The stiffness and collagen control differentiation of osteoclasts with an altered expression of c-Src in podosome.

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.

Effects of 4'-Demethylnobiletin and 4'-Demethyltangeretin on Osteoclast Differentiation In Vitro and in a Mouse Model of Estrogen-Deficient Bone Resorption.

Citrus nobiletin (NOB) and tangeretin (TAN) show protective effects against disease-related bone destruction. We achieved demethylation of NOB and TAN into 4'-demethylnobiletin (4'-DN) and 4'-demethyltangeretin (4'-DT) using enzyme-manufacturing methods. In this study, we examined the effects of 4'-DN and 4'-DT on in vitro osteoclast differentiation, and on in vivo osteoporotic bone loss in ovariectomized (OVX) mice. 4'-DN and 4'-DT clearly suppressed the osteoclast differentiation induced by interleukin IL-1 or RANKL treatment. 4'-DN and 4'-DT treatments resulted in higher inhibitory activity in osteoclasts in comparison to NOB or TAN treatments. RANKL induced the increased expression of its marker genes and the degradation of IκBα in osteoclasts, while these were perfectly attenuated by the treatment with 4'-MIX: a mixture of 4'-DN and 4'-DT. In an in silico docking analysis, 4'-DN and 4'-DT directly bound to the ATP-binding pocket of IKKß for functional inhibition. Finally, the intraperitoneal administration of 4'-MIX significantly protected against bone loss in OVX mice. In conclusion, 4'-DN, 4'-DT and 4'-MIX inhibited the differentiation and function of bone-resorbing osteoclasts via suppression of the NF-κB pathway. Novel 4'-DN, 4'-DT and 4'-MIX are candidates for maintaining bone health, which may be applied in the prevention of metabolic bone diseases, such as osteoporosis.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.