Wrana J L, Kubota T, Zhang Q, Overall C M, Aubin J E, Butler W T, Sodek J
Department of Biochemistry, Faculty of Dentistry, University of Toronto, Ontario, Canada.
Biochem J. 1991 Feb 1;273 ( Pt 3)(Pt 3):523-31. doi: 10.1042/bj2730523.
Secreted phosphoprotein I (SPPI; osteopontin), a highly phosphorylated form of which has been associated with cell transformation, is one of the major phosphorylated proteins in bone. Populations of rat bone cells derived from fetal calvariae, neonatal parietal bone and a rat osteosarcoma cell line (ROS 17/2.8) produce several forms of the protein, the major forms having apparent molecular masses of 55 and 44 kDa by SDS/PAGE on 15% (w/v) cross-linked gels and of 60 and 56 kDa on 10% gels. Northern blot analysis of SPPI mRNA using total cellular RNA revealed a single 1.5 kb mRNA species, indicating that the nascent protein chains of these phosphoproteins are identical. On treatment of the cells with transforming growth factor-beta (TGF-beta; 1 ng/ml), the levels of SPPI mRNA and the synthesis of the 55 kDa phosphoprotein, but not of the 44 kDa phosphoprotein, were increased by 1.8-4.5-fold in the normal osteoblastic cells, the stimulation first being evident at 3 h and reaching a maximum at 12 h. In the transformed ROS 17/2.8 cells, TGF-beta did not alter significantly the SPPI mRNA level or the synthesis of either the 55 kDa or the 44 kDa SPPI over the 24 h period studied. By comparison, neither the steady-state levels of SPARC (secreted protein, acidic, rich in cysteine) mRNA nor the synthesis of SPARC protein were affected significantly by the addition of TGF-beta to any of the osteoblastic bone cells. The half-lives for SPPI and SPARC mRNAs in the osteoblastic calvarial cells were calculated to be 18 h and greater than 50 h respectively, in both the presence and the absence of TGF-beta. Since the stability of the mRNA was unchanged by TGF-beta and the increased expression of SPPI mRNA could be blocked by cycloheximide, TGF-beta appears to increase transcription of the SppI gene indirectly by stimulating the synthesis of a protein that promotes transcription. These results demonstrate that several forms of SPPI are synthesized constitutively by bone cells, and that there are clear differences in the regulation of SppI gene expression by TGF-beta in normal bone cells compared with the tumorigenic ROS 17/2.8 cells. The differential responses of normal osteoblastic cells to TGF-beta in the expression of SPPI and the selective stimulation of specific forms of the SPPI protein may be important in bone repair and remodelling.
分泌型磷蛋白I（SPPI；骨桥蛋白），其高度磷酸化形式与细胞转化相关，是骨中主要的磷酸化蛋白之一。源自胎儿颅骨、新生顶骨的大鼠骨细胞群体以及大鼠骨肉瘤细胞系（ROS 17/2.8）可产生该蛋白的多种形式，在15%（w/v）交联凝胶上进行SDS/PAGE分析时，主要形式的表观分子量为55 kDa和44 kDa，在10%凝胶上则为60 kDa和56 kDa。使用总细胞RNA对SPPI mRNA进行Northern印迹分析显示有一个单一的1.5 kb mRNA种类，表明这些磷蛋白的新生蛋白链是相同的。在用转化生长因子-β（TGF-β；1 ng/ml）处理细胞时，正常成骨细胞中SPPI mRNA水平和55 kDa磷蛋白的合成增加了1.8至4.5倍，但44 kDa磷蛋白的合成未增加，这种刺激在3小时时首次明显，12小时时达到最大值。在转化的ROS 17/2.8细胞中，在研究的24小时期间，TGF-β并未显著改变SPPI mRNA水平或55 kDa或44 kDa SPPI的合成。相比之下，向任何成骨细胞中添加TGF-β对SPARC（分泌型蛋白质，酸性，富含半胱氨酸）mRNA的稳态水平或SPARC蛋白的合成均无显著影响。在成骨颅骨细胞中，无论有无TGF-β，SPPI和SPARC mRNA的半衰期分别计算为18小时和大于50小时。由于TGF-β未改变mRNA的稳定性，且SPPI mRNA表达的增加可被环己酰亚胺阻断，因此TGF-β似乎通过刺激一种促进转录的蛋白质的合成间接增加SppI基因的转录。这些结果表明骨细胞组成性合成多种形式的SPPI，并且与致瘤性ROS 17/2.8细胞相比，正常骨细胞中TGF-β对SppI基因表达的调控存在明显差异。正常成骨细胞对TGF-β在SPPI表达上的差异反应以及对特定形式的SPPI蛋白的选择性刺激在骨修复和重塑中可能很重要。
