The NF-kappa B family of transcription factors has been implicated in regulating cellular processes such as immune response, cell survival, cellular proliferation, and differentiation. This dissertation will focus on the involvement of NF-kappa B in cellular proliferation and differentiation.
Studies support that NF-kappa B functions in cellular proliferation through the transcriptional regulation of cyclin D1, but whether such regulation is attributed to a single NF-kappa B subunit remains unclear. In chapter two we examine endogenous cyclin D1 levels during cell cycle re-entry in mouse embryonic fibroblasts (MEFs) lacking specific NF-kappa B signaling subunits. We demonstrate that each of these subunits is dispensable for regulating cyclin D1 transcription. However, we found that resulting cyclin D1 protein was severely reduced in MEFs lacking only RelA/p65. Cycloheximide treatment revealed that this regulation was due to an increase in protein turnover. Similarly downregulation of cyclin D1 protein, but not RNA, was observed in vivo in multiple tissues lacking p65. Co-immunoprecipitation analysis also showed 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 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 p65-/- myoblasts was adequate to accelerate cell cycle exit and myogenesis in these cells.
A number of studies have identified classical NF-kappa B activity as an inhibitor of the myogenic program. Recent findings reveal that in newborn p65-/- mice, myofiber numbers are increased over that of wild type mice, suggesting that NF-kappa B may be a contributing factor in early postnatal skeletal muscle development. In chapter 3 we show that in addition to p65 deficiency, repression of NF-kappa B with the I kappa B alpha-SR transdominant inhibitor or with muscle specific deletion of IKK beta resulted in similar increases in total fiber numbers, as well as an upregulation of myogenic gene products. Upon further characterization of early postnatal muscle, we observed that NF-kappa B 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-kappa B activity from the myofibers diminishes and stromal fibroblasts become the main cellular compartment within the muscle which contains active NF-kappa B. We find that NF-kappa B functions in these fibroblasts to regulate iNOS expression, which we show is important for myoblast fusion during the growth and maturation process of skeletal muscle.
This dissertation highlights two novel functions of NF-kappa B. We determine that the p65 dependent stabilization of cyclin D1 is necessary for proper cell cycle withdrawal during skeletal myogenesis. We also demonstrate that in addition to NF-kappa B functioning as a negative regulator of myogenesis, it also regulates iNOS expression within stromal fibroblasts to stimulate myoblast fusion and muscle hypertrophy. Taken together these findings demonstrate NF-kappa B can function in multiple capacities in different cell types to regulate skeletal myogenesis.