from permanent focal ischemia(Panahian et al., 1999). The induction of hemeoxygenaseprotein protects neurons in cortex and striatum againsttransient forebrain ischemia (Takizawa et al., 1998). Inthis study, estrogen induction of hemeoxygenase-1 inarteries, glia, and neurons likely protects these cells fromischemic injury and provides a new molecular mecha-nism by which estrogen may protect the brain and heartagainst ischemia.

Estradiol also increased the levels of HSP25/27 inblood vessels of normal brain and in glia and neurons ofischemic brain. This finding is consistent with those ofprevious studies that 17-�-estradiol treatment of MCF7human breast cancer cells increases HSP27 messengerRNA levels twofold for up to 24 hours (Porter et al.,2001). Estrogen increased HSP27 levels in the humanendometrial glandular epithelium (Padwick et al., 1994),and estradiol treatment of endothelial cells resulted in anincrease in both bovine and human HSP27 (Piotrowicz etal., 1995). These data strongly support our results.

The mechanisms of estrogen induction of HSP25/27are still being studied. Because the intron-exon structureof the mouse HSP25 gene is extremely similar to thehuman HSP27 gene, it is likely they represent the same

FIG. 7. Western blots showing heat shock protein (HSP) 70 (up-per band) and Hsc70 protein (lower band) from the female (A)and male rat brain (B). Female rats in A were injected with 17-�-estradiol (lanes 3–8) or vehicle (lanes 1, 2) and allowed tosurvive for 6 (lanes 1, 6–8) or 24 hours (lanes 2, 3–5). 17-�-Estradiol was injected in doses of 46 (lanes 3, 6) or 460 ng/kg(lanes 4, 7) and 4.6 µg/kg (lanes 5, 8), respectively. Male rats inB were treated identically except that lane 2 shows HSP70 in-duced 6 hours after the administration of intraperitoneal kainicacid (10 mg/kg). The band in lane 9 (A and B) shows the purifiedHSP70 and confirms that the upper bands in lane 7 in A and Bare HSP70. Each lane represents a sample from one animal. Areplicate experiment showed similar findings.



gene in these two species and hence are referred to as theHSP25/27 gene herein. The promoter region ofHSP25/27 contains putative transcription factor-bindingelements, including two guanine–cytosine-rich Sp1-

binding domains, two heat-shock elements, and an estro-gen-responsive element half-site in direct proximity tothe TATA box (Gaestel et al., 1993). The Sp1 and half-palindromic estrogen response element and formation of

FIG. 8. HSP70 immunostaining in cortex (A and D), striatum (B and E) and hippocampus (C and F) in male rats 6 hours after injectionof saline (A–C) or 4.6 µg/kg intraperitoneal 17-�-estradiol (D–F). Arrowheads show HSP70 stained vessels in estradiol-injected animals(D–F) that are probably arteries/arterioles.

A. LU ET AL.192


the Sp1/estrogen receptor complex are involved in estro-gen-induced HSP27 gene expression (Porter et al., 1996).However, mutation of the estrogen response elementhalfsite does not result in loss of estrogen responsiveness,suggesting that estrogen inducibility is mediated throughthe Sp1-DNA motif. Moreover, Sp1 and estrogen recep-tor proteins physically interact to enhance Sp1-DNAbinding that is estrogen dependent (Porter et al., 1997).

HSP27 reduces apoptotic and necrotic cell death(Mehlen et al., 1996; Samali and Cotter, 1996; Wagstaffet al., 1999). Estradiol rapidly activates p38� mitogen-activated protein kinase in endothelial cells, which acti-vates the mitogen-activated protein kinase-activated pro-tein kinase-2 and the phosphorylation of HSP27.Estradiol preserves the endothelial cells stress fiber for-mation and actin and membrane integrity in simulatedischemia, prevents hypoxia-induced apoptosis, and in-duces the migration of endothelial cells and the forma-tion of primitive capillary tubes. These effects are re-versed by the inhibition of p38�, by the expression of adominant-negative mitogen-activated protein kinase-activated protein kinase-2 protein, or by the expressionof a phosphorylation site mutant HSP27 (Razandi et al.,2000). Estrogen treatment increases SMER14 cell viabil-ity in the presence of staurosporine. Estrogen decreasesannexin V staining and DNA fragmentation and in-creases HSP27 levels in SMER14 cells (Cooper et al.,2000). HSP27 expression in vascular smooth musclecells induced by mild heat shock prevents inhibition ofproliferation and induction of necrosis by heat shock(Champagne et al., 1999). HSP27 expression in sensoryneurons promotes survival after axotomy or neurotrophinwithdrawal (Lewis et al., 1999). Mild cerebral ischemiainduces HSP27 that correlates with the onset of ischemic

tolerance (Kato et al., 1994). The finding that estrogeninduces HSP27 in blood vessels and glia and neurons inthe brain may indicate an important mechanism for es-trogen protection of cerebral ischemia.

Estrogen has been reported to induce HSP70 in avariety of tissues. Estradiol increased HSP70 messengerRNA in the myometrium and endometrium of non-pregnant ewes (Wu et al., 1996). Estradiol stimulatedHSP70 expression in short-term cultures of steroid-responsive (T47-D) cells (Tang et al., 1995). Estro-gen activated HSF1 and increased HSP72 in isolatedcardiac myocytes (Knowlton and Sun, 2001). Therehave been few suggestions that estrogen could inducethe inducible HSP70 protein in the brain. Estrogenand progesterone induce the constitutive HSP70 (Hsc73)in the pituitary and subregions of the ventromedialhypothalamus, as shown by Northern analysis and insitu hybridization (Krebs et al., 1999). Estrogen inducedHSP72, HSP27, and HSP90, and subsequent granula-tion in astrocytes in estradiol receptor-rich brain re-gions including the arcuate nucleus and the walls ofthe third ventricle (Mydlarski et al., 1995). This is thefirst study to show that estrogen induces HSP70 inmultiple brain regions and that the induction appears tooccur mainly in blood vessels that are likely arteriesand arterioles.

Estrogen induction of HSP70 most likely protects thebrain. Overexpression of HSP70 in cultured cells pro-tects against a variety of different injuries in vitro (Sharpet al., 1999; Yenari et al., 1999). Viral overexpression ofHSP70 protects against ischemia and excitoxicity in vivo(Yenari et al., 1999). Overexpression of HSP70 in trans-genic mice protects the mice against permanent middlecerebral artery occlusions (Rajdev et al., 2000). Estrogen

FIG. 10. Heat-shock Factor1 (HSF1) Western blots of female ratbrain (A) and ischemic male gerbil brain (B). For the female ratbrain in (A) HSF1 was similar at 6h (lane 1) and 24h (lane 2) aftervehicle injections (lanes 1,2), as compared with increased HSF1protein at 6h (lane 3) and 24 hours (lane 4) after 17-�-estradiol(460 ng/kg) injections (lanes 3,4). For the ischemic gerbil brainsin (B) HSF1 protein is shown for 17-�-estradiol–treated animals(lanes 2,4) compared with vehicle-treated animals (lanes 1,3) at6 hours (lanes 1,2) and 24 hours (lanes 3,4) after global isch-emia. Estradiol increased HSF1 at 6h (lane 2) compared withcontrol (lane 1) and estradiol increased HSF1 at 24h (lane 4)compared with control (lane 3). Each lane resents a sample fromone animal. A replicate experiment showed similar findings.

FIG. 9. Western blots showing HSP90 protein (A), HSP47 pro-tein (B) and HSP60 protein (C) from female rat brain after injec-tions with 17-�-estradiol (lanes 3–8) or vehicle (lanes 1,2) andsurvival times of 6 hours (lanes 1, 6–8) or for 24 hours (lanes 2,3–5). 17-�-Estradiol was injected in doses of 46 ng/kg (lanes3,6), 460 ng/kg (lanes 4,7) and 4.6 µg/kg (lanes 5,8). Purifiedproteins are shown in lane 9. Each lane represents a sample fromone animal. A replicate experiment showed similar findings.



induction of HSP70 in vessels might help to preserveblood flow or to decrease reperfusion-induced hemor-rhages after spontaneous or tissue plasminogen activator-induced clot lysis.

In the rat, physiologic plasma 17-�-estradiol levelsrange between 10 and 30 pg/ml. In healthy Wistar malerats, plasma 17-�-estradiol levels reached 10 pg/ml aftera 25-�g estradiol pellet was implanted under the skin for7 to 10 days (Hurn and Macrae, 2000; Toung et al, 1998).Therefore, the 17-�-estradiol doses of 460 ng/kg (115ng/rat) and 4.6 �g/kg used in this study to induce HSPsshould be in the physiologic range. Estradiol in physi-ologic doses protects rats against cerebral ischemia. Im-portantly, the preservation of intraischemic cortical per-fusion by estradiol was achieved by physiologic doses ofestrogen and was lost at supraphysiologic doses (Hurnand Macrae, 2000). Induction of HSPs with low-doseestradiol may contribute to the neuroprotection producedby estrogen.

The mechanism by which estrogen can upregulateHSP32 (hemeoxygenase-1), HSP25/27, and HSP70 incerebral arteries, glia, and neurons is of significant in-terest. Estrogen regulates HSF-1 and -2 at the messenger-RNA and protein levels in the endometrium (Yang et al.,1995). Estrogen and progesterone activate HSF-1 inadult male isolated cardiac myocytes, and this effect isfollowed by an increase in HSP72 protein (Knowlton andSun, 2001). Transcriptional induction of HSP genes isgenerally mediated by binding of heat-shock transcrip-tion factor to the heat-shock element present in the pro-moters of heat-shock genes. In our study, estradiol in-creased HSF1 protein relative to controls at 6 and 24hours. Moreover, estradiol induced hemeoxygenase-1and HSP70 by 6 hours. Therefore, estrogen inductionand activation of HSF1 expression could be one of themechanisms that mediate estrogen induction ofhemeoxygenase-1, HSP25/27, and HSP70 in vessels,glia, and neurons in the brain. Finally, pharmacologicdoses of estrogen would bind HSP90, which would freeHSFs from HSP90 (Zou et al., 1998). This HSP90 bind-ing by estrogen could activate HSFs and HSP transcrip-tion just as the drug geldanamycin appears to bindHSP90 during periods of stress, free HSFs, and activatetranscription of HSPs (Zou et al., 1998).

The estrogen induction of hemeoxygenase-1,HSP25/27, and HSP70 in blood vessels was one of themajor surprise findings of this study. The HSP inductionoccurred mainly in medium-sized arteries to small arte-rioles, which would suggest that estrogen might induceHSPs in the smooth muscle cells or endothelial cellsaround arteries. This observation also suggests that es-trogen likely induces HSPs in arteries throughout thebody including coronary, pulmonary, and renal arteries.The estrogen receptors � and � are found in variousregions of the rat brain (Pelletier, 2000; Commins and

Yahr, 1985). In approximately 70% of the medial smoothmuscle cells of the female rat aorta, tail artery, and uter-ine artery, nuclear immunoreactivity to estrogen receptor� was observed. In these vessels, endothelial cells alsoexpressed estrogen receptor �. In the aorta and tail ar-teries, immunoreactivity for estrogen receptor � was ob-served in medial smooth muscle and endothelial cells ofuterine vessels (Andersson et al., 2001). Why estrogenmight target smooth muscle cells or endothelial cellsaround arteries is not clear. There could be an HSFunique to smooth muscle cells, a particular class of es-trogen receptor in smooth muscle cells or endothelialcells that might signal heat-shock activation in smoothmuscle cells.

In summary, 17-�-estradiol induces HSF1, hemeoxy-genase-1 (HSP32), HSP25/27, and HSP70 in the normaland ischemic brain. Estrogen has an important influenceon the expression of HSPs in normal blood vesselsand under conditions of stress in most other cells. Thiseffect may be an important molecular mechanism of es-trogen protection in the brain and in other organs, in-cluding the heart.

REFERENCES

Andersson C, Lydrup ML, Ferno M, Idvall I, Gustafsson J, Nilsson BO(2001) Immunocytochemical demonstration of oestrogen receptorbeta in blood vessels of the female rat. J Endocrinol 169:241–247

Bouton C, Demple B (2000) Nitric oxide-inducible expression of hemeoxygenase-1 in human cells. Translation-independent stabilizationof the mRNA and evidence for direct action of nitric oxide. J BiolChem 275:32688–32693

Champagne MJ, Dumas P, Orlov SN, Bennett MR, Hamet P, TremblayJ (1999) Protection against necrosis but not apoptosis by heat-stress proteins in vascular smooth muscle cells: evidence for dis-tinct modes of cell death. Hypertension 33:906–913

Chen K, Maines MD (2000) Nitric oxide induces heme oxygenase-1via mitogen-activated protein kinases ERK and p38. Cell Mol Biol(Noisy-le-grand) 46:609–617

Chen K, Gunter K, Maines MD (2000) Neurons overexpressing hemeoxygenase-1 resist oxidative stress-mediated cell death. J Neuro-chem 75:304–313

Commins D, Yahr P (1985) Autoradiographic localization of estrogenand androgen receptors in the sexually dimorphic area and otherregions of the gerbil brain. J Comp Neurol 231:473–489

Cooper LF, Tiffee JC, Griffin JP, Hamano H, Guo Z (2000) Estrogen-induced resistance to osteoblast apoptosis is associated with in-creased hsp27 expression. J Cell Physiol 185:401–407

Elbirt KK, Bonkovsky HL (1999) Heme oxygenase: recent advances inunderstanding its regulation and role. Proc Assoc Am Physicians111:438–447

Foresti R, Sarathchandra P, Clark JE, Green CJ, Motterlini R (1999)Peroxynitrite induces haem oxygenase-1 in vascular endothelialcells: a link to apoptosis. Biochem J 339:729–736

Gaestel M, Gotthardt R, Muller T (1993) Structure and organisation ofa murine gene encoding small heat-shock protein Hsp25. Gene128:279–283

Garcia-Segura LM, Azcoitia I, DonCarlos LL (2001) Neuroprotectionby estradiol. Prog Neurobiol 63:29–60

Green PS, Simpkins JW (2000) Neuroprotective effects of estrogens:potential mechanisms of action. Int J Dev Neurosci 18:347–358

Hurn PD, Macrae IM (2000) Estrogen as a neuroprotectant in stroke. JCereb Blood Flow Metab 20:631–652

Immenschuh S, Ramadori G (2000) Gene regulation of heme oxygen-ase-1 as a therapeutic target. Biochem Pharmacol 60:1121–1128

A. LU ET AL.194


Kato H, Liu Y, Kogure K, Kato K (1994) Induction of 27-kDa heatshock protein following cerebral ischemia in a rat model of isch-emic tolerance. Brain Res 634:235–244

Knowlton AA, Sun L (2001) Heat-shock factor-1, steroid hormones,and regulation of heat-shock protein expression in the heart. Am JPhysiol Heart Circ Physiol 280: H455–464

Krebs CJ, Jarvis ED, Pfaff DW (1999) The 70-kDa heat shock cognateprotein (Hsc73) gene is enhanced by ovarian hormones in theventromedial hypothalamus. Proc Natl Acad Sci U S A 96:1686–1691

Lee PJ, Alam J, Wiegand GW, Choi AM (1996) Overexpression ofheme oxygenase-1 in human pulmonary epithelial cells results incell growth arrest and increased resistance to hyperoxia. Proc NatlAcad Sci U S A 93:10393–10398

Lewis SE, Mannion RJ, White FA, Coggeshall RE, Beggs S, CostiganM, Martin JL, Dillmann WH, Woolf CJ (1999) A role for HSP27in sensory neuron survival. J Neurosci 19:8945–8953

Mehlen P, Schulze-Osthoff K, Arrigo AP (1996) Small stress proteinsas novel regulators of apoptosis. Heat shock protein 27 blocksFas/APO-1- and staurosporine-induced cell death. J Biol Chem271:16510–16514

Mendelsohn ME (2000) Nongenomic, ER-mediated activation of en-dothelial nitric oxide synthase: how does it work? What does itmean? Circ Res 87:956–960

Mydlarski MB, Liberman A, Schipper HM (1995) Estrogen inductionof glial heat shock proteins: implications for hypothalamic aging.Neurobiol Aging 16:977–981

Olazabal UE, Pfaff DW, Mobbs CV (1992) Estrogenic regulation ofheat shock protein 90 kDa in the rat ventromedial hypothalamusand uterus. Mol Cell Endocrinol 84:175–183

Padwick ML, Whitehead M, King RJ (1994) Hormonal regulation ofHSP27 expression in human endometrial epithelial and stromalcells. Mol Cell Endocrinol 102:9–14

Panahian N, Yoshiura M, Maines MD (1999) Overexpression of hemeoxygenase-1 is neuroprotective in a model of permanent middlecerebral artery occlusion in transgenic mice. J Neurochem72:1187–1203

Pelletier G (2000) Localization of androgen and estrogen receptors inrat and primate tissues. Histol Histopathol 15:1261–1270

Piotrowicz RS, Weber LA, Hickey E, Levin EG (1995) Acceleratedgrowth and senescence of arterial endothelial cells expressing thesmall molecular weight heat-shock protein HSP27. FASEB J9:1079–1084

Porter W, Wang F, Wang W, Duan R, Safe S (1996) Role of estrogenreceptor/Sp1 complexes in estrogen-induced heat shock protein 27gene expression. Mol Endocrinol 10:1371–1378

Porter W, Saville B, Hoivik D, Safe S (1997) Functional synergybetween the transcription factor Sp1 and the estrogen receptor. MolEndocrinol 11:1569–1580

Porter W, Wang F, Duan R, Qin C, Castro-Rivera E, Kim K, Safe S(2001) Transcriptional activation of heat shock protein 27 geneexpression by 17beta-estradiol and modulation by antiestrogensand aryl hydrocarbon receptor agonists. J Mol Endocrinol26:31–42

Rajdev S, Hara K, Kokubo Y, Mestril R, Dillmann W, Weinstein PR,Sharp FR (2000) Mice overexpressing rat heat shock protein 70 areprotected against cerebral infarction. Ann Neurol 47:782–791

Razandi M, Pedram A, Levin ER (2000) Estrogen signals to the pres-ervation of endothelial cell form and function. J Biol Chem275:38540–38546

Russell KS, Haynes MP, Caulin-Glaser T, Rosneck J, Sessa WC,Bender JR (2000) Estrogen stimulates heat shock protein 90 bind-ing to endothelial nitric oxide synthase in human vascular endo-thelial cells. Effects on calcium sensitivity and NO release. J BiolChem 275:5026–5030

Samali A, Cotter TG (1996) Heat shock proteins increase resistance toapoptosis. Exp Cell Res 223:163–170

Sharp FR, Massa SM, Swanson RA (1999) Heat-shock protein protec-tion. Trends Neurosci 22:97–99

Takizawa S, Hirabayashi H, Matsushima K, Tokuoka K, Shinohara Y(1998) Induction of heme oxygenase protein protects neurons incortex and striatum, but not in hippocampus, against transient fore-brain ischemia. J Cereb Blood Flow Metab 18:559–569

Tang PZ, Gannon MJ, Andrew A, Miller D (1995) Evidence for oes-trogenic regulation of heat shock protein expression in humanendometrium and steroid-responsive cell lines. Eur J Endocrinol133:598–605

Toung TJ, Traystman RJ, Hurn PD (1998) Estrogen-mediated neuro-protection after experimental stroke in male rats. Stroke 29:1666–1670

Tulis DA, Durante W, Peyton KJ, Evans AJ, Schafer AI (2001) Hemeoxygenase-1 attenuates vascular remodeling following balloon in-jury in rat carotid arteries. Atherosclerosis 155:113–122

Wagstaff MJ, Collaco-Moraes Y, Smith J, de Belleroche JS, Coffin RS,Latchman DS (1999) Protection of neuronal cells from apoptosisby Hsp27 delivered with a herpes simplex virus-based vector. JBiol Chem 274:5061–5069

Wu WX, Derks JB, Zhang Q, Nathanielsz PW (1996) Changes in heatshock protein-90 and -70 messenger ribonucleic acid in uterinetissues of the ewe in relation to parturition and regulation by es-tradiol and progesterone. Endocrinology 137:5685–5693

Xanthoudakis S, Nicholson DW (2000) Heat-shock proteins as deathdeterminants. Nat Cell Biol 2: E163–E165

Yang L, Quan S, Abraham NG (1999) Retrovirus-mediated HO genetransfer into endothelial cells protects against oxidant-induced in-jury. Am J Physiol 277: L127–L133

Yang X, Dale EC, Diaz J, Shyamala G (1995) Estrogen dependentexpression of heat shock transcription factor: implications for uter-ine synthesis of heat shock proteins. J Steroid Biochem Mol Biol52:415–419

Yenari MA, Giffard RG, Sapolsky RM, Steinberg GK (1999) Theneuroprotective potential of heat shock protein 70 (HSP70). MolMed Today 5:525–531

Zou J, Guo Y, Guettouche T, Smith DF, Voellmy R (1998) Repressionof heat shock transcription factor HSF1 activation by HSP90(HSP90 complex) that forms a stress-sensitive complex withHSF1. Cell 94:471–480



17-β-estradiol induces heat shock proteins in brain arteries and potentiates ischemic heat shock...

Documents