Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch

Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis.

Methanol Extract of Croton Pycnanthus Benth. Inhibits Osteoclast Differentiation by Suppressing the MAPK and NF-kappaB Signaling Pathways

BACKGROUND: Osteoclasts are differentiated from monocytes/macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL). Croton pycnanthus Benth. (CPB) is a herbal plant that belongs to Euphorbiaceae family. The aim of this study was to investigate the effects of CPB on osteoclastogenesis and RANKL-dependent signaling pathways. METHODS: Methanol extract of CPB was obtained from International Biological Material Research Center. Osteoclast differentiation was achieved by culturing mouse bone marrow-derived macrophages (BMMs) with M-CSF and RANKL. Osteoclast numbers were evaluated by counting multinuclear cells positive for tartrate-resistant acid phosphatase (TRAP). mRNA and protein levels were analyzed by real-time polymerase chain reaction (PCR) and Western blotting, respectively. The activation of signaling molecules were assessed after acute stimulation of cells with high dose of RANKL by Western blotting with phospho-specific antibodies. RESULTS: CPB reduced the generation of TRAP-positive multinucleated cells and the activation of mitogen-activated protein kinase (MAPK) and NF-kappaB signaling pathways. The induction of the expression of c-Fos, nuclear factor-activated T cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) by RANKL was also suppressed. CONCLUSIONS: CPB exerts negative effects on osteoclast differentiation in response to the RANKL. The inhibitory mechanism involves the suppression of MAPK and NF-kappaB signaling pathways and subsequently the down-regulation of c-Fos and NFATc1 transcription factors.

The Peroxisome Proliferator-Activated Receptor delta Agonist, GW501516, Inhibits Angiogenesis through Dephosphorylation of Endothelial Nitric Oxide Synthase

OBJECTIVE: Peroxisome proliferator-activated receptor delta (PPAR-delta) is an ubiquitously expressed nuclear receptor that has been implicated in adipose tissue formation, brain development, and atherosclerosis. Despite mouse studies demonstrating that PPAR-delta activation has favorable anti-atherogenic properties by improving systemic lipid profiles, the relationship between PPAR-delta agonist and angiogenesis is unknown. We hypothesized that PPAR-delta ligands modulate the angiogenesis. METHODS: To test this hypothesis we treated primary cultures of bovine aortic endothelial cells with PPAR-delta specific ligand, GW501516 (50-800 nM) for 6 h. RESULTS: GW501516 dose-dependently decreased nitric oxide production without alteration in endothelial nitric oxide synthase (eNOS) expression. Analysis with phospho-specific antibodies against eNOS demonstrated that GW501516 significantly decreased the phosphorylation of eNOS at Serine1179 (eNOS-Ser1179). Concurrently, GW501516 also decreased the Akt phosphorylation. GW501516 did not affect endothelial cell proliferation or induce apoptosis. However, GW501516 inhibited endothelial cell migration, and tube formation in a high nanomolar concentration. The inhibition of endothelial cell tube formation by GW501516 was prevented by addition of the nitric oxide donor, DETA NONOate (5 microM). GW501516 was also found to inhibit angiogenesis in vivo in the chicken chorioallantoic membrane assay. CONCLUSION: These results provide that high nanomolar range of GW501516 inhibits angiogenesis by a mechanism involving dephosphorylation of eNOS-Ser1179.