Bisphosphonates are used widely to treat bone disorders associated with increased osteoclast activity. As antiresorptive agents, they are very effective in the treatment of postmenopausal osteoporosis. However, several clinical trials have shown that bisphosphonates fail to prevent local bone loss in patients with inflammatory arthritis (1, 2), but the reasons for this poor efficacy have yet to be elucidated. Bisphosphonates reduce bone resorption by two main mechanisms: inhibition of bone resorption and induction of apoptosis of osteoclasts. At low dosages, they suppress osteoclast function, which is associated with increased osteoclast numbers on bone surfaces, perhaps as compensation for decreased bone resorption (3). High dosages of bisphosphonates reduce osteoclast numbers at bone resorption sites by promoting their apoptosis (4, 5). Thus, regulation of osteoclast apoptosis directly affects the ability of osteoclasts to resorb bone.
Two distinct signaling pathways control cell apoptosis: one is plasma membrane receptor dependent and the other is triggered by intracellular stress. Bisphosphonate-induced apoptosis is triggered by intracellular stress as a consequence of disrupted cholesterol biosynthesis (6, 7). In osteoclasts, this pathway is regulated by the Bcl-2 family of proteins and involves mitochondrial release of cytochrome c, which leads to the activation of caspases 9 and 3 (8). Bcl-2 family members consist of pro- and antiapoptotic proteins, such as Bcl-2, Bcl-xL, Bax, and Bid. The relative ratio and activation states of these molecules determine the fate of cells (9, 10). Osteoclasts express higher levels of Bcl-xL protein than Bcl-2, and thus Bcl-xL is believed to be an important regulator of apoptosis in these cells. Macrophage colony-stimulating factor (M-CSF), a key survival factor for cells in the myeloid lineage, stimulates Bcl-xL expression (11, 12). Osteoclasts generated from bone marrow cells of transgenic mice that overexpress Bcl-xL under the control of the tartrate-resistant acid phosphatase (TRAP) promoter are more resistant to serum withdrawal–induced cell death (13, 14), demonstrating that Bcl-xL is involved in the control of osteoclast apoptosis.
The expression of Bcl-xL is regulated by the transcription factors NF-κB, activator protein 1 (AP-1), and the Ets family members, mainly Ets-1, Ets-2, and PU.1 (15–17). Interestingly, gene knockout studies have demonstrated that NF-κB, c-Fos, and PU.1 are all essential for osteoclastogenesis during development (18, 19). Both NF-κB and c-Fos are critical for the differentiation of CD11b+/c-Fms+/TRAP-osteoclast precursors to TRAP+ osteoclasts, while PU.1 works at a much earlier stage and is essential for the commitment of multipotent cells to become myeloid progenitors (20).
While it appears that these transcription factors do not play a significant role in osteoclast survival (5), the role of Ets-2 in osteoclasts has not been well studied. Ets-2−/− mice die before day 8.5 of gestation due to defective trophoblast function (21). After birth, Ets-2 is highly expressed in late-stage myeloid cells, and its expression is rapidly up-regulated upon induction of macrophage differentiation from progenitors. Ets-2 also mediates M-CSF–dependent macrophage survival through regulation of Bcl-xL expression (11). Recently, increased expression of Ets protein has been observed in blood and synovial samples from arthritic animals and patients, suggesting that cytokines, such as tumor necrosis factor (TNF), which are increased in blood and synovial fluid of patients with inflammatory arthritis, may stimulate the expression of Ets family members (22–24). Thus, it is possible that in erosive inflammatory arthritis, high local levels of TNFα stimulate expression of Ets members in osteoclasts and their precursors, triggering Bcl-xL survival signals that render osteoclasts more resistant to bisphosphonate-induced cell death.
The purpose of this study was to test the hypothesis that the inflammatory microenvironment alters the apoptosis machinery in osteoclasts through the TNF/Ets-2/Bcl-xL pathway, leading to decreased susceptibility to bisphosphonate-induced injury. We used alendronate (ALN), a widely administered bisphosphonate, and TNF-transgenic mice (TNF-Tg) as an animal model of chronic inflammatory, erosive arthritis. ALN-induced apoptosis and expression levels of Bcl-xL were compared between osteoclasts on the eroded joint surfaces of TNF-Tg mice and those in the adjacent metaphyses. The effect of Bcl-xL and Ets-2 overexpression on ALN-induced apoptosis was assessed using a retroviral transfer approach and by proinflammatory cytokine treatment of osteoclast cultures in vitro. The relationship between TNF, Bcl-xL, and Ets-2 was determined, and our findings indicate that under inflammatory conditions, TNF stimulates osteoclasts to up-regulate Ets-2 expression, which leads to increased Bcl-xL expression and osteoclast resistance to ALN-induced apoptosis.
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- MATERIALS AND METHODS
The current study was designed to determine why bisphosphonates are less effective at preventing focal bone loss in patients with inflammatory bone diseases than in patients with osteoporosis. To this end, we examined the response of osteoclasts to ALN in TNF-Tg mice that have established erosive arthritis. We found that ALN diminishes osteoclast numbers in the metaphyses by 97% in both TNF-Tg and WT mice, but by only 46% at sites of focal joint erosion. Expression of the antiapoptotic protein, Bcl-xL, is significantly increased in osteoclasts in the diseased joints of TNF-Tg mice (Figure 2) and patients with RA (results not shown), suggesting that the local environment favors osteoclast survival. In support of this hypothesis, we demonstrated that 1) TNF stimulates Bcl-xL and Ets-2 expression; 2) Bcl-xL overexpression reduces ALN-induced apoptosis; and 3) Ets-2 mediates TNF-induced Bcl-xL expression. Thus, we propose a model to explain the reduced efficacy of bisphosphonate in the treatment of chronic inflammatory bone diseases (1, 2), in which TNF up-regulates Ets-2 expression in osteoclast precursors, which stimulates transcription of the bcl-xL gene and makes osteoclasts more resistant to apoptotic signals.
Our model emphasizes the importance of the microenvironment in determining the response of osteoclasts to certain therapies. Increased osteoclast survival capacity in inflamed joints indicates that a higher concentration or more potent drug may be required locally in arthritic joints. To support this, we show that TNF treatment and Bcl-xL overexpression produce protective effects in osteoclasts only when 20 μM or lower concentrations of ALN are used, but not at high concentrations (Figure 4). Since the local ALN concentration in the inflamed joints and metaphyses of ALN-treated TNF-Tg mice is not known, we cannot exclude the possibility that the concentration of ALN in the joints is lower than that in the growth plate of long bones, given the high affinity of bisphosphonate for bone (36). However, the fact that metaphyseal and joint osteoclasts are separated by only hundreds of microns, and inflamed sites tend to have increased blood flow and thus exposure to potentially higher bisphosphonate concentrations, makes this highly unlikely. Increased Bcl-xL staining in osteoclasts in the inflamed joints and resistance to ALN-induced death of Bcl-xL-infected osteoclasts indicate that inflammation-induced changes within osteoclasts themselves contribute at least in part to the failure of bisphosphonate to prevent local bone loss in inflammatory bone diseases, suggesting a fundamental difference between osteoclasts generated by homeostatic versus inflammatory signals in our model.
We have focused on Bcl-xL in the current study because its role in osteoclast survival has been repeatedly documented (12, 14, 17, 37). In addition, we found that TNF stimulates expression of Bcl-xL, but not the other Bcl-2 family members we tested (Figure 5). However, other Bcl-2 family proteins that are involved in the regulation of cell fate may also mediate TNF-induced osteoclast survival. For instance, Bim, a BH3-only proapoptotic protein, restricts osteoclast lifespan, and its expression is suppressed by M-CSF (38). TNF stimulates the expression of cellular inhibitor of apoptosis 1, an inhibitor of apoptotic proteins, in osteoclast precursors through the NF-κB pathway (39). It will be important to investigate the role of these proteins in osteoclast survival in inflammatory bone disorders.
Our finding that NF-κB and c-Fos overexpression failed to promote osteoclast survival is not surprising given that osteoclasts can be generated from the splenocytes of both NF-κB p50/p52–double-knockout and c-Fos–knockout mice when the knockout cells are infected with certain proteins, such as nuclear factor of activated T cells 2. Miyazaki et al have demonstrated that inhibition of NF-κB activation in osteoclasts by overexpressing the NF-κB superinhibitor, IκBα, does not induce cell death (40). Thus, the major function of NF-κB and c-Fos in osteoclast biology is to control the differentiation of osteoclast precursor cells.
Of the 30 Ets family members, Ets-1, Ets-2 and PU.1 have been reported to be involved in bone cell functions (17). Ets-1 interacts with CbFA1 to regulate osteogenesis (41); Ets-2 is required for cartilage and intramembranous bone formation (42); and PU.1 is essential for osteoclastogenesis (20). Additionally, all of these Ets family members transactivate the bcl-x promoter (11, 37). However, in the current study, along with our unpublished observations, we found that ets-1 mRNA levels are very low in osteoclast precursors and undetectable in mature osteoclasts. While PU.1 is abundantly expressed in osteoclasts, its expression is not regulated by TNF, M-CSF, or RANKL stimulation. Consistent with our findings, So et al recently demonstrated that PU.1 expression is not induced during osteoclast formation, unlike microphthalmia transcription factor and osteoclast-associated receptor (43). These results indicate that Ets-1 and PU.1 are unlikely to play a significant role in mature osteoclast survival.
The significance of our finding that TNF stimulates Ets-2 expression is the implication that Ets-2 may be involved in the regulation of osteoclast function during inflammation-related bone loss. Indeed, Ets-2 is not critical for survival of osteoclasts or osteoclast precursors during development, because transgenic mice carrying a dominant-negative form of Ets-2 under control of the c-Fms promoter have a normal bone phenotype (44). However, it is possible that in pathologic conditions, high levels of Ets-2 alter the intrinsic apoptotic machinery in osteoclasts, leading to enhanced survival, which is supported by increased ets-2 mRNA in osteoclast precursors isolated from TNF-Tg mice (mean ± SEM ets-2:actin ratio of 4.9 ± 0.48 in cells of TNF-Tg mice versus 1 ± 0.05 in cells of WT mice). The TNF signaling pathway used to activate Ets-2 is not clear. TNF activates Akt, Jun, and ERK signaling pathways in osteoclasts, and all of them have been reported to link to Ets-2 activation in various cell types (45–47). We are currently investigating the specific pathway by which TNF mediates Ets-2 activation in osteoclasts.
In summary, we have provided experimental evidence to explain the decreased efficacy of bisphosphonates in preventing local inflammatory bone loss due to changes in the joint microenvironment that favor osteoclast survival and involve both paracrine and autocrine mechanisms. However, our findings do not eliminate the possibility of therapeutic efficacy of bisphosphonates in inflammatory bone diseases, because ALN treatment did reduce osteoclast numbers in inflamed joints, although to a lesser degree compared with cells inside the long bones (Figures 1 and 2). Therefore, more potent bisphosphonates, such as zoledronate, may be efficacious. Recently, 2 groups have reported that zoledronate effectively prevents the bone erosion that occurs in the inflamed joints of TNF-Tg mice or animals with collagen-induced arthritis (48, 49). Unfortunately, in those studies, the authors focused on investigating zoledronate-induced inhibition of osteoclast function, rather than survival. Nevertheless, the effectiveness of zoledronate in preventing focal bone erosion is yet undetermined in patients with inflammatory arthritis and awaits the results of clinical trials. Meanwhile, it may be worth examining the efficacy of intraarticular injections of bisphosphonates to achieve sufficiently high local concentrations to prevent joint erosion.