Interference by adrenaline with chondrogenic differentiation through suppression of gene transactivation mediated by Sox9 family members.

In contrast to osteoblasts, little attention has been paid to the functional expression of adrenergic signaling machineries in chondrocytes. Expression of mRNA was for the first time demonstrated for different adrenergic receptor (AdR) subtypes in chondrogenic ATDC5 cells and mouse metatarsals isolated before vascularization in culture, but not for other molecules related to adrenergic signaling. In neonatal mouse tibial sections, beta(2)AdR and alpha(2a)AdR mRNA expression was found in chondrocytes at different developmental stages by in situ hybridization. Exposure to adrenaline significantly suppressed expression of several maturation markers through the cAMP/protein kinase A pathway activated by beta(2)AdR without affecting cellular proliferation in both cultured ATDC5 cells and metatarsals. Adrenaline also significantly inhibited gene transactivation by sry-type HMG box 9 (Sox9) family members essential for chondrogenic differentiation in a manner prevented by the general betaAdR antagonist propranolol, with a concomitant significant decrease in the levels of Sox6 mRNA and corresponding protein, in ATDC5 cells and primary cultured mouse costal chondrocytes. Systemic administration of propranolol significantly promoted the increased expression of mRNA for collagen I and collagen X, but not for collagen II, in callus of fractured femur in mice. These results suggest that adrenaline may interfere with chondrogenic differentiation through downregulation of Sox6 expression for subsequent suppression of gene transactivation mediated by Sox9 family members after activation of beta(2)AdR expressed by chondrocytes.

Osteoblast protects osteoclast devoid of sodium-dependent vitamin C transporters from oxidative cytotoxicity of ascorbic acid.

The view that ascorbic acid indirectly benefits osteoclastogenesis through expression of receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) by osteoblasts is prevailing. In this study, we have examined the direct effect of ascorbic acid on osteoclastogenesis in cultured mouse osteoclasts differentiated from bone marrow precursors. The absence of alkaline phosphatase and osteoblastic marker genes validated the usefulness of isolation procedures. Sustained exposure to ascorbic acid, but not to dehydroascorbic acid, significantly reduced the number of multinucleated cells positive to tartrate-resistant acid phosphatase (TRAP) staining. In cultured osteoclasts, mRNA expression was seen for glucose transporter-1 involved in membrane transport of dehydroascorbic acid, but not for sodium-dependent vitamin C transporters-1 and -2 that are both responsible for the transport of ascorbic acid. The inhibition by ascorbic acid was completely prevented by catalase, while ascorbic acid or hydrogen peroxide drastically increased the number of cells stained with propidium iodide and the generation of reactive oxygen species, in addition to inducing mitochondrial membrane depolarization in cultured osteoclasts. In pre-osteoclastic cell line RAW264.7 cells, ascorbic acid similarly inhibited the formation of TRAP-positive multinucleated cells, with a significant decrease in RANKL-induced NF-kappaB transactivation. Moreover, co-culture with osteoblastic MC3T3-E1 cells significantly prevented the ascorbic acid-induced decrease in the number of TRAP-positive multinucleated cells in RAW264.7 cells. These results suggest that ascorbic acid may play a dual repulsive role in osteoclastogenesis toward bone remodeling through the direct cytotoxicity mediated by oxidative stress to osteoclasts, in addition to the indirect trophism mediated by RANKL from osteoblasts.