Establishment of assay systems for examining bone metabolism : Studies on differentiation and function of osteoblasts and osteoclasts

点击翻译按钮获取中文摘要

We have established reliable assay systems for investigating signal transduction which is involved in differentiation and function of osteoblasts and osteoclasts. Following findings were obtained from a series of experiments using the assay systems.Studies on Osteoblast Differentiation and Function(1) We have studied the signal trensduction of bone morphogenetic protein 2 (BMP-2) in myoblastic C2C12 cells. C2C12 cells expressed type IA receptor (BMPR-IA) and type II (BMP-RII) for BMP-2, but not type IB receptor (BMPR-IB). (2) C2C12 cells transfected with a kinase domain-truncated BMPR-IA did not differentiate into ALP-positive cells even in the presence of BMP-2. (3) When activated mutant BMPR-IA and BMPR-IB were transfected into C2C12 cells, both mutated receptors induced ALP activity in the absence of BMP-2. (4) C2C12 cells expressed Smad1, Smad2, Smad4, Smad5 mRNAs, and the expression levels were not altered by treatment with BMP-2. When Smads were transfected into C2C12 cells, only … More Smad1 and Smad5 induced ALP activity. Transfection of Cterminal truncated Smad1 and Smad5 (dominant negative Smads) into C2C12 cells, which had been transfected with activated mutant BMPR-IB,induced ALP activity.Studies on Osteoclast Differentiation and Function(1) We established a method for obtaining functionally active osteoclasts from co-cultures of mouse osteoblastic cells and bone marrow cells. Signaling molecules such as phosphatidylinositol-3 kinase, tyrosine kinase, rho p21, and p130^<Cas> have been shown to be involved in ruffled border formation of osteoclasts. (2) Calcitonin and bisphosphonates inhibited osteoclast function through activation of protein kinase A and inhibition of tyrosine phosphatases, respectively. (3) Osteoblasts activated osteoclast function through a mechanism involving cell to cell contact. (4) IL-1 prevented spontaneous apoptosis of osteoclasts and enhanced their survival through activation of NF-kB.(5) The target cells of osteotropic hormones and cytokines such as 1alpha, 25 (OH)_2D_3, PTH and IL-11 in osteoclast formation in the co-culture are osteoblastic cells. Less