Expression of collagen, osteocalcin, and bone alkaline phosphatase in a mineralizing rat osteoblastic cell culture

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Abstract
Rat calvaria bone cells isolated by collagenase digestion form a bone-like matrix which mineralizes in vitro in the presence of β-glycerophosphate, in less than 2 weeks. The purpose of this work was to investigate, in this mineralizing rat osteoblastic cell culture, the synthesis of collagen, osteocalcin, and bone alkaline phosphatase (ALP). The results obtained indicate (1) After 15 days in culture, the extracellular-matrix contains collagen type I, V, and to some extent type III. Metabolic labeling at day 14, during the phase of nodules mineralization as well as new nodules formation, shows that collagen types I and type V are synthesized; (2) During the phase of cell growth, no osteocalcin could be detected in the medium, however, at the point of nodule formation, the osteocalcin level reached values of 3.55±1.39 ng/ml, followed by a 30-fold increase after nodules became mineralized. At day 14, after metabolic labeling, de novo synthesized osteocalcin was chromatographed on an immunoadsorbing column. With urea-SDS PAGE the apparent molecular weight was determined to be 9,000 daltons. (3) Specific activity of ALP was found to be 10 nmol/min/mg of proteins at cell confluence. At day 15, when nodules are mineralized, this activity was increased by 40-fold. The Michaelis constant was 1.58 10-3 M/L. ALP was inhibited by L-homoarginine and levamisole but not by L-phenylalanine. ALP was shown to be heat sensitive at 56°C with two slopes of inhibition. On SDS-PAGE, apparent molecular weight of ALP showed one band at 116,000 daltons (d) when extracted at cell confluence and two bands at 116,000 and 140,000 d when extracted at the 15th day of culture. 32P-labeled subunit of the enzyme migrated as one band at 75,000 d. Sialic acid content was demonstrated by neuraminidase treatment either on the dimeric form or on the 32P-labeled subunit. These data indicate that ALP expressed in this culture is bone specific. The results of the present study show that this mineralizing rat osteoblastic cell culture system synthesizes collagen type I, V, and traces of type III, osteocalcin, and bone ALP isoenzyme. Medium osteocalcin was detected during nodule formation and increased during mineralization. Increase in ALP activity as well as the presence of an additional form of ALP occurred in the mineralization phase. Therefore, this culture may be a useful model for studying the functions of bone-specific proteins during the process of mineralization.