Regulation Of Hepatocyte Proliferation Research Articles

Hepatocyte Nuclear Factor 4 alpha (HNF4α) is involved in regulation of hepatocyte proliferation

HNF4α is the master regulator of hepatocyte differentiation and controls expression of a wide variety of hepatocyte‐specific genes. Here we report that HNF4α is involved in inhibiting hepatocyte proliferation. Decreased HNF4α expression was observed in hepatocyte specific integrin linked kinase knockout mice (ILK‐KO), which have sustained cell proliferation resulting in hepatomegaly. Following partial hepatectomy (PH), the ILK‐KO mice exhibited enhanced hepatocyte proliferation resulting in 60% higher liver mass as compared to their pre‐PH liver mass. The increased proliferation in ILK‐KO mice after PH was accompanied by further decrease in HNF4α expression. Further analysis revealed decrease in expression of HNF4α targets such as p21 and claudin1, which are involved in cell‐cycle regulation in ILK‐KO livers. Hepatocyte specific knockdown of HNF4α in HNF4α‐floxed mice using Cre recombinase carried by a adeno‐associated virus 8 (AAV‐8) vector resulted in induction of cell proliferation accompanied by decreased expression of p21 and claudin1. Over‐expression of rat HNF4α in JM1 cells resulted in decreased proliferation of these cells. Taken together, these data indicate that HNF4α plays an anti‐proliferative role in hepatocytes and may be involved in termination of liver regeneration.

Removal of Integrin Linked Kinase from Hepatocyte Leads to a Prolonged Proliferative Repose to a Mitogenic Stimuli

We have recently demonstrated that disruption of ECM/Integrin signaling via elimination of Integrin linked kinase (ILK) interferes with signals leading to termination of regeneration. This study investigates the role of ILK in hepatocyte proliferation induced by Phenobarbital (PB). PB was administered in drinking water for 10 days. Steady supply of PB led to a modest nuclear translocation of CAR in WT and ILK−/ − mice. CAR target like CYP2B6 was also increased in both the groups. We then looked at the hyperplastic response of PB in both the groups. 10 days of phenobarbital administration led to a 3 times increased liver to body weight ratio as compared to 0 day in the ILK−/ − mice while the WT mice show only a 2 times increase. ILK−/ − mice also have increased percent of PCNA positive cells and mitotic cells even at day 5 and 10 of phenobarbital administration suggestion a prolonged proliferative response. Signaling pathways altered in ILK−/ − mice included enhanced HGF‐cMet signaling, increased downstream activation of mitogenic molecules like cyclin D1, increased activation of YAP pathways and downregulation mitoinhibitory molecules like TGF‐β1. In summarize, ECM proteins communicate with the signaling machinery of dividing cells via ILK to regulate hepatocyte proliferation and termination of the proliferative response. Lack of ILK in the hepatocytes have prolonged proliferative response to a mitogenic stimuli suggesting defect in termination of proliferative response.The work is supported by a R01 grant (5R01CA035373‐26) titled “HGF in Liver Development, Regeneration and Neoplasia”

Bid Regulates Murine Hepatocyte Proliferation by Controlling ER Calcium Homeostasis

Bid, a BH3‐only Bcl‐2 family molecule, is generally known for its importance in activating the mitochondrial apoptosis pathway following death receptor engagement, particularly in hepatocytes. However, Bid also promotes hepatocyte proliferation during liver regeneration and carcinogenesis. This study aimed to determine the mechanism by which Bid regulated hepatocyte proliferation. Wild type and bid‐deficient primary hepatocytes were stimulated with serum and proliferation was measured by BrdU incorporation and immunoblot analysis of key cell cycle molecules. The hypothesis that Bid regulated endoplasmic reticulum (ER) calcium ([Ca2+]ER) homeostasis to affect proliferation was examined by measuring intracellular calcium level, by modulating the calcium level with pharmacological agents and by subcellular localization assay. We found that serum stimulated hepatocyte proliferation was dependent on calcium and depletion of calcium using thapsigargin or EGTA inhibited the proliferation. A portion of Bid was present in the ER and Bid negatively regulated [Ca2+]ER level, which was responsible for the delayed and reduced proliferation in bid‐deficient hepatocytes. Addition of ionomycin or reconstitution of Bid, particularly an ER‐targeted Bid, corrected the deficiency. In conclusion, we have revealed a novel regulatory mechanism of hepatocyte proliferation, exerted by Bid, a Bcl‐2 family protein otherwise known for its pro‐apoptosis activity under stress condition. This mechanism is mediate by the regulation on ER calcium homeostasis. We consider that this mechanism would be important for liver regeneration and carcinogenesis.

Bid Agonist Regulates Murine Hepatocyte Proliferation by Controlling Endoplasmic Reticulum Calcium Homeostasis

BH3-interacting domain death agonist (Bid), a BH3-only B cell lymphoma 2 family molecule, is generally known for its importance in activating the mitochondrial apoptosis pathway after death receptor engagement, particularly in hepatocytes. However, Bid also promotes hepatocyte proliferation during liver regeneration and carcinogenesis. This study was designed to examine the hypothesis that Bid regulates endoplasmic reticulum calcium concentration ([Ca(2+)](ER)) homeostasis to affect hepatocyte proliferation. We found that serum-stimulated hepatocyte proliferation was dependent on calcium, and the depletion of calcium with thapsigargin or ethylene glycol tetraacetic acid (EGTA) inhibited the proliferation. Subcellular fractionation showed that a portion of Bid was inserted into the endoplasmic reticulum (ER)-enriched membranes, and single-cell calcium imaging indicated that Bid was important for maintaining the [Ca(2+)](ER) level. Bid-deficient hepatocytes manifested delayed and reduced serum-stimulated proliferation, which was corrected by ionomycin or reconstitution of Bid, particularly an ER-targeted Bid. Finally, B cell lymphoma 2-associated X protein (Bax) could also be found in the ER-enriched membranes, and Bax deficiency caused the same proliferation defect. However, Bid/Bax double deletion in hepatocytes did not further augment the defect, which suggested that Bid and Bax worked by the same regulatory mechanism in [Ca(2+)](ER) control. Bid regulates hepatocyte proliferation by positively affecting [Ca(2+)](ER) homeostasis, and this could be important for liver regeneration and carcinogenesis.

Micrornas Control Hepatocyte Proliferation During Liver Regeneration

MicroRNAs (miRNAs) constitute a new class of regulators of gene expression. Among other actions, miRNAs have been shown to control cell proliferation in development and cancer. However, whether miRNAs regulate hepatocyte proliferation during liver regeneration is unknown. We addressed this question by performing 2/3 partial hepatectomy (2/3 PH) on mice with hepatocyte-specific inactivation of DiGeorge syndrome critical region gene 8 (DGCR8), an essential component of the miRNA processing pathway. Hepatocytes of these mice were miRNA-deficient and exhibited a delay in cell cycle progression involving the G(1) to S phase transition. Examination of livers of wildtype mice after 2/3 PH revealed differential expression of a subset of miRNAs, notably an induction of miR-21 and repression of miR-378. We further discovered that miR-21 directly inhibits Btg2, a cell cycle inhibitor that prevents activation of forkhead box M1 (FoxM1), which is essential for DNA synthesis in hepatocytes after 2/3 PH. In addition, we found that miR-378 directly inhibits ornithine decarboxylase (Odc1), which is known to promote DNA synthesis in hepatocytes after 2/3 PH. Our results show that miRNAs are critical regulators of hepatocyte proliferation during liver regeneration. Because these miRNAs and target gene interactions are conserved, our findings may also be relevant to human liver regeneration.

Liver-Specific Ablation of Integrin-Linked Kinase in Mice Results in Enhanced and Prolonged Cell Proliferation and Hepatomegaly after Phenobarbital Administration

We have recently demonstrated that disruption of extracellular matrix (ECM)/integrin signaling via elimination of integrin-linked kinase (ILK) in hepatocytes interferes with signals leading to termination of liver regeneration. This study investigates the role of ILK in liver enlargement induced by phenobarbital (PB). Wild-type (WT) and ILK:liver-/- mice were given PB (0.1% in drinking water) for 10 days. Livers were harvested on 2, 5, and 10 days during PB administration. In the hepatocyte-specific ILK/liver-/- mice, the liver:body weight ratio was more than double as compared to 0 h at day 2 (2.5 times), while at days 5 and 10, it was enlarged three times. In the WT mice, the increase was as expected from previous literature (1.8 times) and seems to have leveled off after day 2. There were slightly increased proliferating cell nuclear antigen-positive cells in the ILK/liver-/- animals at day 2 as compared to WT after PB administration. In the WT animals, the proliferative response had come back to normal by days 5 and 10. Hepatocytes of the ILK/liver-/- mice continued to proliferate up until day 10. ILK/liver-/- mice also showed increased expression of key genes involved in hepatocyte proliferation at different time points during PB administration. In summary, ECM proteins communicate with the signaling machinery of dividing cells via ILK to regulate hepatocyte proliferation and termination of the proliferative response. Lack of ILK in the hepatocytes imparts prolonged proliferative response not only to stimuli related to liver regeneration but also to xenobiotic chemical mitogens, such as PB.

Liver Specific Ablation of Integrin Linked Kinase in Mice Results in Enhanced and Prolonged cell proliferation After Phenobarbital Administration

This study investigates the role of ILK in hepatocyte proliferation induced by Phenobarbital (PB). WT (wild type) and ILK‐/‐ mice were given PB( 0.1% in drinking water) for 10 days. Livers were harvested on 2, 5 and 10 day during PB administration. In the ILK‐/‐ mice the liver/body weight ratio was more than double as compared to 0h at day 2 and 5 while at day 10 it was thrice. In the WT mice the increase was not dramatic and seems to have leveled off after day 2. There were slightly increased PCNA positive cells in the ILK‐/‐ animals at day 2 as compared to WT after PB administration. In the WT animals the proliferative response had come back to normal by day 5 and 10 but the ILK‐/‐ mice still had PCNA positive cells at day 5 and 10 suggesting a prolonged proliferative response. ILK‐/‐ mice also showed increased expression of key genes involved in hepatocyte proliferation at different time points during PB administration. In summarize, ECM proteins communicate with the signaling machinery of dividing cells via ILK to regulate hepatocyte proliferation and termination of the proliferative response. Lack of ILK in the hepatocytes have prolonged proliferative response to a mitogenic stimuli suggesting defect termination of proliferative response.The work is supported by a R01 grant (5R01CA035373‐26) titled "HGF in Liver Development, Regeneration and Neoplasia"

Effect of the immunosuppressants on hepatocyte cells proliferation and apoptosis during liver regeneration after hepatectomy – molecular studies

The regeneration and remodeling of the transplanted liver is the result of hepatocyte proliferation and apoptosis (programmed cell death). The purpose of this study was to verify the influence of immunosuppressants on the expression levels of genes: IL-6 (regulator of hepatocyte proliferation), pro-apoptotic (Bak and Bax) and anti-apoptotic (Bcl-Xl and Bcl-2). 36 newborn suckling rats (age 5-7 days, weight 6-10 g) were divided into four groups: hepatectomy, hepatectomy plus methylprednisolone, hepatectomy plus CsA and hepatectomy plus Tac. The same experiments were performed in 24 weaning rats (age 21-23 days, weight 30-50 g). The animals were killed one day after the hepatectomy and the remnant livers were analyzed. The livers of all animals exhibited histological changes of liver regeneration. The immunosuppressants did not promote any alteration on IL-6 gene expression levels. Methylprednisolone and CsA increased the expression levels of Bak gene in newborn rats. However, methylprednisolone and Tac promoted increased expression levels of Bcl-2 in all groups. We hypothesize that these effects explain the efficacy of these drugs on the treatment of acute and chronic liver rejection as the expression of Bcl-2 in cholangiocytes is decreased as a consequence of bile duct lesions

Pregnane X receptor is essential for normal progression of liver regeneration†

Pregnane X receptor (PXR) mediates xenobiotic and endobiotic metabolism as well as hepatocyte proliferation. To determine the role of PXR in liver regeneration, 2/3 partial hepatectomy (PH) was performed on wild-type and PXR-null mice. Our results showed that hepatic steatosis was markedly suppressed in mice lacking PXR 36 hours after PH, concomitant with reduction of hepatocyte proliferation at the same time point. Gene expression analysis revealed the role of PXR in regulating the transcription of genes involved in lipid uptake, transport, biosynthesis, oxidation, and storage during liver regeneration. When PXR was absent, the second wave of hepatocyte proliferation was severely suppressed, which was accompanied by the inactivation of STAT3. Lack of PXR inhibited the second phase of liver growth, leading to 17% less liver mass at the anticipated end point of liver regeneration (day 10). PXR is required for normal progression of liver regeneration by modulating lipid homeostasis and regulating hepatocyte proliferation.

Significance and Therapeutic Potential of Endothelial Progenitor Cell Transplantation in a Cirrhotic Liver Rat Model

We investigated whether endothelial progenitor cell (EPC) transplantation could reduce established liver fibrosis and promote hepatic regeneration by isolating rat EPCs from bone marrow cells. Recipient rats were injected intraperitoneally with carbon tetrachloride (CCl(4)) twice weekly for 6 weeks before initial administration of EPCs. CCl(4) was then readministered twice weekly for 4 more weeks, and EPC transplantation was carried out for these same 4 weeks. At 7 days in culture, the cells expressed Thy-1, CD31, CD133, Flt-1, Flk-1, and Tie-2, suggesting an immature endothelial lineage. Immunohistochemical analyses showed fluorescent-labeled, transplantation EPCs were incorporated into the portal tracts and fibrous septa. Single and multiple EPC transplantation rats had reduced liver fibrosis, with decreased alpha2-(I)-procollagen, fibronectin, transforming growth factor-beta, and alpha-smooth muscle actin-positive cells. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area, in accordance with EPC location. Real-time polymerase chain reaction analysis of multiple EPC-transplantation livers showed significantly increased messenger RNA levels of matrix metalloproteinase (MMP)-2, -9 and -13, whereas tissue inhibitor of metalloproteinase-1 expression was significantly reduced. Expression of hepatocyte growth factor, transforming growth factor-alpha, epidermal growth factor, and vascular endothelial growth factor was increased in multiple EPC-transplantation livers, while hepatocyte proliferation increased. Transaminase, total bilirubin, total protein, and albumin levels were maintained in EPC-transplantation rats, significantly improving survival rates. We conclude that single or repeated EPC transplantation halts established liver fibrosis in rats by suppressing activated hepatic stellate cells, increasing matrix metalloproteinase activity, and regulating hepatocyte proliferation.

Sinusoidal remodeling and angiogenesis: A new function for the liver-specific pericyte?

Sinusoidal remodeling and angiogenesis: A new function for the liver-specific pericyte?

Bile Buildup and Liver Regeneration

Numerous secreted factors, including growth factors and cytokines, have been implicated in regulating hepatocyte proliferation. Huang et al. report that bile acids are essential stimulatory factors for liver regeneration in mice. An increase in bile acids stimulates regeneration and requires the bile acid nuclear receptor FXR. The authors propose a homeostatic mechanism for determination of liver size, in which FXR and perhaps other nuclear receptors sense the levels of endogenous metabolites to determine the liver's functional capacity. When liver function is decreased as a result of injury, the resulting accumulation of bile acids activates FXR, which stimulates signaling pathways to protect the liver from bile acid toxicity and also promotes liver growth to handle the overload. W. Huang, K. Ma, J. Zhang, M. Qatanani, J. Cuvillier, J. Liu, B. Dong, X. Huang, D. D. Moore, Nuclear receptor-dependent bile acid signaling is required for normal liver regeneration. Science 312 , 233-236 (2006). [Abstract] [Full Text]

Growth hormone mediates hepatocyte nuclear factor HNF-6 stimulation of hepatocyte proliferation during bile duct ligation through upregulation of cyclin D1

Background: Hepatocyte Nuclear Factor 6 (HNF6) is a liver enriched transcription factor regulating numerous target genes important in liver homeostatic function. HNF6 has several binding sites to the promoter region of cyclin D1 which regulates G0-S phase cell cycle entry, suggesting that HNF-6 can also regulate hepatocyte proliferation. Prior work in our laboratory has shown that GH administration enhances hepatic HNF6 expression and can be used as an vivo model to increase HNF6 mediated transcriptional activation of its target genes.

Liver‐specific loss of β‐catenin blocks glutamine synthesis pathway activity and cytochrome p450 expression in mice†‡

There is accumulating evidence that Wnt/beta-catenin signaling is involved in the regulation of liver development and physiology. The presence of genetic alterations resulting in constitutive beta-catenin stabilization in human and murine liver tumors also implicates this pathway in hepatocyte proliferation. In the present study, we generated hepatocyte-specific beta-catenin knockout mice to explore the role of beta-catenin in liver function. Conditional knockout mice were born at the expected Mendelian ratio and developed normally to adulthood, indicating beta-catenin is dispensable for essential liver function under normal breeding conditions. However, the liver mass of knockout mice was 20% less than those of mice in the control groups. Expression analysis revealed loss of genes required for glutamine synthesis in knockout mice. Loss of the liver glutamine synthesis pathway did not affect the blood ammonia level in mice fed a standard diet, yet, knockout mice showed significantly elevated blood ammonia levels with high-protein dietary feeding. Furthermore, the expression of two cytochrome P450 enzymes, CYP1A2 and CYP2E1, was almost completely abolished in livers from hepatocyte-specific beta-catenin knockout mice. Consequently, these mice were resistant to acetaminophen challenge, confirming the requirement of these cytochrome P450 enzymes for metabolism of xenobiotic substances. In conclusion, in addition to regulating hepatocyte proliferation, beta-catenin may also control multiple aspects of normal liver function.

Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

To characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells. Fifty-four adult male Wistar rats were randomly divided into three groups: A normal control (NC) group, a partial hepatectomized (PH) group and a sham operated (SO) group. To study the effect of liver regeneration on BMP-2 expression, rats were sacrificed before and at different time points after PH or the sham intervention (6, 12, 24 and 48 h). For each time point, six rats were used in parallel. Expression and distribution of BMP-2 protein were determined in regenerating liver tissue by Western blot analysis and immunohistochemistry. Effects of BMP-2 on cell proliferation of human Huh7 hepatoma cell line were assessed using an MTT assay. In the normal liver strong BMP-2 expression was observed around the central and portal veins. The expression of BMP-2 decreased rapidly as measured by both immunohistochemistry and Western blot analysis. This decrease was at a maximum of 3.22 fold after 12 h and returned to normal levels at 48 h after PH. No significant changes in BMP-2 immunoreactivity were observed in the SO group. BMP-2 inhibited serum induced Huh7 cell proliferation. BMP-2 is expressed in normal adult rat liver and negatively regulates hepatocyte proliferation. The observed down regulation of BMP-2 following partial hepatectomy suggests that such down regulation may be necessary for hepatocyte proliferation.

Forkhead box M1B transcriptional activity requires binding of Cdk-cyclin complexes for phosphorylation-dependent recruitment of p300/CBP coactivators.

Previous liver regeneration studies demonstrated that the mouse forkhead box M1B (FoxM1B) transcription factor regulates hepatocyte proliferation through expression of cell cycle genes that stimulate cyclin-dependent kinase 2 (Cdk2) and Cdk1 activity. In this study, we demonstrated that disruption of the FoxM1B Cdk1/2 phosphorylation site at Thr residue 596 significantly reduced both FoxM1B transcriptional activity and Cdk phosphorylation of the FoxM1B T596A mutant protein in vivo. Retention of this FoxM1B 596 Cdk phosphorylation site was found to be essential for recruiting the histone acetyltransferase CREB binding protein (CBP) to the FoxM1B transcriptional activation domain. Consistent with these findings, dominant negative Cdk1 protein significantly reduced FoxM1B transcriptional activity and inhibited FoxM1B recruitment of the CBP coactivator protein. Likewise, Cdc25B-mediated stimulation of Cdk activity together with elevated levels of the CBP coactivator protein provided a 6.2-fold synergistic increase in FoxM1B transcriptional activity. Furthermore, mutation of the FoxM1B Leu 641 residue within an LXL motif (residues 639 to 641) inhibited recruitment of Cdk-cyclin complexes and caused significant reduction in both FoxM1B transcriptional activity and in vivo Cdk phosphorylation of the FoxM1B Thr 596 residue. We demonstrated that FoxM1B transcriptional activity requires binding of either S-phase or M-phase Cdk-cyclin complexes to mediate efficient Cdk phosphorylation of the FoxM1B Thr 596 residue, which is essential for recruitment of p300/CBP coactivator proteins.

Increased expression of H19 non-coding mRNA follows hepatocyte proliferation in the rat and mouse

Background/Aims H19 is a paternally imprinted gene that is believed to function as non-coding mRNA. While H19 is only faintly expressed in the normal adult liver, it is abundantly expressed during the fetal period. We explored the possibility that H19 might participate in the regulation of hepatocyte proliferation. Methods Adult male rats and mice were subjected to a two-thirds partial hepatectomy, and after various time periods, hepatocytes were isolated and analyzed for H19 gene expression. The expression was also examined in cultured rat hepatocytes. Results The expression of H19 was dramatically increased after 2 days (rat) and 4 days (mouse), peaked at 3 days (rat) and 6 days (mouse), and then gradually declined. In both species, the increase in H19 gene expression was preceded by the induction of proliferating cell nuclear antigen and DNA synthesis. An allele-specific RT-PCR analysis in the mouse showed that the paternally imprinted status of the gene was maintained after a partial hepatectomy. H19 was strongly induced in spheroid cultures after transient hepatocyte proliferation, but not in conventional monolayer cultures, in which persistent proliferation occurred. Conclusions Our results demonstrated that H19 gene expression was dynamically regulated in adult hepatocytes in close association with their proliferation.

Proliferation of Epidermal Growth Factor-stimulated Hepatocytes in a Hormonally Defined Serum-free Medium

The present study shows that adult rat hepatocytes in primary culture, which normally exhibit a restricted capacity to proliferate, can proceed through the cell cycle when cultured in a mixture of minimal essential medium (MEM) and Medium 199 (MEM-M199; 3:1, v/v), containing epidermal growth factor (EGF; 50 ng/ml), low glucose (0.75 g/l) and low levels of inorganic salts, amino acids and vitamins. Under these conditions, hepatocytes flatten and cell extensions appear. In contrast, Dulbecco's modified Eagle's medium (DMEM) containing high glucose (4.5g/l) levels enriched with inorganic salts, amino acids and vitamins favours maintenance of differentiated functional hepatocyte capacities (albumin secretion), but does not allow proliferation or cell spreading. Cultivation of hepatocytes in MEM-M199 (3:1, v/v) results in the onset of DNA synthesis at 48 hours of culture and a concomitant induction of cyclin D1 protein. Under these conditions, cells successively progress through the mitogen-dependent restriction point in mid-late G1 phase, G1/S transition and S phase, as evidenced by Western blot analysis of the markers cyclins E and A and cyclin dependent kinase (CDK)2 and CDK1, respectively. Progression through the cell cycle is accompanied by a decrease in albumin secretion, indicating a decline in differentiated capacities. This study demonstrates that hepatocytes cultured in a mixture of MEM-M199 (3:1) provide a useful in vitro model for studying the regulation of hepatocyte proliferation.

Role of tumor necrosis factor receptor 1 (p55) in hepatocyte proliferation during acetaminophen-induced toxicity in mice

Hepatocyte proliferation represents an important part of tissue repair. In these studies, TNF receptor 1 (TNFR1) knockout mice were used to analyze the role of TNF-α in hepatocyte proliferation during acetaminophen-induced hepatotoxicity. Treatment of wild-type (WT) mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was associated with proliferation of hepatocytes surrounding the damaged areas, which was evident at 24 h. The cell cycle regulatory proteins, cyclin D1 and cyclin A, were also up regulated in hepatocytes. In contrast, in TNFR1−/− mice, which exhibit exaggerated acetaminophen hepatotoxicity, hepatocyte proliferation, and expression of cyclin D1 and cyclin A, as well as the cyclin dependent kinases, Cdk4 and Cdk2, were reduced. The cyclin-dependent kinase inhibitor p21 was also induced in the liver following acetaminophen administration. This was greater in TNFR1−/− mice compared to WT mice. To investigate mechanisms mediating the reduced hepatic proliferative response of TNFR1−/− mice, we analyzed phosphatidyl inositol-3-kinase (PI-3K) signaling. In both WT and TNFR1−/− mice, acetaminophen caused a rapid increase in total PI-3K within 3 h. Acetaminophen also increased phosphorylated PI-3K, but this was delayed 6–12 h in TNFR1−/− mice. Expression of Akt, a downstream target of PI-3K, was increased in both WT and TNFR1−/− mice in response to acetaminophen. However, the increase was greater in WT mice. Acetaminophen-induced expression of phosphorylated STAT3, a key regulator of cytokine-induced hepatocyte proliferation, was also delayed in TNFR1−/− mice relative to WT. These data suggest that TNF-α signaling through TNFR1 is important in regulating hepatocyte proliferation following acetaminophen-induced tissue injury. Delayed cytokine signaling may account for reduced hepatocyte proliferation and contribute to exaggerated acetaminophen-induced hepatotoxicity in TNFR1−/− mice.

Role of tumor necrosis factor receptor 1 (p55) in hepatocyte proliferation during acetaminophen-induced toxicity in mice

Hepatocyte proliferation represents an important part of tissue repair. In these studies, TNF receptor 1 (TNFR1) knockout mice were used to analyze the role of TNF-α in hepatocyte proliferation during acetaminophen-induced hepatotoxicity. Treatment of wild-type (WT) mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was associated with proliferation of hepatocytes surrounding the damaged areas, which was evident at 24 h. The cell cycle regulatory proteins, cyclin D1 and cyclin A, were also up regulated in hepatocytes. In contrast, in TNFR1−/− mice, which exhibit exaggerated acetaminophen hepatotoxicity, hepatocyte proliferation, and expression of cyclin D1 and cyclin A, as well as the cyclin dependent kinases, Cdk4 and Cdk2, were reduced. The cyclin-dependent kinase inhibitor p21 was also induced in the liver following acetaminophen administration. This was greater in TNFR1−/− mice compared to WT mice. To investigate mechanisms mediating the reduced hepatic proliferative response of TNFR1−/− mice, we analyzed phosphatidyl inositol-3-kinase (PI-3K) signaling. In both WT and TNFR1−/− mice, acetaminophen caused a rapid increase in total PI-3K within 3 h. Acetaminophen also increased phosphorylated PI-3K, but this was delayed 6–12 h in TNFR1−/− mice. Expression of Akt, a downstream target of PI-3K, was increased in both WT and TNFR1−/− mice in response to acetaminophen. However, the increase was greater in WT mice. Acetaminophen-induced expression of phosphorylated STAT3, a key regulator of cytokine-induced hepatocyte proliferation, was also delayed in TNFR1−/− mice relative to WT. These data suggest that TNF-α signaling through TNFR1 is important in regulating hepatocyte proliferation following acetaminophen-induced tissue injury. Delayed cytokine signaling may account for reduced hepatocyte proliferation and contribute to exaggerated acetaminophen-induced hepatotoxicity in TNFR1−/− mice.