RNF5 in ER-Related Disorders Figure 7. Delayed Muscle regeneration in RNF5 KO mouse subjected to muscle damage by cardiotoxin treatment. A. Generation of RNF5 KO mouse. Schematic of RNF5 genomic structure (upper) and targeting vector (lower) are shown. Restriction enzyme sites (K: KpnI, Xh: XhoI, S: SalI) are marked. Location of the primers for PCR and the probe for Southern blotting are indicated. Exons 1, 2 and 3 including the translational start site (ATG) and RING finger domain are substituted with neo cassette. Southern blot for analysis of RNF5 gene is shown on the right panel. DNA isolated from the tails of +/+, +/2, and 2/2 mice was digested with EcoRI and resolved by electrophoresis through 0.8% agarose gels. After transferring to nitrocellulose paper, blots were hybridized with radiolabeled cDNA probes corresponding to the fragment indicated in diagram. B. Protein extracts from cardiotoxin and PBS treated tibialis muscles from WT and RNF5 KO animals were analyzed by western blots with the indicated antibodies to monitor changes in endogenous RNF5 as well as ER stress and muscle markers. Ponceau staining is used as a loading control. C. Cross section of tibialis anterior from WT and RNF5 KO mice performed 28 days after cardiotoxin treatment. doi:10.1371/journal.pone.0001609.g007 physiologic ER function and the organization of essential structural muscle components. Further, activation of ER stress was implicated in an animal model of autoimmune myositis with increased expression of MHC class I in muscle fibers and in human myositis patients [38]. ER overload may be sufficient to trigger muscle regeneration prior to, and independently of, the onset of inflammation, supporting ER dysfunction as a primary cause of fiber damage [38,39]. Thus, RNF5 overexpression may impair the ER function, ultimately leading to a defect in the process of muscle maintenance. Independent evidence also supports the fact that ER-associated processes are physiologically relevant during muscle regeneration. The chaperone protein GRP94 and the ER-stress-responsive transcription factor ATF6 were upregulated and required for proper myoblast differentiation in vitro [29,31]. UPR has recently been proposed to be part of a quality control mechanism that determines proper muscle differentiation. In this model, the induction of certain UPR components, such as the transcription factor Xbp1, is used as a threshold to determine the progression of the myoblast into the differentiation program and the survival of PLoS ONE | www.plosone.org 11 February 2008 | Volume 3 | Issue 2 | e1609