Porcine Corneal Ocular Reversibility Assay (PorCORA) predicts ocular damage and recovery for global regulatory agency hazard categories.

In this study, we examined the capacity of the Porcine Corneal Ocular Reversibility Assay (PorCORA) to classify the reversibility of ocular effects for 32 test compounds (20 reversible, 12 irreversible) from various chemical classes. PorCORA predicted 28 of 32 compounds correctly when compared to historical rabbit eye test data. The correlation coefficient for PorCORA versus historical rabbit test data was 0.84, based on the last day of damaged cornea reversal. These results demonstrate a high correlation between corneal irritation recovery time in the PorCORA and the rabbit eye. When compared to historical Modified Maximal Average Score (MMAS) in rabbit eyes, PorCORA yielded a correlation coefficient of 0.80, demonstrating ability to predict MMAS. PorCORA was highly predictive of regulatory agency ocular hazard classification categories, resulting in 91% accuracy for EU R41 and GHS Category 1. PorCORA was also predictive of EPA Category I (88% accuracy). Overall, the accuracy (88-91%), sensitivity (79-86%), specificity (94%), positive predictivity (94%), and negative predictivity (85-89%) for all three regulatory classifications indicate that ocular irritation hazardous effects were well predicted by the PorCORA. This study suggests that PorCORA could help discriminate between EU R36 and R41, GHS Categories 1 and 2, and EPA Categories I and II.

IKK/NF-kappaB regulates skeletal myogenesis via a signaling switch to inhibit differentiation and promote mitochondrial biogenesis.

Nuclear factor kappaB (NF-kappaB) is involved in multiple skeletal muscle disorders, but how it functions in differentiation remains elusive given that both anti- and promyogenic activities have been described. In this study, we resolve this by showing that myogenesis is controlled by opposing NF-kappaB signaling pathways. We find that myogenesis is enhanced in MyoD-expressing fibroblasts deficient in classical pathway components RelA/p65, inhibitor of kappaB kinase beta (IKKbeta), or IKKgamma. Similar increases occur in myoblasts lacking RelA/p65 or IKKbeta, and muscles from RelA/p65 or IKKbeta mutant mice also contain higher fiber numbers. Moreover, we show that during differentiation, classical NF-kappaB signaling decreases, whereas the induction of alternative members IKKalpha, RelB, and p52 occurs late in myogenesis. Myotube formation does not require alternative signaling, but it is important for myotube maintenance in response to metabolic stress. Furthermore, overexpression or knockdown of IKKalpha regulates mitochondrial content and function, suggesting that alternative signaling stimulates mitochondrial biogenesis. Together, these data reveal a unique IKK/NF-kappaB signaling switch that functions to both inhibit differentiation and promote myotube homeostasis.

NF-kappaB regulation of YY1 inhibits skeletal myogenesis through transcriptional silencing of myofibrillar genes.

NF-kappaB signaling is implicated as an important regulator of skeletal muscle homeostasis, but the mechanisms by which this transcription factor contributes to muscle maturation and turnover remain unclear. To gain insight into these mechanisms, gene expression profiling was examined in C2C12 myoblasts devoid of NF-kappaB activity. Interestingly, even in proliferating myoblasts, the absence of NF-kappaB caused the pronounced induction of several myofibrillar genes, suggesting that NF-kappaB functions as a negative regulator of late-stage muscle differentiation. Although several myofibrillar promoters contain predicted NF-kappaB binding sites, functional analysis using the troponin-I2 gene as a model revealed that NF-kappaB-mediated repression does not occur through direct DNA binding. In the search for an indirect mediator, the transcriptional repressor YinYang1 (YY1) was identified. While inducers of NF-kappaB stimulated YY1 expression in multiple cell types, genetic ablation of the RelA/p65 subunit of NF-kappaB in both cultured cells and adult skeletal muscle correlated with reduced YY1 transcripts and protein. NF-kappaB regulation of YY1 occurred at the transcriptional level, mediated by direct binding of the p50/p65 heterodimer complex to the YY1 promoter. Furthermore, YY1 was found associated with multiple myofibrillar promoters in C2C12 myoblasts containing NF-kappaB activity. Based on these results, we propose that NF-kappaB regulation of YY1 and transcriptional silencing of myofibrillar genes represent a new mechanism by which NF-kappaB functions in myoblasts to modulate skeletal muscle differentiation.

Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder associated with dystrophin deficiency that results in chronic inflammation and severe skeletal muscle degeneration. In DMD mouse models and patients, we find that IkappaB kinase/NF-kappaB (IKK/NF-kappaB) signaling is persistently elevated in immune cells and regenerative muscle fibers. Ablation of 1 allele of the p65 subunit of NF-kappaB was sufficient to improve pathology in mdx mice, a model of DMD. In addition, conditional deletion of IKKbeta in mdx mice elucidated that NF-kappaB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to limit regeneration through the inhibition of muscle progenitor cells. Furthermore, specific pharmacological inhibition of IKK resulted in improved pathology and muscle function in mdx mice. Collectively, these results underscore the critical role of NF-kappaB in the progression of muscular dystrophy and suggest the IKK/NF-kappaB signaling pathway as a potential therapeutic target for DMD.

Dystrophin glycoprotein complex dysfunction: a regulatory link between muscular dystrophy and cancer cachexia.

Cachexia contributes to nearly a third of all cancer deaths, yet the mechanisms underlying skeletal muscle wasting in this syndrome remain poorly defined. We report that tumor-induced alterations in the muscular dystrophy-associated dystrophin glycoprotein complex (DGC) represent a key early event in cachexia. Muscles from tumor-bearing mice exhibited membrane abnormalities accompanied by reduced levels of dystrophin and increased glycosylation on DGC proteins. Wasting was accentuated in tumor mdx mice lacking a DGC but spared in dystrophin transgenic mice that blocked induction of muscle E3 ubiquitin ligases. Furthermore, DGC deregulation correlated positively with cachexia in patients with gastrointestinal cancers. Based on these results, we propose that, similar to muscular dystrophy, DGC dysfunction plays a critical role in cancer-induced wasting.