Despite the known importance of cytokines in cartilage destruction and the established effectiveness of exercise or joint mobilization in the restoration of joint function, relatively little is understood about the molecular events underlying the beneficial effects of biomechanical signals on inflamed cartilage. Recent molecular studies have revealed that specific biomechanical stimuli and cell interactions generate intracellular signals that are powerful suppressors of cytokine-mediated proinflammatory gene transcription in chondrocytes, in vitro and in vivo (1, 2). Cytokine production by synovial cells as well as chondrocytes in joints has been linked to the pathogenesis of arthritic diseases (3–5).
Additionally, compelling evidence from studies of transgenic mice genetically engineered for overexpression of tumor necrosis factor α (TNFα) demonstrates that elevated levels of TNFα lead to early and severe induction of rheumatoid arthritis in the joints (6). Similarly, gene delivery of interleukin-1 receptor antagonist (IL-1Ra) or anti-TNFα/IL-1β antibodies to arthritic joints leads to suppression of cartilage destruction, further implicating proinflammatory cytokines in the etiopathology of arthritis (7). Given the importance of the role played by these cytokines in cartilage destruction and the potent effects of biomechanical signals in antagonizing proinflammatory gene transcription, it is critical to characterize the signaling cascade that may be essential in mediating the profound actions of exercise or joint mobilization in preventing joint destruction (8–10).
Several previous studies have identified NF-κB transcription factors as key regulators of TNFα-induced and IL-1β–induced gene activation in chondrocytes (11–15). Furthermore, biomechanical signals have been shown to regulate cytokine gene expression via regulation of the NF-κB signaling pathway. In chondrocytes, biomechanical signals have been found to promote or attenuate proinflammatory gene transcription in a magnitude-dependent manner (11). Biomechanical signals are transduced to cells by surface molecules such as β integrins and focal adhesion kinases (FAKs) (16). At high magnitudes these signals trigger activation of the NF-κB signaling cascade to induce proinflammatory gene transcription (11). Notably, at low magnitudes, biomechanical signals fail to activate NF-κB transcription factors, and act as potent inhibitors of IL-1β– and TNFα-dependent proinflammatory gene transcription (11, 17, 18). However, the molecular components of this signaling cascade regulated by biomechanical signals have not yet been described.
Members of the NF-κB family of transcription factors are sequestered in the cytoplasm of unstimulated cells as heterodimers or homodimers by binding to their inhibitory units, IκBα and IκBβ. Multiple cytokine-induced proinflammatory pathways converge at the signalsome comprising IKKα, IKKβ, and IKKγ/NF-κB–essential modulator, to activate downstream events in the NF-κB cascade. Upon phosphorylation by IKK, IκB proteins are ubiquitinated and marked for proteosomal degradation. The liberation of NF-κB from IκB complexes is followed by phosphorylation of NF-κB at multiple sites in a stimulant-dependent manner and eventual translocation to the nucleus.
The binding of NF-κB to its consensus sequences leads to transcription of a plethora of genes, including proinflammatory cytokines and mediators, as well as several of the molecules required for the activation of the NF-κB signaling cascade. Although this classic model of NF-κB activation by TNFα or IL-1β is well documented, its complexity evolves from its regulation at multiple intracellular levels, in a cell-dependent as well as stimulus-dependent manner.
In an effort to identify the key signaling and regulatory mechanisms that allow biomechanical signals to inhibit IL-1β–induced NF-κB activation, we examined the expression of NF-κB–controlled genes, such as tumor necrosis factor receptor–associated factor 1 (TRAF1) and TRAF2 and IL-1 receptor type I (IL-1RI) and IL-1RII, as transcription biomarkers. We focused on IL-1β–induced NF-κB nuclear translocation and prerequisite upstream events involved in its activation, in order to identify the key target molecule(s) that are regulated by biomechanical signals.
The results of the present study showed that, although IKK is the central target of biomechanical signals, these signals intercept multiple sites along the NF-κB signal transduction pathway to block IL-1β–induced proinflammatory gene expression. Recently, there has been a substantial effort to develop drugs and gene therapy approaches targeted at inhibiting NF-κB or IKK in order to prevent cartilage and bone destruction in arthritis (14, 19–21). Notably, appropriate biomechanical signals can inhibit both NF-κB and IKKβ, suggesting that these signals can be used as an effective therapeutic intervention, without side effects, to suppress joint inflammation in arthritic diseases. Furthermore, these signals, while functioning as antiinflammatory cues, also act as powerful inducers of genes that contribute to cartilage repair and matrix synthesis (19, 20). Hence, understanding the mechanisms of the intracellular actions by which biomechanical signals attenuate NF-κB–induced proinflammatory gene transcription to limit cartilage destruction is of critical importance.
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In this study, we demonstrated that biomechanical signals at appropriate magnitudes attenuate IKK activity that could suppress the cytokine-mediated NF-κB signaling cascade. Previous studies have demonstrated inhibition of IL-1β and TNFα-induced proinflammatory gene induction through exposure of chondrocytes to low physiologic levels of tensile or compressive forces (1, 17, 33). In the present study, we examined the mechanisms of the antiinflammatory effects of tensile forces, because under physiologic loading cartilage undergoes both tension and compression, due to its intrinsic viscoelasticity (22, 34).
The signals generated by cyclic tensile strain are perceived by cells via cell surface molecules such as β integrins and FAKs (35–37). However, cyclic tensile strain does not down-regulate IL-1β receptors during the initial hour of its activity (18). Rather, cyclic tensile strain acts by modulating those signal transduction pathways that ultimately result in the induction of proinflammatory gene transcription. Many of the downstream target genes of NF-κB function in the initiation and subsequent amplification of the NF-κB signaling cascade (38). Thus, the NF-κB family of transcription factors forms a particularly important target for biomechanical signals to mediate its antiinflammatory actions (Figure 5).
Figure 5. Schematic representation of the mechanisms of intracellular action of cyclic tensile strain (CTS). Cyclic tensile strain suppresses interleukin-1β (IL-1β)–induced proinflammatory gene induction by intercepting salient steps in the NF-κB signaling cascade to inhibit transcription activity. Cyclic tensile strain suppresses IL-1β–induced IKK activation, and thus phosphorylation and proteosomal degradation of IκBα and IκBβ. This leads to the inhibition of nuclear translocation of NF-κB. During the initial stages of IL-1β–mediated activation of cells, cyclic tensile strain up-regulates IκBα nuclear translocation to prevent NF-κB binding to DNA and facilitate export of nuclear NF-κB, which may enter the nucleus. Cyclic tensile strain represses IL-1β–induced IκBα and IκBβ mRNA expression. Collectively, these actions of cyclic tensile strain inhibit proinflammatory gene induction as well as expression of multiple molecules involved in the regulation of the NF-κB signaling cascade to suppress IL-1β–induced inflammation. IL-1R = IL-1 receptor; TRAF1 = tumor necrosis factor receptor–associated factor 1; iNOS = inducible nitric oxide synthase; COX-2 = cyclooxygenase 2; MMPs = matrix metalloproteinases; TNF = tumor necrosis factor. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.
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Because of its dynamic control of many critical genes, the transcription activity of NF-κB is regulated at multiple levels, ranging from subcellular localization to posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. In the present study, EMSA demonstrated that biomechanical signals generated by cyclic tensile strain inhibited IL-1β–induced binding of NF-κB to its consensus sequences. IF analysis confirmed that cyclic tensile strain abrogates IL-1β–induced nuclear translocation of NF-κB. Following activation of cells by IL-1β, NF-κB rapidly translocates to the nucleus, is retransported to the cytoplasm, and is resynthesized, resulting in a net increase in the total level of NF-κB in cells (11).
Cyclic tensile strain inhibited IL-1β–induced increases in cellular NF-κB levels. Since the NF-κB promoter region does not have NF-κB binding sites, it is not clear how inhibition of NF-κB nuclear translocation by cyclic tensile strain results in the down-regulation of NF-κB. The 2 major regulatory IL-1β–inducible phosphorylation sites of NF-κB p65 are at the serine 276 and serine 536 residues. Phosphorylation of serine 276 greatly increases NF-κB transactivation and promotes DNA binding and interactions with CBP/p300, whereas phosphorylation of serine 536 increases the transcriptional efficacy of NF-κB (27, 39, 40). Our experimental results suggest that, although cyclic tensile strain strongly modulates NF-κB signaling, it does not regulate the IL-1β–induced phosphorylation of NF-κB p65 at either serine 276 or serine 536.
At the intracellular localization level, both IκBα and IκBβ act as natural inhibitors of NF-κB activation by sequestering NF-κB in the cytoplasm. After exposure to IL-1β, both IκBα and IκBβ are phosphorylated, ubiquitinated, and targeted for proteosomal degradation, consequently allowing import of free cytoplasmic NF-κB into the nucleus. Characterization of the actions of cyclic tensile strain showed a consistent inhibition of IL-1β–induced degradation of both IκBα and IκBβ. Further experiments revealed the important finding that cyclic tensile strain abrogates the massive IL-1β–induced increase in IκBα and Iκββ gene expression to control levels.
While IκBα expression is controlled by NF-κB transcription activity, IκBβ has not been found to be under the transcriptional control of NF-κB (41). Since the pathways that culminate in IκBβ resynthesis after its degradation are incompletely characterized, the main conclusion that can be drawn from these findings is that cyclic tensile strain abrogates IL-1β–dependent IκBα and IκBβ degradation and synthesis, which inhibits NF-κB transcription activity. Moreover, these findings suggest that the signals generated by cyclic tensile strain likely interact with proteins other than those controlled by NF-κB to regulate proinflammatory gene induction.
We have observed that IL-1β induces a significant increase in IκBα and IκBβ mRNA expression; however, measurement of total proteins did not reveal a parallel increase in levels of IκBα and IκBβ proteins. This may be due to the simultaneous synthesis and degradation of both of these proteins in the presence of IL-1β (42). Furthermore, while cyclic tensile strain inhibited IL-1β–induced IκBα and IκBβ degradation during the first 30 minutes of exposure, the total levels of IκBα and IκBβ were found to have decreased at 60 and 90 minutes. This reduction in the levels of IκBα and IκBβ proteins may be due to cyclic tensile strain–mediated abrogation of IL-1β–induced IκBα and IκBβ mRNA expression, and to the constitutive loss of IκBα and IκBβ due to a relatively short half-life of 30–40 minutes in cells (42, 43). Thus, by inhibiting IκBα mRNA expression, cyclic tensile strain may reduce the synthesis of IκBα, while the reduction of the level of IκBα due to its short half-life in cells may lead to a total reduction of IκBα levels in cells treated with IL-1β and cyclic tensile strain.
More importantly, in the present study the levels of IκBα or IκBβ mRNA were not completely suppressed by cyclic tensile strain, as evidenced by the presence of IκBα and IκBβ in the cytoplasm at all time points tested (Figures 2B and 3B). In cells treated with IL-1β and subjected to cyclic tensile strain, the presence of NF-κB in the cytoplasm and its absence in the nuclei (Figure 1B) further confirmed that IκBα and IκBβ played an active role in sequestering NF-κB in cells exposed to cyclic tensile strain. Thus, suppression of IL-β–induced IκBα and IκBβ mRNA expression, as well as inhibition of nuclear translocation of NF-κB, may be the key regulatory actions involved in the suppression of proinflammatory gene induction by cyclic tensile strain.
IL-1β–induced activation of NF-κB is balanced by the positive transcriptional regulation of the IκBα gene under the direct control of NF-κB proteins. Newly synthesized IκBα down-regulates NF-κB activity by interacting with transcriptionally active NF-κB in the nucleus and in the cytoplasmic compartments of cells (44). The results presented here suggest that, in addition to inhibiting IκBα degradation, signals generated by cyclic tensile strain regulate the trafficking and/or interactions of IκBα with NF-κB to down-regulate NF-κB binding to DNA. For example, we have observed a marked nuclear translocation of IκBα at 10 and 30 minutes following the application of cyclic tensile strain, and then again at 90 minutes.
This rapid entry of IκBα may be important in inhibiting the binding of any residual nuclear NF-κB to DNA, and thus terminating its transcriptional activation. Additionally, simultaneous migration of IκBα along with NF-κB to the nucleus and cytoplasm, demonstrated by IF analysis, suggests that nuclear IκBα may also be involved in facilitating the export of nuclear NF-κB–IκBα complexes to the cytoplasm (45). Furthermore, EMSA demonstrated that, even though some NF-κB translocates to the nucleus while costimulated with IL-1β and cyclic tensile strain (Figure 1B), it fails to productively bind to its consensus sequences (Figure 1A), suggesting that IκBα trafficking may be another mechanism by which cyclic tensile strain inhibits NF-κB activity.
Because the IκBα promoter is under the control of NF-κB, it is not surprising that inhibition of NF-κB nuclear translocation leads to suppression of IκBα mRNA expression and synthesis. Thus, inhibition of nuclear NF-κB binding to DNA by IκBα, and inhibition of IκBα degradation and its synthesis, reflect collective mechanisms by which cyclic tensile strain may attenuate IL-1β–induced NF-κB transcription activity.
IL-1β–induced regulation of IκB is a tightly controlled event that is regulated by phosphorylation by IKK (46). In the canonical pathway, IKKβ is essential for the phosphorylation of IκBα and IκBβ (47). Upon phosphorylation, IκBα and IκBβ are ubiquitinated and degraded to allow the nuclear translocation of NF-κB. In the in vitro assay system used in the present study, IL-1β induced a rapid and sustained phosphorylation of GST-IκBα proteins, reflecting the presence of activated IKK in chondrocytes. In contrast, cyclic tensile strain markedly abrogated IL-1β–dependent IKK activation, as reflected by the drastic reduction in the phosphorylation of exogenous IκBα in chondrocytes treated with IL-1β and subjected to cyclic tensile strain.
Recently, NF-κB regulation has been shown to be mediated by activation of IKK in a transforming growth factor β–activated kinase 1 (TAK-1)– or MEKK-3–dependent manner (48). TAK-1 activates IKKβ and phosphorylates IκBα to promote their degradation. The MEKK-3–dependent pathway phosphorylates IKKα, which results in NF-κB activation without degradation of IKKα (48). Since IL-1β–induced IκBα phosphorylation results in IκBα degradation, and cyclic tensile strain intercepts IL-1β–induced IκBα phosphorylation and degradation, it is likely that cyclic tensile strain actions involve IKK activation that is mediated by TAK-1.
NF-κB directly regulates several genes that are essential for the regulation of its own signaling cascade. By repressing IKK activity, cyclic tensile strain inhibits transcriptional up-regulation of such genes as TRAF1 and TRAF2, which are required for IKK activation. Thus, by suppressing the expression of these genes, cyclic tensile strain causes a multistep inhibition of IL-1β activation in chondrocytes, which may help in inhibiting proinflammatory responses in a sustained manner.
In conclusion, our findings represent the first demonstration that biomechanical signals generated by appropriate magnitudes of cyclic tensile strain attenuate IL-1β–dependent activation of IKK to inhibit NF-κB transcription activity. These signals act at multiple steps within the NF-κB signaling cascade to inhibit the transcription activity of NF-κB itself by preventing its nuclear import, as well as inhibiting the activation and gene expression of the NF-κB inhibitors IκBα and IκBβ (Figure 5). Cyclic tensile strain–enforced dynamic distribution of NF-κB–IκBα complexes may represent another level of regulation, which could inhibit the activity of NF-κB and subsequent downstream signaling events involved in proinflammatory gene induction.
The NF-κB signal transduction pathway has been exploited in many pharmacologic agents to reduce inflammation of the joints. For example, acetylsalicylic acid inhibits synthesis of proinflammatory mediators by inhibiting IKKβ (49). Additionally, several studies have shown that the application of inhibitors of IKK or NF-κB may be efficacious for the treatment of arthritic joints, suggesting that inhibition of this pathway is an attractive therapeutic target for joint inflammation (14, 21, 50, 51). However, these approaches may provide only temporary relief, and are limited due to the transient nature of their effects. Our findings demonstrate that the effects of appropriate biomechanical signals are more substantial and generate a sustained inhibition of proinflammatory gene induction (1).
Thus, the present findings provide insight into how biomechanical signals regulate and reduce inflammation. Further studies may assist in targeting specific treatment strategies by the use of appropriate exercise to enhance the ability of chondrocytes to curb inflammation in diseases affecting the joint.