Bcl-2 Expression in Synovial Fibroblasts Is Essential for Maintaining Mitochondrial Homeostasis and Cell Viability1 Harris Perlman,† Constantinos Georganas,†‡ Lisa J. Pagliari,† Alisa E. Koch,† Kenneth Haines III,† and Richard M. Pope2† The regulation of proliferation and cell death is vital for homeostasis, but the mechanism that coordinately balances these events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining thickens in part through increased proliferation and/or decreased synovial fibroblast cell death. Here we demonstrate that the anti-apoptotic protein, Bcl-2, is highly expressed in RA compared with osteoarthritis synovial tissues, particularly in the CD68-negative, fibroblast-like synoviocyte population. To determine the importance of endogenous Bcl-2, an adenoviral vector expressing a hammerhead ribozyme to Bcl-2 (Ad-Rbz-Bcl-2) mRNA was employed. Ad-Rbz-Bcl-2 infection resulted in reduced Bcl-2 expression and cell viability in synovial fibroblasts isolated from RA and osteoarthritis synovial tissues. In addition, Ad-Rbz-Bcl-2-induced mitochondrial permeability transition, cytochrome c release, activation of caspases 9 and 3, and DNA fragmentation. The general caspase inhibitor zVAD.fmk blocked caspase activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation, but not loss of transmembrane potential or viability, indicating that cell death was independent of caspase activation. Ectopically expressed Bcl-xL inhibited Ad-Rbz-Bcl-2-induced mitochondrial permeability transition and apoptosis in Ad-Rbz-Bcl-2-transduced cells. Thus, forced down-regulation of Bcl-2 does not induce a compensatory mechanism to prevent loss of mitochondrial integrity and cell death in human fibroblasts. The Journal of Immunology, 2000, 164: 5227–5235.
A
poptosis is a complex process that may be divided into three phases: initiation, commitment, and execution (1). The Bcl-2 family of proteins regulates the commitment phase, while caspases regulate the execution of apoptosis. Bcl-2 homologous proteins may function as protectors (Bcl-2, Bcl-xL, Mcl-1, A1, and Bcl-w) or accelerators (Bax, Bad, Bcl-xS, Mtd, Bak, Bik, Bok, Bim, Bip, Bid, Diva, Hrk, and Blk) of apoptosis (2). Mice null homozygous for Bcl-2 alleles exhibit normal fetal development, but postnatally develop polycystic kidney disease, fulminant lymphoid apoptosis, and hypopigmented hair (3, 4). Thus, Bcl-2 is not necessary for development, and Bcl-2 homologous proteins may function in a redundant fashion prenatally. However, few studies have examined the effects of forced reduction of Bcl-2 postnatally. The prevailing hypothesis concerning the Bcl-2 protein family is that the balance between proliferation and death is dependent on the ratio of the levels of apoptotic protectors to accelerators (5).
*Division of Rheumatology, Northwestern University Medical School, and †Veterans Administration Chicago Healthcare System, Lakeside Division, Chicago, IL 60611; and ‡Rheumatology Department, 251 Hellenic Airforce Veterans Affairs General Hospital, Athens, Greece Received for publication December 27, 1999. Accepted for publication March 1, 2000. 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 the Northwestern Memorial Foundation; by Grant T32AI07476-03 (to H.P.), by National Institutes of Health Grants AR43642 and AR30692 and Contract AR62229 (to R.M.P.) and Grants AR41492 and AI40987, funds from the Veterans Affairs Research Service, and by the Gallagher Professorship for Arthritis Research (to A.E.K.). 2 Address correspondence and reprint requests to Dr. Richard M. Pope, Division of Rheumatology, Department of Medicine. Northwestern University Medical School. 303 East Chicago Avenue, Ward 3-315, Chicago, IL 60611. E-mail address:
[email protected]
Copyright © 2000 by The American Association of Immunologists
Rheumatoid arthritis (RA)3 is an autoimmune disease characterized by fibroblastic proliferation, infiltration of the synovial lining by lymphocytes and macrophages, and a paucity of apoptosis (6). In RA, fibroblasts that reside in the synovial lining markedly increase in number, display a transformed phenotype, and invade and destroy adjacent cartilage. In contrast, osteoarthritis (OA) fibroblasts do not proliferate in vivo or contribute to articular cartilage degradation (7). These data suggest that the decision by synovial fibroblasts to undergo apoptosis or proliferate may be linked through the actions of common nodal point regulators, one of which may be the apoptotic antagonist, Bcl-2. A critical target of the Bcl-2 family proteins is the mitochondria. Ectopic Bcl-2 or Bcl-xL expression inhibits mitochondrial dysfunction induced by death receptor-mediated apoptosis, growth factor withdrawal, chemotherapeutic reagents, or gamma irradiation (8). Forced Bcl-2 expression blocks cytochrome c release (9 – 11) induced by either growth factor withdrawal or Bax overexpression and prevents loss of mitochondrial membrane potential in reconstituted liposomes (12). Additionally, Bcl-2 overexpression may inhibit apoptosis following cytochrome c release (13, 14), although the mechanism remains unknown. Once released, cytochrome c binds apoptotic protease-activating factor 1 (Apaf1) (15), pro-caspase 9, and ATP and induces the conversion of procaspase 9 into the active heterotetrameric protease (16). Activated caspase 9 induces the activation of caspase 3 (17), which results in the cleavage of cytoskeletal, nuclear scaffold, DNA repair, and cell cycle proteins (18). Although most investigations have focused on the inhibitory mechanism of Bcl-2/Bcl-xL overexpression, little is known about the effects of forced reduction of endogenous Bcl-2 on mitochondria function, caspase activation, and cellular survival.
3 Abbreviations used in this paper: RA, rheumatoid arthritis; ST, synovial tissue; OA, osteoarthritis; Adeno-Bcl-2 ribozyme, Ad-Rbz-Bcl-2; Ad--gal, adeno--galactosidase; zVAD.fmk, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone; PARP, poly(ADP-ribose) polymerase.
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5228 Here, we documented the expression of Bcl-2 in RA and OA synovial tissues (ST). Although, a marked increase in Bcl-2 was observed in RA ST compared with OA ST in vivo, particularly in the synovial fibroblast population, cultured RA and OA synovial fibroblasts displayed similar levels of Bcl-2, suggesting that the factor(s) responsible for increased Bcl-2 expression in vivo was no longer present. The effects of forced reduction of Bcl-2 in fibroblast-like synoviocytes by adenoviral-mediated hammerhead ribozyme to Bcl-2 were examined. Ad-Rbz-Bcl-2 infection induced apoptosis, as indicated by the reduction in fibroblast cell number, loss of mitochondrial membrane potential, and fragmented DNA. No differences in the induction of apoptosis by Ad-Rbz-Bcl-2 were observed in RA or OA synovial fibroblasts, suggesting that Bcl-2 is essential for fibroblast viability. Cytosolic cytochrome c was detected, and caspases 9 and 3 were activated in the Ad-Rbz-Bcl2-infected cultures compared with uninfected and Ad--gal-infected cultures. The pan-caspase inhibitor benzyloxycarbonyl-ValAla-Asp fluoromethyl ketone (zVAD.fmk) suppressed caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage in Ad-Rbz-Bcl-2-transduced cultures. Ectopic expression of Bcl-xL, but not zVAD.fmk, inhibited Ad-Rbz-Bcl-2-induced permeability transition and cell death. Thus, forced disruption of endogenous Bcl-2 in human RA synovial fibroblasts resulted in mitochondrial dysfunction and cell death, independent of caspase activation.
Materials and Methods Immunohistochemistry Synovial tissue for immunohistochemistry was obtained at the time of arthroplasty from 11 patients with RA and 12 patients with OA. All the patients met the American College of Rheumatology classification criteria for RA and OA, respectively (19, 20). Five-micron sections from ST fixed in methyl Carnoy were deparaffinized and blocked in 10% goat serum. Sections were incubated with rabbit anti-Bcl-2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG (Sigma, St. Louis, MO). Peptide inhibition was conducted using a 10-fold excess of the immunogenic peptide and incubating the peptide with the Ab for 2 h. Rat 3-day postcastration prostates were used as a positive control (21). A biotinylated goat anti-rabbit secondary Ab (BioGenex, San Ramon, CA) followed by alkaline phosphatase (BioGenex) conjugated to streptavidin was used to detect primary Ab complexes. Visualization was accomplished using the Fast Red alkaline phosphatase substrate kit (BioGenex), and counterstaining was performed using hematoxylin. Specimens were examined and photographed on a Nikon ES400 microscope (Nikon, Garden City, NY) equipped for phase contrast visualization. Sublining synovial fibroblasts were identified by their spindle-shaped nuclei, while the sublining macrophage-like cells were recognized by their abundant cytoplasm and rounded nuclei (22). The synovial lining thickness (median cell number); inflammatory score (0 to ⫹5), which was estimated by the degree of sublining inflammatory cell infiltrate composed of macrophages, lymphocytes, and neutrophils; and percentage of cells positive for Bcl-2 were scored by a blinded pathologist as previously described (22–24).
Cell culture OA and RA ST samples were obtained from patients undergoing total joint replacement who met the American College of Rheumatology criteria (19, 20). Isolated synovial tissues were digested with collagenase, dispase, and DNase I, and single-cell suspensions were obtained (23, 24). Human RA and OA synovial fibroblasts (passages 4 –9) and normal dermal fibroblasts (CRL1475, American Type Culture Collection, Rockville, MD) were cultured in 10% FBS/DMEM. For infections, cells were plated in growth medium (10% FBS) and allowed to attach before being transferred to low serum medium. Cultures were serum starved in 0.1– 0.5% FBS/DMEM for 3 days before infection. Cells were then counted, and cultures were incubated at a multiplicity of infection of 750 PFU/cell with Ad--gal (25) or Ad-Rbz-Bcl-2 (26, 27), or with a multiplicity of infection of 250 PFU/cell of Ad-Bcl-xL for 12 h in low serum medium. At the end of the infection period the virus was removed by washing with PBS and was returned to low serum medium for an additional 12 h. The cultures were then stimulated for 72–96 h by the addition of medium containing 10% FBS, and 50 M zVAD-fmk (Enzyme System Products, Livermore, CA) was added 24 h postinfection as indicated.
ENDOGENOUS Bcl-2 REGULATES FIBROBLAST SURVIVAL Flow cytometry Mitochondrial permeability transition measured by retention of the cationic dye, Rh123 (0.1 g/ml; Molecular Probes, Eugene, OR), which was added to cultures for 30 min before analysis by flow cytometry, and number of live cells were determined by propidium iodide exclusion. Experiments using a dose-response curve revealed that 0.1 g/ml Rh123 was the optimum concentration to detect alterations in mitochondrial membrane potential. Medium-treated cultures incubated with the mitochondrial uncoupling reagent CCCP (9) did not retain 0.1 g/ml Rh123, documenting that mitochondrial function was required for Rh123 retention at 0.1 g/ml. For subdiploid DNA content, cultures were harvested by trypsinization, fixed in 70% ethanol overnight, and stained with propidium iodide (Roche Biochemical, Indianapolis, IN) as previously described (28). The subdiploid peak, immediately adjacent to the G0/G1 peak (2N), was determined by flow cytometry using a Beckman-Coulter EPICS XL flow cytometer and system 2 software (see Fig. 1B). Objects with minimal light scatter were excluded, because they may represent debris and would have inappropriately enhanced our estimate of the subdiploid population (29). No differences were detected in the subdiploid DNA content in samples analyzed in linear or logarithmic scale (28, 29). Flow cytometry was conducted at the Robert H. Lurie Comprehensive Cancer Center, Flow Cytometry Core Facility of the Northwestern University Medical School (Chicago, IL).
Western blot analysis Whole cell extracts were prepared as previously described (25, 28) from uninfected and infected cultures. Extracts (25 g) were analyzed by SDSPAGE on 12.5% polyacrylamide gels and transferred to Immobilon-P (Millipore, Bedford, CA) by semidry blotting. For cytosolic extraction, cells were resuspended in lysis buffer (20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF) and incubated for 3 min on ice as previously described by Walter et al. (30). Lysates were homogenized for 1 min and centrifuged for 15 min (750 ⫻ g), and supernatants containing the cytosolic fraction (15 g) were electrophoresed on 15% SDS-PAGE polyacrylamide gels and transferred to Immobilon P membranes by semidry transfer. Filters were blocked for 1 h at room temperature in PBS/0.2% Tween 20/5% nonfat dry milk. The filters were then incubated with mouse anti-Bcl-2 (Transduction Laboratories, Lexington, KY), anti-PARP (PharMingen, San Diego, CA), anticytochrome c (PharMingen), or anti-tubulin (Calbiochem, La Jolla, CA) Abs at a concentration of 0.25– 0.4 g/ml. Rabbit anti-caspase 8 (Chemicon, Temecula, CA), anti-caspase 9 (Chemicon), or anti-caspase 3 (Upstate Biotechnology, Lake Placid, NY) Abs were used at a concentration of 0.25– 0.4 g/ml. All primary Abs were incubated overnight at 4°C in PBS/ 0.2% Tween 20/2% nonfat dry milk. Filters were washed in PBS/0.2% Tween 20/2% nonfat dry milk and incubated with donkey anti-rabbit or anti-mouse secondary Ab (1/2000 dilution) conjugated to HRP (Amersham, Arlington Heights, IL). Visualization of the immunocomplex was conducted by enhanced chemiluminescence (ECL Plus, Amersham).
Caspase 3 activity Cell pellets from uninfected and infected cells were placed in lysis buffer provided by the manufacturer (Clontech, Palo Alto, CA), and caspase 3 activity was measured by the ApoAlert CPP32/Caspase-3 fluorescent assay kit (Clontech). Briefly, lysed cells were incubated with benzyloxycarbonylAsp-Glu-Val-Asp-AFC (DEVD-AFC) for 30 min and examined for protease activity by measuring fluorescence with a fluorometer equipped with a 400-nm excitation filter and a 505-nm emission filter.
Statistical analysis Results were expressed as the mean ⫾ SE. Differences between groups were analyzed using unpaired two-tailed Student’s t test. Correlations were determined by regression analysis.
Results Increased Bcl-2 expression in RA ST compared with OA ST Previous investigations have demonstrated a lack of apoptotic cells in RA ST. Therefore, we characterized the expression of the antiapoptotic protein Bcl-2 to define a potential mechanism responsible for resistance to apoptosis in these tissues. Immunohistochemical analysis revealed enhanced Bcl-2 expression in RA ST compared with OA ST (Fig. 1A). The staining pattern for Bcl-2 was a granular cytoplasmic staining with pseudo-nuclear inclusions, consistent with mitochondrial staining. Increased numbers
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FIGURE 1. Bcl-2 expression in RA and OA ST and in synovial fibroblasts. A, Bcl-2 expression is enhanced in RA-ST compared with OA-ST. Representative photomicrographs of RA and OA ST were stained for Bcl-2 (red) and counterstained with hematoxylin (blue). Eleven RA and 12 OA sections were incubated with rabbit anti-Bcl-2 Ab, Bcl-2 peptide plus rabbit anti-Bcl-2 Ab (10-fold excess of peptide to Ab), or control normal IgG (not shown). Sections were then incubated with biotinylated anti-rabbit Ab. Streptavidin-conjugated alkaline phosphatase and the Fast Red alkaline phosphatase substrate kit were used to visualize Bcl-2-anti-Bcl-2 complexes (red). B, Expression of Bcl-2 in RA and OA synovial fibroblasts. Quiescent RA and OA synovial fibroblasts were cultured in 10% FBS/DMEM for 96 h and harvested, and whole cell extracts (25 g) were subjected to immunoblot analysis with Abs to Bcl-2 and tubulin.
of RA synovial lining (76 ⫾ 7.6 vs 37 ⫾ 15%; p ⬍ 0.02) and sublining (73 ⫾ 4.5 vs 32 ⫾ 11%; p ⬍ 0.002) cells were positive for Bcl-2 compared with OA ST (Table I). Bcl-2-positive cells in the sublining were phenotypically fibroblasts and macrophages. Vascular smooth muscle cells were comparably positive for Bcl-2 in RA and OA ST (Fig. 1A). Lymphoid follicles in the RA ST also contained cells positive for Bcl-2 (not shown). Staining with normal control IgG was negative for both RA (not shown) and OA ST (Fig. 1A). Ab specificity was demonstrated by preincubation of the anti-Bcl-2 Ab with the Bcl-2 immunogenic peptide (Fig. 1A),
Table I. Increased Bcl-2 expression in RA compared to OA-STs
Median synovial lining thickness Inflammatory score Vessel score Synovial lining: Bcl2 staining (%) Sublining: Bcl-2 staining (%) a
RA (n ⫽ 11)
OA (n ⫽ 9)
p Valuea
2.4 ⫾ 0.3
1.3 ⫾ 0.3
⬍0.01
2.9 ⫾ 0.5 3.0 ⫾ 0.2 76.0 ⫾ 7.6
1.2 ⫾ 0.1 1.9 ⫾ 0.2 37.0 ⫾ 15.0
⬍0.0001 ⬍0.0001 ⬍0.02
73.0 ⫾ 4.5
32.0 ⫾ 11.0
⬍0.002
The p values were determined by comparing RA to OA by Student’s t test.
which abrogated the Bcl-2-positive signal. In addition, immunoblot analysis performed on extracts from fibroblasts overexpressing human Bcl-2 demonstrated a single 26-kDa band (not shown), further supporting the specificity of these observations using anti-Bcl-2 Ab. The enhanced expression of Bcl-2 in the synovial lining correlated with increased synovial lining thickening (r ⫽ 0.55; p ⬍ 0.01) and inflammatory score (r ⫽ 0.46; p ⬍ 0.04) in the combined ST. In contrast, Bcl-2 positivity in the sublining region did not correlate significantly with the inflammatory score (r ⫽ 0.40) or with lining thickness (r ⫽ 0.13). These observations suggest a relationship between Bcl-2 expression and synovial lining thickness and degree of inflammation. Two-color immunofluorescence was used to more definitively identify which cell types were Bcl-2 positive. Employing an anti-CD68 Ab to detect macrophages and anti-Bcl-2 Ab revealed that the vast majority of CD68-negative RA synovial lining and sublining cells were Bcl-2 positive (not shown). To confirm the presence of Bcl-2 in synovial fibroblasts, immunoblot analysis was performed on RA and OA synovial fibroblasts (passages 4 –9). In contrast to the immunohistochemical data, which demonstrated enhanced expression of Bcl-2 in RA ST, immunoblot analyses on extracts prepared from isolated RA and OA synovial fibroblasts demonstrated that Bcl-2 was comparably ex-
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FIGURE 2. Ad-Rbz-Bcl-2 reduces synovial fibroblast viability. A, Ad-Rbz-Bcl-2 reduces Bcl-2 expression in synovial fibroblasts. Primary cultures of RA (not shown) and OA synovial fibroblasts were made quiescent for 72 h in 0.5% FBS/DMEM. Cells were transduced with either Ad--gal or Ad-Rbz-Bcl-2 for 12 h, after which the virus was removed, and cultures were returned to 0.5% FBS/DMEM for an additional 12 h. Ten percent FBS/DMEM was then added for 96 h to stimulate proliferation, and whole cell extracts (25 g) were subjected to immunoblot analysis with Abs to Bcl-2 and tubulin. Medium control (mock) cultures are denoted with a bar. B, RA and OA synovial fibroblasts infected with Ad-Rbz-Bcl-2 round-up and detach from the plate surface. C and D, Quantitative analysis of RA (C) and OA (D) synovial fibroblast viability following infection. Triplicate synovial fibroblast cultures were harvested by trypsinization following serum stimulation for 96 h. Cells were then fixed and counted with a hemocytometer using the trypan blue exclusion method. Values represent the mean ⫾ SE from a representative of eight independent experiments, which were compared for statistical significance by unpaired two-tailed Student’s t test. ⴱ, p ⬍ 0.02 relative to the quiescent control.
pressed (Fig. 1B). Immunoblot analysis of Bcl-2 homologous proteins revealed an undetectable level of A1 and Mcl-1 and faint expression of Bcl-xL in RA synovial fibroblasts (not shown). These data demonstrate that Bcl-2 is highly expressed in RA compared with OA ST in vivo; however, following isolation and culture, consistent differences in Bcl-2 were no longer observed. Ad-Rbz-Bcl-2 reduces fibroblast cell number To determine whether Bcl-2 was essential for RA synovial fibroblast survival, we examined the effects of forced ablation of Bcl-2 by a hammerhead ribozyme directed against bcl-2 (26, 27). The affects of Bcl-2 down-regulation in RA synovial fibroblasts were compared with those in OA synovial fibroblasts and normal dermal fibroblasts. Immunoblot analyses on extracts prepared from mock-, Ad--gal-, or Ad-Rbz-Bcl-2 infected RA (not shown) and OA synovial fibroblasts (Fig. 2A) demonstrated Bcl-2 down-regulation in Ad-Rbz-Bcl-2-treated cultures compared with Ad--gal. Comparable levels of tubulin were detected in the Ad--gal- and Ad-RbzBcl-2-infected cultures, indicating equal loading of protein. Between 72 and 96 h following the addition of serum, the AdRbz-Bcl-2-infected RA and OA synovial and dermal (not shown) fibroblasts displayed an altered morphology that differed markedly from that of control infected cells. Light microscopic analysis revealed cytoplasmic shrinkage and detachment from the plate surface in the Ad-Rbz-Bcl-2-infected cultures (Fig. 2B). The mediumtreated (mock; not shown) and Ad--gal-treated cultures remained viable and attached to the plate surface under these conditions. Trypan blue exclusion demonstrated a significant (Fig. 2, C and D) decrease in cell viability in the Ad-Rbz-Bcl-2-infected RA (48%;
p ⬍ 0.03) and OA (48%; p ⬍ 0.001) synovial fibroblasts and dermal fibroblasts (66%; p ⬍ 0.001; not shown) compared with that in quiescent control cultures. In contrast, compared with the quiescent cells, there was an increase ( p ⬍ 0.002) in the number of viable cells in mock- and Ad--gal-treated synovial and dermal fibroblasts over this time course. These results demonstrate no consistent differences between the RA and OA synovial fibroblasts, suggesting that Bcl-2 is necessary for survival. Ad-Rbz-Bcl-2 induces apoptosis The decreased cell viability and the phenotypic characteristics displayed by the Ad-Rbz-Bcl-2-transduced fibroblasts suggested that these cells were undergoing apoptosis. Therefore, flow cytometric analysis (FACS) was used to determine DNA content in uninfected and infected cultures. Ad-Rbz-Bcl-2-infected RA and OA synovial and dermal fibroblasts (not shown) exhibited hypodiploid DNA (⬍2N), indicative of apoptosis, while Ad--gal-transduced cultures displayed normal DNA profiles (Fig. 3). To further support the apoptotic data obtained by FACS, TUNEL analysis revealed increased TUNEL-positive cells that displayed condensed nuclei in the Ad-Rbz-Bcl-2 infected cultures compared with Ad--galtreated cells (not shown). Forced Bcl-2 down-regulation results in mitochondrial dysfunction An early event in some forms of apoptosis is the loss of mitochondrial function, which is regulated by the Bcl-2 family proteins (31, 32). Quantitative loss of mitochondrial transmembrane potential was determined by the inability of apoptotic cells to accumulate
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FIGURE 3. Ad-Rbz-Bcl-2 induces subdiploid DNA in RA and OA synovial fibroblasts. Fibroblasts infected with Ad-Rbz-Bcl-2 display DNA fragmentation. Triplicate cultures of quiescent RA (n ⫽ 8) and OA (n ⫽ 7) synovial fibroblasts were uninfected (mock) or infected by the indicated adenoviral constructs for 12 h, after which the virus was removed, and cultures were returned to 0.5% FBS/DMEM for an additional 12 h. Cultures were transferred to 10% FBS/DMEM for 96 h and then analyzed by flow cytometry to determine DNA content. Values represent the mean ⫾ SE and were compared for statistical significance by unpaired two-tailed Student’s t test. ⴱ, p ⬍ 0.001 relative to mock or Ad--gal.
the charged cationic green fluorochrome rhodamine 123 (Rh123). A significant ( p ⬍ 0.05) reduction of the inner ion transmembrane potential was observed in Ab-Rbz-Bcl-2-infected cultures compared with Ad--gal-transduced RA and OA synovial fibroblasts (Fig. 4A), demonstrating that Bcl-2 ablation induced mitochondrial dysfunction independent of the origin of the fibroblast. Mitochondrial factors, including cytochrome c, are released following the induction of apoptosis, although cytochrome c release may precede or occur before mitochondrial permeability transition (10, 11, 33). Immunoblot analyses of cytosolic extracts prepared from mock-, Ad--gal-, or Ad-Rbz-Bcl-2-infected dermal fibroblasts demonstrated an increase in the presence of cytosolic cytochrome c in the Ad-Rbz-Bcl-2-infected compared with the Ad-gal-infected cultures (Fig. 4B). Equal loading of protein was determined by tubulin expression. Thus, the forced down-regulation of Bcl-2 resulted in the loss of mitochondrial membrane potential and release of cytochrome c into the cytosol. zVAD.fmk blocks caspase activation and subsequent downstream events, but does not inhibit Ad-Rbz-Bcl-2 induction of mitochondrial permeability transition Caspase activation, specifically caspase 3, has been shown to be responsible for induction of the endonuclease involved in DNA fragmentation (34, 35). Analysis of caspase 3 activity by fluorometric assay revealed that the Ad-Rbz-Bcl-2-transduced cultures
5231 exhibited a significant ( p ⬍ 0.01) increase in caspase 3 activity relative to mock- or Ad--gal-infected cultures (Fig. 5A). Additionally, activation of caspase 3 was detected by immunoblot analysis in Ad-Rbz-Bcl-2-infected but not in Ad--gal-infected cells (Fig. 5B). To define the mechanism of caspase 3 activation, the effect of Ad-Rbz-Bcl-2 infection on caspases 8 and 9 was examined. Immunoblot analysis of caspase 9 revealed caspase 9 cleavage (Fig. 5B) in the Ad-Rbz-Bcl-2-transduced cultures compared with that in mock- or Ad--gal-infected cells. In contrast to caspase 9, expression of the active isoform of caspase 8 was not detected in the Ad-Rbz-Bcl-2-infected cultures (not shown). To determine whether caspases were necessary for Rbz-Bcl-2-induced cell death, the general caspase inhibitor, zVAD.fmk, was used. Mock-, Ad--gal-, and Ad-Rbz-Bcl-2-infected synovial fibroblasts were cultured in the presence or the absence of zVAD.fmk and assayed for caspase activation and mitochondrial dysfunction. Immunoblot analyses of caspases 9 and 3 revealed that the appearance of the activated isoforms of caspases 9 and 3 in Ad-Rbz-Bcl2-infected synovial fibroblasts was prevented by the general caspase inhibitor zVAD.fmk (Fig. 5B). Furthermore, zVAD.fmk blocked PARP cleavage and suppressed DNA fragmentation (not shown) in the Ad-Rbz-Bcl-2-infected cultures. However, Ad-RbzBcl-2 induced mitochondrial permeability transition (Fig. 5C) was not abrogated by zVAD.fmk, suggesting that caspase activation did not precede mitochondrial dysfunction (36, 37). Additionally, Ad-Rbz-Bcl-2 infected synovial fibroblasts treated with zVAD.fmk detached from the plate surface (not shown) and displayed reduced cell numbers (Fig. 5D), indicating that the infected cells were indeed undergoing cell death. These data demonstrate that in this system a tripeptide caspase inhibitor was ineffective in preventing the commitment phase of apoptosis that resulted from the reduction of Bcl-2, but was able to block the caspase-mediated execution phase. Bcl-xL overexpression rescues Bcl-2-ablated fibroblasts from apoptosis Forced Bcl-xL expression prevents the loss of mitochondrial permeability transition and apoptosis (9). Endogenous Bcl-xL was unchanged in Ad-Rbz-Bcl-2-infected cells (not shown), suggesting that expression of this anti-apoptotic protein did not compensate for the decrease in Bcl-2 in these cells. Therefore, the effect of the expression of Bcl-xL by a replication defective adenovirus was examined in Ad-Rbz-Bcl-2-transduced cultures. In contrast to the RA synovial fibroblasts infected with the Ad-Rbz-Bcl-2 alone, coinfection with Ad-Bcl-xL reduced the number of cells rounding
FIGURE 4. Bcl-2 down-regulation induces mitochondrial permeability transition. A, Ad-Rbz-Bcl-2 disrupts mitochondrial function. RA and OA synovial fibroblasts were treated as described above and, before analysis by flow cytometry, were pulsed for 30 min with 0.1 g/ml of Rh123. Values represent the mean ⫾ SE of six independent experiments and were compared for statistical significance by unpaired two-tailed Student’s t test. ⴱ, p ⬍ 0.05 relative to mock or Ad--gal. B, Ad-Rbz-Bcl-2 induces cytochrome c release into the cytosol. Cytoplasmic extracts of dermal fibroblasts infected with the indicated adenoviral constructs were subjected to immunoblot analysis with an Ab to cytochrome c or tubulin.
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ENDOGENOUS Bcl-2 REGULATES FIBROBLAST SURVIVAL
FIGURE 5. zVAD.fmk inhibits caspase activation, but not mitochondrial membrane permeability induced by Ad-Rbz-Bcl-2. A, Ad-Rbz-Bcl-2-transduced cultures displayed caspase 3 activity. Quiescent OA synovial fibroblast cultures were infected by the indicated adenoviral construct. Relative caspase 3 activity is indicated by the ratio of relative light units from infected to medium control-treated cultures. Values represent the mean ⫾ SE of three independent experiments and were compared for statistical significance by unpaired two-tailed Student’s t test. ⴱ, p ⬍ 0.01 relative to Ad--gal. B, zVAD.fmk blocked caspase 9 and 3 activation and PARP cleavage. Triplicate cultures of quiescent OA synovial fibroblasts were infected by the indicated adenoviral constructs. Cultures were either transferred to 10% FBS/DMEM with the addition of 50 M zVAD.fmk or cultured in the presence of 10% FBS/DMEM alone for 96 h. Immunoblot analyses were performed on 25 g of whole cell extracts with Abs directed against tubulin, caspases 9 and 3, and PARP. The pro and active forms of caspase 3 are from the same immunoblot. The immunoblots are representative of three independent experiments. C, zVAD.fmk was ineffective at inhibiting mitochondrial permeability transition. Quiescent RA and OA synovial and dermal fibroblasts were infected by the indicated adenoviral construct or medium control (mock). Cultures were then transferred to 10% FBS/DMEM with or without 50 M zVAD.fmk for 96 h. Cultures were harvested to analyze retention of Rh123 by flow cytometry. The values of dermal, RA, and OA fibroblasts from individual experiments were combined because no difference was observed among these cell types. Values represent the mean ⫾ SE of four independent experiments and were compared for statistical significance by unpaired two-tailed Student’s t test. ⴱ, p ⬍ 0.02 relative to control treated cultures. D, Ad-Rbz-Bcl-2 induces synovial fibroblast cell death independent of caspases. Quiescent RA and OA synovial and dermal fibroblasts were infected by the indicated adenoviral construct or medium control (mock) as described above. Cell number was determined by flow cytometry gating on live cells by forward and side scatter. Values represent the mean ⫾ SE of three independent experiments and were compared for statistical significance by unpaired two-tailed Student’s t test. ⴱ, p ⬍ 0.02 relative to control treated.
up and detaching from the plate surface (not shown). FACS analyses of cultures infected with either Ad-RbzBcl-2 or Ad-RbzBcl-2 and Ad-Bcl-xL revealed that ectopic Bcl-xL expression inhibited the loss of Rh123 incorporation (Fig. 6A). Furthermore, subdiploid DNA was reduced by two-thirds in Ad-Bcl-xL/Ad-RbzBcl-2-transduced compared with Ad-Rbz-Bcl-2-transduced cultures (Fig. 6B). These data demonstrate that expression of Bcl-xL, but not that of zVAD.fmk, inhibits the apoptosis that results from forced down-regulation of Bcl-2.
Discussion Protection against synovial fibroblast apoptosis may contribute to the synovial hyperplasia that is associated with RA. Previous investigations have demonstrated few apoptotic cells in the synovial lining or the pannus in RA ST (38, 39), suggesting that an antiapoptotic protein may be up-regulated in RA. Here, we demonstrated by immunohistochemistry that Bcl-2 was highly expressed in RA compared with OA ST. Dual immunofluorescence showed that although macrophages were variably positive, the majority of the Bcl-2-positive cells in the synovial lining and the sublining region were CD68-negative, synovial fibroblasts. In contrast, other
studies revealed little Bcl-2 expression in the synovial lining (38) or sublining (40). The observed differences between these investigations and ours may have been technical. Prior studies employed mAbs and frozen (40) or glutaraldehyde-fixed (38) sections, while we used methanol-fixed tissues and a monospecific, polyclonal anti-Bcl-2 Ab. A number of observations support the validity of our data: the peptide competition experiments; limited expression of Bcl-2 in RA synovial lymphocytes, similar to an earlier study (41); high Bcl-2 expression in vascular smooth muscle cells in both RA and OA ST; and a single band on an immunoblot. Consistent with our data, higher levels of bcl-2 mRNA in RA synovial tissues compared with OA were demonstrated by in situ hybridization (42), suggesting that bcl-2 is up-regulated at the transcriptional level in RA. Thus, the enhanced expression of Bcl-2 in RA compared with OA synovial fibroblasts in vivo suggests that Bcl-2 may function to inhibit apoptosis in these cells. In contrast to the immunohistochemical data, following isolation and culture, differences in Bcl-2 expression between RA and OA synovial fibroblasts were no longer observed. A similar phenomenon was seen in studies that examined NF-B activity in RA and OA ST and synovial fibroblasts. Increased NF-B binding activity
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FIGURE 6. Bcl-xL overexpression rescues fibroblasts from Ad-Rbz-Bcl-2-induced apoptosis. Triplicate cultures of quiescent RA synovial fibroblasts were either uninfected (mock) or infected with Ad-Rbz-Bcl-2 or Ad-Bcl-xL and Ad-Rbz-Bcl-2. Cultures were transferred to 10% FBS/DMEM for 96 h before analysis by flow cytometry. A, Ad-Bcl-xL/Ad-Rbz-Bcl-2-infected cultures retain Rh123. Representative FACS analysis of unfixed, uninfected, Ad-Rbz-Bcl-2-infected, and Ad-Rbz-Bcl-2/Ad-Bcl-xL-infected cultures determining mitochondrial permeability transition. The y-axis represents cells stained positively for propidium iodide (PI; dead), and the x-axis shows cells retaining Rh123. Quadrant 4 demarcates cells normally retaining Rh123 and no PI incorporation. Quadrant 3 shows the loss of Rh123 retention and PI exclusion. Quadrant 2 are cells that are PI positive and yet retain Rh123. Quadrant 1 represents the dead cells that incorporate PI and fail to retain Rh123. B, Ad-Bcl-xL/Ad-Rbz-Bcl-2-infected cultures display reduced subdiploid DNA content. Representative FACS analyses of fixed and PI-stained, uninfected, Ad-Rbz-Bcl-2-infected, and Ad-Rbz-Bcl-2/Ad-Bcl-xL-infected cultures. The y-axis represents the number of events (cell number), while the x-axis represents the extent of propidium iodide incorporation. The data represent one of three replicate samples from two representative experiments.
in synovial tissue extracts by electrophoretic mobility shift assays and enhanced nuclear NF-B localization in the synovial lining have been demonstrated in RA compared with OA (43– 45). However, a recent study showed that I-kinases that activate NF-B were equally expressed in cultured RA and OA synovial fibroblasts (46). Collectively, these data suggest that Bcl-2, like NF-B, may be regulated by the inflammatory cytokine milieu. Indeed, TNF-␣ (47), IL-1 (47, 48), or TGF (47, 48), cytokines present in the rheumatoid joint, induced Bcl-2 expression in synovial fibroblasts. Thus, the enhanced expression of Bcl-2 in vivo may not be attributed to an intrinsic property of these cells, but, rather, is due to environmental regulation by inflammatory cytokines. RA and OA synovial fibroblasts equally underwent Ad-RbzBcl-2-induced apoptosis. Permeability transition, cytochrome c release, and activation of caspases 9 and 3 were detected in both cell types, demonstrating that the mechanism of Ad-Rbz-Bcl-2-induced apoptosis was similar in RA and OA synovial fibroblasts. The induction of apoptosis following the inhibition of Bcl-2 in fibroblasts, regardless of the source or conditions of culture, suggests that compensatory Bcl-2 anti-apoptotic family proteins were not up-regulated. In contrast to Ad-Rbz-Bcl-2-treated fibroblasts, Bcl-2 null mice were viable (3, 4, 49), suggesting functional redundancy among Bcl-2 family members during development. Postnatally however, the thymus and spleen underwent fulminant apoptosis in these mice (3, 4, 49). To date, no study has described defects in fibroblast apoptosis in these Bcl-2⫺/⫺ mice. These observations suggest that following development, no internal feedback mechanism exists by which the fibroblast senses the forced
reduction in Bcl-2 and up-regulates other anti-apoptotic proteins to prevent cell death. During apoptosis, mitochondrial permeability transition is an early and deciding event, which may result in the activation of caspases (50). Addition of zVAD.fmk to Ad-Rbz-Bcl-2 infected fibroblasts inhibited the characteristics of apoptosis, including activation of caspases 9 and 3, PARP cleavage, and DNA fragmentation. In contrast, zVAD.fmk had no affect on the loss of mitochondrial membrane potential induced by Ad-Rbz-Bcl-2, suggesting that the induction of permeability transition by Bcl-2 ablation resulted in a “point of no return” (32) in the apoptotic mode of death. zVAD.fmk has also been shown to block caspase activation and the nuclear characteristics of apoptosis induced by chemicals, including etoposide (51) and staurosporine (33), or UVB irradiation (33), while mitochondria permeability transition still occurred, supporting the idea of caspase-independent apoptosis. These data indicate that following reduction of Bcl-2, fibroblasts undergo apoptosis due to mitochondrial dysfunction. Furthermore, depending on the apoptotic stimulus, zVAD.fmk may prevent the degradative phase of apoptosis that gives rise to the characteristic phenotype (52), but may have no effect on the commitment phase or on cell death. The relative stoichiometries between the antagonist and agonist Bcl-2 protein family members may function as a molecular rheostat regulating cell survival. The mechanism of apoptosis induced by Bcl-2 down-regulation is similar to that observed with Bax overexpression (14, 52–55). Both Ad-Rbz-Bcl-2 infection and Bax overexpression induced apoptosis, cytochrome c release, and
5234 caspase activation. zVAD.fmk prevented cleavage of nuclear and cytosolic substrates and DNA degradation, but the fall of mitochondrial membrane potential still occurred after Bcl-2 down-regulation or Bax overexpression (14, 52–55). Overexpression of Bcl-xL prevented loss of mitochondrial permeability transition and DNA fragmentation in cultures infected with Ad-Rbz-Bcl-2 or overexpressing Bax (14, 52, 55). Additionally, ectopic Bcl-xL has been shown to inhibit cytochrome c release, which may be regulated by Bax through the opening of voltage-dependent anion channels (56). Thus, the inhibition of Bcl-2 may allow Bax or other pro-apoptotic Bcl-2 family members to induce mitochondrial permeability transition and bind voltage-dependent anion channels to alter cytochrome c subcellular localization. These observations support the hypothesis that disruption of the homeostatic balance between apoptotic agonistic and antagonistic members of the Bcl-2 family results in cell death. The expression of Bcl-2 has been investigated in a variety of disease states, but little is known about its significance in RA. High Bcl-2 expression has been associated with a poorer prognosis in transitional cell carcinoma of the bladder (57), Hodgkin’s disease (58), testicular carcinoma (59), and prostate cancer (60, 61). Additionally, the ratio of Bcl-2 to Bax was demonstrated to be of predictive value for response to radiotherapy to treat prostate cancer (62) and for chemotherapy to treat gastric cancer (63) and acute myeloid leukemia (64). Because synovial lining thickness has been shown to correlate with radiographic outcome (65), and lining thickness correlated with the frequency of Bcl-2-positive cells, these data suggest that increased Bcl-2 expression in the synovial lining may be associated with a worse outcome in RA. Thus, the in vivo down-regulation of Bcl-2 by a hammerhead ribozyme has potential as a novel therapeutic agent that may enhance apoptosis in the RA synovium, potentially limiting disease progression.
Acknowledgments We thank Kariman Dadbeh for his assistance with the adenoviral preparation, Dr. Kenneth Walsh for the Ad-Bcl-xL, and Mary Paniagua for the flow cytometry that was conducted at the Robert H. Lurie Comprehensive Cancer Center, Flow Cytometry Core Facility of the Northwestern University Medical School (Chicago, IL).
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