Regulation of transformation-sensitive secreted phosphoprotein (Sppi/osteopontin) expression by transforming growth factor- . Comparisons with expression of Sparc (secreted acidic cysteine-rich protein)
Coimbra, Joao Carlos; Pinto, Iraja Damiani; Wurdig, Norma Luiza; Do Carmo, Dermeval Aparecido
Biochemical Journal 273(3): 523-531
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.