1,25 dihydroxyvitamin-D-3 attenuates the confluence-dependent differences in the osteoblast characteristic proteins alkaline phosphatase, procollagenI peptide, and osteocalcin

In the present study a cell culture model of primary human osteoblasts base d on degrees of confluence was investigated by measuring basal and 1,25(OH) (2)D-3-stimulated levels of the osteoblast characteristic proteins alkaline phosphatase (AP), procollagen I-peptide (PICP), and osteocalcin (OC), as w ell as the corresponding gene expression. Primary osteoblast-like cell cult ures from seven donors were treated in the second passage with 1,25(OH)(2)D -3 (5 x 10(-8) M for 48 hours) and investigated at four stages of confluenc e (stage I 50%, stage IT 75%, stage III 100%, and stage IV 7 days postconfl uence). In untreated cultures passing through the different stages of confl uence, we saw a 1.8-fold increase of AP activity, a 2.3-fold increase of OC secretion, but a decrease of PICP levels to 0.36-fold. Gene expression sho wed only minor variation between the different confluence stages. 1,25(OH)( 2)D-3 did not significantly affect PICP production. Alkaline phosphatase pr otein was stimulated during proliferation until confluence, with no effect thereafter. Surprisingly, OC secretion and mRNA expression were stimulated in all four stages to the same absolute level independent of basal values. We conclude that our results correspond to other studies showing differenti ation-stage dependent changes of basal levels of osteoblast-specific protei ns. However, 1,25(OH)(2)D-3 stimulation decreased the confluence-dependent difference for AP and abolished it for osteocalcin, thus leading to a more differentiated phenotype of the osteoblast. Therefore, 1,25(OH)(2)D-3 stimu lation might improve the reproducibility of results obtained at different c onfluence stages from cultures of clinical samples.