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

我们建立了可靠的检测系统，用于研究成骨细胞和破骨细胞分化和功能的信号转导。成骨细胞分化与功能的研究（1）我们研究了成肌细胞C2 C12中骨形态发生蛋白2（BMP-2）的信号转导。C2 C12细胞表达BMP-2的IA型受体（BMPR-IA）和II型受体（BMP-RII），但不表达IB型受体（BMPR-IB）。(2)用激酶结构域截短的BMPR-IA转染的C2 C12细胞即使在BMP-2存在下也不分化成ALP阳性细胞。(3)当激活的突变体BMPR-IA和BMPR-IB转染到C2 C12细胞中时，两种突变的受体在不存在BMP-2的情况下诱导ALP活性。(4)C2 C12细胞表达Smad 1，Smad 2，Smad 4，Smad 5 mRNA，并且表达水平不受BMP-2处理的改变。当Smads转染C2 C12细胞时， ...更多信息 Smad 1和Smad 5诱导ALP活性。将C端截短的Smad 1和Smad 5（显性失活Smads）转染到转染了活化突变体BMPR-IB的C2 C12细胞中，可诱导ALP活性。破骨细胞分化和功能的研究（1）建立了一种从小鼠成骨细胞和骨髓细胞共培养中获得具有功能活性的破骨细胞的方法。信号分子如磷脂酰肌醇-3激酶、酪氨酸激酶、rho p21和p130 α<Cas>已显示参与破骨细胞的皱褶边缘形成。(2)降钙素和双磷酸盐抑制破骨细胞功能，通过激活蛋白激酶A和抑制酪氨酸磷酸酶，分别。(3)成骨细胞通过细胞与细胞接触的机制激活破骨细胞的功能。(4)IL-1通过激活NF-κ B阻止破骨细胞自发凋亡并增强其存活。(5)促骨激素和细胞因子如1 α，25（OH）_2D_3、PTH和IL-11在共培养中破骨细胞形成的靶细胞是成骨细胞。少

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