Safadi F F, Xu J, Smock S L, Rico M C, Owen T A, Popoff S N
Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
J Cell Biochem. 2001;84(1):12-26. doi: 10.1002/jcb.1259.
Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N-linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3- to 4-fold overexpressed in op versus normal bone. RT-PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non-osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast-specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function.
成骨细胞发育是一个复杂的过程，涉及特定生长因子和调节蛋白的表达，这些因子和蛋白控制细胞增殖、分化和成熟。在本研究中，我们利用大鼠骨石化（op）突变体来检测突变体op与正常同窝仔鼠之间骨骼基因表达的差异。从长骨和颅骨分离的总RNA用作mRNA差异显示的模板。在正常或突变骨中选择性表达的众多cDNA之一被克隆和测序，发现与人nmb和Pmel 17基因有一些同源性。这个新的cDNA被命名为骨激活素。骨激活素具有1716 bp的开放阅读框，编码一个572个氨基酸的蛋白质，预测分子量为63.8 kD。蛋白质序列分析显示存在一个信号肽和位于第23位的切割位点。该蛋白质还有13个预测的N-连接糖基化位点和一个位于第556位的潜在RGD整合素识别位点。Northern印迹分析证实，与正常骨相比，骨激活素在op骨中过表达3至4倍。RT-PCR分析表明，与所检测的任何其他非骨组织相比，骨激活素在骨中表达最高。正常骨中骨激活素的原位杂交分析表明，它主要在积极参与骨基质产生和矿化的成骨细胞中表达。在原代大鼠成骨细胞培养物中，骨激活素呈现出一种时间表达模式，在基质成熟和矿化的后期阶段表达水平最高，并与碱性磷酸酶和骨钙素的表达相关。我们的研究结果表明，骨激活素在骨中的表达是成骨细胞特异性的，并表明它可能在成骨细胞分化和基质矿化中起重要作用。此外，op突变骨中骨激活素的过表达可能是骨吸收和形成解偶联的继发结果，导致成骨细胞基因表达和功能异常。
