Tumor necrosis factor (TNF) receptor-2-mediated DNA synthesis and proliferation in primary cultures of adult rat hepatocytes: The involvement of endogenous transforming growth factor-alpha.
We investigated the effects of tumor necrosis factor (TNF)-alpha on DNA synthesis and proliferation, and its signal transduction pathways in primary cultures of adult rat hepatocytes. TNF-alpha induced time- and dose-dependent increases in hepatocyte DNA synthesis and proliferation. The hepatocyte proliferation stimulated by 30 ng/ml TNF-alpha was significantly inhibited by anti-TNF receptor 2 antibody, but not by anti-TNF receptor 1 antibody. TNF-alpha-induced hepatocyte DNA synthesis and proliferation were blocked by AG1478 (10(-7) M), PD98059 (10(-6) M), LY 294002 (10(-7) M), and rapamycin (100 ng/ml). TNF-alpha at 30 ng/ml significantly increased phosphorylation of receptor tyrosine kinase (175 kDa) and p42 mitogen-activated protein (MAP) kinase. This data suggests that the proliferative signal for primary cultured hepatocytes induced by TNF-alpha is mediated by TNF receptor 2 and the receptor tyrosine kinase/MAP kinase pathway. In addition, TNF-alpha-induced hepatocyte mitogenesis was significantly blocked by somatostatin (10(-6) M), adenylate cyclase inhibitor dideoxyadenosine (10(-7) M), protein kinase A inhibitor H-89 (10(-7) M), and neutralizing antibody to transforming growth factor (TGF)-alpha in culture. Indeed, 30 ng/ml TNF-alpha was found to rapidly stimulate secretion of TGF-alpha, and this secretion was also blocked by anti-TNF receptor 2 antibody. Moreover, TGF-alpha secretion induced by TNF-alpha was suppressed by dideoxyadenosine, H-89, and somatostatin. Together, these results indicate that stimulation of TNF receptor 2 by 30 ng/ml TNF-alpha induces autocrine secretion of TGF-alpha via the adenylate cyclase/protein kinase A pathway, after which TGF-alpha induces hepatocyte DNA synthesis and proliferation through the TGF-alpha receptor-linked tyrosine kinase (175 kDa)/MAP kinase signaling system.
Okamoto
Hiroshi
H
Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Josai University. 1-1 Keyakidai, Sakado City, Saitama 350-0295, Japan.
Kimura
Mitsutoshi
M
Watanabe
Noriyuki
N
Ogihara
Masahiko
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D016211:Transforming Growth Factor alpha / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D047889:Receptors, Tumor Necrosis Factor, Type II; D016211:Transforming Growth Factor alpha; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases