TRANSFORMING GROWTH-FACTOR-BETA STIMULATES ASCORBATE TRANSPORT ACTIVITY IN OSTEOBLASTIC CELLS

Transforming growth factor-beta (TGF-beta) modulates the proliferation and differentiation of a number of cell types, including osteoblasts. TGF-beta has been shown to stimulate matrix synthesis by connective tissue cells, but its mechanism of action is poorly understood. Because ascorbate (reduced vitamin C) also influences osteoblastic differentiation and is required as a cofactor for collagen synthesis, the present study examined the effect of TGF-beta on osteoblastic ascorbate uptake. Saturable Na+-dependent uptake of ascorbate by cultures of UMR-106 rat osteosarcoma cells proceeded linearly with time for at least 10 min at 37 C. Exposure of cultures to TGF-beta(1) stimulated initial rates of saturable Na+-dependent ascorbate transport, but did not affect nonspecific uptake or binding of the vitamin. Cells pretreated for 24 h with either vehicle or TGF-beta(1) (3 ng/ml) and then assayed for transport of L-[C-14] ascorbate (10-mu-M) showed significantly different transport activities (vehicle, 30 +/- 2; TGF-beta(1), 44 +/- 3 nmol ascorbate/g protein/min; n = 14; P < 0.005). Kinetic studies revealed that TGF-beta(1) increased the maximum velocity of ascorbate transport without changing the affinity of the transporter for the vitamin, since the apparent maximum velocity increased from 83 to 106 nmol ascorbate/g protein/min, while the apparent K(m) remained unchanged at 20-mu-M L-ascorbate. The effect of this growth factor on ascorbate transport appeared to require protein synthesis, because it was completely blocked by cycloheximide. These results are consistent with TGF-beta(1) increasing the rate of synthesis of either new Na+ ascorbate cotransporters or a regulatory protein that interacts with existing transporters to increase their turnover number. Enhanced uptake of ascorbate may contribute to the increase in collagen synthesis induced by TGF-beta.