The human inflammatory myopathies are muscle diseases in which myofiber damage appears to be the consequence of an immune process (1–4). The precise pathogenesis of the immune process leading to muscle damage, however, is unknown. The degree of inflammation does not consistently correlate with severity of the structural changes in the muscle fibers or the severity of clinical disease (5), suggesting that nonimmune processes also play a role in the pathogenesis. Several observations provide further evidence of this. First, marked structural changes in the muscle fibers occur in the absence of any inflammatory cells (6, 7). Second, there is a lack of correlation between the degree of inflammation and the degree of muscle weakness (8). Third, in some myositis patients the disease does not respond even to powerful antiinflammatory therapy (9, 10). Fourth, steroid treatment may eliminate inflammatory cells in myositis muscle tissue, but this removal alone may not substantially improve clinical disease (11), suggesting that immunosuppressive therapies modulate disease activity but do not change other mediators of the disease process. Finally, clinical disease may progress even when identifiable inflammation has subsided (12).
To investigate these and other pathogenic processes, we have generated a conditional transgenic mouse model in which class I major histocompatibility complex (MHC) is overexpressed in skeletal muscle. These mice develop clinical, biochemical, histologic, and immunologic features similar to those of human myositis, providing a close model of the human disease. The disease is inflammatory, limited to skeletal muscles, self-sustaining, more severe in females, and is often accompanied by autoantibodies (13).
The following observations in human myositis patients and in the mouse model of myositis suggest that class I MHC molecules themselves may potentially mediate muscle fiber damage and dysfunction in the absence of lymphocytes: 1) in human myositis, induction of class I MHC antigen in muscle fibers occurs early, preceding inflammatory cell infiltration (14, 15); 2) class I MHC staining of biopsy specimens from patients with myositis reveals both a cell surface and a sarcoplasmic reticulum pattern of internal reactivity, demonstrating that some of the class I MHC may be retained in the endoplasmic reticulum (ER) of these fibers (7, 16, 17); 3) overexpression of class I MHC in muscle fibers may persist in the absence of an inflammatory infiltrate (12); 4) the controlled induction of class I MHC in the murine model was followed by muscle weakness occurring before mononuclear cell infiltration (13); and 5) it has recently been shown that in vivo gene transfer of class I MHC plasmids attenuates muscle regeneration and differentiation (18). Together, these observations—particularly the obvious retention of class I MHC within the cell in both the human and murine diseases—indicate that the muscle fiber damage seen in myositis may be mediated not solely by immune attack (e.g., cytotoxic T lymphocytes and autoantibodies), but may also be mediated through nonimmunologic mechanisms such as the ER stress response. Since class I MHC up-regulation in myositis muscle fibers is widespread even in the absence of visible inflammatory infiltrates, we propose that ER stress may play a potential role in the muscle fiber damage and dysfunction in human myositis.
The ER has several critical roles in the folding, export, and processing of newly synthesized proteins. When there is an imbalance between the load of proteins in the ER and the cell's ability to process that load, a collection of signaling pathways that adapt cells to ER stress will commence. The ER stress response can be provoked by a variety of pathophysiologic conditions, including ischemia, hyperhomocysteinemia, viral infections, mutations that impair protein folding, and excess accumulation of proteins in the ER (19, 20). Cells self-protect against ER stress by initiating several responses. At least 4 functionally distinct responses have been identified: 1) responses involving up-regulation of the NF-κB pathway (ER overload response), 2) up-regulation of genes encoding ER chaperone proteins such as BiP/Grp78 and Grp94, to increase protein folding activity and to prevent protein aggregation, 3) translational attenuation to reduce the load of protein synthesis and to prevent further accumulation of unfolded proteins (unfolded protein response), and 4) cell death, which occurs when functions of the ER are severely impaired. This cell death event is mediated by transcriptional activation of the gene for cholesterol oxidase-peroxidase C/EBP homologous protein (CHOP)/growth arrest and DNA damage 153 (GADD153), a member of the CCAAT/enhancer binding protein family of transcription factors (21), and by activation of ER-associated caspase 12 (22).
Results of the present study revealed evidence for activation of the ER stress response pathway in human myositis. Furthermore, it was demonstrated that the ER stress response pathway is also activated in the conditional class I MHC–transgenic mouse model of myositis, suggesting a potential role for this pathway in both human inflammatory myopathies and the murine model of myositis.
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- MATERIALS AND METHODS
In this study we demonstrated that 2 major components of the ER stress response pathway, ER overload response and unfolded protein response, are highly activated in muscle both in human myositis and in the mouse model. We further showed that overexpression of mouse class I MHC induces an ER stress response in transiently transfected C2C12 skeletal muscle cells.
The expression of class I MHC molecules on the surface of a cell is the end point of a complicated process of assembly and transport initiated in the ER. Class I MHC molecules are trimeric, consisting of a transmembrane glycoprotein which is the product of the MHC-linked gene, a small protein called β2m, and a short peptide of 8–10 amino acids. Assembly of class I MHC molecules has much in common with the folding and assembly of other multimeric glycoproteins in the ER. Housekeeping chaperones (calnexin, calreticulin, ERp57, tapasin, and Grp78) assist the assembly process. When certain murine class I MHC alleles are expressed in β2m-deficient cells, misfolding is associated with the formation of heavy chain homodimers in the ER via an unpaired Cys in the cytoplasmic tail. In those studies heavy chain dimerization was also observed in cells expressing β2m, but only after β2m had dissociated from previously assembled complexes (36).
In HLA–B27 (human class I heavy chain)–transgenic mice, spontaneous arthritis develops in the absence of β2m, indicating that cell surface expression of classic trimeric HLA–B27 (heavy chain–peptide–β2m) complexes is not required (37). Interestingly, the same spontaneous arthritis phenotype has been found in the absence of HLA–B27, in mice with a mixed genetic background that were deficient for either β2m or transporter-associated protein (38). These findings raise the possibility that class I heavy chain misfolding and the intracellular sequelae may be a critical component of the disease mechanism in myositis, perhaps through the generation of ER stress (39).
Skeletal muscle cells do not constitutively express or display class I MHC molecules, although they can be induced to do so by proinflammatory cytokines such as interferon-α and tumor necrosis factor α (TNFα) (6, 17, 40, 41). In human myositis, the early and widespread appearance of class I MHC in non-necrotic muscle cells, even distant from lymphocytic infiltration, is a striking feature (6, 7, 42). The pathologic significance of these observations has not been fully studied. We propose that class I MHC molecules themselves may potentially mediate muscle fiber damage and dysfunction in the absence of lymphocytes, via the ER stress response pathway. In particular, we propose that transgenic overexpression of class I MHC in muscle fibers may initiate 2 overlapping ER stress response pathways: the unfolded protein response and ER overload response. Under normal transient stress conditions, the unfolded protein response activates 3 proximal ER sensors, Ire-1, PERK, and ATF-6. These, in turn, control the rate of protein translation, with prolonged unfolded protein response activation ultimately initiating death pathways through CHOP and caspase 12. ER overload response activates NF-κB, and activated NF-κB induces NF-κB target genes (including up-regulation of endogenous class I MHC), which in turn will induce the ER stress response, thus initiating a self-sustaining loop.
It has recently been suggested that Grp78 is the master regulator of the ER stress response, the concert of signaling pathways that alters gene transcription in response to an accumulation of unfolded or misfolded proteins in the ER (30). Grp78 regulates the activation of at least 3 transducers of ER stress, ATF-6, Ire-1, and PERK (43). Grp78, which negatively regulates the unfolded protein response, interacts with all 3 sensors under nonstressed conditions and probably plays an essential regulatory role in unfolded protein response activation (43). In stressed cells, Grp78 binds to accumulating unfolded or misfolded proteins, releasing these 3 transducers and thus allowing activation of downstream nuclear events (44). We have shown that Grp78 is highly up-regulated both in human myositis and in the mouse model. Using global gene expression profiling, we have further demonstrated that several members of the ER stress response pathway are greatly up-regulated in myositis patients compared with healthy controls. These data suggest that the ER stress response pathway is highly activated in myositis. Activation of ER stress and unfolded protein response pathways has also been demonstrated in sporadic inclusion body myositis (45).
To further test the effects of class I MHC induction, we transiently transfected C2C12 muscle cells with WT and mutant H-2Kb constructs and showed that WT, but not mutant, H-2Kb induced Grp78 expression. This may be due to their inability to fold and to interact properly with chaperones such as calnexin and calreticulin in the ER. These mutants may be translocated from the ER lumen to cytosol for degradation by the proteosome (46). The data demonstrate that class I MHC overexpression in muscle cells can lead to ER stress.
We have previously demonstrated that muscle disease in class I MHC–transgenic mice is self-sustaining (13). The disease in HT double-transgenic mice could not be reversed by readministering doxycycline after the onset of clinical disease, probably because of the activation of transgene-independent genes such as endogenous H-2Kb, H-2Db, ICAM-1, and the proinflammatory chemokines and cytokines macrophage inflammatory protein 1α, monocyte chemotactic protein 1 (MCP-1), and interleukin-15 (IL-15) (13). We tested the possibility that the initial transgenic class I MHC overexpression in the sarcoplasmic reticulum of muscle fibers may trigger activation of the NF-κB pathway through ER overload response, and NF-κB would in turn activate several proinflammatory target genes (cytokines, cytokine receptors, cell adhesion molecules, chemoattractant proteins, and growth regulators) including endogenous class I MHC, thus initiating a self-sustaining loop. The ER overload response partly overlaps with, but is distinct from, the unfolded protein response (47). Some, but not all, of the conditions that evoke the unfolded protein response will also trigger the ER overload response. In contrast to the unfolded protein response, the ER overload response activates the nuclear transcription factor NF-κB (32). The promoter sequences of the ER chaperones Grp78 and Grp94 do not contain NF-κB binding sites; accordingly, the ER overload response–associated NF-κB induction does not increase their expression levels.
Since the ER overload response can be triggered by high levels of normal or mutant proteins processed through or retained in the ER, it is believed to have a broad role in maintaining cellular homeostasis under stress conditions (32). It is possible that in human myositis, NF-κB may activate both classical (proinflammatory cytokines IL-1 and TNFα) and nonclassical (ER stress response) pathways (48). The downstream target genes (e.g., class I MHC, ICAM, VCAM, E-selectin, MCP-1) regulated by the NF-κB pathway are highly up-regulated in myositis patients (16). These results further confirm the previous finding of NF-κB activation in inflammatory myopathies (49). Thus, we have shown that NF-κB p65 is activated both in human myositis and in the mouse model, suggesting that this pathway may be directly involved in muscle fiber damage or dysfunction.
Mechanisms underlying the class I MHC–induced muscle damage in myositis may be proposed based on earlier findings and the results obtained in the present study. Class I MHC overexpression (e.g., transgenic or infection or nerve injury in an autoimmune-prone genetic background) in muscle fibers may initiate 2 overlapping ER stress response pathways: ER overload and unfolded protein response. The ER overload response will activate NF-κB, and activated NF-κB will induce NF-κB target genes including endogenous class I MHC up-regulation, which in turn will induce the ER stress response, thus initiating a self-sustaining loop. NF-κB is known to suppress myoblast differentiation by inhibiting MyoD (50). It is also known to induce proinflammatory cytokine expression and consequently, muscle fiber damage. Under normal transient stress conditions, the unfolded protein response will activate 3 proximal ER sensors, Ire-1, PERK, and ATF-6, and these in turn control the rate of protein translation. Prolonged unfolded protein response activation will ultimately initiate death pathways through CHOP and caspase 12.
Therefore, we propose that class I MHC–initiated ER stress response may be one of the nonimmune mechanisms of muscle fiber damage in myositis (Figure 7). Interestingly, we have observed significant muscle wasting and reduction in body weight in the self-sustaining phase of the disease in our mouse model (13). It therefore appears that skeletal muscle cell death in class I MHC–expressing muscle fibers can be induced partly by cytotoxic T lymphocytes, which initiate the “extrinsic” death pathway, and partly by perturbation of intracellular homeostasis (ER stress response), the “intrinsic” damage pathway. These results suggest that therapeutic interventions that target inflammatory and ER stress pathways may be useful in myositis. These effector pathogenic mechanisms may also be relevant in other muscle diseases in which overexpression of class I MHC, or possibly other molecules, occurs.
Figure 7. Potential mechanisms of muscle fiber damage in class I major histocompatibility complex (MHC)–overexpressing muscle fibers in the mouse myositis model. Overexpression of class I MHC (transgene or infection or denervation in a susceptible individual) in the skeletal muscle cells will lead to endoplasmic reticulum (ER) accumulation of class I MHC and activation of the ER stress response pathway. Two overlapping pathways of ER stress response are activated (ER overload [EOR] and unfolded protein response [UPR]). EOR activates the NF-κB pathway, which up-regulates expression of endogenous class I MHC (a NF-κB target), and consequently, more class I MHC accumulates in the sarcoplasmic reticulum of skeletal muscle fibers, and more NF-κB is activated (self-sustaining loop [red arrows]). UPR activates several ER sensors (Ire-1, PKR-like ERK [PERK], and ATF-6) through Grp78. These sensors will down-regulate translational machinery and reduce protein accumulation in the ER. Persistent activation of EOR through NF-κB may lead to the activation of proinflammatory cytokines and muscle damage; likewise, prolonged activation of UPR will also activate cell death through the C/EBP homologous protein and caspase 12 pathways.
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