Functional Expression of the Human Receptor for Colony-Stimulating Factor 1 (CSF-1) in Hamster Fibroblasts: CSF-1 Stimulates Na⁺/H⁺ Exchange and DNA-Synthesis in the Absence of Phosphoinositide Breakdown

Abstract

The human CSF-1 receptor (c-fms protooncogene product) was introduced into CSF-1-unresponsive Chinese hamster lung fibroblasts (CCL39 cell line) in order to study its coupling to biochemical signal-transducing systems and to compare the growth-regulating properties of CSF-1 to those of other growth factors. Independent clones expressing different levels of CSF-1 receptors were isolated and characterized. CSF-1 increased [³H]thymidine incorporation in serum-starved cells and potentiated the mitogenic effects of FGF and thrombin. As already observed for other growth factors activating receptor tyrosine kinases (EGF, FGF, IGF-I), CSF-1 alone did not trigger inositol phosphate formation, but slightly enhanced the activity of phospholipase C agonists (thrombin, AIF₄- complex). Activation of the CSF-1 receptor by its ligand was evidenced by the rapid activation of the Na⁺/H⁺ exchanger resulting in amiloride-sensitive cytoplasmic alkalinization (0.1–0.2 pH units) within minutes after stimulation. Whereas pertussis toxin does not affect the action of EGF, FGF, or IGF-I in CCL39 cells, it partially inhibited both DNA synthesis reinitiation and activation of Na⁺/H⁺ exchange by CSF-1, indicating that the CSF-1 receptor can communicate with a signal-transducing GTP binding protein. A point-mutated form of the c-fms gene product, in which Tyr 969, a residue negatively modulating signal transduction, had been replaced with Phe [fms (F969)], did not generate responses significantly different from those obtained with the wild-type c-fms gene product. In the absence of CSF-1, cells expressing either wild-type or fms (F969) showed a considerably higher basal level of thymidine incorporation and decreased anchorage dependence compared with parental CCL39 cells. Monoclonal antibodies that interfere with signal transduction by the human CSF-1 receptor inhibited both basal [³H]thymidine incorporation and soft agar colony formation, indicating that relaxation of growth control was dependent on CSF-1 receptor expression.

Key Words:

• receptor tyrosine kinase
• signal transduction
• growth control
• transformed phenotype
• fibroblasts