During liver regeneration quiescent hepatocytes undergo one or two rounds of replication and then return to a nonproliferative state. Growth factors regulate this process by providing both stimulatory and inhibitory signals for cell proliferation. EGF, TGF alpha, and HGF stimulate DNA synthesis in hepatocytes in vivo and in culture but the sensitivity of cultured hepatocytes to the mitogenic effects of these factors is much higher than that of quiescent hepatocytes in intact livers. We have proposed that after partial hepatectomy, hepatocytes enter a state of replicative competence ("priming") before they can fully respond to growth factors. The priming step is an initiating event in liver regeneration that involves the activation and DNA binding of NF-kappa B and other transcription factors, which could be induced by TNF or other cytokines. EGF, TGF alpha, and HGF have major effects on liver growth. TGF alpha expression correlates with hepatocyte DNA synthesis during liver development and growth and the constitutive expression of the factor confers proliferative activity to adult hepatocytes in vivo and in culture. The data indicate that the activity of stimulatory and inhibitory growth factors such as TGF beta 1 and activin is low in normal livers but that the expression of both types of factors increase during liver regeneration.

We used KO mice lacking either TNF receptor 1 (TNFR-1) or receptor 2 (TNFR-2) to determine whether signaling at the start of liver regeneration after partial hepatectomy (PH) involves only one or both TNF receptors and to analyze in more detail the abnormalities caused by lack of TNFR-1 receptor, which is required for the initiation of liver regeneration. Lack of TNFR-2 had little effect on NF-B and STAT3 binding, and no effect in interleukin-6 production after PH, but caused a delay in AP-1 and C/EBP binding and in the expression of c-jun and c-myc messenger RNA (mRNA). In contrast to mice lacking TNFR-1, which had deficient hepatocyte DNA synthesis and massive lipid accumulation in hepatocytes, TNFR-2 KO mice had normal liver structure and similar levels of hepatocyte DNA replication as those of wild type mice. We conclude that TNFR-1, but not TNFR-2, is necessary for liver regeneration, and that NF-B and During liver regeneration after partial hepatectomy, more than 95% of hepatocytes replicate in a semisynchronous process that ultimately restores the liver mass.

Signaling through tumor necrosis factor receptor type 1 (TNFR-1) using a pathway that involves nuclear factor B (NF-B), interleukin-6 (IL-6), and STAT3 is required for the initiation of liver regeneration. We have proposed that TNF primes hepatocytes to respond to the mitogenic effect of growth factors, but so far, there has been no experimental demonstration that TNF enhances growth factor responses of hepatocytes. To test this hypothesis, we infused hepatocyte growth factor (HGF) and transforming growth factor ␣ (TGF-␣) (40 g/24 h) directly into the portal vein of rats for 24 hours using osmotic pumps and determined whether TNF injection (5 g per rat) would significantly increase hepatocyte DNA labeling in these animals. All rats received 5-bromo-2Ј-deoxyuridine (BrdU) by intraperitoneal delivery during a 48-hour period (i.e., BrdU infusion continued for 24 hours after the end of growth factor administration). Although highly differentiated and normally quiescent, hepatocytes retain an impressive potential for proliferation. This is well demonstrated in vivo by the regeneration of the liver following the removal of tissue by partial hepatectomy (PH) or in response to hepatocyte destruction by toxic agents. 1-3 Hepatocyte proliferation during liver regeneration is mediated by several growth factors, of which hepatocyte growth factor (HGF) and transforming growth factor ␣ (TGF-␣) (a member of the epidermal growth factor [EGF] family) appear to be most important. These growth factors are potent mitogens for hepatocytes in culture, each capable of stimulating hepatocyte DNA synthesis by fivefold or more. [1][2][3][4][5][6][7] Direct infusion of these growth factors into the liver of rats does not, however, cause a major wave of DNA synthesis. 8,9 Although it is possible to obtain hepatocyte replication after infusion of HGF into intact rats, quantitatively significant effects are only achieved by prolonged infusion in the presence of dextran or by multiple injections. 10,11 We have shown previously that a 24-hour infusion of up to 80 µg of HGF, TGF-␣, or EGF into rat livers elicited DNA synthesis in no more than 1% to 2% of hepatocytes at the end of the infusion period. 8 We also demonstrated that a much higher mitogenic response could be obtained by infusion of HGF in rats in which a 30% hepatectomy had been performed immediately before the infusion. 8 In contrast to the standard 70% hepatectomy, removal of 30% of the liver 12 by itself did not cause a wave of DNA synthesis under the conditions of the experiment. 8 Based on these data, we proposed that hepatocytes in vivo have low sensitivity to growth factors and that to effectively respond to these factors, hepatocytes must undergo a set of changes referred to as ''priming. '' 13 Other work has shown that a priming state can be obtained in normal rat liver hepatocytes by nutritional modification 14 or by perfusing livers with small amounts of collagenase. 15

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Although growth factor effects have been studied in cultured hepatocytes, little information exists as to whether these factors can trigger hepatocyte replication in vivo. In this study we infused epidermal growth factor, transforming growth factor-alpha and hepatocyte growth factor directly into the portal vein of rats for 24 hr to see whether they could induce DNA synthesis in normal livers or in livers subjected to one-third hepatectomy. Infusion of transforming growth factor-alpha or epidermal growth factor at doses up to 80 micrograms/24 hr had little effect on hepatic DNA synthesis in normal liver, whereas the monomeric and heterodimeric forms of hepatocyte growth factor generally produced increases of less than threefold in hepatic DNA synthesis. In contrast, after one-third hepatectomy infusion of epidermal growth factor, transforming growth factor-alpha or hepatocyte growth factor produced dose-dependent increases in hepatic DNA synthesis. At a dose of 40 micrograms/24 hr, epidermal growth factor increased DNA synthesis threefold, whereas transforming growth factor-alpha or hepatocyte growth factor increased DNA synthesis to greater than six times that in rats that had undergone hepatectomy alone. Furthermore, infusion of these growth factors, with or without one third-hepatectomy, induced the expression of transforming growth factor-alpha mRNA in the liver. The pattern of protooncogene expression induced by one-third hepatectomy was studied to determine the effect of this procedure in sensitizing the liver to the growth factors. Compared with the well-characterized two-thirds hepatectomy system, there was a similar but smaller increase in c-myc expression but no induction of c-jun expression.(ABSTRACT TRUNCATED AT 250 WORDS)