Research for role of bone morphogenetic protein in the differentiation and activation of osteoclast and osteoblast

尽管骨形态发生蛋白(BMP)在成骨细胞分化中的作用已被广泛研究，但其对破骨细胞分化的影响尚不清楚。本研究检测了BMP-2在体外对破骨细胞分化和骨吸收的影响。BMP-2(1~100 ng/ml)可显著促进小鼠骨细胞的骨吸收，而对体外培养的兔破骨细胞的骨吸收无明显影响。在骨细胞培养中，BMP-2也能刺激破骨细胞分化。接下来，我们利用来自脾细胞的造血母细胞，研究了在没有基质细胞的情况下，BMP-2对破骨细胞前体细胞的直接影响。在这些破骨细胞前体细胞中检测到BMP 1型受体的mRNA。和BMP-2刺激GM-…支持的造血母细胞形成破骨细胞。本研究首次表明BMP-2通过直接刺激破骨细胞对CFF的作用促进骨吸收在体外，可能通过基质细胞，更多地形成和激活成熟的破骨细胞。接下来，我们提出了一个编码硫酸盐转运蛋白(设计为来自成骨细胞的硫酸盐转运蛋白的st-ob)的cDNA，该基因被克隆为成骨细胞中与成骨细胞分化相关的诱导基因。基于BMP-2在未成熟的成纤维细胞C3H10T1/2中诱导成骨表型的事实，我们对BMP-2处理的细胞和未处理的细胞进行了消减杂交，并分离到了一个在成骨细胞中持续表达的BMP-2诱导的克隆。其推导的氨基酸序列和推测的结构与人营养不良硫酸盐转运蛋白基因产物(DTDST)基本一致。ST-ob基因在胸腺、睾丸、颅骨和成骨细胞MC3T3-E1中大量表达，而在C3H10T1/2细胞中表达较弱。BMP-2使C3H10T1/2细胞的硫酸盐掺入增加约为基线水平的两倍。蛋白多糖是骨和软骨细胞外基质的主要成分之一，它们诱导产生大量的硫酸盐。在骨骼中，成骨细胞主动吸收硫酸盐合成蛋白多糖。与Sat-1不同，成骨细胞的硫酸盐转运系统部分由st-ob介导，st-ob在成骨细胞分化过程中表达，是成骨细胞的基本特征。较少

Although the action of bone morphogenetic protein (BMP) on osteoblast differentiation has been extensively investigated, its effect on osteoclast differentiation remained unknown. In this study, in vitro effects of BMP-2 on osteoclast differentiation and bone resorption were examined. BMP-2 (1-100ng/ml) significantly stimulated bone resorption in mouse bone cell culture, whereas it did not affect the bone-resorbing activity of isolated rabbit osteoclasts. BMP-2 also stimulated osteoclast differentiation in bone cell culture. We next examined the direct effect of BMP-2 on osteoclast precursors in the absence of stromal cells, using hemopoietic blast cells derived from spleen cells. The mRNA for BMP type 1 receptor was detected in these osteoclast precursor cells. And BMP-2 stimulated osteoclast formation from these hemopoietic blast cells supported by GM-CSF.The present data were the first to indicate that BMP-2 stimulates bone resorption through both direct stimulation of osteoclast fo … More rmation and activation of mature osteoclasts, possibly via stromal cells, in vitro. Next, we present a cDNA encoding a sulfate transporter (designed as st-ob for sulfate transporter from osteoblast) that was cloned as an induced gene in osteoblasts in association with osteoblastic differentiation. Based on the fact that BMP-2 induces osteoblastic phenotypes in immature fibroblastic C3H10T1/2 cells, we performed a subtraction hybridization between BMP-2-treated and -untreated cells, and have isolated one clone induced by BMP-2 to be constantly expressed in osteoblastic cells. The deduced amino acid sequence and proposed structure of st-ob were mostly identical to human diastrophic dysplasia sulfate transporter gene product (DTDST). St-ob mRNA was abundantly expressed in the thymus, testis, calvaria and osteoblastic MC3T3-E1 cells, while its expression was faint in C3H10T1/2 cells. BMP-2 increased sulfate incorporation in C3H10T1/2 cells about two fold of the baseline level. Proteoglycan is one of major components of extracellular matrix of bone and cartilage, and they induce a large amount of sulfate. In bone, osteoblasts actively uptake sulfate to synthesize proteoglycans. The sulfate transporter system in osteoblasts is mediated partly by st-ob distinct from sat-1, and st-ob is expressed during differentiation as a fundamental feature of osteoblasts. Less