1,25-DIHYDROXY VITAMIN-D-3 AND TRIIODOTHYRONINE STIMULATE THE EXPRESSION OF A PROTEIN IMMUNOLOGICALLY RELATED TO OSTEOCALCIN

Osteocalcin (OC), a bone-specific protein, is a marker of late osteobl astic differentiation. Its expression is influenced by various growth factors and hormones. We investigated the effect of 1,25-dihydroxy vit amin D-3 (D-3) and tri-iodothyronine (T-3) on OC expression in osteobl ast-like MC3T3-E1 cells. A heterologous OC green fluorescence protein (GFP) fusion vector was established and expressed to study possible ef fects on protein transport. Immunostaining of endogenous OC revealed a significant increase in the percentage of positive cells after D-3 an d T-3 treatment. This was consistent for MC3T3-E1 cells as well as non osteogenic NIH-3T3 and mammary carcinoma cells, but not for neuroblast oma cells. The perinuclear immunostaining corresponded to the NBD C-6 ceramide Golgi staining. Conversely, we found a strong induction of OC in MC3T3-E1 cells at the mRNA and protein levels only with T-3 and no t with D-3 OC mRNA and protein expression was not detected in NIH fibr oblasts. OC GFP transfection experiments indicate rapid transport and secretion of OC, because OC CFP was not found to be accumulated at int racellular compartments after hormone treatment. We conclude that the strong perinuclear immunostaining does not represent OC but a protein immunologically related to OC, as indicated by preabsorption experimen ts. The expression of this OC epitope-sharing protein is regulated by both D-3 and T-3 in the osteoblastic MC3T3-E1 and in nonosteogenic cel ls.