Mechanical strain-mediated reduction in RANKL expression is associated with RUNX2 and BRD2.

Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.

Classical NF-κB activation impairs skeletal muscle oxidative phenotype by reducing IKK-α expression.

BACKGROUND: Loss of quadriceps muscle oxidative phenotype (OXPHEN) is an evident and debilitating feature of chronic obstructive pulmonary disease (COPD). We recently demonstrated involvement of the inflammatory classical NF-κB pathway in inflammation-induced impairments in muscle OXPHEN. The exact underlying mechanisms however are unclear. Interestingly, IκB kinase α (IKK-α: a key kinase in the alternative NF-κB pathway) was recently identified as a novel positive regulator of skeletal muscle OXPHEN. We hypothesised that inflammation-induced classical NF-κB activation contributes to loss of muscle OXPHEN in COPD by reducing IKK-α expression. METHODS: Classical NF-κB signalling was activated (molecularly or by tumour necrosis factor α: TNF-α) in cultured myotubes and the impact on muscle OXPHEN and IKK-α levels was investigated. Moreover, the alternative NF-κB pathway was modulated to investigate the impact on muscle OXPHEN in absence or presence of an inflammatory stimulus. As a proof of concept, quadriceps muscle biopsies of COPD patients and healthy controls were analysed for expression levels of IKK-α, OXPHEN markers and TNF-α. RESULTS: IKK-α knock-down in cultured myotubes decreased expression of OXPHEN markers and key OXPHEN regulators. Moreover, classical NF-κB activation (both by TNF-α and IKK-ß over-expression) reduced IKK-α levels and IKK-α over-expression prevented TNF-α-induced impairments in muscle OXPHEN. Importantly, muscle IKK-α protein abundance and OXPHEN was reduced in COPD patients compared to controls, which was more pronounced in patients with increased muscle TNF-α mRNA levels. CONCLUSION: Classical NF-κB activation impairs skeletal muscle OXPHEN by reducing IKK-α expression. TNF-α-induced reductions in muscle IKK-α may accelerate muscle OXPHEN deterioration in COPD.