Fetal hepatic maturation consists of multisteps and is regulated by several cytokines and cell–cell or cell–matrices interactions. In the mid-to-late fetal stage, hepatocytes have few metabolic functions associated with adult liver homeostasis. Cultured fetal hepatocytes acquire the expression of several mature liver-specific genes through stimulation with hepatic maturation factor oncostatin M (OSM) and matrigel. Tumor necrosis factor-α (TNFα) regulates fetal hepatic maturation stimulated by OSM and matrigel. TNFα suppressed expression of mature liver-specific genes such as tyrosine aminotransferase and apolipoproteins. In addition, the expression of hematopoietic cytokines and cyclin A2, repressed by OSM and matrigel, is induced by TNFα in the fetal hepatic cultures coincident with cell division. TNFα inhibited the induction of hepatocyte nuclear factor 4α induced by OSM and matrigel, suggesting that down-regulation of hepatocyte nuclear factor 4α expression is involved in the mechanism of suppression of hepatic maturation by TNFα. Interestingly, TNFα is expressed in the prenatal and postnatal liver but not in adult liver, whereas TNFR1, a TNFα receptor, is expressed in both fetal and adult livers. In conclusion, TNFα is a suppressive factor of hepatic maturation. The balance between hepatic maturation factor (OSM and extracellular matrices) and TNFα is important for liver development. (HEPATOLOGY 2004;40:527–536.)
Tumor necrosis factor-α (TNFα) was first identified as a factor capable of killing tumor cells in vitro and causing hemorrhagic necrosis of transplantable tumors in mice.1, 2 TNFα is involved in homeostatic and physiological functions by influencing cell proliferation and differentiation, while causing apoptotic cell death in certain cell types. These responses are mediated through binding of TNFα to its receptor, TNFR1. TNFα binds to a trimer of TNFR1 and activates three distinct signaling pathways: death-signaling, nuclear factor (NF)-κB activation, and SEK1- and MKK7-mediated JNK activation.
Adult liver is a central organ for intermediary metabolism, whereas fetal liver has few metabolic functions associated with adult liver. Instead, it functions as the major hematopoietic organ in the mid-gestation to late-gestation fetus.3–5 Embryonic liver formation consists of multiple stages and is regulated by hormonal factors as well as intercellular and matrix cellular interactions. In the beginning of liver development, the liver primordium proliferates and invades the mesenchyme of the septum transversum to give rise to the hepatic cords and buds.6, 7 Then, fetal hepatocytes proceed through a series of maturation steps that are accompanied by a decrease in hematopoietic activity and an increase in the expression of several genes associated with liver maturation such as glucose-6-phosphatase (G6Pase) and tyrosine aminotransferase (TAT).8, 9 The final stage of differentiation takes place after birth, and the fully matured liver expresses adult liver-specific enzymes.
The liver has a unique capacity to regulate cell growth.10, 11 Hepatocytes rarely divide in their normal state, whereas their proliferative capacity and the ability of the liver to adapt to variable metabolic functions are not lost. It was reported that several growth factors and cytokines are important for liver regeneration in vivo. In particular, TNFα is important for the priming step in liver regeneration.12, 13 In addition, several studies using knockout mice revealed that TNFα signaling molecules are involved in fetal liver development. Knockout mice lacking expression of genes regulating the NF-κB signal pathways exhibit massive liver degeneration and apoptosis during midfetal gestation.14–19 Interestingly, the embryonic lethality and liver apoptosis in these knockout mice can be rescued by inactivation of TNFR1, indicating that the TNFα signaling can result in two opposing pathways, depending on cellular context: one initiating programmed cell death whereas the other activating survival responses through NF-κB.18, 20
It was previously determined that oncostatin M (OSM), an interleukin 6 (IL-6) family cytokine, in the presence of glucocorticoid hormones promotes the maturation of fetal hepatic cells derived from the embryonic day 14 (E14) livers in vitro.21 Extracellular matrices such as matrigel enhance hepatic maturation induced by OSM.22 However, although midfetal hepatocytes support hematopoiesis in vitro, induction of hepatic maturation by OSM reduces such activity.23 As described previously,24 the matured bone marrow may attract hematopoietic cells by secreting chemotactic factors to establish adult-type bone marrow hematopoiesis near birth. OSM is produced by the hematopoietic cells expanding in the fetal liver and induces fetal hepatic maturation. Thus, OSM induces a mature liver microenvironment unfavorable for hematopoietic cells, resulting in a shift of hematopoietic organs from the fetal liver to the adult bone marrow and other organs. However, it remains unknown whether another cytokine existing in the midfetal and postnatal livers, such as TNFα, influences functions of the fetal liver.
In this report, the effect of TNFα in the liver development was studied using primary fetal hepatic cultures. The addition of TNFα in the culture induced the suppression of hepatocyte nuclear factor 4α (HNF4α) and several adult liver-specific genes. Stimulation by OSM and matrigel suppressed expression of cyclin A2 required for the S-G2 progression. However, TNFα inhibited the suppression of this gene by OSM and matrigel. During embryonic development, the expression of TNFα is detected from midfetal to postnatal livers. These results suggest that TNFα is involved in the fetal liver development through the suppression of hepatic maturation.
TNFα, tumor necrosis factor-α; NF-κB, nuclear factor-κB; G6Pase, glucose-6-phosphatase; TAT, tyrosine aminotransferase; OSM, oncostatin M; IL-6, interleukin 6; E, embryonic day; HNF, hepatocyte nuclear factor; C/EBP, CCAAT/enhancer-binding protein; BrdU, bromo-2′-deoxyuridine; mRNA, messenger RNA; Bsep, bile salt export pump; CPS, carbamoylphosphate synthetase-1; M-CSF, macrophage colony-stimulating factor; MCP-1, macrophage chemoattractant protein 1.
Materials and Methods
Liver-specific HNF4α and CCAAT/enhancer-binding protein α (C/EBPα) knockout mice were generated by Cre-loxP–mediated deletion (in which the Cre gene is under the control of the albumin promoter).25, 26 All embryonic hepatocyte culture experiments were performed with HNF4α-flox/flox (FLOX) E14 mice. Dulbecco's modified eagle medium, modified eagle medium nonessential amino acid solution, antibiotic-antimycotic solution, and insulin-transferrin-selenium X were purchased from Gibco-BRL (Rockville, MD). Fetal bovine serum was purchased from HyClone (Logan, UT). Collagenase, dexamethasone, bromo-2′-deoxyuridine (BrdU), and mouse TNFα were purchased from Sigma (St. Louis, MO). Mouse OSM was from R&D Systems (Minneapolis, MN). Matrigel (Growth Factor Reduced Matrigel Matrix) was purchased from BD Biosciences (Bedford, MA). The recombinant adenovirus expressing Cre was described earlier.26
Production of fetal hepatic cultures derived from E14 mouse livers was described previously.27 Briefly, minced embryonic liver tissues were dissociated with liver digest medium (0.05% collagenase solution) followed by hemolysis with hypotonic buffer. Cells were cultured in hepatocyte culture media (Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1× modified eagle medium nonessential amino acid solution, 1× insulin-transferrin-selenium X, 1× antibiotic-antimycotic solution, and 10−7 M dexamethasone) on 0.1% gelatin-coated tissue culture dishes. Several hours later, the cells were washed with culture medium to remove contaminating hematopoietic cells and cell debris. The culture medium was replaced every 2 days. For induction of fetal hepatic maturation, 10 ng/mL OSM was added on the first day and matrigel was then overlaid on day 3 (5 days culture) or 5 (7 days culture). To overlay matrigel, the culture medium was removed and matrigel, diluted in ice-cold hepatocyte culture media at a volume ratio of 1:7 (matrigel/medium), was added to the culture dishes. Control cultures received ice-cold media only. Cells were harvested on day 5 or 7 for analyses of gene expression. The reproducibly of this study was checked by two independent experiments.
All adenoviruses used in this study were E1a deletion recombinants and were propagated in 293 cells.28 Ad-LacZ encodes for the LacZ reporter gene and Ad-Cre for Cre-recombinase. Adenovirus infection at an moi of 3 was performed as shown previously.27
Northern Blot Analysis.
Cellular and tissue total RNA samples were extracted with Trizol reagent (Invitrogen, Carlsbad, CA). Ten micrograms of total RNA from each sample was separated by electrophoresis on a 1.5% agarose gel containing 2% formaldehyde. RNA was transferred to GeneScreen Plus membranes (Dupont, Wilmington, DE), and the blots were hybridized with 32P-labeled cDNA probes generated by random-prime labeling reactions. The membranes and probes were incubated at 65°C in PerfectHyb Plus hybridization buffer (Sigma), and after washing, the blot was exposed to a PhosphorImager screen cassette and the signals were visualized using a Molecular Dynamic Storm 860 PhosphorImager system (Sunnyvale, CA). All probe cDNAs were amplified from a mouse liver cDNA library by using gene-specific primers and cloned into pCR TOPOII (Invitrogen). The identity of the probes was confirmed by nucleotide sequencing.
Messenger RNA Detection of Reverse-Transcriptase Polymerase Chain Reaction.
Total RNA was extracted from livers derived from E14, E16, neonatal, 9-, 14-, 21-day-old and adult mice using Trizol. First-strand cDNA was synthesized from 5 μg of total RNA using the Superscript II reverse transcriptase (Invitrogen) and was used as a template for polymerase chain reaction amplification. Murine TNFα primers (forward primer: 5′-CAC-GTC-GTA-GCA-AAC-CAC-CAA-3′; reverse primer: 5′-CCC-ATC-CCC-TTC-ACA-GAG-CAA-3′) and murine TNFR1 primers (forward primer: 5′-TAC-ATC-CAT-CAG-GGG-TCA-CTG-G-3′; reverse primer: 5′-TGT-CCT-TGT-CAG-CTT-GGC-AAG-3′) were used for polymerase chain reactions. The primers were annealed at 50°C for 45 seconds and amplification was repeated for approximately 40 to 45 cycles. Amplified products were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide.
Cell Proliferation Assays.
BrdU incorporation was used to monitor cell proliferation. Fetal hepatocytes were cultured for 3 days with or without OSM, and hepatic maturation was stimulated by the overlay of matrigel in the presence or absence of TNFα. After 5 days of culture, the cells were treated with 10 μM of BrdU for 2 hours. After fixation with 3.7% formaldehyde in phosphate-buffered saline, the cells were incubated with mouse anti-BrdU antibody (DAKO, Glostrup, Denmark) in 0.5% bovine serum albumin and phosphate-buffered saline. Subsequently, the cells were incubated with biotin-conjugated goat anti-mouse immunoglobulin G using the Vectastain ABC Elite Kit (Burlingame, CA). Finally, cells were counterstained with hematoxylin.
DAPI Fluorescence for Detecting Apoptotic Cells.
Cells were fixed with 4% formaldehyde for 20 minutes. After two washing steps with 1 mg/mL bovine serum albumin in phosphate-buffered saline, hepatocytes were permeabilized using the 0.2% Triton X-100, 1 mg/mL bovine serum albumin in phosphate-buffered saline. DNAs were stained in a solution containing 10 μg/mL DAPI. The number of cells with an apoptotic nuclear morphological features was counted.
Western Blot Analyses.
Fetal hepatic cells were cultured for 5 days, and nuclear extracts were purified using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL). Eight micrograms of extract protein was mixed with sodium dodecyl sulfate sample loading buffer containing β-mercaptoethanol, electrophoresed on a 15% sodium dodecyl sulfate-polyacrylamide gel and electrotransferred onto a Hybond-P membrane (Amersham, Piscataway, NJ). The membranes were blocked with Tris-buffered saline with Triton X-100 (20 mM Tris-HCl, 150 mM NaCl, 0.05% Triton X-100) containing 3% bovine serum albumin and incubated with a primary antibody in Tris-buffered saline with Triton X-100. The membranes then were washed with Tris-buffered saline with Triton X-100 and incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After another wash with Tris-buffered saline with Triton X-100, the immunoreactive proteins were developed by use of the ECL reagent (Amersham), and blots were exposed to a Bio-Max film (Kodak, Rochester, NY). Goat polyclonal anti-HNF4α and rabbit polyclonal anti-C/EBPα antibodies (Santa Cruz) were used as primary antibodies.
Expression of TNFα and TNFR1 in the Developing Liver.
In the adult liver, TNFα is mainly expressed during liver injury or inflammation.29 In contrast, TNFα is involved in embryonic liver development, because knockout mice lacking the expression of several proteins involved in NF-κB signal transduction are embryonically lethal because of enhance hepatic apoptosis, and this lethality can be rescued by inactivation of TNFR1.18, 20 We therefore examined whether the expression of TNFα and TNFR1 is detected in developing mouse livers. Total RNAs isolated from E14, E16, neonatal, 9-, 14-, 21-day-old, and adult livers revealed the presence of both TNFα and TNFR1 messenger RNA (mRNA; Fig. 1A). TNFα mRNA was detected from midfetal livers and continued to be expressed in 14-day-old mouse livers. In contrast, 21-day-old and the adult liver express low levels of TNFα mRNA. However, TNFR1 mRNA expression continued to be expressed in the adult liver. These data suggest that TNFα is expressed mainly in the prenatal and postnatal livers.
Suppression of Expression of Mature Liver-Specific Genes in Fetal Hepatic Cultures Stimulated by TNFα
The expression of mature liver-specific genes is strictly regulated during hepatic development, and striking changes occur around the perinatal stage. The expression of TAT was detected in the neonatal liver, and this expression was increased in the postnatal and adult livers (Fig. 1B). Carbamoylphosphate synthetase-1 (CPS) was expressed transiently in the neonatal liver, whereas its expression was low in the postnatal liver and was upregulated along with liver development. In contrast, the expression of bile salt export pump (Bsep) was not significantly changed in neonatal, postnatal, and adult livers.
To determine the effects of TNFα on fetal liver maturation, a primary hepatocyte culture system derived from E14 fetal livers was used. Cells were cultured for 5 or 7 days in the presence or absence of 50 ng/mL TNFα in combination with hepatic maturation factors OSM and matrigel, and mRNAs for mature liver-specific genes were analyzed by Northern blotting. As shown previously, the expression of glucose-6-phosphatase, TAT, and CPS was barely detected in E14 fetal hepatocytes; however, the expression of these genes was induced significantly by the addition of OSM and matrigel, showing that hepatic maturation was induced by these factors.22 TNFα treatment repressed the expression of these genes after 5 and 7 days in culture of fetal hepatic cells (Fig. 2). Other mature liver-specific genes, such as those encoding Bsep and apolipoproteins AI and AIV, also were suppressed by TNFα. Interestingly, glucokinase was not changed by TNFα treatment, suggesting that the suppression of liver maturation was not caused by nonspecific cell death and that the mechanism of expression of mature liver-specific genes was different between these genes. In addition, the suppression of hepatic maturation by TNFα was dose dependent (Fig. 2A, right panel). To determine whether continuous stimulation by TNFα was required for the suppression of hepatic maturation, fetal hepatocytes were cultured with OSM and matrigel and were treated with TNFα for 1 to 3 days or 3 to 5 days of culture (Fig. 2B). The 1 to 3 day stimulation by TNFα did not result in the downregulation of expression of mature liver-specific genes. However, TNFα treatment between day 3 to 5 was sufficient for suppression of liver maturation in fetal hepatic cells (Fig. 2B). These data suggested that in vitro hepatic maturation was repressed by only a short period of stimulation by TNFα.
OSM and TNFα Have Opposite Roles in Fetal Liver Development.
Fetal hepatocytes have low level expression of the metabolic functions found in fully differentiated adult livers. However, the fetal liver environment is important for hematopoiesis. The growth and chemotactic activities of hematopoietic cells are regulated by several cytokines and chemokines. Previous results indicated that OSM inhibited the expression of macrophage colony-stimulating factor (M-CSF) and macrophage chemoattractant protein 1 (MCP-1) mRNA in fetal hepatic cells.23 Therefore, the effects of TNFα on expression of M-CSF and MCP-1 mRNA were examined. Stimulation by OSM and matrigel downregulated the expression of M-CSF and MCP-1 mRNA (Fig. 3A). In contrast, the expression of these genes was significantly induced by the addition of TNFα in the fetal hepatic cultures. OSM is an IL-6 family cytokine, and TNFα and IL-6 are known as proinflammatory cytokines; however, these results suggest that OSM and TNFα have opposite roles in fetal liver development. As shown above, the inhibitory effect of TNFα in fetal hepatic maturation was detected by the last 2 days of stimulation. Expression of M-CSF and MCP-1 also was induced by a short-term stimulation by TNFα (Fig. 3B), suggesting the existence of a correlation between the suppression of hepatic maturation and the induction of hematopoietic cytokines and chemokines by TNFα.
TNFα Regulates Hepatic Maturation Induced by OSM and Matrigel Through Modulation of the Cell Cycle.
It is known that cell cycle regulation is important for the induction and maintenance of cell differentiation. During cell differentiation, cell growth is suppressed and the expression of several cyclins is downregulated. Expression of cyclins D1 and D2, the G1-S cyclins, is decreased by OSM in fetal hepatic culture.30 In addition, when hepatic maturation is induced by OSM and matrigel in fetal hepatic cells, cyclin A2, an S-G2 cyclin, was downregulated (Fig. 4A), suggesting that OSM and matrigel affect progression of the cell cycle and induce hepatic maturation. The expression of cyclins D1 and D2 was not significantly changed by treatment with TNFα, whereas stimulation of cells with TNFα resulted in elevated expression of cyclin A2 suppressed by OSM and matrigel in fetal hepatic primary cultures. Next, cell proliferation activity in this culture system was assessed using the BrdU incorporation assay that measures DNA synthesis. Numerous BrdU-positive cells were detected in hepatocytes cultured with only dexamethasone. In contrast, stimulation with OSM and matrigel significantly reduced the relative proliferation of fetal hepatocytes (Fig. 4B). The addition of TNFα recovered the proliferation of fetal hepatocytes inhibited by OSM and matrigel. These data suggest that TNFα regulates hepatic maturation through the control of cell proliferation.
The Suppression of Fetal Liver Maturation by TNFα Was Not the Result of Elevated Apoptosis of Hepatocytes.
TNFα mediated apoptosis in lymphocytes and other cells.31 In cultured hepatocytes infected with an adenovirus expressing a dominant NF-κB repressor, TNFα-induced apoptosis and necrosis is preceded by the opening of high-conductance mitochondrial pores.32 In contrast, TNFα alone has no activity on cell survival in primary hepatocytes and in a murine hepatocyte cell line.33, 34 To determine whether the effect of TNFα in fetal hepatocytes is the result of apoptosis of hepatocytes, chromosomal DNA was stained using DAPI (Fig. 5A). In a previous report, AML12 cells, a nontransformed murine hepatocyte cell line, were found to be insensitive to cell death induced by TNFα alone, but died by apoptosis when exposed to TNFα and a small dose of actinomycin D.34 Similar to these results, fetal hepatic cells exposed to a combination of TNFα and actinomycin D were killed by apoptosis as revealed by apoptotic nuclear morphology visualized by DAPI fluorescence (Fig. 5). In contrast, TNFα alone did not significantly induce cell death in fetal hepatic primary cultures. These results suggest that the suppression of hepatic maturation induced by TNFα in this culture system was not the result of nonspecific cell death.
Role of Liver-Enriched Transcription Factors in the Suppression of Fetal Hepatic Maturation by TNFα.
The expression of mature liver-specific genes is regulated mainly by several liver-enriched transcription factors such as HNF1α, 3α, β, γ, 4α, 6, and the C/EBPs. Several studies revealed that HNF4α is important for both liver development and adult liver functions. As examples, adult liver-specific disruption of the HNF4α gene results in several defects in liver function.25, 35 HNF4α is induced by OSM and matrigel in this culture system.27 However, HNF4α induction was suppressed by the addition of TNFα (Fig. 6A). In contrast, other liver-enriched transcription factors, such as HNF1α, C/EBPα, and C/EBPβ, were not changed significantly by treatment of fetal hepatocytes with TNFα. OSM and matrigel induced the production of HNF4α protein, and TNFα inhibited this upregulation (Fig. 6B). In contrast, the expression of C/EBPα was not affected by TNFα. Thus, TNFα may suppress hepatic maturation through direct or indirect regulation of the liver-enriched transcription factor, HNF4α.
The Inactivation of HNF4α in Fetal Hepatocytes Inhibits Expression of Several Mature Liver-Specific Genes.
The expression of HNF4α is induced by OSM and matrigel and TNFα suppressed this induction of HNF4α production. To determine whether the expression of mature liver-specific genes is regulated by HNF4α in this culture system, the in vitro HNF4α conditional knockout system was used.27 E14 fetal hepatocytes derived from HNF4α-floxed mice were cultured, and HNF4α inactivation was carried out with a recombinant adenovirus carrying the Cre gene (Ad-Cre). Fetal hepatic maturation was then induced by OSM and matrigel. HNF4α inactivation significantly suppressed the expression of apolipoprotein AIV and G6Pase similar to the suppression of expression in fetal hepatocytes stimulated by TNFα (Fig. 7). However, other TNFα-repressed genes such as TAT and apolipoprotein AI were not inhibited by the inactivation of HNF4α, suggesting that the HNF4α suppression partially contributed the inhibition of hepatic maturation by TNFα.
In this study, fetal hepatic maturation induced by hepatic maturation factor, OSM, and matrigel was suppressed by the addition of TNFα to fetal hepatic primary cultures. The expression of several adult liver-specific genes was downregulated by TNFα; however, the expression of some cytokines and cell cycle molecules, inhibited during in vitro fetal hepatic maturation were induced by TNFα. It is noteworthy that TNFα is expressed primarily during the perinatal and postnatal stages in liver. However, OSM, expressed in the late-fetal liver, and matrigel are important for postnatal liver development.21 Thus, both the hepatic maturation inducer (OSM and matrigel) and the putative hepatic maturation suppressor (TNFα) coexist in the developing perinatal liver. A balance between these factors may control the extent of maturation of fetal hepatocytes (Fig. 8).
The molecular mechanism of suppression of hepatic maturation by TNFα is an interesting question. The downregulation of HNF4α mRNA and protein by TNFα is one potential mechanism suppressing fetal hepatic maturation. In addition, TNFα may regulate the activation of HNF4α directly. In this connection, treatment of HepG2 cells with TNFα reduced transcription of the rat CYP7A1 promoter.36 This repression could be the result of the suppression of the HNF4α activity by phosphorylation through the MEKK1 signaling pathway. However, some genes, such as TAT and apolipoprotein AI, are controlled by transcription factors distinct from HNF4α (Fig. 7).25 STAT3 is important for hepatic maturation induced by OSM such as expression of TAT.30, 37 However, TNFα did not change the phosphorylation status of STAT3 in fetal hepatocytes induced by OSM (data not shown). Thus, there may be another mechanism, independent of STAT3 and HNF4α, involved in the inhibition of hepatic maturation by TNFα.
TNFα is expressed in the perinatal and postnatal (until 14 days old) livers and decreased in the adult mouse. During these stages of development, the expression of several mature liver-specific genes such as TAT and CPS were detected and were found to increase coincident with liver maturation. During the postnatal stage, the liver is significantly expanded coincident with development growth of the mature mouse. Thus, it is possible that expression of TNFα in the postnatal liver is involved in the proliferation of hepatocytes during the early postnatal stages of liver development and that the downregulation of TNFα in 21-day-old mice is associated with increased of expression of TAT and CPS in vivo. The cell cycle is important for regulating cell differentiation and is tightly controlled by cell cycle regulatory molecules including cyclins. Expression of cyclin D1 is downregulated by OSM in fetal hepatocytes,30 whereas OSM and matrigel-induced suppression of cyclin D1 and D2 could not be recovered by treatment with TNFα. In contrast, the expression of cyclin A2, an S-G2 cyclin, was suppressed by the addition of OSM and matrigel, and this suppression was recovered by the addition of TNFα. A previous report revealed that TNFα induces cell proliferation in rat primary hepatocytes.38 In this study, TNFα regulated DNA synthesis in fetal hepatocytes treated with OSM and matrigel. These results suggest that direct or indirect control of cell cycle by TNFα is involved in the suppression of fetal hepatic maturation.
Regeneration is a unique capacity of liver to regulate its growth and mass. Under normal conditions, hepatocytes rarely divide and are stimulated by several cytokines and mitogens expressed during liver regeneration. A large number of genes exhibit increased expression during the first step of liver regeneration, and TNFα is thought to be required for priming liver regeneration.12, 13, 39 Indeed, TNFR1-null mice have markedly reduced DNA replication after partial hepatectomy and significant mortality 24 to 40 hours after the operation. This severe inhibition of liver regeneration by the lack of TNFR1 signaling was rescued by a single injection of IL-6. In addition, liver regeneration is impaired in IL-6 knockout mice and IL-6 administration corrected the defects in DNA replication by STAT3 activation during liver regeneration.40 These results suggested that TNFα is important for the first step of liver regeneration through the induction of IL-6. In addition, TNFα acts as a primer to sensitize hepatocytes to the proliferative effects of growth factors. Previous reports revealed that preinjection of TNFα into the portal vein of rats significantly induced hepatocyte DNA replication induced by the injection of hepatocyte growth factor and transforming growth factor.41 Hepatocytes exist in G0in vivo but retain the capacity to enter the cell cycle in response to specific stimuli. The present study revealed that TNFα has a suppressing activity for hepatic maturation, suggesting that TNFα sensitizes the cells to growth factors through the induction of cell dedifferentiation. Furthermore, the addition of TNFα rescued the proliferation of fetal hepatocytes inhibited by the hepatic maturation factors, OSM and matrigel. This control of cell differentiation and dedifferentiation by TNFα may be important for both fetal liver development and adult liver regeneration. However, as noted above, TNFα mainly induces the priming of hepatocytes through the induction of IL-6 production during liver regeneration. OSM, an IL-6-related cytokine, has an opposite role in fetal liver development compared to TNFα. Interestingly, IL-6 and other IL-6-related cytokines, such as IL-11 or leukemia inhibitory factor, in contrast to OSM, have no effect on fetal liver development, indicating that the activity to induce hepatic maturation is specific to OSM.21 Adult hepatocytes are sensitivity to both OSM and IL-6. OSM, in addition to IL-6 and TNFα, is significantly induced during the first step of liver regeneration.42 In conclusion, these studies reveal that TNFα has a suppressive activity on hepatic maturation induced by OSM. The interaction of OSM and TNFα also may be important in the control of adult liver functions, in particular, liver regeneration.