The Binding of rhBMP‐2 to the Receptors of viable MC3T3‐E1 Cells and the Question of Cooperativity

The binding of rhBMP‐2 to its receptors, the signal transduction cascade and the final responses of bone cells, osteoprogenitor cells and derived cell lines is of high fundamental and clinical interest. In this report concentration‐response curves of the osteoblast cell line MC3T3‐E1 under influence of rhBMP‐2 was investigated. The biological response of the cells (corresponding to a down‐stream effect of the receptor state‐function) was monitored in pilot experiments by the MC3T3‐cell alkaline phosphatase‐induction test (MC3T3‐cell ALP‐induction test). It is shown that the MC3T3‐cell ALP‐induction test is a good tool for measuring biologically active recombinant human BMP‐2 (rhBMP‐2) in crude extracts of E. coli as well as in highly purified form. In addition this test is very sensitive to chemically induced structural changes of rhBMP‐2 such as those resulting from a radiolabeling of rhBMP‐2 by the Bolton‐Hunter procedure. The latter procedure reduces the biological activity of rhBMP‐2 by a factor of 3–4. The measured concentration‐response curves could all be non‐linearly fitted to a rectangular hyperbola. The half‐maximal saturation, K_0.5, is found between 30–100 nM rhBMP‐2 (= 0.8–2.5 μg/ml). The effect of rhBMP‐2 shows a plateau i.e. maximal response at ca. 300–1000 nM rhBMP‐2 (= 8–25 μg/ml). The data thus indicate a non‐cooperative binding‐response behavior. This was unexpected since BMP‐2 binds simultaneously to two cooperating receptors of type 1 and type 2. However in the very low concentration range of employed rhBMP‐2 a variable response of the cells was measured so that a full exclusion of cooperativity cannot be concluded at the present time. This will be clarified by future experiments.