Tumor necrosis factor-alpha acts as a complete mitogen for primary rat hepatocytes

The cytokine tumor necrosis factor (TNF)-alpha has previously been shown to prime hepatocytes to a state of replicative competence, but has not been shown to act as a complete mitogen for these cells. In the present study we have altered our previously described long-term dimethyl sulfoxide culture system to exclude all known hepatocyte mitogens from the culture media and enable us to directly examine the effects of TNF-alpha on primary rat hepatocytes. We have shown that cells maintained under these culture conditions retain the biochemical and morphological features of well-differentiated hepatocytes. Treatment with TNF-alpha induced DNA synthesis relative to control, to a level not significantly different from that induced by the known hepatocyte mitogen, epidermal growth factor (EGF). Maximal DNA synthesis was induced by treatment with 250 U/ml TNF-alpha for 24 hours. Mitotic figures were observed in cultures treated with TNF-alpha or EGF but not in untreated controls. Treatment of cultures with TNF-alpha, but not EGF, induced activation of both nuclear factor-kappaB p50 homodimers and p50/p65 heterodimers. DNA synthesis induced by TNF-alpha was inhibited by treatment with transforming growth factor-beta. Based on the results of our studies, we conclude that TNF-alpha acts as a complete mitogen for rat hepatocytes.