The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 178: 6100-6108.
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kuo, C.-C.
Right arrow Articles by Liang, S.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuo, C.-C.
Right arrow Articles by Liang, S.-M.

Involvement of Heat Shock Protein (Hsp)90beta but Not Hsp90{alpha} in Antiapoptotic Effect of CpG-B Oligodeoxynucleotide1

Cheng-Chin Kuo*, Chi-Ming Liang2,{dagger},{ddagger},§, Chen-Yen Lai* and Shu-Mei Liang2,*,{dagger}

* Agricultural Biotechnology Research Center, {dagger} Genomics Research Center, and {ddagger} Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; and § Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli County, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune responses in a TLR9-dependent manner. In this study, we found that stimulation of mouse macrophages and dendritic cells with B-type CpG ODN (CpG-B ODN) increased the cellular level of heat shock protein (Hsp) 90beta but not Hsp90{alpha} and prevented apoptosis induced by serum starvation or staurosporine treatment. The CpG-B ODN-induced Hsp90beta expression depended on TLR9, MyD88, and PI3K. Inhibition of Hsp90beta level by expressing small-interfering RNA suppressed not only Hsp90beta expression but also PI3K-dependent phosphorylation of Akt and CpG-B ODN-mediated antiapoptosis. Additional studies demonstrated that as described by other group in mast cells, Hsp90beta but not Hsp90{alpha} was associated with Bcl-2. Inhibition of Hsp90beta suppressed the CpG-B ODN-induced association of Hsp90beta with Bcl-2 and impaired the inhibitory effect of CpG-B ODN in the release of cytochrome c and activation of caspase-3. This study thus reveals the involvement of Hsp90beta but not Hsp90{alpha} in CpG-B ODN-mediated antiapoptotic response and that Hsp90beta is distinct from Hsp90{alpha} in regulation of the cellular function of immune cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
An unmethylated CpG motif in bacterial DNA (bacterial CpG DNA) activates immune responses and exhibits antitumor activity in mammals (1). The immunostimulatory effects of bacterial DNA can be mimicked by synthetic oligodeoxynucleotides containing a CpG motif (CpG ODN)3 (2). Bacterial CpG DNA and biologically active CpG ODN stimulate macrophages and immature dendritic cells, thereby increasing the expression of MHC class II and costimulatory molecules to transcribe cytokine mRNAs and produce proinflammatory cytokines (3).

The binding of bacterial CpG DNA to TLR9 (4, 5, 6) and the subsequent endosomal maturation are thought to be essential for bacterial CpG DNA-driven immunostimulatory activity (7). After CpG DNA binding, TLR9 signaling is initiated by recruitment of the adaptor molecule MyD88. Recruitment of MyD88 is followed by the engagement of IL-1R-associated kinases (e.g., IRAK-1 and IRAK-4) and the adaptor protein TNFR-associated factor 6. Oligomerization of TNFR-associated factor 6 can activate the I{kappa}B kinase complex (8, 9, 10) and subsequently activates the NF-{kappa}B-dependent genes, such as TNF-{alpha}, IL-1, and IL-6, leading to increased production of these cytokines (3).

CpG ODNs can be classified into two major classes: type A (CpG-A ODNs) and type B (CpG-B ODNs) (3). CpG-A ODNs are effective in activating NK cells and stimulating plasmacytoid dendritic cells (pDCs) and macrophages to produce high levels of IFN-{alpha} (11, 12). In contrast, CpG-B ODNs primarily stimulate proliferation of B cells as well as secretion of Igs, IL-6, and IL-10. CpG-B ODNs also induce maturation and activation of pDCs and macrophages (11, 13). CpG-B ODNs protect B cells and pDCs against spontaneous apoptosis and have been shown to rescue WEHI-231 B cells from apoptosis induced by IgM (14, 15, 16). They also protect mouse spleen cells as well as RAW264.7 macrophage and human RPMI 8226 B cells against gamma irradiation-induced apoptosis (17). However, the molecular mechanisms of the antiapoptotic effects of CpG-B ODNs remain to be elucidated.

By using microarray and proteomic approaches, we have recently shown that treating cells with CpG-B ODN results in increased expression of heat shock proteins (Hsp), notably Hsp90beta (18). Hsp90, coded by two distinct genes, Hsp90{alpha} and Hsp90beta (19, 20), may cause antiapoptosis by associating with apoptotic protease activation factor-1 (Apaf-1) to prevent the formation of a functionally competent apoptosome (21, 22) or by interacting with phosphorylated serine/threonine kinase Akt/protein kinase B, a protein that generates a survival signal in response to cell stimulation (23, 24, 25). Some reports describe the formation of the Bcl-2 and Hsp90beta complex preventing the release of cytochrome c from mitochondria and activation of caspase-3 in mast cells (26, 27). However, a similar result in pDCs and macrophages, the cells activated by CpG-B ODN (11, 13), has not yet been documented.

In this study, we found that CpG-B ODN treatment increased the expression of Hsp90beta but not Hsp90{alpha} in mouse spleen pDCs and monocytes/macrophages, as well as the mouse macrophage RAW264.7 cell line. We further investigated the mechanisms of CpG-B ODN-induced expression of Hsp90beta via the TLR9/MyD88/PI3K signaling pathway. In addition, we evaluated the potential role of the formation of the complex Hsp90beta and Bcl-2 in CpG-B ODN-mediated cellular antiapoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Phosphorothioate-modified CpG-B ODN and GpC ODN were synthesized by MWG Biotech. The sequences of ODN we used on human cells were as follows: CpG-B ODN, 5'-TCG TCG TTT TGT CGT TTT GTC GTT-3'; and GpC-ODN, 5'-TGC TGC TTT TGT GCT TTT GTG CTT-3'. The sequence of CpG-B ODN we used on mouse cells was 5'-TCC ATG ACG TTC CTG ATG CT-3' and on GpC-ODN was 5'-TCC ATG AGC TTC CTG ATG CT-3'.

Chloroquine, radicicol, staurosporine (STS), SB203580, LY294002, and anti-cytochrome c were purchased from Sigma-Aldrich. Anti-Hsp90{alpha} and anti-Hsp90beta mAbs were obtained from Stressgen Biotechnologies. Anti-Akt, anti-phospho-Akt (Ser473), and anti-procaspase-3 Abs were purchased from New England Biolabs. Anti-Bcl-2 Abs were purchased from Santa Cruz Biotechnology.

Cell culture and treatment

Human THP-1 monocytic leukemia cells, mouse RAW264.7 macrophage, and human embryonic kidney 293 cells (HEK293) were obtained from the American Type Culture Collection. RAW264.7 and HEK293 were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 200 mmol/L L-glutamine, and 50 µM 2-ME in a humidified atmosphere of 5% CO2 at 37°C. Human THP-1 monocytic leukemia cells, shown to express TLR9 and respond to CpG-DNA stimulation (28), were cultured in RPMI 1640 with the same supplements as for RAW264.7 cell cultures. The medium was changed every 2 days for all experiments.

Cells were typically preincubated with or without inhibitors for 1 h before CpG-B ODN treatment, unless specified otherwise. The duration of CpG-B ODN treatment varied depending on the experiment.

pDCs and monocyte/macrophage preparation

Normal 8-wk-old male BALB/c mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The mice were sacrificed by instant neck dislocation. Monocytes/macrophages and pDCs were then prepared from splenocytes. In brief, spleen cells were depleted of erythrocytes in 0.25x HBSS for 15 s, followed by the addition of 2x HBSS and centrifugation. The cell pellet was suspended in RPMI 1640 medium, then pDCs and monocytes/macrophages were isolated with use of a pDC isolation kit and CD11b MicroBeads, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Plasmid constructions

The mouse TLR9 cDNA was generated by RT-PCR with total RNA of the mouse RAW264.7 cell line used as a template and the following oligonucleotides as primers: 5'-AAG CTT ATG GTT CTC CGT CGA AGG ACT-3' and 5'-CTC GAG CTA TTC TGC TGT AGG TCC-3'. Because the primers incorporate HindIII and XhoI sites, the PCR product was then cloned into HindIII- and XhoI-digested pcDNA3.0 (Invitrogen Life Technologies) to generate pcDNA3.0-mTLR9.

The MyD88 cDNA was generated by RT-PCR with total RNA of the THP-1 cell line used as a template and the following oligonucleotides as primers: 5'-GGA TCC ATG GCT GCA GGA GGT CCC GGC-3' and 5'-AAG CTT CTC AGG GCA GGG ACA AGG CCT-3'. Because the primers incorporate BamHI and HindIII sites, the PCR product was then cloned into BamHI- and HindIII-digested pcDNA3.1(–) to generate pcDNA3.1(–)-MyD88. To create the MyD88 dominant-negative construct pcDNA3.1(–)-MyD88-DN, a truncated version of MyD88 expressing the N-terminal death domain, pcDNA3.1(–)-MyD88 was used as a template for PCR amplification with the forward primer containing the BamHI restriction site (5'-GGA TCC ATG GCT GCA GGA GGT CCC GGC-3') and a reverse primer containing the HindIII restriction site (5'-AAG CTT AAT GCT GGG TCC CAG CTC CAG). The PCR product was cloned into BamHI- and HindIII-digested pcDNA3.1(–). The class I PI3K kinase-deficient plasmid M-p110-{Delta}kin-myc was provided by Dr. A. Klippel (Atugen AG, Berlin, Germany).

Small-interfering RNA (siRNA) transfection

dsRNAs were synthesized by Invitrogen Life Technologies. The target sequence for mouse Hsp90beta was 5'-GAG CTG ATA CCT GAG TAC CTC AAC T-3' and its control sequence was 5'-GAG AGA TCC GTG AAT CCC TAT CAC T-3'. The siRNA sequence for mouse Hsp90{alpha} is 5'-GCTGTATTGTCACAAGCACAT-3'. The sequences were cloned between the BamHI and HindIII sites downstream of the U6 promoter in the pSilencer 2.1-U6 neo (Ambion) plasmid. The plasmid was transfected into RAW264.7 cells with use of FuGENE 6 (Roche Molecular Biochemicals). After 48 h, a G418 antibiotic (0.6 mg/ml) was used for clonal selection. pDCs were transfected with siRNA duplexes with use of Lipofectamine 2000 (Invitrogen Life Technologies). The transfection medium was removed 6 h later. The cells were then washed twice with PBS and maintained in culture medium for 48–72 h.

RT-PCR analysis

Total cellular RNAs were isolated with TRIzol (Invitrogen Life Technologies) and the cDNA was produced with use of Superscript II reverse transcriptase (Invitrogen Life Technologies) and an oligo(dT)15 primer for 1 h at 42°C. PCR of cDNA was performed with specific primers for the genes of Hsp90{alpha}, Hsp90beta, and control beta-actin. All PCR products were electrophoresed on 1.5% agarose gel and DNA bands were visualized by staining the gel with ethidium bromide.

Protein extraction and Western blotting

The cells (106/well) were treated on ice for 15 min with 300 µl of lysis buffer (Pierce) supplemented with protease inhibitor mixture (Sigma-Aldrich). The lysates underwent centrifugation at 12,000 x g for 15 min at 4°C, and protein concentrations in the supernatants were determined by use of Bio-Rad Protein Assay (Bio-Rad). The supernatants (50~80 µg of protein/lane) were applied to 12% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences) according to the manufacturer’s instructions.

Cell viability assay

An MTT assay was used to measure cell viability in terms of metabolic turnover, as indicated by the oxidation of MTT to purple formazan by mitochondria (29). In some cases, trypan blue exclusion assay was also used. In brief, 20 µl of cell suspension was mixed with 20 µl of trypan blue (Sigma-Aldrich) and cells were then counted with use of a hemacytometer. The ratio of trypan blue-stained cells to total cells in each experiment was determined by counting four different fields.

Cellular DNA fragmentation ELISA

The level of apoptotic cell death was quantified by cellular DNA fragmentation ELISA (Roche Molecular Biochemicals). The assay was based on the incorporation of the nonradioactive thymidine analog BrdU into the genomic DNA. BrdU was added to the cell medium at the time of seeding, and the BrdU-labeled DNA fragments were released from the cells into the cytoplasm during apoptosis. The DNA fragments were then detected immunologically by ELISA with the use of an anti-DNA Ab-coated microplate to capture the DNA fragment, and an anti-BrdU Ab peroxidase conjugate to detect the BrdU contained in the captured DNA fragments. The degree of apoptosis (cytosolic DNA fragments) was quantified following the manufacturer’s recommendations.

Caspase-3 activity assay

Caspase-3 activity was determined by the cleavage of the fluorometric substrate z-DEVD-AMC (Upstate Biotechnology) according to the manufacturer’s instructions. In brief, cells were harvested and washed twice in PBS, and lysed in a lysis buffer (Pierce) supplemented with protease inhibitor mixture (Sigma-Aldrich). The lysates underwent centrifugation at 12,000 x g for 15 min at 4°C, and protein concentrations in the supernatants were determined by use of Bio-Rad Protein Assay. An amount of 100 µg of the cell lysates were incubated with 72 µM z-DEVD-AMC at room temperature for 15 min in triplicate. Cleavage of z-DEVD-AMC was determined by measurement of emission at 460 nm after excitation at 380 nm with the fluorescence plate reader.

Cell fractionation

Mitochondria and cytosolic fractions for cytochrome c studies were prepared by use of a mitochondria/cytosol fraction kit (BioVision). In brief, cells were harvested and washed twice in PBS, and lysed in a cytosol extraction buffer supplemented with protease inhibitor mixture (Sigma-Aldrich) on ice. After a 10-min incubation, homogenization was performed in a Dounce homogenizer. The homogenate was centrifuged at 700 x g spun for 10 min at 4°C, and the supernatant was then spun at 10,000 x g for 30 min. The supernatant is the cytosol fraction and the pellet is the intact mitochondria.

Immunoprecipitation assays

Cells were lysed in cold lysis buffer (Pierce) supplemented with protease inhibitor mixture (Sigma-Aldrich). After homogenization, the lysates were incubated with anti-Hsp90{alpha} or anti-Hsp90beta Abs (2 µg/ml) and protein A-Sepharose beads (Pierce) overnight at 4°C. The immunocomplexes were collected by centrifugation and then the pellet was washed with cold lysis buffer. After three washes, recovered immunocomplexes were solubilized in SDS sample buffer and separated on 4–20% Novex Tris-Glycine gel (Invitrogen Life Technologies). The protein was visualized on Western blotting with a specific Ab.

Statistical analysis

All values were given as means ± SD. For all data, one-way ANOVA with subsequent Scheffé test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Up-regulation of Hsp90beta but not Hsp90{alpha} in CpG-B ODN-stimulated cells

We have recently shown that treating THP-1 cells with CpG-B ODN increases the expression of Hsps, notably Hsp90beta (18). In this study, we further evaluated the time-dependent influence of CpG-B ODN on the expression profile of Hsp90beta in THP-1 cells and the mouse macrophage cell line RAW264.7. RT-PCR showed that after 8–24 h of CpG-B ODN treatment, the cellular mRNA level of Hsp90beta but not HSP90{alpha} was increased in RAW264.7 cells as compared with cells treated with medium alone (Fig. 1A). Western blotting analysis of the supernatant and cell lysates of THP-1 cells (data not shown) or RAW264.7 cells (Fig. 1B) revealed that 8 or 16 h CpG-B ODN treatment increased the protein level of neither HSP90{alpha} nor Hsp90beta in the supernatant but did increase the cellular level of Hsp90beta but not HSP90{alpha}. A similar increase in cellular Hsp90beta level was found in mouse spleen pDCs and monocytes/macrophages after 16 h CpG-B ODN treatment (Fig. 1C). In addition, we investigated whether the ligands of TLR3 (poly inosine:cytosine (poly IC)) or TLR4 (LPS) had any effect on the induction of Hsp90beta and Hsp90{alpha} in RAW264.7 cells. After 8 h stimulation, neither poly IC nor LPS had any influence on the level of Hsp90{alpha}; however, LPS but not poly IC increased the mRNA level and expression of Hsp90beta (Fig. 1, D and E).


Figure 1
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 1. Enhancement of Hsp90beta expression by CpG-B ODN. A, Mouse macrophage RAW264.7 cells were stimulated with medium alone for 24 h or CpG-B ODN for indicated times. RT-PCR was then performed to analyze Hsp90{alpha} and Hsp90beta expression. beta-actin was used as an internal control. B, RAW264.7 cells were stimulated for 8 or 16 h with medium alone, GpC ODN, or CpG-B ODN as indicated. The level of Hsp90{alpha} and Hsp90beta in culture medium (supernatant) or cell lysates was measured by Western blotting analysis. C, Cell lysates of mouse spleen pDCs and monocytes/macrophages as well as RAW264.7 cells incubated in culture medium (left lanes) or medium supplemented with 1 µM CpG-B ODN (right lanes) for 16 h underwent Western blotting analysis. Anti-Hsp90beta Abs were used and the level of Hsp90beta was quantified by densitometry. Densitometry of Hsp90beta expression level was normalized by using actin. Data represent the mean ± SD from three experiments. *, p < 0.001 for CpG-B ODN vs medium alone. D and E, RAW264.7 cells were stimulated for 8 h with medium alone, LPS (100 ng/ml), poly IC (25 µg/ml), GpC ODN or CpG-B ODN as indicated, RT-PCR (D) and Western blotting analysis (E) was then performed to analyze Hsp90{alpha} and Hsp90beta expression. beta-actin was used as an internal control.

 
CpG-B ODN up-regulates Hsp90beta via the TLR9/MyD88/PI3K pathway

After being endocytosed, bacterial CpG DNA colocalizes in an endosomal compartment with TLR9 (30). To evaluate whether TLR9 and endosomal maturation play a role in CpG-B ODN-mediated induction of Hsp90beta, we treated a TLR9-deficient HEK293T cell line with CpG-B ODN and found that CpG-B ODN caused little, if any, increase in the cellular Hsp90beta level (data not shown). However, after the mouse TLR9 gene was stably integrated into HEK293T cells (mTLR9–293T), the cellular level of Hsp90beta was greatly increased by CpG-B ODN and was modulated by the addition of chloroquine (Fig. 2A), an inhibitor of endosomal maturation known to affect TLR9 function (7).


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 2. CpG-B ODN-mediated up-regulation of Hsp90beta depends on TLR9 and MyD88 expression. A, HEK293T transfectants, stably transfected with mouse TLR9 (mTLR9–293T), were stimulated with GpC ODN or CpG-B ODN (1 µM) for 16 h in the presence or absence of chloroquine (CQ, 1 µg/ml) as indicated. B, RAW264.7 cells were transiently transfected with empty vector (1 µg/ml) or various concentrations of MyD88-DN plasmids for 48 h. The cells were then treated with or without CpG-B ODN for 16 h as indicated. C, RAW264.7 cells were treated with or without CpG-B ODN (1 µM) for 16 h in the presence of various concentrations of LY294002 as indicated. D, RAW264.7 cells were transiently transfected with empty vector (1 µg/ml) or various concentrations of DN-PI3K (M-p110-{Delta}kin-myc) plasmids for 48 h, and then stimulated with or without CpG-B ODN for 16 h as indicated. Cell lysates were immunoblotted with Abs specific for Hsp90{alpha}, Hsp90beta, TLR9, MyD88-myc, PI3K-myc, or actin as indicated. The experiment was repeated three times with similar results.

 
The signaling of all known TLRs except TLR3 occurs via an MyD88-dependent pathway (31). We thus further investigated whether the dominant-negative mutant of MyD88 (MyD88-DN) would affect the level of Hsp90beta induced by CpG-B ODN. RAW264.7 cells were transiently transfected with various concentrations of MyD88-DN plasmids for 48 h, then cell lysates underwent Western blotting analysis to detect the expression of Hsp90beta. CpG-B ODN-mediated up-regulation of Hsp90beta was inhibited by MyD88-DN but not an empty vector in a dose-dependent manner (Fig. 2B). Taken together, these results suggest that CpG-B ODN up-regulates the expression of Hsp90beta via a TLR9/MyD88 signaling pathway.

Because CpG DNA/ODN activates PI3K to regulate various cellular responses (32, 33) and the PI3K/Akt pathway is involved in Hsp induction under certain stress conditions (34, 35), we treated RAW264.7 cells with the PI3K inhibitor LY294002 before CpG-B ODN treatment. CpG-B ODN-induced Hsp90beta expression was inhibited by LY294002 (Fig. 2C) in a concentration-dependent manner. Consistent with the findings of inhibition analysis, CpG-B ODN-mediated increase of Hsp90beta was inhibited by overexpressing class I PI3K kinase-deficient plasmid (DN-PI3K, i.e., M-p110-{Delta}{kappa}in-myc) (Fig. 2D), which inhibited the CpG-B ODN-mediated phosphorylation of the PI3K substrate Akt (data not shown). Thus, the PI3K/Akt pathway might be instrumental for CpG-B ODN-mediated induction of Hsp90beta.

Hsp90beta is associated with CpG ODN-mediated cytokine production

To evaluate the influence of Hsp90beta on the CpG-B ODN-mediated response, we established RAW264.7-Hsp90betaRNAi, a RAW264.7 cell line whose Hsp90beta level was markedly knocked down by siRNA designed to target the Hsp90beta gene (Hsp90betaRNAi; Fig. 3A). Cytokine production (e.g., IL-12 and TNF-{alpha}) induced by CpG-B ODN was impaired in RAW264.7- Hsp90betaRNAi cells as compared with cells transfected with scrambled RNA interference (RNAi) (MockRNAi; Fig. 3B). Similar results were observed in mouse pDCs and macrophages transfected with siRNA directed to Hsp90beta (Fig. 3).


Figure 3
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 3. Effect of Hsp90beta inhibition on CpG ODN-mediated cytokine production. A, Lysates of scrambled siRNA-transfected RAW264.7 cells (MockRNAi), RAW264.7 cells stably transfected with Hsp90beta siRNA (Hsp90betaRNAi), and scrambled siRNA-transfected pDCs (MockRNAi) or pDCs transiently transfected with Hsp90beta siRNA (Hsp90betaRNAi) underwent Western blotting with Abs to Hsp90{alpha} and Hsp90beta. B, Scrambled siRNA- or Hsp90beta siRNA-transfected RAW264.7 cells, pDCs, and macrophages were incubated in medium alone or medium supplemented with 1.5 µM CpG-B ODN for 20 h. IL-12 and TNF-{alpha} levels in the culture supernatants were measured by ELISA. Data represent the mean ± SD of three experiments. *, p < 0.001.

 
Up-regulation of Hsp90beta via PI3K is positively associated with CpG-B ODN-induced antiapoptosis

Incubation of THP-1 (data not shown) or RAW264.7 cells (Fig. 4A) in serum-starved medium (0% FBS) for 60 h resulted in an ~32 or 35% decrease, respectively, in cell viability. The addition of CpG-B ODN to cells cultured in serum-starved medium improved the cellular viability in a dose-dependent manner, whereas GpC ODN (negative control) had no effect on viability (data not shown). Incubation of RAW264.7 cells in serum-free medium for longer than 60 h–7 days–resulted in even more cell death (>70%) which was, nonetheless, still preventable by the addition of CpG-B ODN (Fig. 4A). This effect of CpG-B ODN was accompanied by a decrease in cellular DNA fragmentation (Fig. 4, D and E), which indicates the decline of apoptosis. A similar antiapoptotic effect of CpG-B ODN was observed in pDCs (data not shown) and RAW264.7 cells pretreated with the proapoptotic agent STS. The antiapoptotic effect of CpG-B ODN was blocked by treatment with the PI3K inhibitor LY294002 but not the p38 MAPK inhibitor SB203580 (Fig. 4B), which suggests that PI3K plays a more important role than p38 MAPK in mediating the antiapoptotic effect of CpG-B ODN.


Figure 4
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 4. Inhibition of Hsp90beta or PI3K decreases the antiapoptotic effect of CpG-B ODN. A, RAW264.7 cells were cultured in serum-free (0% FBS) medium with or without GpC ODN or CpG-B ODN (1 µM) for the indicated times in the presence or absence of 0.15 µM radicicol as indicated. B, RAW264.7 cells were cultured for 60 h in serum (10% FBS) or serum-free (0% FBS) medium with or without GpC ODN or CpG-B ODN (1 µM) in the presence or absence of 30 µM LY294002 and 20 µM SB203580 as indicated. *, p < 0.05. C, RAW264.7 cells were cultured for 60 h in serum (10% FBS) or serum-free (0% FBS) medium in the presence of GpC ODN, CpG-B ODN (1.0 µM), chloroquine (CQ, 1 µg/ml), and radicicol as indicated. Trypan blue exclusion assay was used to determine cell viability. Data represent means ± SD of at least three independent experiments. *, p < 0.05 for the difference in viability among cells treated with CpG-B ODN alone and cells treated with CpG-B ODN together with CQ or radicicol. D, RAW264.7 cells were cultured for 60 h in serum (10% FBS) or serum-free (0% FBS) medium with 1 µM CpG-B ODN or GpG ODN in the presence or absence of 0.15 µM radicicol (Radi.) as indicated. The extent of apoptotic DNA fragmentation was analyzed by 2% DNA agarose gel. E, Cells were cultured as in (D) and the amount of cytosolic BrdU-labeled DNA fragments was determined by ELISA. *, p < 0.05 for the increase in DNA fragmentation after adding radicicol to the CpG-B ODN-treated cells.

 
Because Hsp are known to regulate apoptosis (22), we then examined whether Hsp90beta plays a critical role in CpG-B ODN-mediated antiapoptosis. Radicicol, an Hsp90 inhibitor that binds to the ATP-binding pocket of Hsp90 to disrupt its function by preventing ATP hydrolysis (36), did not affect cell viability (Fig. 4, A and C) and DNA fragmentation of RAW264.7 cells (Fig. 4, D and E), but it modulated the antiapoptotic effect of CpG-B ODN in these cells (Fig. 4, D and E). Additional studies involving RAW264.7 and pDCs stably or transiently transfected with Hsp90betaRNAi showed that even though the basal level of Hsp90beta was greatly decreased (Fig. 3A) in Hsp90betaRNAi-transfected cells, the viability was similar to that for cells transfected with MockRNAi (Fig. 5). However, unlike MockRNAi cells, the Hsp90betaRNAi-transfected RAW264.7 (RAW264.7Hsp90betaRNAi) cells and pDCs were not protected by CpG-B ODN against apoptosis induced by either serum deprivation or the addition of 1 mM STS (Fig. 5). These results indicate that inducible Hsp90beta, rather than the basal level of Hsp90beta, is positively associated with the CpG-B ODN-mediated antiapoptosis.


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 5. Inhibition of Hsp90beta by expressing specific siRNA decreases the antiapoptotic effect of CpG-B ODN. A, RAW264.7MockRNAi or RAW264.7Hsp90betaRNAi cells were cultured for 60 h in serum (10% FBS) or serum-free (0% FBS) medium with or without CpG-B ODN (1 µM). B, RAW264.7MockRNAi or RAW264.7Hsp90betaRNAi cells were pretreated with or without CpG-B ODN for 14 h as indicated. The cells were then treated with 1 mM STS for 8 h. C, MockRNAi or Hsp90betaRNAi-transfected pDCs were incubated in serum-free medium (0% FBS) with or without CpG-B ODN (1 µM) for the indicated times. An MTT assay was used to determine cell viability. Data represent the mean ± SD of at least three independent experiments (*, p < 0.05).

 
Hsp90beta induced by CpG-B ODN modulates Akt activity and down-regulates active caspase-3

To further elucidate the involvement of Hsp90beta in the CpG-B ODN-mediated responses, we examined the effect of Hsp90 inhibition on phosphorylation of Akt. Akt phosphorylation induced by CpG-B ODN was suppressed by the PI3K inhibitor LY294002 or the Hsp90 inhibitor radicicol but not the p38 MAPK inhibitor SB203580 (Fig. 6A). To further evaluate the influence of Hsp90beta on CpG ODN-mediated Akt phosphorylation, we measured the phosphorylation level of Akt in RAW264.7Hsp90betaRNAi cells and found CpG ODN-induced Akt phosphorylation was impaired in these cells as compared with RAW264.74 cells or cells transfected with scrambled RNAi (RAW264.7MockRNAi; Fig. 6B). These results imply that Hsp90beta may play an important role in CpG-B ODN-mediated activation of Akt.


Figure 6
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 6. Inhibiting Hsp90beta decreases CpG-B ODN-induced Akt activation. A, RAW264.7 cells were treated with GpC ODN or CpG-B ODN (1 µM) in the presence or absence of 0.15 µM radicicol, 30 µM LY294002, or 20 µM SB203580 as indicated. B, RAW264.7, RAW264.7MockRNAi, or RAW264.7Hsp90betaRNAi cells were treated with or without CpG-B ODN (1 µM) as indicated. An amount of 60 µg of cell lysates underwent Western blotting analysis with Abs against Akt and phospho-Akt. beta-actin was used as an internal control. The experiment was repeated two times with similar results.

 
Bacterial CpG DNA induces dendritic cell survival via the PI3K pathway by preventing the cleavage of procaspase-3 and down-regulating the formation of active caspase-3 fragments such as p17 (16). We therefore investigated whether Hsp90beta induced by CpG-B ODN has a similar effect. Western blotting analysis revealed that the level of procaspase-3 in THP-1 lysates was increased after CpG-B ODN stimulation. The PI3K inhibitor LY294002 and Hsp90 inhibitor radicicol significantly suppressed the effect of CpG-B ODN on the level of procaspase-3, whereas the p38 MAPK inhibitor SB203580 did not (data not shown). To further examine the effect of Hsp90beta on CpG-B ODN-regulated caspase-3 activation, we used siRNA directed to Hsp90beta. The induction of caspase-3 activation resulting from serum deprivation or STS treatment was decreased by CpG-B ODN in RAW264.7MockRNAi but not RAW264.7Hsp90betaRNAi cells (Fig. 7, A and B). Thus, CpG-B ODN may increase and prolong cell survival by preventing the activation of caspase-3 via a PI3K/Hsp90beta-dependent pathway.


Figure 7
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 7. Inhibiting Hsp90beta decreases the inhibitory effect of CpG-B ODN on cytochrome c release from mitochondria and caspase-3 activation. A, Cells were cultured for 60 h in serum (10% FBS) or serum-free (0% FBS) medium with or without 1 µM CpG-B ODN or GpC-ODN as indicated. B, Cells were pretreated with CpG-B ODN or GpC ODN for 14 h followed by 1 mM STS stimulation for 8 h. The caspase-3 activity was measured by fluorogenic substrate as described under Materials and Methods. Data represent means ± SD of at least three independent experiments. *, p < 0.05. C, Cells were cultured for 60 h in serum (10% FBS) or serum-free (0% FBS) medium with or without 1 µM CpG-B ODN or GpC-ODN. Cytochrome c in the mitochondria (mitochondria) or cytosolic (cytoplasm) fractions was detected by anti-cytochrome c Ab. The experiment was repeated two times with similar results.

 
Once cells are exposed to apoptotic stimuli such as STS, cell-apoptosis signals induce the release of cytochrome c from mitochondria, which then binds to Apaf-1 and initiates caspase activation and cell death (21, 22). We found that compared with cells cultured in 10% FBS medium, cells cultured in serum-starved medium were induced to release cytochrome c from mitochondria to cytoplasm, and this cytochrome c release was modulated by CpG-B ODN but not GpC ODN (Fig. 7C). The inhibition of Hsp90beta by Hsp90beta siRNA impaired the inhibitory effect of CpG-B ODN on cytochrome c release (Fig. 7C). Taken together, these results suggest that CpG-B ODN may induce Hsp90beta to inhibit the release of cytochrome c from mitochondria and prevent the activation of caspase-3.

CpG-B ODN increases the association of Hsp90beta with Bcl-2

The release of cytochrome c from mitochondria to cytoplasm is controlled by Bcl-2 (21, 22, 37). Previous reports have indicated that the formation of a Bcl-2-Hsp90 complex prevents the release of cytochrome c from mitochondria and activation of caspase-3 in mast cells (26, 27). To investigate whether CpG-B ODN may induce the association of Bcl-2 with Hsp90beta or Hsp90{alpha}, anti-Hsp90beta and anti-Hsp90{alpha} Abs were used to immunoprecipitate the Bcl-2-Hsp90 complex in cells treated with or without CpG-B ODN. Hsp90beta but not Hsp90{alpha} was associated with Bcl-2 in RAW264.7 cells as well as pDCs and macrophages (Fig. 8, A and B). The association of Bcl-2 with Hsp90beta was increased in CpG-B ODN-treated cells as compared with cells not treated with CpG-B ODN or treated with GpC ODN even though CpG-B ODN did not affect the cellular level of Bcl-2 (Fig. 8, A and B). The addition of radicicol to CpG-B ODN-treated cells did not decrease the cellular level of Hsp90beta but caused a marked decrease in the association of Bcl-2 with Hsp90beta (Fig. 8C), which indicates that radicicol affects the function rather than the expression of Hsp90beta. These results suggest that CpG-B ODN may increase not only the expression of Hsp90beta but also the association of Bcl-2 with Hsp90beta.


Figure 8
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 8. CpG-B ODN increases the association of Hsp90beta with Bcl-2 in RAW264.7 cells. A, RAW264.7 cells were treated with or without GpC ODN or CpG-B ODN (1 µM) for 16 h. Hsp90{alpha} or Hsp90beta was immunoprecipitated from the cell lysates by anti-Hsp90{alpha} or anti-Hsp90beta Abs. The association and coimmunoprecipitation of Bcl-2, Bcl-xL, and Bax with Hsp90beta or Hsp90{alpha} was determined by Western blot analysis with anti-Bcl-2, anti-Bcl-xL, or anti-Bax Abs. B, pDCs and macrophages were treated with or without GpC ODN or CpG-B ODN (1 µM) for 16 h. Hsp90beta was immunoprecipitated from the cell lysates by anti-Hsp90beta Abs. The association of Bcl-2 or Bcl-xL with Hsp90beta was determined by Western blot analysis with anti-Bcl-2 or anti-Bcl-xL Abs. C, RAW264.7 cells were treated with GpC ODN or CpG-B ODN for 16 h in the absence or presence of radicicol (0.15 µM). Hsp90beta was immunoprecipitated from the cell lysates by anti-Hsp90beta Abs. The association of Bcl-2 with Hsp90beta was determined by Western blot analysis with anti-Bcl-2 Abs. The experiments were repeated two times with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is well-known that the innate immune system of mammals relies on a set of pattern recognition receptors such as TLRs to distinguish the self from foreign molecular structures such as LPS and unmethylated bacterial CpG ODN (9). Among the TLRs, TLR9 recognizes unmethylated microbial CpG DNA and CpG ODN and TLR4 recognizes LPS (9). Several studies involving exogenous Hsps showed that Hsps are associated with TLR2 or TLR4 signal pathways (38, 39, 40). The linkage of Hsp90 to the TLR9 signaling pathway, however, has not been well-addressed. In this study, using Western blotting and RT-PCR, we demonstrated for the first time that stimulation of macrophages and pDCs with CpG-B ODN resulted in up-regulation of cellular Hsp90beta but not Hsp90{alpha} in a TLR9/MyD88-dependent manner (Figs. 1 and 2). Because Hsp90{alpha} is an isoform known to be expressed extracellularly (41), we also measured the level of both Hsp90beta and Hsp90{alpha} in the supernatant (Fig. 1B) and found them not increased in level. Our results thus indicate clearly that CpG-B ODN selectively increases the expression of Hsp90beta intracellularly via the TLR9 pathway.

After the selective binding of LPS and CpG ODN to TLR4 and TLR9, respectively, both TLR4 and TLR9 signaling is initiated by the recruitment of the adaptor molecule MyD88 (8, 9). Hsp90beta is induced after the activation of MyD88, which may explain why the factor induces a similar response when cells are exposed to LPS or CpG-B ODN (Fig. 1). The induction of Hsp90beta in immune cells by both LPS and CpG-B ODN suggests that inducible Hsp90beta may serve as an effector in prolonging the lifespan and function of immune cells regardless of whether the signal of microbial invasion is via a TLR4 (by LPS) or TLR9 (by CpG) pathway.

CpG-B ODN has previously been shown to protect B cells, macrophages, and pDCs against apoptosis (14, 15, 16). Its mechanism of action, however, remains to be further elucidated. In the present study, the increase in Hsp90beta level was positively associated with the antiapoptotic effect of CpG-B ODN in cells cultured in serum-starved or STS-supplemented medium (Figs. 4 and 5). Inhibition with the specific Hsp90 inhibitor radicicol and Hsp90beta siRNA did not affect cell viability (Fig. 4, A and C–E) but did interfere with CpG-B ODN-mediated antiapoptosis (Fig. 4 and 5). Thus, these results strongly suggest that the induction of Hsp90beta by CpG-B ODN may contribute to the antiapoptotic effect of CpG-B ODN.

Of note, although we found that radicicol and Hsp90beta siRNA did not affect viability of the immune cells (Fig. 4, A and C–E), Hooven et al. (42) reported that radicicol induced apoptosis by inhibiting the activity of Hsp90beta in epidermal cells of cavefish. Plausible explanations for the variation in the effect of radicicol on cell viability could be the difference in cell type used (i.e., epidermal cell vs immune cells) or concentrations of radicicol. Hooven et al. (42) used 1–2.5 µM radicicol, which was much higher than our concentration (0.15 µM).

Activation of the PI3K/Akt but not p38 MAPK pathway is believed to be positively associated with antiapoptosis (16). Recently, in studying the relation between Hsps and hypoxia-induced apoptosis, Zhou et al. (34) reported that PI3K/Akt contributes to stabilization of hypoxia-inducible factor 1-{alpha} by inducing the expression of Hsps. Our findings have demonstrated that CpG-B ODN up-regulated Hsp90beta primarily via the PI3K pathway (Fig. 2, C and D) and that the inhibition of PI3K but not p38 MAPK modulated the CpG-B ODN-mediated antiapoptosis (Fig. 4B). In addition, inhibition of Hsp90beta decreased CpG-B ODN-mediated Akt activation (Fig. 6). Taken together, these results provide further evidence for the importance of Hsp90beta in antiapoptosis mediated by CpG-B ODN via the Akt/PI3K pathway.

Cell apoptosis signals initiate the release of cytochrome c from mitochondria, which then binds to Apaf-1 and induces caspase 3 activation (21, 22). Our findings that CpG-B ODN treatment prevented cytochrome c release and caspase-3 activation (Fig. 7) suggest that CpG-B ODN may exert its antiapoptotic effect via the binding of Bcl-2 to Hsp90beta. The release of cytochrome c is guarded by Bcl-2, which locates in the outer mitochondrial membrane (21, 22, 37). Hsp90beta has been shown before to interact with Bcl-2, thereby increasing the antiapoptotic activity of Bcl-2 to inhibit cytochrome c release and prevent caspase-3 activation in mast cells (26, 27). Our studies demonstrate, as previously described by others in mast cells (27), that antiapoptosis was closely related to the association of Hsp90beta with Bcl-2 in pDCs and macrophages (Fig. 8, A and B). Of note, even though CpG-B ODN increased both the expression of Hsp90beta and the association of Hsp90beta with Bcl-2, CpG-B ODN affected neither the expression nor association of Hsp90{alpha} with Bcl-2 (Fig. 8A). These results reveal, for the first time, that Hsp90beta but not Hsp90{alpha} is an important mediator of CpG-B ODN-induced cell survival, a mechanism that involves binding of Bcl-2 to Hsp90beta and inhibition of cytochrome c release and caspase-3 activation. Recently, we evaluated the effect of CpG-B ODN on Hsps other than Hsp90beta and found that it increased the cellular level of Hsp70 (43). The elevated level of Hsp70 increases Bcl-xL and decreases nuclear translocation of apoptosis-inducing factor to induce antiapoptosis (43). It will be interesting to evaluate under what kind of conditions these Hsps play a more critical role in the antiapoptotic effect of CpG-B ODN.

In summary, although the whole process of CpG-B ODN-mediated antiapoptosis remains to be elucidated in more detail, our results demonstrate that CpG-B ODN increases the expression of Hsp90beta but not Hsp90{alpha} via a TLR9/MyD88/PI3K signaling pathway in human THP-1 cells, mouse spleen pDCs, and macrophages. The enhanced Hsp90beta protein expression may be positively associated with CpG-B ODN-mediated antiapoptosis primarily via increasing the formation of the Hsp90beta-Bcl-2 complex, inhibiting cytochrome c release from mitochondria, and preventing activation of caspase-3.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Academia Sinica (94F006) and the National Science Council (NSC93-2317-B-001-003) of Taiwan, Republic of China. Back

2 Address correspondence and reprint requests to Dr. Shu-Mei Liang, Agricultural Biotechnology Research Center, Academia Sinica, 128 Academia Road, Section 2, Taipei, Taiwan; E-mail address: smyang{at}gate.sinica.edu.tw or Dr. Chi-Ming Liang, Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Taipei, Taiwan; E-mail address: cmliang{at}gate.sinica.edu.tw Back

3 Abbreviations used in this paper: ODN, oligodeoxynucleotide; pDC, plasmacytoid dendritic cell; Hsp, heat shock protein; Apaf, apoptotic protease activation factor; siRNA, small interfering RNA; STS, staurosporine; DN, dominant negative; RNAi, RNA interference. Back

Received for publication October 20, 2006. Accepted for publication March 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Shimada, S., O. Yano, H. Inoue, E. Kuramoto, T. Fukuda, H. Yamamoto, T. Kataoka, T. Tokunaga. 1985. Antitumor activity of the DNA fraction from Mycobacterium bovis BCG. II. Effects on various syngeneic mouse tumors. J. Natl. Cancer Inst. 74: 681-688. [Medline]
  2. Krieg, A. M., A. K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G. A. Koretzky, D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374: 546-549. [Medline]
  3. Krieg, A. M.. 2002. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 20: 709-760. [Medline]
  4. Bauer, S., C. J. Kirschning, H. Hacker, V. Redecke, S. Hausmann, S. Akira, H. Wagner, G. B. Lipford. 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. USA 98: 9237-9242. [Abstract/Free Full Text]
  5. Lund, J., A. Sato, S. Akira, R. Medzhitov, A. Iwasaki. 2003. Toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells. J. Exp. Med. 198: 513-520. [Abstract/Free Full Text]
  6. Krug, A., A. R. French, W. Barchet, J. A. Fischer, A. Dzionek, J. T. Pingel, M. M. Orihuela, S. Akira, W. M. Yokoyama, M. Colonna. 2004. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function. Immunity 21: 107-119. [Medline]
  7. Hacker, H., H. Mischak, T. Miethke, S. Liptay, R. Schmid, T. Sparwasser, K. Heeg, G. B. Lipford, H. Wagner. 1998. CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation. EMBO J. 17: 6230-6240. [Medline]
  8. Hacker, H., R. M. Vabulas, O. Takeuchi, K. Hoshino, S. Akira, H. Wagner. 2000. Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J. Exp. Med. 192: 595-600. [Abstract/Free Full Text]
  9. Akira, S., H. Hemmi. 2003. Recognition of pathogen-associated molecular patterns by TLR family. Immunol. Lett. 85: 85-95. [Medline]
  10. Kobayashi, T., M. C. Walsh, Y. Choi. 2004. The role of TRAF6 in signal transduction and the immune response. Microbes Infect. 6: 1333-1338. [Medline]
  11. Verthelyi, D., R. A. Zeuner. 2003. Differential signaling by CpG DNA in DCs and B cells: not just TLR9. Trends Immunol. 24: 519-522. [Medline]
  12. Lenert, P., W. Rasmussen, R. F. Ashman, Z. K. Ballas. 2003. Structural characterization of the inhibitory DNA motif for the type A (D)-CpG-induced cytokine secretion and NK-cell lytic activity in mouse spleen cells. DNA Cell Biol. 22: 621-631. [Medline]
  13. Hartmann, G., J. Battiany, H. Poeck, M. Wagner, M. Kerkmann, N. Lubenow, S. Rothenfusser, S. Endres. 2003. Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-{alpha} induction in plasmacytoid dendritic cells. Eur. J. Immunol. 33: 1633-1641. [Medline]
  14. Yi, A. K., P. Hornbeck, D. E. Lafrenz, A. M. Krieg. 1996. CpG DNA rescue of murine B lymphoma cells from anti-IgM-induced growth arrest and programmed cell death is associated with increased expression of c-myc and bcl-xL. J. Immunol. 157: 4918-4925. [Abstract]
  15. Yi, A. K., M. Chang, D. W. Peckham, A. M. Krieg, R. F. Ashman. 1998. CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry. J. Immunol. 160: 5898-5906. [Abstract/Free Full Text]
  16. Park, Y., S. W. Lee, Y. C. Sung. 2002. Cutting edge: CpG DNA inhibits dendritic cell apoptosis by up-regulating cellular inhibitor of apoptosis proteins through the phosphatidylinositide-3'-OH kinase pathway. J. Immunol. 168: 5-8. [Abstract/Free Full Text]
  17. Sohn, W. J., K. W. Lee, S. Y. Choi, E. Chung, Y. Lee, T. Y. Kim, S. K. Lee, Y. K. Choe, J. H. Lee, D. S. Kim, H. J. Kwon. 2006. CpG-oligodeoxynucleotide protects immune cells from {gamma}-irradiation-induced cell death. Mol. Immunol. 43: 1163-1171. [Medline]
  18. Kuo, C. C., C. W. Kuo, C. M. Liang, S. M. Liang. 2005. A transcriptomic and proteomic analysis of the effect of CpG-ODN on human THP-1 monocytic leukemia cells. Proteomics 5: 894-906. [Medline]
  19. Hickey, E., S. E. Brandon, G. Smale, D. Lloyd, L. A. Weber. 1989. Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein. Mol. Cell. Biol. 9: 2615-2626. [Abstract/Free Full Text]
  20. Rebbe, N. F., W. S. Hickman, T. J. Ley, D. W. Stafford, S. Hickman. 1989. Nucleotide sequence and regulation of a human 90-kDa heat shock protein gene. J. Biol. Chem. 264: 15006-15011. [Abstract/Free Full Text]
  21. Beere, H. M.. 2001. Stressed to death: regulation of apoptotic signaling pathways by the heat shock proteins. Sci. STKE 2001: RE1[Medline]
  22. Garrido, C., S. Gurbuxani, L. Ravagnan, G. Kroemer. 2001. Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem. Biophys. Res. Commun. 286: 433-442. [Medline]
  23. Basso, A. D., D. B. Solit, G. Chiosis, B. Giri, P. Tsichlis, N. Rosen. 2002. Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function. J. Biol. Chem. 277: 39858-39866. [Abstract/Free Full Text]
  24. Fortugno, P., E. Beltrami, J. Plescia, J. Fontana, D. Pradhan, P. C. Marchisio, W. C. Sessa, D. C. Altieri. 2003. Regulation of survivin function by Hsp90. Proc. Natl. Acad. Sci. USA 100: 13791-13796. [Abstract/Free Full Text]
  25. Sato, S., N. Fujita, T. Tsuruo. 2000. Modulation of Akt kinase activity by binding to Hsp90. Proc. Natl. Acad. Sci. USA 97: 10832-10837. [Abstract/Free Full Text]
  26. Dias, S., S. V. Shmelkov, G. Lam, S. Rafii. 2002. VEGF(165) promotes survival of leukemic cells by Hsp90-mediated induction of Bcl-2 expression and apoptosis inhibition. Blood 99: 2532-2540. [Abstract/Free Full Text]
  27. Cohen-Saidon, C., I. Carmi, A. Keren, E. Razin. 2006. Antiapoptotic function of Bcl-2 in mast cells is dependent on its association with heat shock protein 90beta. Blood 107: 1413-1420. [Abstract/Free Full Text]
  28. Akhtar, M., J. L. Watson, A. Nazli, D. M. McKay. 2003. Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NF-{kappa}B-independent pathway. FASEB J. 17: 1319-1321. [Abstract/Free Full Text]
  29. Tada, H., O. Shiho, K. Kuroshima, M. Koyama, K. Tsukamoto. 1986. An improved colorimetric assay for interleukin 2. J. Immunol. Methods 93: 157-165. [Medline]
  30. Ahmad-Nejad, P., H. Hacker, M. Rutz, S. Bauer, R. M. Vabulas, H. Wagner. 2002. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32: 1958-1968. [Medline]
  31. Moynagh, P. N.. 2005. TLR signalling and activation of IRFs: revisiting old friends from the NF-{kappa}B pathway. Trends Immunol. 26: 469-476. [Medline]
  32. Fukao, T., S. Koyasu. 2003. PI3K and negative regulation of TLR signaling. Trends Immunol. 24: 358-363. [Medline]
  33. Kuo, C. C., W. T. Lin, C. M. Liang, S. M. Liang. 2006. Class I and III phosphatidylinositol 3'-kinase play distinct roles in TLR signaling pathway. J. Immunol. 176: 5943-5949. [Abstract/Free Full Text]
  34. Zhou, J., T. Schmid, R. Frank, B. Brune. 2004. PI3K/Akt is required for heat shock proteins to protect hypoxia-inducible factor 1{alpha} from pVHL-independent degradation. J. Biol. Chem. 279: 13506-13513. [Abstract/Free Full Text]
  35. Guo, F., C. Sigua, P. Bali, P. George, W. Fiskus, A. Scuto, S. Annavarapu, A. Mouttaki, G. Sondarva, S. Wei, et al 2005. Mechanistic role of heat shock protein 70 in Bcr-Abl-mediated resistance to apoptosis in human acute leukemia cells. Blood 105: 1246-1255. [Abstract/Free Full Text]
  36. Neckers, L.. 2002. Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol. Med. 8: S55-S61. [Medline]
  37. Kroemer, G., J. C. Reed. 2000. Mitochondrial control of cell death. Nat. Med. 6: 513-519. [Medline]
  38. Vabulas, R. M., P. Ahmad-Nejad, C. da Costa, T. Miethke, C. J. Kirschning, H. Hacker, H. Wagner. 2001. Endocytosed HSP60s use Toll-like receptor 2 (TLR2) and TLR4 to activate the Toll/interleukin-1 receptor signaling pathway in innate immune cells. J. Biol. Chem. 276: 31332-31339. [Abstract/Free Full Text]
  39. Vabulas, R. M., P. Ahmad-Nejad, S. Ghose, C. J. Kirschning, R. D. Issels, H. Wagner. 2002. HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J. Biol. Chem. 277: 15107-15112. [Abstract/Free Full Text]
  40. Vabulas, R. M., S. Braedel, N. Hilf, H. Singh-Jasuja, S. Herter, P. Ahmad-Nejad, C. J. Kirschning, C. Da Costa, H. G. Rammensee, H. Wagner, H. Schild. 2002. The endoplasmic reticulum-resident heat shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway. J. Biol. Chem. 277: 20847-20853. [Abstract/Free Full Text]
  41. Eustace, B. K., T. Sakurai, J. K. Stewart, D. Yimlamai, C. Unger, C. Zehetmeier, B. Lain, C. Torella, S. W. Henning, G. Beste, et al 2004. Functional proteomic screens reveal an essential extracellular role for hsp90{alpha} in cancer cell invasiveness. Nat. Cell Biol. 6: 507-514. [Medline]
  42. Hooven, T. A., Y. Yamamoto, W. R. Jeffery. 2004. Blind cavefish and heat shock protein chaperones: a novel role for hsp90{alpha} in lens apoptosis. Int. J. Dev. Biol. 48: 731-738. [Medline]
  43. Kuo, C. C., S. M. Liang, C. M. Liang. 2006. CpG-B oligodeoxynucleotide promotes cell survival via up-regulation of Hsp70 to increase Bcl-xL and to decrease apoptosis-inducing factor translocation. J. Biol. Chem. 281: 38200-38207. [Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kuo, C.-C.
Right arrow Articles by Liang, S.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuo, C.-C.
Right arrow Articles by Liang, S.-M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS