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ORIGINAL ARTICLE

GATA-3 Transduces Survival Signals in Osteoblasts Through Upregulation of bcl-xL Gene Expression Ruei-Ming Chen , 1,2 Yi-Ling Lin , 2 and Chih-Wei Chou1 1 2

Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan Cell Physiology and Molecular Image Research Center and Department of Anesthesiology, Taipei Medical University–Wan Fang Medical Center, Taipei, Taiwan

ABSTRACT GATA-3, a transcription factor, participates in regulating cell development, proliferation, and death. This study was aimed at evaluating the roles of GATA-3 in protecting osteoblasts against oxidative stress–induced apoptotic insults and their possible mechanisms. Pretreatment with nitric oxide (NO) for 24 hours protected osteoblasts, prepared from neonatal rat calvaria, against oxidative stress– induced apoptotic insults. Such protection involved enhancement of Bcl-XL messenger (m)RNA and protein syntheses and the translocation of this antiapoptotic protein from the cytoplasm to mitochondria. GATA-3 was detected in rat osteoblasts, and GATA3-specific DNA-binding elements exist in the promoter region of the bcl-xL gene. NO preconditioning attenuated oxidative stress–caused suppression of GATA-3 mRNA and protein synthesis and the translocation of this transcription factor from the cytoplasm to nuclei. Application of GATA-3 small interfering (si)RNA into osteoblasts decreased the levels of this transcription factor and simultaneously inhibited Bcl-XL mRNA synthesis. Pretreatment with NO lowered the oxidative stress–caused alteration in the binding of GATA-3 to its specific DNA motifs. Oxidative stress–inhibited Runx2 mRNA expression, but NO preconditioning decreased such inhibition. NO pretreatment time-dependently enhanced the association of GATA-3 with Runx2. Knocking down the translation of GATA-3 using RNA interference significantly decreased the protection of NO preconditioning against oxidative stress–induced alterations of cell morphologies, DNA fragmentation, and cell apoptosis. In comparison, overexpression of GATA-3 could promote NO preconditioning–involved Bcl-XL expression and cell survival. Therefore, this study shows that GATA-3 plays critical roles in mediating survival signals in osteoblasts, possibly through upregulating bcl-xL gene expression. ß 2010 American Society for Bone and Mineral Research. KEY WORDS: GATA-3; OSTEOBLASTS; NO PRECONDITIONING; OXIDATIVE STRESS; APOPTOSIS; BCL-XL

Introduction

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one is maintained by a dynamic process called bone remodeling that involves bone removal and replacement.(1) Osteoblasts play crucial roles in bone formation.(2) The survival and activity of osteoblasts are related to the maintenance of bone remodeling and the occurrence and recovery of bone diseases. Meanwhile, there are a number of local and systemic factors that contribute to regulating osteoblast survival, development, and even death.(3,4) Reactive oxygen species (ROS) are one of such critical factor.(5) In bone loss–related diseases, ROS-induced oxidative stress can stimulate expression of key osteoclastogenic factors such as the receptor activator of nuclear factor-kB ligand to regulate osteoblast activities.(6) Nitric oxide (NO), one of the ROS, has biphasic effects on osteoblast metabolism.(7,8) Under stimulation of inflammatory cytokines or mechanical stress, NO can be overproduced and

lead to osteoblast injuries.(9,10) In comparison, constitutive NO can be an effective mediator that regulates 17b-estradiolstimulated proliferation and differentiation of human and rodent osteoblasts.(11) In ischemia and reperfusion injury, NO preconditioning was shown to produce protective effects on cardiomyocytes, hepatocytes, and endothelial cells.(12) Our previous studies showed that pretreatment with NO protects osteoblasts from oxidative stress–induced apoptotic insults via a mitochondriondependent mechanism.(8,13) Thus NO preconditioning may be applied as a cell model to investigate the survival signals or as an effective treatment of inflammation-induced bone diseases. However, the molecular mechanisms of NO pretreatment–caused osteoblast protection are still little known. GATA-DNA-binding proteins (GATAs), a family of transcriptional regulators, contain two zinc fingers with the Cys-X2-CysX17-Cys-X2-Cys motif that binds directly to the nucleotide sequence element (A/T)GATA(A/G).(14,15) There are six members

Received in original form August 18, 2009; revised form March 24, 2010; accepted April 15, 2010. Published online April 30, 2010. Address correspondence to: Ruei-Ming Chen, PhD, Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan. E-mail: [email protected] Part of these data was presented in abstract form at the 33rd Federation of European Biochemical Societies Congress and 11th International Union of Biochemistry and Molecular Biology Conference, Athens, Greece, June 28 to July 3, 2008. Journal of Bone and Mineral Research, Vol. 25, No. 10, October 2010, pp 2193–2204 DOI: 10.1002/jbmr.121 ß 2010 American Society for Bone and Mineral Research

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in the GATA family of transcription factors. In general, GATA-1, -2, and -3 are known to regulate critical events in hematopoietic lineages, whereas GATA-4, -5, and -6 are reported to be expressed mainly in nonhematopoietic tissues, including the heart and gut.(16) GATA-3, one of the GATA family proteins, transcriptionally regulates interleukin-4, -5, and -13 gene expressions in T-helper 2 cells, which control cell differentiation and mediate allergic inflammation.(17,18) In addition to hematopoietic cells, transcription factors GATA-3 and GATA-2 also were detected during early stages of embryonic development in the central nervous system of the mouse.(19) Dysfunction of GATA-3 is associated with the occurrence of such human diseases as human hypoparathyroidism, sensorineural deafness, and renal anomalies syndrome.(20) Thus GATA-3 is an important transcription factor expressed in both hematopoietic and nonhematopoietic tissues and can regulate the function of these tissues/cells. Meanwhile, the expression of GATA-3 and its function in bones and skeletal muscle are still unknown. Our preliminary data revealed that GATA-3 messenger (m)RNA and protein were detected in osteoblasts, and NO preconditioning or oxidative stress altered the levels of this transcription factor in bone cells. Apoptosis, a type of energy-dependent cell death, is involved in physiologic and pathophysiologic regulation of tissue homeostasis and cell activities.(21) A previous study showed that apoptosis determines the osteoblast population and bone turnover in the adult skeleton.(22) A plethora of pro- and antiapoptotic proteins are involved in the homeostatic balance between cell survival and apoptosis. Bcl-XL and Bax are two multidomain Bcl-2 family members with homology in the BH1 to BH3 domains.(23) When exposed to apoptotic stimuli, the Bax protein is specifically translocated from the cytoplasm to mitochondria and triggers a series of apoptotic events.(24) By comparison, Bcl-XL is determined to possess antiapoptotic effects because it can associate with Bax to prevent apoptotic insults.(25) In ischemia-reperfusion-induced heart injury, overexpression of the bcl-xL gene was shown to suppress Bax translocation, leading to a reduction in cytochrome C release and cardiac cell apoptosis.(26) Thus expression of the bcl-xL gene is regulated by various stimuli, and its intracellular levels drive cells to undergo survival or apoptosis. A previous study reported that GATA-DNA-binding elements are found in the 5’-end promoter region of the bcl-xL gene.(27) After searching using a bioinformatic approach, we further found that there are at least three GATA-3specific DNA-binding elements in the promoter region of the bclxL gene. Our previous studies demonstrated that NO preconditioning can protect osteoblasts from oxidative stress–induced apoptosis.(8,13) In this study, we further used an NO preconditioning model to evaluate the roles of GATA-3 in transducing the survival signals in primary neonatal rat osteoblasts and its possible molecular mechanisms.

Materials and Methods Preparation of rat osteoblasts Rat osteoblasts were prepared from 3-day-old Wistar rat calvaria according to a previously described collagenase digestion

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Journal of Bone and Mineral Research

method.(10) All procedures were performed according to the National Institutes of Health Guidelines for Use of Laboratory Animals and were preapproved by the Institutional Animal Care and Use Committee of Taipei Medical University. Osteoblasts were seeded in DMEM (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated FBS, L-glutamine, penicillin (100 IU/mL), and streptomycin (100 mg/mL) in 75-cm2 flasks at 378C in a humidified atmosphere of 5% CO2. Osteoblasts were grown to confluence prior to drug treatment. Only the first passage of rat osteoblasts was used in this study.

Establishment of NO preconditioning and oxidative stress models Our previous studies showed that pretreatment with 0.3 mM sodium nitroprusside (SNP), an NO donor, for 24 hours caused about a 30% increase in levels of NO in osteoblasts but did not affect cell viability.(8,13) Meanwhile, exposure of osteoblasts to 2 mM SNP increased oxidative stress, leading to cell insults. Thus, in this study, osteoblasts were treated with 0.3 mM SNP for 24 hours as a condition of NO preconditioning and were exposed to 2 mM SNP as a condition of oxidative stress.

Assay of cell viability Cell viability was determined by a colorimetric 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously.(28) Briefly, osteoblasts (2 104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, osteoblasts were cultured with new medium containing 0.5 mg/mL of MTT for a further 3 hours. The blue formazan products in the osteoblasts were dissolved in DMSO and measured spectrophotometrically at a wavelength of 550 nm.

Determination of DNA fragmentation DNA fragmentation was quantified as described previously.(29) The BrdU-labeled histone-associated DNA fragments in the cytoplasm of cell lysates were detected according to the instructions of the cellular DNA fragmentation enzyme-linked immunosorbent assay kit (Boehringer Mannheim, Indianapolis, IN, USA). Briefly, osteoblasts (2 105) were subcultured in 24-well tissue culture plates and labeled with BrdU overnight. Cells were harvested and suspended in the culture medium. Then 100 mL of cell suspension was added to each well of 96-well tissue culture plates. Osteoblasts were cocultured with drugs for another 8 hours at 378C in a humidified atmosphere of 5% CO2. Amounts of BrdU-labeled DNA in the cytoplasm were quantified using an Anthos 2010 microplate photometer (Anthos Labtec Instruments, Lagerhausstrasse, Wals/Salzburg, Austria) at a wavelength of 450 nm.

Analysis of apoptotic cells Apoptotic osteoblasts were determined using propidium iodide (PI) according to a previously described method.(30) After drug treatment, osteoblasts were harvested and fixed in cold 80% ethanol. Following centrifugation and washing, fixed cells were stained with PI and analyzed by a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) on the basis of a 560-nm CHEN ET AL.

dichromic mirror and a 600-nm bandpass filter. SNP was freshly dissolved in PBS buffer (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). Control cells were exposed to PBS only. Our previous study has shown that hydrogen peroxide could induce osteoblast apoptosis.(31) Thus PBS and hydrogen peroxide were used in this study as negative and positive controls for the apoptosis assay.

Confocal microscopic analysis of Bcl-XL translocation and GATA-3 localization Bcl-XL in osteoblasts was recognized by a specific antibody and visualized using confocal microscopy following a previously described method.(32) Briefly, after drug treatment, osteoblasts were fixed with a fixing reagent (acetone- methanol 1:1) at 208C for 10 minutes. Following rehydration, cells were incubated with 0.2% Triton X-100 at room temperature for 15 minutes. The mouse monoclonal antibody (mAb) used in this study was generated against human Bcl-XL (Santa Cruz Biotechnology, Santa Cruz, CA, USA). This antibody can detect Bcl-XL in whole cells, including the cytoplasm and mitochondria. Immunodetection of Bcl-XL in osteoblasts was carried out at 48C overnight. After washing, cells were reacted sequentially with the second antibodies and biotin-SP-conjugated AffiniPure goat anti-rabbit immunoglobulin G (IgG; Jackson ImmunoResearch, West Grove, PA, USA) at room temperature for 1 hour. After washing, the third antibody with Cy3-conjugated streptavidin (Jackson ImmunoResearch) was added to osteoblasts and reacted at room temperature for 30 minutes. Mitochondria of fixed osteoblasts were stained with 3,3’-dihexyloxacarbocyanine (DiOC6; Molecular Probes, Eugene, OR, USA), a positively charged dye, at 378C for 30 minutes. A confocal laser scanning microscope (Model FV500, Olympus, Tokyo, Japan) was used for sample observation. The excitation wavelength was set to 568 nm, whereas a 585-nm longpass filter was used to collect the emitted light. Illumination for the existence of Bcl-XL protein was demonstrated by the appearance of ‘‘hot spots’’ in both the cytoplasm (red signals) and membranes (yellow signals). Images were acquired and quantified using FLUOVIEW software (Olympus). The increased densities of hot spots were analyzed by automated recordings within the same region in a cell. The average density of hot spots was the average of values for 10 areas within a cell. Localization of GATA-3 also was immunodetected and observed by confocal microscopy.

Real-time polymerase chain reaction (PCR) assays mRNA from osteoblasts was prepared for real-time PCR analyses of Bcl-XL, GATA-3, Runx2, and b-actin mRNA. The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were, respectively 5’-TTGGACAATGGACTGGTTG3’ and 3’-GTGACTGGTAGGTGAGATG-5’ for Bcl-XL, 5’-CCTACCGGGTTCGGATGTAA-3’ and 3’-TGTCTTCCGTCCCTCACACAC-5’ for GATA-3, 5’-GACAGAAGCTTGATGACTCTAAACC-3’ and 5’-CTGTAATCTGACTCTGTCCTTGTG-3’ for Runx2, and 5’-TATGGAGAAGATTTGGCACC-3’ and 3’-ATGAGACACACCTAACCACC-5’ for bactin.(8,13,33) A real-time PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad). ROLES OF GATA-3 IN OSTEOBLAST SURVIVAL

Immunoblotting analyses of Bcl-XL, GATA-3, Runx2, and b-actin Protein analyses were carried out according to a previously described method.(34) After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris-HCl, pH 7.2, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). To avoid degradation of the cytosolic proteins by proteinases, a mixture of 1 mM phenyl methyl sulfonyl fluoride, 1 mM sodium orthovanadate, and 5 mg/mL leupeptin was added to the radioimmunoprecipitation assay buffer. Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 mg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Bcl-XL, GATA-3, and Runx2 were immunodetected using mouse mAbs or rabbit polyclonal antibodies (pAbs) (Santa Cruz Biotechnology). Cellular b-actin protein was immunodetected using a mouse mAb against mouse b-actin (Sigma, St. Louis, MO, USA) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK).

Extraction of nuclear proteins and immunodetection Nuclear components were extracted and immunodetected following the method of Lee and colleagues.(35) After drug treatment, nuclear extracts of osteoblasts were prepared. Protein concentrations were quantified with a bicinchonic acid protein assay kit (Pierce). Nuclear proteins (50 mg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, nuclear GATA-3 was immunodetected using a mouse mAb against human recombinant GATA-3 (Santa Cruz Biotechnology). Proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (UVtec).

GATA-3 knockdown Translation of GATA-3 mRNA in osteoblasts was knocked down using an RNA interference (RNAi) method following a small interfering (si)RNA transfection protocol provided by Santa Cruz Biotechnology, as described previously.(36) GATA-3 siRNA was purchased from Santa Cruz Biotechnology and is a pool of three target-specific 20- to 25-nt siRNAs designed to knock down GATA-3’s expression. Briefly, after culturing osteoblasts in antibiotic-free DMEM at 378C in a humidified atmosphere of 5% CO2 for 24 hours, the siRNA duplex solution, which was diluted in the siRNA transfection medium (Santa Cruz Biotechnology), was added to the osteoblasts. Scrambled siRNA, purchased from Santa Cruz Biotechnology, was applied to control cells as a negative standard. After transfection for 24 hours, the medium was replaced with normal DMEM, and osteoblasts were treated with NO or oxidative stress. Journal of Bone and Mineral Research

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Expression of Lox-1 full-length complementary (c)DNA pFLAG-GATA-3 plasmids, which were constructed with fulllength mouse GATA-3 cDNA, were purchased from Addgene, Inc. (Cambridge, MA, USA). pFLAG plasmids were purchased from Sigma. pFLAG-GATA-3 plasmids were transfected into osteoblasts as described previously.(37) Empty pFLAG vectors were transfected as the negative control. After transfection for 48 hours, osteoblasts were exposed to NO or oxidative stress. Then cells were harvested for analyses of Bcl-XL mRNA expression and cell apoptosis.

Electrophoretic mobility shift assay (EMSA) An EMSA was performed using a DIG gel shift kit (Roche Diagnostics, Mannheim, Germany) as described previously.(38) Briefly, GATA-3 consensus oligonucleotides, purchased from Santa Cruz Biotechnology, were labeled with digoxigenin (DIG). The nuclear extract (10 mg) was allowed to react with DIG-labeled oligonucleotides at room temperature for 25 minutes. To show the specificity of GATA-3–DNA interactions, mutated GATA-3 consensus oligonucleotides (cold probes) were labeled with DIG and reacted with the nuclear extracts. These complexes were subjected to nondenatured PAGE and transferred to positively charged nylon membranes. After cross-linking at 120 mJ and blocking, membranes were immunoreacted with the anti-DIG-GATA-3. Following washing and chemiluminescent detection, membranes were exposed to X-ray films. Intensities of these DNA-protein complex bands were quantified with the aid of the UVIDOCMW Version 99.03 digital imaging system (UVtec).

Coimmunoprecipitation and immunoblotting assays Analyses of immunoprecipitation and immunoblot were carried out using the ExtractCruz kit provided by Santa Cruz Biotechnology. Osteoblasts were extracted in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% TritonX-100, and 100 mg/mL PMSF) supplemented with protease inhibitor cocktails for 60 minutes on ice. After centrifugation, the supernatant was incubated with a rabbit pAb against human recombinant GATA-3 (Santa Cruz Biotechnology) and subsequently with protein A-Sepharose (50% slurry) at 48C overnight and then was washed three times. The pellet was resuspended in the same volume of SDS sample buffer and boiled to remove the Sepharose beads. Then the cells lysates and immunoprecipitates were analyzed by an immunoblotting analysis of Runx2 using a rabbit pAb against mouse Runx2.(39) A normal rabbit IgG purchased from Santa Cruz Biotechnology was used for the pull-down assay as the negative control.

Statistical analysis Statistical differences between the control and drug-treated groups were considered significant when the p value of Duncan’s multiple-range test was less than .05. Statistical analysis between drug-treated groups was carried out using two-way analysis of variance (ANOVA).

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Results Exposure of osteoblasts to 0.05, 0.1, and 0.3 mM SNP for 24 hours did not affect their viability (Fig. 1A). When the concentrations reached 0.5, 1, 1.5, and 2 mM, SNP caused significant 20%, 31%, 47%, and 91% mortality of osteoblasts, respectively. After treatment with 2 mM SNP for 24 hours, the viability of osteoblasts was reduced by 84% (Fig. 1B). Pretreatment of osteoblasts with 0.05 and 0.1 mM SNP for 24 hours did not influence 2 mM SNP–induced osteoblast death. Meanwhile, pretreatment with 0.3 and 0.5 mM SNP caused 60% and 42% decreases in 2 mM SNP–induced osteoblast death, respectively (Fig. 1B). Pretreatment with 0.3 mM SNP for 6, 12, 18, and 24 hours did not change osteoblast viability (Fig. 1C). The viability of osteoblasts was decreased significantly by 22%, 49%, and 84% following exposure to 2 mM SNP for 6, 12, and 24 hours, respectively. Pretreatment of osteoblasts with 0.3 mM SNP for 6, 12, and 24 hours, respectively, caused significant 100%, 28%, and 56% decreases in 2 mM SNP–induced osteoblast death (Fig. 1C). Thus, in this study, pretreatment with 0.3 mM SNP for 24 hours and exposure to 2 mM SNP, respectively, were applied as the sources of NO and oxidative stress (Fig. 1D, E). Exposure to oxidative stress–induced DNA fragmentation by 5.7-fold (Fig. 1E) Genomic DNA in osteoblasts pretreated with NO for 24 hours was not affected. Meanwhile, pretreatment with NO led to a significant 66% attenuation in oxidative stress– induced DNA fragmentation (Fig. 1D). Treatment with oxidative stress induced 88% of osteoblasts to undergo apoptosis (Fig. 1E). Pretreatment of osteoblasts with NO for 24 hours did not cause cell apoptosis but significantly lowered oxidative stress– induced cell apoptosis by 67%. Treatment with PBS did not affect cell apoptosis. Meanwhile, hydrogen peroxide induced osteoblast apoptosis by 56% (Fig. 1E). Exposure of osteoblasts to oxidative stress decreased the translocation of Bcl-XL from the cytoplasm to mitochondria (Fig. 2A). Pretreatment with NO for 24 hours increased the translocation of Bcl-XL from the cytoplasm to mitochondria and simultaneously attenuated the oxidative stress–caused reduction of this antiapoptotic protein’s translocation (Fig. 2A). Levels of Bcl-XL in osteoblasts decreased following exposure to oxidative stress (Fig. 2B, top panel, lane 2). NO preconditioning for 24 hours increased Bcl-XL production in osteoblasts and simultaneously decreased the oxidative stress–caused reduction of this antiapoptotic protein (lanes 3 and 4). Amounts of bactin were immunodetected as the internal standard (Fig. 2B, bottom panel). These immunorelated protein bands were quantified and analyzed (Fig. 2C). Oxidative stress decreased Bcl-XL by 64% in osteoblasts. Pretreatment with NO for 24 hours caused an 81% enhancement of Bcl-XL production and completely attenuated the oxidative stress–induced reduction in cellular Bcl-XL levels (Fig. 2C). When exposed to oxidative stress, the expression of Bcl-XL mRNA in osteoblasts was inhibited by 71% (Fig. 2D). NO preconditioning induced 2.1-fold Bcl-XL mRNA expression and lowered the oxidative stress–involved inhibition of Bcl-XL mRNA expression by 100% (Fig. 2D). In untreated osteoblasts, the transcription factor GATA-3 was detected in the cytoplasm (Fig. 3A, top panel). Three GATA-3responsive DNA elements were found in the promoter region of CHEN ET AL.

Fig. 1. Protective effects of nitric oxide (NO) preconditioning on oxidative stress (OS)–induced insults. Pretreatment of osteoblasts, prepared from neonatal rat calvaria, with 0.3 mM SNP for 24 hours was used as NO preconditioning, and exposure to 2 mM was applied as the source of OS. Cell viability was analyzed using a colorimetric MTT assay (A–C). DNA fragmentation was quantified using an enzyme-linked immunosorbent assay (D). Apoptotic cells were determined with aid of a flow cytometer (E). Osteoblasts were treated with PBS and hydrogen peroxide (H2O2) as negative and positive controls for the apoptotic assay. Each value represent the mean SEM for n ¼ 6. The symbols and # indicate that the values significantly ( p < .05) differed from the respective control and OS-treated groups. C ¼ control; OD ¼ optical density.

the bclxL gene (bottom panel). Exposure of osteoblasts to oxidative stress inhibited the expression of GATA-3 mRNA in osteoblasts by 75% (Fig. 3B). Pretreatment with NO for 24 hours enhanced the GATA-3 mRNA expression by 2.5-fold and caused a significant 100% decrease in oxidative stress–involved suppression of this transcription factor’s mRNA production. NO preconditioning increased GATA-3 production by osteoblasts (Fig. 3C, top panel, lane 2). After exposure to oxidative stress, the amounts of GATA-3 in osteoblasts obviously decreased (lane 3). Meanwhile, pretreatment with NO for 24 hours attenuated the oxidative stress–caused reduction in GATA3 production (lane 4). b-Actin was immunodetected as the internal standard (Fig. 3C, bottom panel). These protein bands ROLES OF GATA-3 IN OSTEOBLAST SURVIVAL

were quantified and analyzed (Fig. 3D). Oxidative stress decreased the amounts of GATA-3 in osteoblasts by 69%. NO preconditioning increased GATA-3 synthesis by 2.1-fold and completely attenuated the oxidative stress–caused suppression of GATA-3 production (Fig. 3D). Furthermore, the translocation of GATA-3 from the cytoplasm to nuclei was determined (Fig. 3E, F). Exposure of osteoblasts to oxidative stress decreased the levels of nuclear GATA-3 but increased the amounts of this transcription factor in the cytoplasm (Fig. 3E, top 1 and 3 panes, lane 2). NO preconditioning increased nuclear GATA-3 levels but decreased its levels in the cytoplasm (lane 3). Meanwhile, pretreatment with NO reduced oxidative stress–induced alterations in levels of GATA-3 Journal of Bone and Mineral Research

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Fig. 2. Bcl-XL involved in the regulation of osteoblast survival. Osteoblasts were pretreated with nitric oxide (NO) for 24 hours and then exposed to oxidative stress (OS). Translocation of Bcl-XL from the cytoplasm to mitochondria (Mit) was observed and analyzed using confocal microscopy (A). Cellular proteins and mRNA were prepared for immunoblotting and RNA analyses. Amounts of Bcl-XL were immunodetected (B, top panel). Levels of b-actin were determined as the internal standard (bottom panel). These protein bands were quantified and analyzed (C). Real-time PCR analysis of Bcl-XL mRNA was carried out (D). Each value represents the mean SEM for n ¼ 6. The symbols and # indicate that the values significantly ( p < .05) differed from the respective control and OS-treated groups. C ¼ control.

in nuclei and the cytoplasm (lane 4). Amounts of nuclear PCNA and cytosolic b-actin were immunodetected as the internal standards (Fig. 3E, top 2 and bottom panels). These immunorelated protein bands were quantified and analyzed (Fig. 3F). Oxidative stress caused a significant 53% decrease in levels of nuclear GATA-3 but increased cytosolic levels by 90%. NO preconditioning enhanced nuclear GATA-3 amounts by 2.2-fold but reduced cytosolic levels by 37%. After pretreatment with NO, the oxidative stress–caused alterations completely changed (Fig. 3F). Application of GATA-3 siRNA to osteoblasts for 24 and 48 hours decreased the amounts of this transcription factor (Fig. 4A, top panel, lanes 2 and 3). b-Actin was immunodetected as the internal standard (bottom panel). These protein bands were quantified and analyzed (Fig. 4B). Application of GATA3 siRNA for 24 and 48 hours significantly reduced the levels of GATA-3 in osteoblasts by 31% and 70%, respectively. Treatment of osteoblasts with GATA-3 siRNA for 48 hours did not affect Bcl-XL mRNA expression (Fig. 4C). NO preconditioning lowered the oxidative stress–caused inhibition of Bcl-XL mRNA expression, but this suppressive effect was attenuated

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by 52% following application of GATA-3 siRNA. Transfection with scrambled siRNA did not affect Bcl-XL mRNA expression (Fig. 4C). An EMSA was carried out to determine the binding of nuclear GATA-3 to its specific DNA motifs (Fig. 4D). When nuclear extracts were reacted with GATA-3-specific DNA motifs, one protein-DNA band appeared on the blot (Fig. 4D, left panel, lane 1). Such a DNA-protein reaction apparently was attenuated when mutated GATA-3-specific DNA probes were used (lane 2). Amounts of free probes were determined as the internal standard (Fig. 4D, left panel). These protein-DNA bands were quantified and analyzed (Fig. 4D, right panel). Application of mutated GATA-3-specific DNA probes caused a significant decrease in the specificity of DNA-protein interactions. Exposure of osteoblasts to oxidative stress decreased the binding of nuclear extracts to GATA-3-specific DNA-binding elements (Fig. 4E, lane 2). Pretreatment with NO for 24 hours did not affect the binding of GATA-3 to its specific DNA-binding elements but decreased oxidative stress–caused suppression of this protein-DNA binding (lanes 3 and 4). Free probes were quantified as the internal standards (Fig. 4E). These protein-DNA bands were quantified and analyzed (Fig. 4F). CHEN ET AL.

Fig. 3. Roles of GATA-3 in mediating osteoblast survival. Osteoblasts were pretreated with nitric oxide (NO) for 24 hours and then exposed to oxidative stress (OS). In untreated osteoblasts, GATA-3 was expressed and distributed in the cytoplasm (A, top panel), and its specific DNA-binding elements were found in the promoter region of the bcl-xL gene (bottom panel). Real-time PCR analysis of GATA-3 mRNA was carried out (B). Amounts of total GATA-3 were immunodetected (C, top panel). Levels of b-actin were determined as the internal standard (bottom panel). These protein bands were quantified and analyzed (D). Nuclear and cytosolic proteins were prepared for immunoblotting analyses. Nuclear (nGATA-3) and cytosolic GATA3 (cGATA-3) were immunodetected (E). Amounts of PCNA and b-actin were determined as the nuclear and cytosolic internal standards, respectively. These protein bands were quantified and analyzed (F ). Each value represents the mean SEM for n ¼ 6. The symbols and # indicate that the values significantly ( p < .05) differed from the respective control and OS-treated groups. C ¼ control.

Oxidative stress caused a significant 68% decrease in the binding of GATA-3 to its specific DNA motifs, but this reduction was completely alleviated following pretreatment with NO (Fig. 4F). Exposure of osteoblasts to oxidative stress inhibited Runx2 mRNA expression by 64% (Fig. 5A). NO preconditioning did not affect Runx2 mRNA synthesis but caused a significant 49% decrease in the oxidative stress–induced suppression of the mRNA expression of this transcription factor (Fig. 5A). Pretreatment of osteoblasts with NO for 1, 6, and 24 hours increased the association between GATA-3 and Runx2 (Fig. 5B, top panel, lanes 2–4). Levels of total GATA-3 were augmented following NO preconditioning for 24 hours (middle panel, lane 4). Amounts of the IgG heavy chain were determined as the internal ROLES OF GATA-3 IN OSTEOBLAST SURVIVAL

standard (bottom panel). These protein bands were quantified and analyzed (Fig. 5C). NO preconditioning for 1, 6, and 24 hours, respectively, increased the association of GATA-3 and Runx2 by 80%, 227%, and 305%. The complex of GATA-3 associated with Runx2 could not be immunoprecipitated using a normal rabbit IgG (Fig. 5D). Oxidative stress decreased cell numbers and induced cell shrinkage, whereas pretreatment with NO for 24 hours reduced these insults (Fig. 6A). Meanwhile, application of GATA-3 siRNA to osteoblasts attenuated the protection of NO preconditioning against oxidative stress–induced changes in cell morphologies. Protective effects of NO preconditioning against oxidative stress–caused alteration of cell viability were decreased significantly by 60% following exposure to GATA-3 siRNA Journal of Bone and Mineral Research

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Fig. 4. GATA-3 contributes to the regulation of bcl-xL gene expression. Application of GATA-3 small interfering (si)RNA into osteoblasts for 24 and 48 hours. Levels of GATA-3 were immunodetected (A, top panel). Amounts of b-actin were determined as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Real-time PCR analysis of Bcl-XL mRNA was carried out (C). Scrambled siRNA, containing nontargeting 20to 25-nt siRNA, was applied to control cells as a negative standard. Nuclear proteins were prepared and reacted with normal (N) or mutated (M) GATA-3specific DNA probes, and an electrophoretic mobility shift assay (EMSA) was conducted (D, left panel). Amounts of free probes were determined as the internal standards. These DNA-protein bands were quantified and analyzed (right panel). Osteoblasts were pretreated with nitric oxide (NO) for 24 hours and then exposed to oxidative stress (OS). Nuclear proteins were prepared for an EMSA to determine the binding of GATA-3 to its specific DNA motifs, and free probes were determined as the internal standard (E). These DNA-protein bands were quantified and analyzed (F). Each value represents the mean SEM for n ¼ 6. The symbols , #, and y indicate that the values significantly ( p < .05) differed from the respective control, OS-, and NO þ OS–treated groups. C ¼ control.

(Fig. 6B). Application of GATA-3 siRNA caused significant 73% and 43% increases in NO preconditioning–involved protection against oxidative stress–induced DNA fragmentation and cell apoptosis, respectively (Fig. 6C, D). Treatment with GATA-3 siRNA alone did not affect cell morphologies, alkaline phosphatase (ALP) activity, cell viability, DNA fragmentation, or cell apoptosis (Fig. 6C, D). Transfection of pFLAG-GATA-3 plasmids into osteoblasts for 48 hours caused a significant 3.5-fold increase of GATA-3 (data not shown). In parallel, overexpression of GATA-3 induced Bcl-XL mRNA expression by 2.8-fold (Fig. 6E). Protection of NO preconditioning against oxidative stress– induced alterations of Bcl-XL mRNA expression and cell apoptosis was significantly promoted (Fig. 6E, F).

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Discussion Pretreatment with NO can protect osteoblasts against oxidative stress–induced apoptotic insults. SNP is decomposed to NO under light exposure or in the presence of a biologic reducing system.(40) In this study, exposure of osteoblasts to a high concentration of SNP (2 mM) was used to produce massive amounts of NO. SNP-decomposed NO and its derivative, peroxynitrite, an oxidative product of NO with superoxide, can be major sources of oxidative stress. In addition, NO is such an active radical that it can attack the cellular membrane bilayer to produce lipid peroxidation.(41) Thus diverse sources of SNPinduced ROS can cause oxidative stress to osteoblasts and incur CHEN ET AL.

Fig. 5. Participation of Runx2 in the regulation of osteoblast survival. Osteoblasts were pretreated with nitric oxide (NO) for 24 hours and then exposed to oxidative stress (OS). Cellular mRNA was prepared for a real-time PCR analysis of Runx2 mRNA (A). The complex of GATA-3 associating with Runx2 was immunoprecipitated (IP) using an antibody against GATA-3, electrophoretically separated, and finally immunodetected by a Runx2 antibody (B, top panel). Levels of total GATA-3 were immunodetected (middle panel). Amounts of IgG heavy chains (IgG-H) were determined as the internal standard (bottom panel). Control cells were exposed to PBS only. These immunorelated protein bands were quantified and analyzed (C). A normal rabbit IgG was used as the negative control (D). After reacting with the normal rabbit IgG, the complex was separated electrophoretically and finally immunodetected by Runx2 and GATA-3 antibodies, respectively. Each value represents the mean SEM for n ¼ 6. The symbols and # indicate that the values significantly ( p < .05) differed from the respective control and OS-treated groups. C ¼ control.

cell death. Pretreatment with a low concentration of SNP (0.3 mM) for 24 hours did not affect cell viability but significantly attenuated the 2 mM SNP–caused decrease in cell survival. Thus pretreatment with 0.3 mM SNP for 24 hours and exposure to 2 mM SNP can be used as sources of NO preconditioning and oxidative stress, respectively. Under such a model, NO preconditioning lowered oxidative stress–induced DNA fragmentation and cell cycle arrest at the sub-G1 phase. DNA fragmentation and the appearance of the sub-G1 phase are two typical characteristics of cells undergoing apoptosis.(42,43) In cardiomyocytes, hepatocytes, and endothelial cells, NO preconditioning was shown to have antiapoptotic effects in ischemia and reperfusion injury.(12) This study further showed that NO preconditioning produces antiapoptotic effects on oxidative stress–induced osteoblast apoptosis. Bcl-XL contributes to the protection of NO pretreatment from oxidative stress–induced apoptotic insults to osteoblasts. Bcl-XL is thought to have antiapoptotic effects because it can be translocated to mitochondrial membranes from the cytoplasm and associates with the proapoptotic Bax protein.(24) This study showed that the oxidative stress–caused reduction in Bcl-XL translocation from the cytoplasm to mitochondria in ROLES OF GATA-3 IN OSTEOBLAST SURVIVAL

osteoblasts was attenuated following pretreatment with NO. Our previous studies reported that pretreatment with NO can increase the translocation of Bcl-2, the other antiapoptotic protein, to mitochondria.(13,44) Thus the NO pretreatment– caused protection against osteoblast injuries involves the antiapoptotic proteins Bcl-XL and Bcl-2. Besides mitochondria, the results by confocal microscopy revealed that pretreatment with NO also enhanced signals of Bcl-XL antibody recognition in the cytoplasm of osteoblasts. This phenomenon was further confirmed by an immunoblotting analysis; that is, NO pretreatment increased the levels of Bcl-XL in osteoblasts. In addition, exposure to oxidative stress decreased the expressions of Bcl-XL mRNA and protein in osteoblasts, but NO preconditioning significantly decreased this inhibition. A previous study showed that overexpression of the bcl-xL gene inhibits Bax translocation and cytochrome C release and prevents cardiac cells from apoptosis.(26) Thus NO preconditioning can induce expression of the bcl-xL gene and its translocation, leading to protection against oxidative stress-caused osteoblast insults. GATA-3 mediates NO preconditioning–caused osteoblast protection. Traditionally, GATA-3 was thought to be expressed mainly in hematopoietic cells and to function as an important Journal of Bone and Mineral Research

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Fig. 6. Effects of GATA-3 small interfering (si)RNA and overexpression on nitric oxide (NO) preconditioning–involved protection against oxidative stress (OS)–induced insults. GATA-3 siRNA was applied to osteoblasts, which then were exposed to NO preconditioning, OS, or a combination of NO preconditioning and OS. Morphologies of osteoblasts were observed and photographed (A). Cell viability was analyzed using a colorimetric assay (B). DNA fragmentation was quantified using an enzyme-linked immunosorbent assay (C). Apoptotic cells were determined with the aid of a flow cytometer (D). Osteoblasts were transfected with pFLAG-GATA-3 plasmids for 48 hours and then exposed to NO or OS. Bcl-XL mRNA expression (E) and cell apoptosis ( F) were determined. Empty pFLAG was transfected into osteoblasts as the negative control. Each value represents the mean SEM for n ¼ 6. The symbols , #, and y indicate that the values significantly ( p < .05) differed from the respective control, OS-, and NO þ OS–treated groups. C ¼ control; OD ¼ optical density.

transcription factor regulating cell development.(16–18) Meanwhile, this is the first study to show that GATA-3 was detected in osteoblasts and was localized in the cytoplasm. Exposure of osteoblasts to oxidative stress decreased the levels of GATA-3 in osteoblasts. GATA-3 can affect cell survival and tissue function.(45) In parallel with the reduction in cellular GATA-3 levels, this study demonstrated that oxidative stress can decrease osteoblast viability. Meanwhile, the oxidative stress– caused suppression of GATA-3 production in osteoblasts was attenuated significantly by NO pretreatment. Simultaneously, NO preconditioning also protected osteoblasts against oxidative stress–induced death. We further used RNA interference technology to knock down the translation of GATA-3 in

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osteoblasts, and our results reveal that application of GATA-3 siRNA significantly attenuated the NO preconditioning–involved protection from oxidative stress–induced alterations of cell morphologies, DNA fragmentation, cell apoptosis, and cell viability. In addition, this study showed that overexpression of GATA-3 could promote NO preconditioning–caused stimulation of Bcl-XL expression and osteoblast survival. These results provide significant evidence to validate the critical roles of GATA3 in mediating the survival signals of NO-pretreated osteoblasts. GATA-3 can regulate the expression of the bcl-xL gene and participates in osteoblast protection. Oxidative stress decreased the levels of nuclear GATA-3, but pretreatment with NO lowered this suppression. GATA-3 functions as a CHEN ET AL.

transcription factor when it is activated and translocated into nuclei from the cytoplasm.(17,18) This study shows that NO preconditioning can decrease the oxidative stress–caused reduction of GATA-3 translocation from the cytoplasm to nuclei. In addition, the NO preconditioning–caused induction of Bcl-XL mRNA expression was inhibited after transfection of GATA-3 siRNA into osteoblasts. Yu and colleagues (2005) reported the existence of the GATA DNA-binding elements (A/T)GATA (A/G) in the 5’-end promoter region of the bcl-xL gene.(27) We used a biomedical approach to further find that there are at least 3 GATA-3-specific DNA-binding elements in the bcl-xL gene’s promoter. Analysis by EMSA was carried out to validate that exposure to oxidative stress decreased the binding of nuclear extracts to GATA-3-specific DNA motifs. Pretreatment of osteoblasts with NO for 24 hours lowered the oxidative stress–caused suppression of GATA-3 binding to its specific DNA elements. In parallel with the inhibition of Bcl-XL mRNA expression, knocking down the translation of GATA-3 using RNA interference decreased the protection of NO preconditioning from oxidative apoptotic insults. This is the first study to demonstrate that GATA-3 has antiapoptotic effects on NO’s protection against oxidative stress–induced osteoblast apoptosis via upregulation of bcl-xL gene expression. Runx2 contributes to the regulation of osteoblast survival via its association with GATA-3. Runx2, a transcription factor, was reported to participate in the processes of osteoblast growth and differentiation.(46) This study showed that exposure of osteoblasts to oxidative stress inhibited Runx2 mRNA expression, but pretreatment with NO decreased such inhibition. In heavy metal–induced osteotoxicity, cadmium inhibited Runx2 mRNA expression and triggered apoptotic death of cells of the Saos-2 human osteoblast-like cell line.(47) Eliseev and colleagues (2008) showed that Runx2 mediates activation of the bax gene and enhances the sensitivity of human osteosarcoma cells to apoptotic death.(48) Our previous study provided in vitro evidence verifying the role of Runx2 in regulating bcl-2 gene expression in osteoblasts.(44) Thus Runx2 is involved in regulating osteoblast survival and death by modulating the expressions of Bcl-2 family proteins. Our results reveal that pretreatment with NO time-dependently increased the association of Runx2 and GATA-3. Runx2 was reported to interact directly with SATB2, a nuclear matrix protein, and to enhance the activity of Runx2.(49) Therefore, the association of GATA-3 and Runx2 in osteoblasts may be important in the protection afforded by NO pretreatment against oxidative stress–induced apoptotic insults. In conclusion, this study showed that pretreatment of osteoblasts with NO can attenuate oxidative stress–induce insults via an antiapoptotic mechanism. Expression of the bcl-xL gene and translocation of Bcl-XL are both involved in NO preconditioning–caused protection against oxidative stress– induced apoptosis of osteoblasts. GATA-3 was detected in osteoblasts and was localized mainly in the cytoplasm. After exposure to oxidative stress, the expressions of GATA-3 mRNA and protein were inhibited. Meanwhile, pretreatment with NO lowered oxidative stress–caused suppression of GATA-3 mRNA and protein syntheses. GATA-3-specific DNA-binding elements exist in the promoter region of the bcl-xL gene. When knocking down the translation of GATA-3 using RNA interference, the ROLES OF GATA-3 IN OSTEOBLAST SURVIVAL

expression of Bcl-XL mRNA was inhibited. Pretreatment of osteoblasts with NO significantly decreased the oxidative stress– triggered reduction in Runx2 mRNA production. NO preconditioning time-dependently stimulated the association of GATA-3 and Runx2. In parallel with the inhibition of Bcl-XL mRNA expression, application of GATA-3 siRNA to osteoblasts attenuated the NO preconditioning–caused protection from oxidative stress–induced alterations in cell morphologies, ALP activities, DNA fragmentation, cell apoptosis, and cell death damage. Therefore, GATA-3 plays critical roles in mediating the survival signals in osteoblasts by upregulating bcl-xL gene expression.

Disclosures All the authors state that they have no conflicts of interest.

Acknowledgments This study was supported by the National Science Council (NSC95-2314-B-038-029-MY3 and NSC96-2628-B-038-005-MY3) and Wan-Fang Hospital (99WF-eva-06), Taipei, Taiwan.

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