ALPHA-2-MACROGLOBULIN AND SERUM PREFERENTIALLY COUNTERACT THE MITOINHIBITORY EFFECT OF TRANSFORMING GROWTH FACTOR-BETA-2 IN RAT HEPATOCYTES

Abstract

Transforming growth factors-.beta.1 and .beta.2 (TGF-.beta.1 and TGF-.beta.2) are potent, multifunctional modifiers of cellular proliferation and differentiation in many cell types. To evaluate factors which may alter the activity of the TGF-.beta.s during hepatocyte proliferation, we examined the influences of bovine serum and purified bovine or human .alpha.2-macroglobulin (.alpha.2M) on the mitoinhibitory effects of the TGF-.beta. in primary cultures of rat hepatocytes. The inhibitory activity of TGF-.beta. was evaluated by autoradiographic labeling index at 48 hours in hepatocyte cultures exposed to [3H]thymidine between hours 24 and 48 in culture. In the absence of serum or .alpha.2M, TGF-.beta.1 and TGF-.beta.2 were equivalently potent in inhibiting S-phase DNA synthesis in hepatocytes cultured with or without epidermal growth factor. However, bovine serum and purified bovine or human .alpha.2M consistently counteracted the mitoinhibitory effects of TGF-.beta.2. S-phase DNA synthesis increased five- to six-fold when bovine serum (15%) or .alpha.2M (200 .mu.g/ml) were included with TGF-.beta.2 (0.1 ng/ml) and epidermal growth factor (20 ng/ml). The mitoinhibitory effect of TGF-.beta.1 was not influenced by the addition of purified bovine .alpha.2M or bovine serum counteracted inhibition by TGF-.beta.1 to a lesser extent. [125I]TGF-.beta.1 and 125I]TGF-.beta.2 formed complexes with purified bovine .alpha.2M and serum proteins migrating at identical positions to purified .alpha.2M during nondenaturing polyacrylamide gel electrophoresis. However, [125I]TGF-.beta.1 associated preferentially with the "fast" migrating form of .alpha.2M, whereas [125I]TGF-.beta.2 associated with both the "slow" and "fast" migrating forms. Inhibitory activity of TGF-.beta.1 and TGF-.beta.2 coeluted with .alpha.2M in the high molecular weight fractions from a Sephacryl S-200 column. These results support the hypothesis that purified .alpha.2M and .alpha.2M in bovine serum binds both TGF-.beta.s but preferentially counteracts the mitoinhibitory effect of TGF-.beta.2 on rat hepatocytes.