Vol.
2, 583-591,
November
1991
Cell Growth
& Differentiation
583
Release of H202 and Phosphorylation of 30 Kilodalton Proteins as Early Responses of Cell Cycle-dependent Inhibition of DNA Synthesis by Transforming Growth Factor fi,'
by TGF-31 was reported to be mediated by an increase in collagen secretion (16). The inhibition of growth of epithelial cells by TGF-31 is suggested to be associated with inhibition of c-myc expression (5). Recently, Laiho et a!. (4) found that TGF-f31 influenced the phosphorylation state of RB protein, which might be one cause of growth inhibition. However, many problems about its mechanism of action, including its primary mediators, are still unresolved. Active oxygens have been shown to be involved in the physiological responses of cells. At higher concentrations, they damage cellular components or structures, and thus are toxic to cells (17-19). However, accumulating evidence now suggests that active oxygens at low concentrations act as mediators of cellular responses and growth signals. Addition of H2O2, like addition of insulin, stimulated glucose transport and lipid synthesis in isolated rat adipocytes (20, 21). Pancreatic islet cells generate H2O2 on elevation of the cytoplasmic free Ca2 level and activation of protein kinase C (22). We reported previously that active oxygens induce the expression of c-los and c-myc mRNA and also DNA synthesis cooperatively with insulin (23, 24). Crawford et a/. (25) found that low concentrations of oxidants could act as a mitogenic signal in jB6 mouse epidermal cells. They found that ribosomal S6 proteins were phosphorylated in response to H2O2 or growth factors (26). We also observed phosphorylation of a specific protein in quiescent BALB/ 313 cells treated with H,O, (24). Generation of active oxygens by tumor promoters and growth factors has been observed in various experimental systems including ours (27-32). It thus seemed reasonable to speculate that H,O, acts as a physiological mediator of the actions of growth factors and tumor promoters. The present study showed that TGF-/31 inhibited DNA synthesis of MC3T3 mouse osteoblastic cells (33) in a cell cycle-dependent manner and that the signal may be mediated by release of H2O2 into the medium and phosphorylation of 30 kilodalton proteins.

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