sarcolemma-associated b-catenin for degradation and controls growth and maintenance of myofibrils [11]. A similar role in myofibrillogenesis has been proposed for the MuRF2 E3 ligase [12]. These studies support the notion that E3 ligases are an integral component of muscle dynamics during maintenance of normal muscle function. In muscle disease, recent evidence supports a role of the UPS in the pathogenesis of select myopathies. Mutations in TRIM32 RING finger E3 ligase have been associated with Limb-Girdle Muscular Dystrophy (LGMD) type 2H [13] and sarcotubular myopathy [14]. Although the mechanism linking TRIM32 ligase activity to LGMD type 2H is unknown, TRIM32 has been shown to interact with myosin and to ubiquitinate actin [15], suggesting a role in myofibrillar turnover during muscle adaptation. Accumu- lation of ubiquitin-containing cytoplasmic aggregates has been associated with both sporadic and hereditary forms of inclusion- body myositis (sIBM and hIBM). These findings suggest an important role of the UPS in accumulation, misfolding and aggregation of muscle proteins [16]. This has been confirmed by genetic and histopathological analysis that links IBM-like muscle disorders with impairment of the endoplasmic reticulum associat- ed degradation (ERAD) machinery, a degradation pathway important in removal of misfolded proteins [17]. The sporadic form of IBM (sIBM) is the most common acquired myopathy that occurs in the older human population. sIBM is a slowly progressive myopathy that combines both a T-cell mediated autoimmune inflammatory response and myodegenerative features including vacuolar degeneration, protein aggregation and inclusion formation [16]. Remarkably, sIBM patients are poorly responsive to anti-inflammatory or immunosuppressive treatments, suggesting that inflammation per se may not be a primary cause of the disease [16]. Among current hypotheses, accumulation of malfolded proteins such as amyloid b Precursor Protein (abPP) and its processed forms, and ER overload associated with proteasomal dysfunction are thought to be key players in sIBM pathogenesis [16]. This hypothesis has been further substantiated by the fact that different ER stress markers, such as GRP78 and GRP94 are overexpressed and present in amyloid containing aggregates in sIBM muscles [16,17], most likely as a consequence of the activation of the Unfolded Protein Response (UPR). Therefore, ER stress induction and impaired clearance of malfolded proteins are thought to be part of the pathogenic process occurring in s-IBM. We have previously shown that the RING finger ubiquitin ligase RNF5 (also known as RMA-1; [18,19]) plays an important role in muscle physiology using the C. elegans model [20]. RNF5 is a membrane-bound E3 ligase that is conserved from worm to human. In C. elegans, RNF-5 localizes to the dense bodies and the M-line of the body wall muscles, where it regulates the levels of the LIM domain protein UNC-95. In C. elegans, UNC-95 has been genetically associated with uncoordinated movement [21] and shown to be important for formation of muscle attachment sites (M-lines and Z- lines) associated with the downstream process of sarcomere organization [20]. In a mammalian cell culture system, RNF5 has been shown to ubiquitinate and regulate the localization of the protein paxillin [22], a critical component of focal adhesion, involved in cell adhesion and motility. More recently, RNF5 has been described as a new component of the ERAD machinery, where it contributes to the ubiquitination-dependent degradation of mal- folded proteins as part of cell protein quality control [23]. Here, we have established and characterized inducible trans- genic mouse over-expressing RNF5 either ubiquitously or in a muscle specific manner. We show that general as well as tissue specific overexpression of RNF5 induces myofiber degeneration associated with an alteration of endoplasmic reticulum (ER) RNF5 in ER-Related Disorders function. Conversely, RNF5 KO mice exhibit delayed repair of muscle damage associated with attenuated ER stress response. Screening for RNF5 expression in muscle biopsies from patients suffering from muscular disorders including classical myopathies such as Duchenne and Becker myopathy as well as other myopathies with unknown etiology identified upregulation and mislocalization of RNF5 to aggregates in muscles from sIBM patients, as well as in a mouse model for hereditary IBM. Our findings establishes the importance of RNF5 in muscle physiology and in ER stress associated muscular disorders while pointing to the possible use of RNF5 transgenic mouse as a unique model to study the role of ER function in the pathogenesis of degenerative muscle diseases. Results Aberrant RNF5 expression in human myopathies To assess whether RNF5 expression or localization would be deregulated in certain human myopathies associated with ER impairment, muscle biopsies from patients affected by different forms of myopathies were screened for possible changes in pattern or level of RNF5 expression using RNF5 antibodies with confirmed specificity developed in our laboratory (Fig. S1). An alteration of RNF5 expression in both vacuolated muscle fibers and in apparently normal fibers (2–20% of total fibers) was detected in biopsies from 10 patients with sIBM. Compared to control muscle, RNF5 in biopsies from sIBM patients was increased in amount and mislocalized, most frequently in apparently non vacuolated muscle fibers and diffusely within the cytoplasm (Fig. 1A). Strikingly, in 3 out of 10 biopsies from sIBM patients, intensive staining for RNF5 was evident with formation of giant aggregates inside some muscle fibers (Fig. 1B). Interest- ingly, RNF5 inclusions partly co-localized with beta-amyloid positive aggregates (Fig. 1C) but not with phospho-tau containing structures (Fig. 1D). Given the limited expression of RNF5 in skeletal muscle, endogenous RNF5 was not detectable by straight western blots in extracts from human biopsies. Elevated RNF5 protein levels could be observed after RNF5 immunoprecipitation in biopsies from sIBM patients (Fig. 1E). The specificity of the immunoprecipitation reaction was confirmed using muscle extracts from RNF5 KO and WT mice (Fig. 1E right panel). Unlike in sIBM, common muscular dystrophies associated with mutations in proteins of the dystrophin glycoprotein complex (DGC) such as Duchenne or Becker forms of muscular dystrophy, did not exhibit any alteration in the pattern or amount of RNF5 expression (Fig. S2). Since beta-amyloid accumulation and impairment of ERAD and UPR functions are thought to be upstream events in sIBM development [16], our data points to RNF5 deregulation as an early event in the onset of the disease. To determine whether changes in amount or localization of RNF5 was a common occurrence in IBM type degenerative myopathies, additional analysis was performed in a mouse model of hereditary IBM, the distal myopathy with rimmed vacuoles (DMRV) mutant mouse [24]. Transgenic animals, but not their control littermates, exhibited a strong increase in RNF5 expression in muscle fibers within areas of regeneration including rimmed vacuoles and centrally located nuclei (Fig. 1F), characteristic of this model [24].These findings reveal altered RNF5 expression in specific degenerative myopathies associated with dysfunction of the ER or the ERAD machinery such as sporadic and hereditary IBM. Construction of RNF5 transgenic mouse In order to address the function of RNF5 deregulation in degenerative myopathies, we constructed a transgenic mouse model which allowed conditional RNF5 overexpression. A construct containing the RNF5 gene was cloned downstream of PLoS ONE | www.plosone.org 2 February 2008 | Volume 3 | Issue 2 | e1609