Osteopontin is an acidic phosphoprotein containing casein kinase II (CKII) phosphorylatable sites and an acidic amino acid cluster. The metabolically 32P-labelings of both serines and threonines in vitro in osteopontin immunoprecipitated from rat osteoblast-like ROS 17/2.8 cells may suggest that casein kinase II catalyzes this modification. The enzyme occurs in microsomal fractions of rat osteoblast-like ROS 17/2.8 cells. Subcellular fractions containing endoplasmic reticulum and Golgi apparatus were isolated by differential centrifugation and were identified according to their ultrastructures and the presence of marker enzymes such as glucose-6-phosphatase and thiamine pyrophosphatase, respectively. both fractions phosphorylated the partially dephosphorylated osteopontin and the specific substrate peptide RRREEETEEE. Endoplasmic reticulum-catalyzed peptide phosphorylation was 2.7 times lower than that of Golgi although both endoplasmic reticulum- and Golgi-catalyzed peptide reactions were 50% inhibited by 20 and 100 ng/ml heparin, respectively. Western blot analysis revealed that both fractions contained osteopontin and microsomal CKII. Furthermore, microsomal CKII was immunogold-labeled in endoplasmic reticulum and Golgi apparatus. Heparin inhibition and utilization of [gamma-32P]GTP as a phosphate donor by both fractions confirmed their capacity to phosphorylate osteopontin. The results suggest that microsomal CKII modifies the acidic matrix proteins during transportation. These matrix phosphoproteins may participate in the mineralization process of hard tissues.

Phenytoin (diphenylhydantoin, DPH) , an anticonvulsant drug for epileptic patients, has several adverse effects that include calvarial thickening and coarsening of the facial features. These effects often occur with chronic DPH therapy.While previous studies have shown DPH to heve an anabolic action on bone cells in vivo and in vitro, the basis of these effects is not fully understood.In this study, the effect of DPH on osteoblastic differentiation in cultured MC 3 T 3-E 1 cells was investigated by measuring mineralized bone nodule (BN) formation, alkaline phosphatase (ALPase) activity, cell growth, and the expression of matrix proteins, such as osteocalcin (OCN) , and osteopontin (OPN) . Continuous treatment of MC 3 T 3-E 1 cells with DPH for 20 days increased the BNs (9.1 to 19.7-fold) in a dose-dependent manner for concentrations of 10-50 , u M DPH. ALPase activity was also stimulated by DPH (1.4-3.2 hold) in a dose-dependent manner for concentrations of 10-100 , u M. Although a concentration 50 , u M DPH increased the DNA content of the cells on days 5, 10, and 15, the final cell saturation density was not affected by any concentration of DPH. When DPH was added at two different culture stages, days 1-10 (growth stage) and days 11-20 (matrix development stage) , BN formation was markedly stimulated in the case of the latter addition. A maximal effect was observed at a concentration of 200 AtNI DPH. The expression of OCN and OPN mRNA was also augmented by continuous treatment at a concentration of 200 du M DPH on days 1-20, and a remarkable effect was also observed when DPH was added on days 11-20. These findings demonstrate that DPH stimulates osteoblastic markers, such as BNs, ALPase activity, and OCN and OPN expression, mainly during the stage of matrix development in MC 3 T 3-E 1 cells.