Leukemia Inhibitory Factor Binds with High Affinity to Preosteoblastic RCT-1 Cells and Potentiates the Retinoic Acid Induction of Alkaline Phosphatase

Abstract

This study examines the effect of leukemia inhibitory factor (LIF) on preosteoblastic rat calvaria (RCT-1) cells, which acquire osteoblastic properties when treated with retinoic acid (RA). LIF potentiated the increase in alkaline phosphatase (AP) activity produced by RA. The LIF effect was time and dose dependent (EC50, approximately 1 pM). The earliest effects on AP activity were detected at 48 h, and maximal effects were observed after 72 h. RA increased AP mRNA about 2-fold at 3 h and 6-fold at 6 and 12 h. LIF further increased AP mRNA to 18-fold at 12 h. After RA treatment AP mRNA returned to control levels at 24 h, but in the presence of LIF, AP mRNA remained elevated at 24 and 72 h of treatment. When given alone, LIF had no effect on either AP activity or mRNA levels. Tumor necrosis factor-alpha and 1,25-dihydroxyvitamin D3 also potentiated the RA induction of AP, and interleukin-6 had a small effect, whereas granulocyte macrophage colony-stimulating factor had no effect. LIF alone had a small inhibitory effect on type 1 collagen mRNA, but did not oppose the stimulatory effect of RA. Consistent with these biological actions, LIF receptors were demonstrated on these cells. [125I]LIF bound to RCT-1 cells at 0 C with an apparent dissociation constant of 20 pM, and it was found that these cells express an average of 300 receptors/cell. Scatchard analyses showed a single class of high affinity binding site. LIF was internalized with an endocytic rate constant for occupied receptors of 0.03 min-1, and the apparent equilibrium dissociation constant at 37 C was 358 pM. These findings suggest that osteoblast precursor cells are among the target cells of LIF.