A NUMBER OF OSTEOCALCIN ANTISERA RECOGNIZE EPITOPES ON PROTEINS OTHERTHAN OSTEOCALCIN IN CULTURED SKIN FIBROBLASTS - IMPLICATIONS FOR THE IDENTIFICATION OF CELLS OF THE OSTEOBLASTIC LINEAGE IN-VITRO

Rabbit antisera to bovine osteocalcin were produced independently in t wo laboratories and their specificities established by western blot an alysis. By immunohistochemistry each of the five polyclonal antisera p roduced an intense cytoplasmic staining in human bone-derived cells. S taining intensity was strongly attenuated by preabsorption of the anti sera with osteocalcin. No staining was observed using nonimmune rabbit serum. However, the choice of skin cells as negative controls for ost eocalcin synthesis yielded an unexpected positive staining pattern sim ilar to that seen with the bone-derived cells over a range of antiseru m dilutions. This was not caused by the uptake of exogenous osteocalci n from the culture medium because a similar pattern of staining was ob served when medium was supplemented with osteocalcin-depleted fetal ca lf serum. Treatment with 1,25-dihydroxyvitamin D-3 induced osteocalcin mRNA expression and osteocalcin secretion in cultures of bone-derived cells but not in skin fibroblasts. The results demonstrate that these polyclonal antisera also recognize epitopes shared with other protein s synthesized in culture by skin fibroblasts. Furthermore, three mouse monoclonal antibodies to distinct regions of the osteocalcin molecule show differential staining of human bone-derived cells, skin cells, a nd osteosarcoma cells (MG63). These observations indicate that the sha red epitope resides in the central region of osteocalcin and are consi stent with the specific synthesis of osteocalcin by bone cells alone. The observed nonspecificity of many osteocalcin antisera may compromis e immunocytochemical studies of the osteoblast phenotype in studies in vitro when based solely on reactivity with inadequately characterized osteocalcin antisera.