BASIC FIBROBLAST GROWTH-FACTOR REGULATES IGF-I BINDING-PROTEINS IN THE CLONAL OSTEOBLASTIC CELL-LINE MC3T3-E1

In previous studies, we reported that basic fibroblast growth factor ( bFGF) regulates insulin-like growth factor messenger RNAs and protein levels in the osteoblastic MC3T3-E1 cells. In the present study, we ex amined the expression of insulin-like growth factor binding proteins ( IGFBPs) in MC3T3-E1 cells and determined whether bFGF altered IGFBP mR NAs and protein levels. Since previous studies suggested that IGFBPs c an inhibit DNA synthesis stimulated by IGF-I,,Ve wondered whether the mitogenic effect of bFGF was altered by exogenous IGFBP-3. Confluent M C3T3-E1 cells were serum-deprived for 24 h and then treated with bFGF for 6-24 h. In control cultures, MC3T3-E1 cells expressed the mRNAs fo r IGF-I, IGF-II, and IGFBP-2, 4, 5, and 6 but not IGFBP-1 or 3. A 24 h treatment with bFGF at 10(-8) M decreased IGF-I mRNA by 97%, IGF-II m RNA by 73%, IGFBP-2 by 64%, IGFBP-4 by 73%, IGFBP-5 by 95%, and IGFBP- 6 by 65%. The inhibitory effect of bFGF on IGF-I and IGFBP mRNA levels was not altered by aphidicolin, an inhibitor of cell replication. bFG F 10 nM decreased IGF-I levels determined by radioimmunoassay after ac idification by 45% and 72% at 24 and 48 h, respectively. Western ligan d blot for IGF binding proteins revealed that MC3T3-E1 cells expressed IGFBPs of 24, 30, and 34 kD. Treatment with bFGF 10(-8) M decreased t he levels of the 24 and 30 kD band at 24 h but increased the 34 kD ban d. Western immunoblot revealed that the 24 kD protein was IGFBP-4 and the 34 kD band was IGFBP-2. bFGF at 10(-9)-10(-8) M increased thymidin e incorporation into DNA (TdR) in a dose-dependent manner. When exogen ous IGFBP-3 was added to the cultures there was a significant reductio n in DNA synthesis while the mitogenic effect of bFGF was not blocked. In summary, bFGF not only regulates IGF-I mRNA and protein levels but it also regulates the IGF-II mRNA and mRNA and protein levels of the IGFBPs expressed in MC3T3-E1 cells. However, the mitogenic effect of b FGF may be independent of endogenous IGF-I. These results may be impor tant in understanding the role of bFGF in bone cell function.