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B Blocks Bone Erosion Associated with Inflammatory Arthritis1
Departments of
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Orthopaedic Research Laboratory,
Medicine,
Pathology, and
Cell Biology and Physiology, Barnes-Jewish Hospital at Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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B plays a central role in both processes, the function of this transcription factor was examined. Using a mouse model of autoreactive Ig transfer that engenders inflammatory arthritis, we show numerous osteoclasts in the articular joint tissue associated with progressive periarticular osteolytic lesions. Moreover, cells retrieved from these joints exhibit heightened NF-B activity. Importantly, direct administration of dominant negative*I-B or tyrosine 42-mutated I-B (Y42F*I-B) proteins into mice before induction of the disease attenuates in vivo activation of the transcription factor. More importantly, these I-B mutant forms significantly inhibit in vivo production of TNF and receptor activator of NF-B ligand, and block joint swelling, osteoclast recruitment, and osteolysis. Thus, NF-B appears to be the centerpiece of inflammatory-osteolytic arthritis and direct inhibition of this transcription factor by unique and novel I-B mutant proteins blocks manifestation of the disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (RANK),3 a molecule essential for osteoclast formation, is required for bone erosion associated with arthritis. Osteoclasts are hemopoietic cells formed by fusion of precursors derived from the monocyte/macrophage lineage. They are the principal cells regulating bone resorption and remodeling, and absence of these cells ultimately leads to osteopetrosis (6).
Differentiation of osteoclasts depends primarily on the bioavailability of two hemopoietic cytokines; M-CSF and receptor activator of NF-B ligand (RANKL) (6). This is evident by the osteopetrotic phenotype of the M-CSF-deficient op/op mouse, and the RANKL-deficient mouse that lacks osteoclasts (7, 8, 9). RANKL is a member of the TNF family and recent studies unveiled that RANKL-transmitted intracellular signals are very similar to the TNF prototype (10). The cytokine induces activation of multiple signals, most notably the NF-B activation cascade. This latter transcription factor is also required for osteoclastogenesis since combined deletion of its p50 and p52 subunits ablates osteoclast formation and leads to osteopetrosis (11, 12, 13). In addition, NF-B is a known proinflammatory transcription factor that regulates a number of genes that invoke inflammation and osteolysis (14, 15, 16, 17). In fact, NF-B is considered as the centerpiece of the inflammatory process and induces expression and production of proinflammatory and osteoclastogenic mediators (14, 18, 19). NF-B activation is permitted following its release from its inhibitory protein I-B. The inactive I-B/NF-B complex is normally present in the cytosol. Subsequent signals transmitted by proinflammatory and osteoclastic cytokines such as TNF and RANKL lead to phosphorylation of I-B N-terminal moieties. These events lead eventually to the release of NF-B, its nuclear translocation and transactivation (18, 20). In this regard, deletion of I-B phosphorylation sites renders the protein insensitive to proteolytic processing and attenuates NF-B activation (21, 22). More importantly, work by our group has shown previously that introduction of such I-B superrepressor that lacks its N-terminal phosphorylation sites, or another form in which tyrosine 42 has been mutated, blocks osteoclast differentiation and activation in vitro (23).
In the current study, we document that NF-B is activated in vivo in cells residing in the inflamed extremity joints of arthritic mice. More importantly, direct administration of dominant-negative (DN) form of I-B (DN*I-B), lacking N-terminal phosphorylation sites, significantly blocks NF-B activation and attenuates osteoclast recruitment and bone erosion. Of special significance is the finding that tyrosine 42-mutated I-B (Y42F*I-B) is also a potent in vivo inhibitor of inflammatory osteolysis. This novel observation provides a unique opportunity for therapeutic intervention.
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Materials and Methods |
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Abs used in this study were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ECL kit was obtained from Pierce (Rockford, IL). Wild-type (WT) and Y42F*I-B
cDNAs were a gift of Dr. J.-F. Peyron (Nice, France). DN*I-B (superrepressor) mutant was generated by N-terminal deletion of residues 1–45 using a standard PCR approach. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Mice
KRN-TCR (K/B) transgenic mice on a B6 background were kindly provided by D. Mathis and C. Benoist (Harvard Medical School, Boston, MA). K/BxN mice were generated by breeding KRN-TCR with nonobese diabetic mice (Taconic Farms, Germantown, NY). C3H/Hej mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed under controlled barrier facility at Washington University (St. Louis, MO).
Arthritogenic serum
Serum was obtained from K/BxN mice (6- to 12-wk-old), pooled and stored in aliquots in -70°C until used. A single dose of 150 µl of serum per mouse was selected as optimal to induce arthritis in 100% of the mice.
Three- to 4-wk-old BALB/c mice were injected i.p. with PBS serum (150 µl), TAT-TK, TAT-I-B proteins (1 mg/Kg body weight (BW) established as optimal dose) alone, or combination of serum and TAT-fused proteins. TAT-fusion proteins were injected daily for the entire course of each experiment. Each condition contained four mice and was repeated at least three times.
Tissue collection
Knee and ankle joints were excised and skin and soft tissues were removed. These joints were snap-frozen immediately in liquid nitrogen for further analysis. For cellular and nuclear isolation, extracts were prepared from mouse paws following Han et al. (24). In brief, whole mouse joints and paws were flash frozen in liquid nitrogen, crushed using a stainless steel mortar and pestle, then pulverized using CertiPrep Freezer Mill 6570 (Spex Industries, Metuchen, NJ) under liquid nitrogen to achieve a fine powder of 
2 x 1 mm. Equal amounts were then subjected to lysis or nuclear isolation as described elsewhere. For histology, intact limbs were skinned and processed for histology as described elsewhere.
pTAT construct and protein coupling
Construction of the pTAT-I-B protein expression and purification was previously described (23). Briefly, we have cloned the TAT transduction domain followed by a multiple cloning site into the pREST (Invitrogen, San Diego, CA) bacterial expression vector. The plasmid expresses a six histidine tag for purification, hemagglutinin (HA) tag for detection followed by the TAT transduction domain (RKKRRQRRRPP) and finally the I-B sequence. This plasmid was then used to express the TAT-coupled I-B in the BL21 (DE3) strain of Escherichia coli. The protein was purified and misfolded by switching the ionic exchange column in one step from 4 M urea to aqueous buffer (20 mM HEPES). pTAT-coupled and misfolded proteins remain highly concentrated and resistant to freeze-thaw denaturation, and readily enter the cells upon incubation. Proteins are folded properly after administration to cells by cellular chaperons such as heat shock protein-90 (25).
Histology and computed tomography
For histology, intact limbs were preserved in 10% buffered formalin (24 h), skinned, and subjected to a decalcification process using 10% EDTA, pH 7.0, for 7 days with gentle rocking, and daily replacement of solution. Decalcified bones were then dehydrated in graded alcohol, cleared through xylene, and embedded in paraffin. Paraffin blocks were sectioned longitudinally. Five micrometer sections were then stained with either H&E or histochemically for tartrate-resistant acid phosphatase (TRAP) to determine osteoclasts. For tomography, three-dimensional images from intact mouse legs (knee joints, ankle, and entire foot) were obtained from microcomputed (µC) µCT40 scanner (Scanco Medical, Switzerland).
Immunostaining
Histological sections embedded on slides were subjected to immunostaining using primary Abs as indicated in each experiment and detected with HRP-conjugated secondary Ab following standard procedures.
Immunoblotting
Crude cell lysates were boiled in the presence of 2x SDS-sample buffer (0.5 M Tris-HCl (pH 6.8), 10% (w/v) SDS, 10% glycerol, 0.05% (w/v) bromophenol blue, and distilled water) for 5 min and subjected to electrophoresis on 8–12% SDS-PAGE (26). Proteins were transferred to nitrocellulose membranes using a semidry blotter (Bio-Rad, Richmond, CA) and incubated in blocking solution (10% skim milk prepared in PBS containing 0.05% Tween 20), to reduce nonspecific binding. Membranes were washed with a PBS/Tween buffer and exposed to primary Abs (1 h at room temperature), washed again four times, and incubated with the respective secondary HRP-conjugated Abs (1 h at room temperature). Membranes were washed extensively (5 x 15 min), and an ECL detection assay was performed following manufacturer’s directions.
EMSA
Nuclear fractions were prepared as previously described (27, 28). In brief, cells retrieved from joint tissue were washed twice with ice-cold PBS. Cells were then resuspended in hypotonic lysis buffer A (10 mM HEPES (pH 7.8), 10 mM KCl, 1.5 mM MgCl, 0.5 mM DTT, 0.5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), and 5 µg/ml leupeptin) and incubated on ice for 15 min. Nonidet P-40 was added to a final concentration of 0.64%. Nuclei were pelleted and the cytosolic fraction was carefully removed. The nuclei were then resuspended in nuclear extraction buffer B (20 mM HEPES (pH 7.8), 420 mM NaCl, 1.2 mM MgCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, 0.5 mM AEBSF, 5 µg/ml pepstatin A, and 5 µg/ml leupeptin), vortexed for 30 s and rotated for 30 min in 4°C. The samples were then centrifuged and the nuclear proteins in the supernatant were transferred to fresh tubes and protein content was measured using standard BCA kit (Pierce). Nuclear extracts (10 µg) were incubated with an end-labeled double stranded oligonucleotide probe containing the sequence 5'-AAA CAG GGG GCT TTC CCT CCT C-3' (29) derived from the B3 site of the TNF promoter. The reaction was performed in a total of 20 µl of binding buffer (20 mM HEPES (pH 7.8), 100 mM NaCl, 0.5 mM DTT, 1 µg of poly dI-dC, and 10% glycerol) for 30 min at room temperature. Samples were then fractionated on a 4% polyacrylamide gel and visualized by exposing dried gel to film.
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Results |
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This set of experiments included five groups of three mice each and was repeated three times. These groups were injected with saline (control group) or with arthritogenic (K/BxN-derived) serum for 3, 5, 7, and 12 days. Following injection (150 µl, i.p.) of mice, limbs were collected at different time points (3–12 days), photographed, and processed for histology. The knee joints from serum-injected mice were 2.5 times larger in diameter compared with the PBS-injected control group (6.5 and 2.7 mm, respectively). The swelling of joints was consistent at each time point and was greater at 3, 5, and 7 days compared with 12 days postserum injection. However, this difference was not significant. Specifically, the average diameter of joints collected from 3 to 7 days treatment was 6.3–7.4 mm while that of 12 day-treated mice was 5.7–6.5 mm. These observations may suggest that the acute response is followed by reduction of limb swelling, an observation that will require a longer time course in future studies. Other features were also examined. As expected, the mice developed paw redness, swelling, and ankylosis (Fig. 1A). Histological analysis of distal joint sections shows a pronounced increase in synovial space, typical tissue breakdown, and massive cellular infiltration (Fig. 1B, denoted by double-head arrow and asterisk). Further examination of TRAP-stained sections shows a striking increase (8- to 20-fold) in osteoclast recruitment in articular cartilage, in subchondral bone, as well as in trabecular bone (Fig. 1, B and C arrows). This increase in osteoclast numbers is accompanied with substantial bone erosion (Fig. 1C, asterisks), evident by numerous bone erosions in sections from joints of arthritic mice compared with controls.
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B, TNF, and RANKL are elevated in cells retrieved from arthritic joints
To better understand the mechanism underlying bone erosion in serum-induced inflammatory arthritis, we investigated expression and activation of likely mediators of the process. In this regard, earlier studies documented that TNF, RANKL, and NF-B are central to osteoclast function and development (6, 10, 19). To this end, expression levels of TNF and RANKL were measured systemically in the serum (by ELISA), in joint tissue (by immunoblots), and by histoimmunostaining of tissue sections. The findings indicate that while systemic levels of the cytokines were slightly, but not significantly, different in arthritic compared with control mice (not shown), expression of TNF in cells retrieved from the arthritic joints was abundant (Fig. 2A). High level expression of the cytokine was evident three days post arthritis induction and was sustained up to 7 days postinduction, the longest time examined. We also examined expression levels of RANKL, the cytokine essential for osteoclast development. Like TNF, expression of RANKL was increased in arthritic mice compared with controls, evident by abundant reactive staining (Fig. 2B, brown color indicated by arrows). More importantly, we also found that NF-B is highly activated in nuclei of cells derived from the joints of arthritic mice (Fig. 3, lanes 4–7) compared with controls (lane 3). This activation was evident 2 days postserum injection and persisted up to 12 days (Fig. 3).
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B or Y42F*I-B blocks NF-B activation in arthritic cells, attenuates osteoclast recruitment and bone erosion, and inhibits signs of inflammation
Having identified NF-B as a possible mediator of the serum-induced arthritic response, we asked whether direct administration of mutant I-B proteins blocks NF-B activation and subsequent inflammatory and osteolytic events.
Purified TAT-fused DN*I-B or Y42F*I-B were injected intraperitonealy (1 mg/Kg BW) at the time of serum-injection and once a day thereafter. Specifically, eight mouse groups, comprised of four mice in each group, were injected with TAT alone, WT I-B, DN*I-B, Y42F*I-B, or arthritogenic serum alone or in combination with the various I-B forms. Localization of the exogenously administered TAT-I-B proteins was documented by immunostaining of histological sections using anti-HA Ab (Fig. 4). Having established that I-B proteins are successfully delivered in vivo, NF-B activation in nuclear extracts from control, arthritic, and TAT-I-B-treated arthritic joints were examined using gel shift analysis. The data indicate that DN*I-B as well as Y42F*I-B markedly inhibit NF-B activation in arthritic mice (Fig. 5, lanes 7 and 8). This inhibition was better that 90% compared with NF-B levels in serum-induced mice. A significant decrease (75%) was also observed in WT*I-B-treated animals (Fig. 5, lane 9). Consistent with its effect on NF-B activation in vivo, we also found that high expression of TNF in cell extracts from arthritic joints was vastly inhibited in the presence of TAT-I-B proteins (Fig. 6) and levels of the cytokine were barely detectable under inhibitory conditions (<10% of TNF levels in arthritic joints).
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B-treated mice compared with their respective controls. The data indicate that limbs from arthritic mice are significantly larger than control (not shown). Ankle diameter of arthritic mice was 3.9 ± 0.4 mm compared with 1.9 ± 0.3 mm in control mice, an 200% increase. Pretreatment with WT-I-B, DN*I-B, or Y42F*I-B significantly reduced swelling of joints. Specifically, serum injected into these animals caused a respective modest 35, 29, and 27% increase in ankle diameter compared with control. Overall, joint swelling and redness were significantly reduced in limbs of all mice treated with I-B proteins. Further examination of histological sections indicates that while ample osteoclasts (indicated by arrows) and resorbed lacunae are present in arthritic bone sections (compare Fig. 7B to control Fig. 7A), TAT-I-B proteins significantly reduced bone erosion to the level of control. It is also evident in these sections that TAT-DN*I-B (Fig. 7C) and TAT-Y42F*I-B (Fig. 7D) significantly prevented osteoclast recruitment (arrows), and reduced bone erosion. Altogether, in vivo administered I-B proteins appear effective in blocking cytokine expression, transcriptional activation of NF-B, osteoclast recruitment, bone erosion, and joint swelling. More convincingly, µCT analysis of joints from control, arthritic, and IB-treated mice, indisputably indicate that DN*IB and Y42F*IB significantly block bone erosion in serum-induced arthritis (Fig. 8A, d and e, respectively). No such inhibition was observed in mice injected with TAT control protein (Fig. 8Ab) and very subtle effects were seen in WT I-B-treated animals (panel c), the latter I-B protein is subject to normal proteolysis. Further support for these findings was gained from two-dimensional µCT analysis of intact limbs. The data depicts that serum-induced arthritis leads to >50% decrease in bone volume (BV) relative to total tissue volume (TV) (Fig. 8B, compare control and serum bars). Reflective of the data presented in Fig. 8A, pretreatment of animals with DN*I-B and Y42F*I-B significantly blocks serum induction of bone erosion (Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that NF-B is essential for osteoclastogenesis, skeletal remodelling, and a key mediator of inflammatory processes (11, 12, 14, 31). In this regard, we have shown that blocking activation of NF-B in osteoclastogenic cultures, by its inhibitory protein I-B, attenuates osteoclastogenesis and bone resorption, in vitro (23). Mechanistically, we herein provide direct evidence that at least the transcription factor NF-B is mediating inflammatory arthritis. Levels of NF-B were elevated in synovial tissue collected from arthritic mice joints as early as two days postserum transfer and persists for 12 days, the longest time examined. This heightened transcriptional activity was accompanied by localized increase in the levels of TNF and RANKL, both are known for their potent osteoclastogenic effects. The former cytokine is well documented as a mediator of inflammatory responses, and is also thought to be responsible for exacerbation of RANKL-mediated osteoclast formation and activation reminiscent of inflammatory osteolysis (10). Low levels of circulating RANKL and TNF, as opposed to abundant local expression in the joints, further substantiate the joint-localized phenomenon attributed to the development of the disease (32, 33). This phenomenon may be due to high expression of membrane-anchored (uncleaved) RANKL and TNF, an issue that requires further investigation.
Taking advantage of the proposed role of NF-B as central mediator of bone erosion in inflammatory arthritis we designed an experimental approach in which NF-B inhibition in vivo is achieved by direct administration of DN forms of I-B. Successful protein delivery was possible using the TAT fusion peptide, which others and we have successfully used for in vitro and in vivo protein transduction (23, 25, 34).
DN*I-B resists phosphorylation, therefore, proteolysis and degradation of this form of the protein is much slower than its endogenous WT counterpart. In view of these observations, it appears that a likely mechanism by which DN*I-B inhibits NF-B activation and osteoclast recruitment entails displacement of endogenous I-B. Thus, administration of excessive amounts of exogenous I-B protein forms a stable, long-lived TAT-I-B/NF-B complex, resulting in reduced NF-B activity (23, 27).
Similarly, data from our recent studies suggest that mutation of I-B tyrosine 42 residue renders the protein resistant to degradation in osteoclast progenitors, thus prolonging its half-life (23). As a result Y42F*I-B is sufficient to arrest NF-B activation in osteoclast progenitors and attenuates osteoclastogenesis, in vitro (23). We, herein, provide evidence that Y42F*I-B is as potent inhibitor of in vivo osteolysis as DN*I-B. Although functional differences and tissue specificity between the two forms of I-B mutants have not been established yet, the data present Y42F*I-B as a potential inhibitor of inflammatory osteolysis. Our data show that WT I-B only partially blocks signs of osteolysis. This observation is in agreement with our previous in vitro findings indicating partial inhibition of osteoclastogenesis (23). Incomplete inhibition of osteoclastogenesis and osteolysis using WT I-B is most likely due to degradation of the protein that, unlike the mutant forms, does not resist proteolysis.
TNF plays a crucial role in exacerbation of osteolytic and inflammatory lesions (1, 35, 36, 37). In fact the cytokine, acting through its type 1 receptor, heightens basal RANKL-generated osteoclasts in vitro, and permissive levels of the latter cytokine are sufficient to mediate TNF-induced osteoclast recruitment (10, 38, 39). Our data point out that administration of NF-B inhibitors in vivo blocks production of inflammatory mediators, such as TNF. This finding indicates that regulation of TNF gene transcription and production is under the aegis of NF-B. Given the multiple roles that TNF plays in inflammation and osteoclastogenesis, it is not surprising that DN*I-B proteins attenuate both events. Interestingly, however, our findings point out that inhibition of NF-B blocks heightened expression of RANKL under inflammatory conditions (our unpublished data). These findings implicate NF-B in regulating RANKL production or expression and suggest that TAT-I-B proteins may also target RANKL-producing cells.
The NF-B family members are ubiquitous and regulate a wide range of genes in healthy and diseased tissues. Thus, a concern arises regarding undesirable side effects following global inhibition of NF-B. Contrary to this notion, our observations indicate that healthy or arthritic mice treated with various forms of I-B showed no obvious signs of health retardation and were indistinguishable from controls. This may be due to localized effects of I-B proteins in inflammatory sites in which concentrations of active NF-B are the highest. Another reason may be due to the relatively short course of treatment with I-B protein, which was limited to seven days.
Although, NF-B appears to be central to bone erosion in this model, other parallel pathways may be involved. One such candidate is the c-Jun/AP-1 pathway. In this regard, recent evidence implicates c-Jun N-terminal kinase/c-Jun signaling in the development of RA (24). However, blocking the c-Jun N-terminal kinase pathway by a selective pharmacological agent resulted only in a partial inhibition of joint destruction in rats (40). These findings suggest that other pathways contribute to the arthritic process. Preliminary evidence from our studies points out that indeed c-Jun is activated in arthritic samples (not shown). However, further studies are underway to examine the possible interplay between this and the NF-B pathway as mediators of bone erosion in inflammatory arthritis.
In summary, our studies show that NF-B plays a profound role in the development of a joint localized form of inflammatory arthritis engendered by serum-Ig derived from K/BxN mice. More importantly, data presented here show that direct inhibition of NF-B by mutated I-B proteins in the form of TAT-fused compounds are sufficient to block generalized symptoms of the diseases and to specifically arrest bone erosion.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yousef Abu-Amer, Washington University School of Medicine, Department of Orthopedic Surgery, One Barnes Hospital Plaza, 11300 West Pavilion, Campus Box 8233, St. Louis, MO 63110. E-mail address: abuamery{at}msnotes.wustl.edu
3 Abbreviations used: RANK, receptor activator of NF-B; RANKL, RANK ligand; DN, dominant negative; WT, wild type; TRAP, tartrate-resistant acid phosphatase; BV, bone volume; TV, total tissue volume; RA, rheumatoid arthritis; BW, body weight; HA, hemagglutinin.
Received for publication May 15, 2003. Accepted for publication September 15, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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