NF-kappaB functions in stromal fibroblasts to regulate early postnatal muscle development.

Classical NF-kappaB activity functions as an inhibitor of the skeletal muscle myogenic program. Recent findings reveal that even in newborn RelA/p65(-/-) mice, myofiber numbers are increased over that of wild type mice, suggesting that NF-kappaB may be a contributing factor in early postnatal skeletal muscle development. Here we show that in addition to p65 deficiency, repression of NF-kappaB with the IkappaB alpha-SR transdominant inhibitor or with muscle-specific deletion of IKKbeta resulted in similar increases in total fiber numbers as well as an up-regulation of myogenic gene products. Upon further characterization of early postnatal muscle, we observed that NF-kappaB activity progressively declines within the first few weeks of development. At birth, the majority of this activity is compartmentalized to muscle fibers, but by neonatal day 8 NF-kappaB activity from the myofibers diminishes, and instead, stromal fibroblasts become the main cellular compartment within the muscle that contains active NF-kappaB. We find that NF-kappaB functions in these fibroblasts to regulate inducible nitric-oxide synthase expression, which we show is important for myoblast fusion during the growth and maturation process of skeletal muscle. Together, these data broaden our understanding of NF-kappaB during development by showing that in addition to its role as a negative regulator of myogenesis, NF-kappaB also regulates nitric-oxide synthase expression within stromal fibroblasts to stimulate myoblast fusion and muscle hypertrophy.

The RelA/p65 subunit of NF-kappaB specifically regulates cyclin D1 protein stability: implications for cell cycle withdrawal and skeletal myogenesis.

Studies support that NF-kappaB functions in cellular growth through the transcriptional regulation of cyclin D1, but whether such regulation is attributed to a single NF-kappaB subunit remains unclear. To address this issue we examined endogenous cyclin D1 levels during cell cycle re-entry in mouse embryonic fibroblasts (MEFs) lacking specific NF-kappaB signaling subunits. Results showed that each of these subunits were dispensable for regulating cyclin D1 transcription. However, we found that resulting cyclin D1 protein was severely reduced in MEFs lacking only RelA/p65. Cyclohexamide treatment revealed that this regulation was due to an increase in protein turnover. Similar downregulation of cyclin D1 protein, but not RNA, was observed in vivo in multiple tissues lacking RelA/p65. Co-immunoprecipitation analysis also showed that RelA/p65 and cyclin D1 were capable of interacting, thus providing a possible explanation for cyclin D1 protein stability. In addition, although the decrease in cyclin D1 in RelA/p65(-/-) MEFs was concomitant with lower CDK4 activity during cell cycle re-entry, this was not sufficient to affect S phase progression. Nevertheless, similar decreases in cyclin D1 protein in primary RelA/p65(-/-) myoblasts was adequate to accelerate cell cycle exit and differentiation of these cells. Based on these findings we conclude that RelA/p65 functions as a specific regulator of cyclin D1 protein stability, necessary for proper cell cycle withdrawal during skeletal myogenesis.

Stress exacerbates neuropathic pain via glucocorticoid and NMDA receptor activation.

There is growing recognition that psychological stress influences pain. Hormones that comprise the physiological response to stress (e.g., corticosterone; CORT) may interact with effectors of neuropathic pain. To test this hypothesis, mice received a spared nerve injury (SNI) after exposure to 60 min restraint stress. In stressed mice, allodynia was consistently increased. The mechanism(s) underlying the exacerbated pain response involves CORT acting via glucocorticoid receptors (GRs); RU486, a GR antagonist, prevented the stress-induced increase in allodynia whereas exogenous administration of CORT to non-stressed mice reproduced the allodynic response caused by stress. Since nerve injury-induced microglial activation has been implicated in the onset and propagation of neuropathic pain, we evaluated cellular and molecular indices of microglial activation in the context of stress. Activation of dorsal horn microglia was accelerated by stress; however, this effect was transient and was not associated with the onset or maintenance of a pro-inflammatory phenotype. Stress-enhanced allodynia was associated with increased dorsal horn extracellular signal-regulated kinase phosphorylation (pERK). ERK activation could indicate a stress-mediated increase in glutamatergic signaling, therefore mice were treated prior to SNI and stress with memantine, an N-methyl-D-aspartate receptor (NMDAR) antagonist. Memantine prevented stress-induced enhancement of allodynia after SNI. These data suggest that the hormonal responses elicited by stress exacerbate neuropathic pain through enhanced central sensitization. Moreover, drugs that inhibit glucocorticoids (GCs) and/or NMDAR signaling could ameliorate pain syndromes caused by stress.

Zebrafish alpha-crystallins: protein structure and chaperone-like activity compared to their mammalian orthologs.

PURPOSE: The vertebrate small heat shock proteins alphaA- and alphaB-crystallin contribute to the transparency and refractive power of the lens and may also prevent the aggregation of non-native proteins that would otherwise lead to cataracts. We previously showed that zebrafish (Danio rerio) and human alphaB-crystallin have diverged far more in primary structure and expression pattern than the orthologous alphaA-crystallins. In this current study we further compare the structure and function of zebrafish and mammalian alpha-crystallins. METHODS: Near UV CD spectroscopy was used to analyze the tertiary structure and thermal stability of recombinant zebrafish alpha-crystallins. The chaperone-like activities of zebrafish and human alpha-crystallins were compared by assaying their ability to prevent the chemically induced aggregation of several target proteins at temperatures between 25 degrees C and 40 degrees C. RESULTS: Zebrafish and human alphaA-crystallin showed very similar tertiary structures, while the alphaB-crystallin orthologs showed differences related to the presence of additional aromatic amino acids in the zebrafish protein. The denaturation temperatures of zebrafish crystallins were lower than those of mammals. The chaperone-like activities of the two zebrafish alpha-crystallins were highly divergent, with alphaA-crystallin showing much greater activity than alphaB-crystallin. CONCLUSIONS: alphaA-crystallin serves a similar physiological function in both zebrafish and mammals as a lens specific chaperone-like molecule. The reduced chaperone-like function of zebrafish alphaB-crystallin and its lack of extralenticular expression indicates that it plays a different physiological role from its mammalian ortholog. Future comparative studies of alpha-crystallin from closely related vertebrate species can help identify specific structural changes that lead to alterations in chaperone-like activity.

Detection of NF-κB activity in skeletal muscle cells by electrophoretic mobility shift analysis.

An electrophoretic mobility shift assay (EMSA) is a common and invaluable technique which can be utilized to study the affinity of proteins to a specific DNA or RNA sequence. These assays are performed in vitro with protein extracts isolated from either cultured cells or isolated tissues. Here, we describe the methodology used to isolate the cytoplasmic and nuclear protein extracts from both cultured cells and tissues and utilize the nuclear protein fraction to assess NF-κB DNA-binding activity by EMSA analysis.

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.