The effect of bone morphogenetic protein-7 on the expression of type I inositol 1,4,5-trisphosphate receptor in G-292 osteosarcoma cells and primary osteoblast cultures

Abstract

Bone morphogenetic protein-7 (BMP-7) affects differentiation of preosteoblasts enabling the resultant cells to respond optimally to acutely acting regulators. As the phosphoinositide cascade and, particularly, the calcium-mobilizing inositol 1,4,5-trisphosphate (InsP 3) receptor are integral to stimulus–secretion coupling in osteoblasts, the hypothesis that BMP-7 affects InsP 3 receptor expression was examined in the G-292 human osteosarcoma cell line and in primary cultures of human osteoblasts. G-292 osteosarcoma cells were found to be a valid experimental model for primary human osteoblasts, expressing osteoblastic mRNAs encoding osteocalcin, bone sialoprotein, alkaline phosphatase, α1-collagen, epidermal growth-factor receptor, and BMP type II receptor. When cultured long term in the presence of ascorbic acid and β-glycerophosphate, G-292 cells underwent further osteoblastic differentiation, forming nodules and exhibiting restricted mineralization. G-292 cells responded to BMP-7 with an increase in InsP 3 receptor density. Ligand-binding studies established that BMP-7 (50 ng/ml) treatment of G-292 cells increased InsP 3 receptor density 2.4-fold with no apparent change in affinity. Immunoblot analysis with antibodies specific for type I, type II, and type III InsP 3 receptors revealed that BMP-7 (50 ng/ml) treatment resulted in a specific increase (206±8%) in the type I receptor. Reverse transcription-polymerase chain reaction and Northern blot analyses of G-292 and primary human osteoblasts confirmed an increase in type I InsP 3 receptor mRNA upon BMP-7 treatment. These results demonstrate that G-292 cells respond to BMP-7 with an increase InsP 3 receptor density, consistent with the enhanced capacity of these cells to respond to Ca 2+-mobilizing secretory hormones during osteoblast differentiation.