Potential conflict of interest: Nothing to report.
During the evolution of cirrhosis, there is a relative decrease in volume percentage of hepatocytes and a relative increase in biliary epithelial cells and myofibroblasts. This is recognized histopathologically as a ductular reaction and leads to gradual distortion of the normal hepatic architecture. The final or decompensated stage of cirrhosis is characterized by a further decline in hepatocyte proliferation and loss of functional liver mass that manifests clinically as ascites, encephalopathy, and other signs of liver failure. In this report, we tested the hypothesis that p21-mediated hepatocyte mito-inhibition accelerates the evolution of cirrhosis using an established mouse model of decompensated biliary cirrhosis, p21-deficient mice, and liver tissue from humans awaiting liver replacement. Despite the same insult of long-term (12-week) bile duct ligation, mice prone to decompensation showed significantly more oxidative stress and hepatocyte nuclear p21 expression, which resulted in less hepatocyte proliferation, an exaggerated ductular reaction, and more advanced disease compared with compensation-prone controls. Mice deficient in p21 were better able than wild-type controls to compensate for long-term bile duct ligation because of significantly greater hepatocyte proliferation, which led to a larger liver mass and less architectural distortion. Mito-inhibitory hepatocyte nuclear p21 expression in humans awaiting liver replacement directly correlated with pathological disease stage and model of end-stage liver disease scoring. In conclusion, stress-induced upregulation of hepatocyte p21 inhibits hepatocyte proliferation during the evolution of cirrhosis. These findings have implications for understanding the evolution of cirrhosis and associated carcinogenesis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005.)
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The evolution of cirrhosis can be divided into three clinical-pathological stages. First, the injury and proliferative phase is characterized by liver damage and hepatocyte proliferation that results in a net increase of hepatocytes and liver enlargement. Second, the compensated phase is characterized by ongoing injury and regeneration, but impaired growth of some cell types and enhanced growth of others changes gradually the relative volume proportions and spatial distribution of various cell types. A relative decrease in volume percentage of hepatocytes and a relative increase in biliary epithelial cells and myofibroblasts is recognized histopathologically as a ductular reaction, which gradually distorts the normal architecture. Third, the decompensated stage is characterized by a decline in hepatocyte proliferation1, 2 and loss of functional liver mass that manifests clinically as ascites, encephalopathy, and other signs of liver failure.
Understanding molecular mechanisms that control hepatocyte proliferation during the evolution of cirrhosis has the potential to even more accurately stage the cirrhotic process and provide insights into treatment that could stabilize or slow the progression of disease. Currently, liver allograft candidates are prioritized for transplantation according to disease severity, or the stage of cirrhosis, based on the Model for End-Stage Liver Disease (MELD).3–5 Inclusion of only three laboratory values—serum bilirubin, creatinine, and international normalized ratio of prothrombin3—reflects the functional hepatocyte mass and avoids obtaining an invasive biopsy in unstable patients.
Cellular division is a highly regulated process involving the coordination of many proteins that work together to ensure accurate and controlled replication. Cell cycle progression is mediated by cyclin family members that complex with and activate cyclin-dependent kinases (cdk). Individual cyclin/cdk complexes facilitate specific stages of the cell cycle by migrating to the nucleus and interacting with additional proteins—including proliferating cell nuclear antigen (PCNA), a component of the DNA polymerase holoenzyme—that facilitate DNA replication. Conversely, two families of cdk inhibitors, the broad-acting cip/kip family: p21, p27, p57, and the cdk4-specific ink4 family: p15, p16, p18, and p19,6 bind to cyclin-cdk-PCNA complexes and impede cell cycle progression under unfavorable or stressful conditions. Examples include exposure to ultraviolet irradiation or high levels of reactive oxygen species.7 In the liver, hepatocyte growth factors, regenerative stimuli, stress, and transforming growth factor β can upregulate hepatocyte p21 expression.8 Disruption of p21-mediated mechanisms that inhibit cell cycle progression in the presence of noxious stimuli can result in replication of damaged or potentially mutated genes and neoplastic transformation.
In this study, we tested the hypothesis that p21-mediated mito-inhibition of hepatocytes—possibly related to multifactorial stress—blocks hepatocyte proliferation, which in turn, leads to an exaggerated ductular reaction and more rapid architectural distortion and decompensation. The hypothesis was tested using an established mouse model of decompensated biliary cirrhosis after long-term (12-week) bile duct ligation (BDL).9 In this model, IL-6−/− mice advance to decompensated cirrhosis more rapidly than IL-6+/+ controls and exhibit increased mortality and serum bilirubin along with decreased hepatocyte proliferation and liver mass. Our hypothesis was also tested in p21-deficient (p21−/−) mice subjected to long-term BDL and in liver specimens from humans with biliary and nonbiliary liver disease awaiting hepatic transplantation.
MELD, Model for End-Stage Liver Disease; cdk, cyclin-dependent kinase; PCNA, proliferating cell nuclear antigen; BDL, bile duct ligation; IL, interleukin; RT-PCR, reverse-transcriptase polymerase chain reaction; mRNA, messenger RNA; BrdU, bromo-deoxyuridine; RNS, reactive nitrogen species.
Materials and Methods
Animals and Operative Procedures.
Eight- to twelve-week-old male interleukin (IL)-6+/+,9 IL-6−/−,9 p21−/−,10 and B6129F1 (Taconic, Tarrytown, NY) mice were used for all experiments. Animals were subjected to BDL as previously described.9 No differences were observed between the two strains of wild-type mice used (B6129F1 and IL-6+/+); therefore, the data from these two strains were combined. Mice were sacrificed at 0 and 12 weeks after BDL (n ≥ 5 in each group), and liver tissues and serum were collected. Liver mass index was calculated as the percentage of the liver weight normalized to the body weight. Animal experiments were conducted under University of Pittsburgh Institutional Animal Care and Use Committee guidelines.
Protein was extracted from liver tissue using either RIPA11 or Triton X-100 buffer.8 Fifty micrograms of total protein was separated on 10% to 20% or 4% to 15% Tris-HCl gels (Bio-Rad, Hercules, CA) and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked in 5% nonfat milk in TBST for 1 hour and probed for p21 (SXM30; Pharmingen, San Diego, CA), cyclin A (C-19), cyclin E (M-20), cyclin D1 (72-13G), cdk2 (M2), cdk4 (H-22), or cdk6 (B-10) (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Anti-mouse immunoglobulin G-HRP or anti-rabbit immunoglobulin G-HRP (Amersham Pharmacia Biotech, Piscataway, NJ) were used as secondary antibodies for 1 hour and were developed using Western Lightning chemiluminescence reagent (Perkin Elmer Life Sciences, Boston, MA). The intensity of the individual bands was determined using National Institutes of Health ImageJ analysis software (NIH, Bethesda, MD).
Triton X-100 insoluble fractions of lysates were prepared according to Cayrol et al.12 Liver tissue was homogenized in Triton X-100 lysis buffer as above. Following centrifugation, the residual pellet was resuspended in lysis buffer supplemented with 1% SDS and incubated on ice for 30 minutes. The sample was centrifuged at 14,000 rpm for 15 minutes, and the supernatant was collected as the Triton X-100 insoluble fraction.
Quantitative Real-Time Polymerase Chain Reaction.
Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was performed as previously reported.13 Mouse p21 and GAPDH messenger RNA (mRNA) expression was quantified by SYBR Green two-step, real-time RT-PCR on an ABI PRISM 7000 Sequence Detection System using SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, CA). The forward and reverse primers, which were selected using Primer Express software (PE Applied Biosystems), were as follows: p21-forward, TTGCACTCTGGTGTCTGAGC; p21-reverse, AATCTGTCAGGCTGGTCTGC; GAPDH-forward, TGGCAAAGTGGAGATTGTTGCC; GAPDH-reverse, AAGATGGTGATGGGCTTCCCG. Expression of p21 was normalized to GAPDH mRNA.
Formalin-fixed, paraffin-embedded sections were deparaffinized, and antigen retrieval was performed by steam or microwave in citrate buffer (pH 6.0) or Target Antigen Retrieval Buffer (Dako, Carpinteria, CA). Slides were blocked using either a mouse-on-mouse blocking kit (Vector, Burlingame, CA) or 10% normal serum (Vector) diluted in Protein Blocking Agent (Immunon, Pittsburgh, PA) and incubated overnight using antibodies to p21 (SX-118; Dako), Ki67 (AbCam, Cambridge, MA), or PCNA (PC10, Dako). An avidin-biotin-peroxidase technique (Vectastain ABC Elite Kit, Vector) was used for detection. Bromo-deoxyuridine (BrdU) incorporation was analyzed as previously reported.9 For determination of mouse hepatocyte labeling indexes, the total number of positive nuclei were counted blindly in 20 randomly selected high-power fields (×400) containing approximately 2,000 total hepatocytes.
p21/Ki67 double-labeling was assessed via immunofluorescence. Tissue sections were prepared as described in the previous paragraph. p21 antibody was incubated overnight and then for 1.5 hours with biotinylated horse anti-mouse immunoglobulin G (Vector). Avidin conjugated FITC (Vector) was applied for 1.5 hours. Slides were reblocked with Protein Blocking Agent (Immunon) and incubated with Ki67 antibody for 1.5 hours followed by Texas red conjugated anti-rabbit immunoglobulin G (Vecor) for 1 hour. Sections were counterstained with Hoescht and mounted with gelvatol.
Localization of copper was performed using rhodanine stain. A stock solution of 5-4-demethylaminobenzylidene rhodanine (Sigma, St. Louis, MO) was prepared in ethanol and diluted to 20% in distilled water. Paraffin sections were incubated at 37°C overnight in the rhodanine working solution.
Analysis of Reactive Nitrogen Species.
Reactive nitrogen species were analyzed via the formation of 3-nitrotyrosine in Triton X-100 liver lysates by way of ELISA according to the manufacturer's protocol (Oxis International, Portland, OR).
Patient Selection, Histopathological Staging, and p21/Ki67 Scoring.
Native hepatectomy specimens from adult patients with biliary cirrhosis (i.e., primary biliary cirrhosis or sclerosing cholangitis; n = 22) or nonbiliary cirrhosis (i.e., autoimmune hepatitis or chronic hepatitis C; n = 16) at the time of liver replacement during the last 10 years were considered for analysis under an exempt institutional review board protocol #0404010. Patients with coexistent disease, such as hepatitis C virus infection or bile duct or hepatocellular carcinoma, and patients subjected to transjugular intrahepatic portosystemic shunting were excluded because of the potential confounding influence of these coexistent conditions.14 An average MELD score was calculated for the month before transplantation. Patients representing a distribution of relatively high (>20) and relatively low (<15) MELD scores were chosen randomly for histopathological staging and p21 and Ki67 immunostaining.
Routine hemotoxylin-eosin–stained sections were staged histopathologically15 and scored blindly for cholestasis, p21, and Ki-67 staining by A. J. D. Initial review of the p21-stained sections showed that p21-positive hepatocyte nuclei were preferentially located in periportal/periseptal hepatocyte nuclei, varied throughout the section, and in some cases were too numerous to count. Therefore, a semiquantitative scale was used for scoring: 0, minimal or no p21-positive hepatocyte nuclei; 1, rim of p21-positive hepatocyte 1 to 2 cell layers thick near occasional portal tracts/septae; 2, rim of p21-positive hepatocyte 1 to 2 cell layers thick near most portal tracts/septae; 3, rim of p21-positive hepatocyte 1 to 2 cell layers thick near most portal tracts/septae with occasional areas of 3 or more layers; 4, rim of p21-positive hepatocyte 3 or more cell layers thick near most portal tracts/septae with occasional complete staining of small nodules.
Ki67 staining was graded on a semiquantitative scale. The number of Ki67-positive nuclei was counted in 20 random ×200 fields. The distribution of these counts was separated into quartiles and scored as such.
Cholestasis was also scored on a scale of 0 to 4, with 0 representing minimal or no cholestasis and 4 representing cholestasis at the periphery of virtually all nodules and pan-nodular staining in more than an occasional nodule.
Data are presented as the mean ± SD and were analyzed using SPSS version 11.0 software (SPSS, Chicago, IL). To assess statistical significance, paired data was analyzed using the Student t test; a P value of less than .05 was considered significant. Data of abnormal distribution or of unequal variance were analyzed using the Mann-Whitney U test. Correlations were determined via Pearson or Spearman rank correlation.
Diminished Hepatocyte Proliferation and Liver Mass Index in Decompensated Cirrhosis.
As in humans,1, 2, 14 hepatocyte proliferation decreased during progression from compensated to decompensated cirrhosis in our mouse model of decompensated biliary cirrhosis.9 Despite similar early phases, between 6 and 12 weeks after BDL, IL-6−/− mice, which are decompensation-prone, begin to die earlier and more often than compensation-prone wild-type IL-6+/+ controls. Consistent with our previous study,9 significantly decreased hepatocyte proliferation in decompensated cirrhosis was apparent using three different markers of proliferation: BrdU incorporation (Fig. 1A), PCNA (Fig. 1B), and Ki67 (Fig. 1C). Livers with compensated cirrhosis show a threefold increase in BrdU labeling, but only a 15% increase in liver mass compared with livers with decompensated cirrhosis (Fig. 1D).
Elevated p21 in Decompensated Cirrhosis.
Analysis of positive regulators of cell cycle progression revealed no substantial differences that might explain impaired hepatocyte proliferation in decompensated cirrhosis (Supplemental Fig. 1). We therefore focused on the cell cycle inhibitor p21Waf-1/Cip-1 because of our previous experience11 and because of increased hepatocyte p21 expression in models of impaired hepatocyte proliferation after partial hepatectomy.16–21
p21 expression was negative in normal livers8, 22 (Fig. 2), as expected. In contrast, by 12 weeks after BDL, mice with decompensated cirrhosis showed significantly more p21 expression via Western blot analysis (Fig. 2A), quantitative immunohistochemistry (Fig. 2B), and real-time RT-PCR (Fig. 2C). Immunohistochemistry analysis also showed that the increased p21 protein localized predominantly to hepatocyte nuclei with a predilection for hepatocytes near the fibrous septae (Fig. 2B,D).
Relationship Between Hepatocyte p21 Expression and Markers of Cell Cycle Progression.
p21 plays a complex role in cell cycle progression, DNA repair, and mito-inhibition.6, 7, 23 During synchronized proliferation induced by partial hepatectomy, p21 shows biphasic upregulation during the G1 phase and after the S phase.22 Expression during the cell cycle may either prevent premature progression through G1,22 or p21 could function as a bridging molecule, facilitating the formation and nuclear translocation of cyclin/cdk complexes.24 However, at a high stoichiometric ratio, p21 can clearly inhibit cell cycle progression at the G1-S interface by binding to and inhibiting cdk/cyclin complexes and PCNA.6, 7 A diagram relating the expression of cyclins, cdks, markers of proliferation, and p21 to the cell cycle is shown in Fig. 3A.
Because of the complex role of p21 in cell cycle control and DNA repair, hepatocyte p21 expression was related to three different markers of hepatocyte proliferation: (1) BrdU (labels cells in S-phase), (2) Ki-67 (labels cells in late G1, S, and G2/M-phase), and (3) PCNA (labels all cycling cells that have left G0).6, 7, 23 All three proliferation markers labeled a significantly smaller fraction of hepatocytes in decompensated compared with compensated cirrhosis (Fig. 1). Conversely, p21 labeling was significantly higher in decompensated versus compensated cirrhosis (Fig. 2B). Thus, the p21/BrdU, p21/Ki-67, and p21/PCNA ratios were significantly higher in decompensated versus compensated cirrhosis (Fig. 3B-D). These data suggest that hepatocyte p21 expression in compensated cirrhosis may be in part related to hepatocyte proliferation or to cell cycle progression, whereas in decompensated cirrhosis, p21 inhibits hepatocyte proliferation.
We next hypothesized that if p21 upregulation was related to hepatocyte proliferation or cell cycle progression, p21 and Ki-67 should localize to the same hepatocytes and would therefore be detectable via double immunofluorescence labeling. In contrast, mito-inhibitory hepatocyte p21 expression should cause hepatocytes to exit the cell cycle, and fewer p21 and Ki-67 double-positive hepatocytes should be seen in decompensated cirrhosis. Indeed, double-positive hepatocytes simultaneously expressing p21 and Ki-67 (Fig. 3E, yellow arrow) were detected only in livers with compensated cirrhosis. In contrast, despite numerous p21-positive hepatocyte nuclei in decompensated cirrhosis, no double-labeling for Ki-67 was detected (Fig. 3F).
The functional activity of p21 in decompensated cirrhosis can be further evaluated by examining the p21 interaction with PCNA. PCNA can exist in a soluble nuclear form present in all cycling non–S-phase cells and an insoluble form that binds to DNA during replication and DNA excision repair6, 7, 23; p21 binds directly to the N-terminal portion of PCNA in both forms.7 The insoluble p21–PCNA complex blocks the replicative function of PCNA, while the DNA excision/repair function of PCNA proceeds unchecked.6, 7, 23 We postulated that if p21 was inhibiting the DNA replication function of PCNA in decompensated cirrhosis, p21 should be increased in the Triton X-100 insoluble fraction of livers with decompensated cirrhosis.
Western blot analysis of Triton X-100 insoluble liver lysates revealed significantly more p21 in livers with decompensated cirrhosis compared with compensated controls (Fig. 3G). Furthermore, the ratio of p21/PCNA in the Triton X-100 insoluble fraction was significantly higher in decompensated than in compensated cirrhosis. Because PCNA is an integral component of the functional DNA polymerase holoenzyme, inhibition by p21 should be reflected in the number of S-phase cells. The reduced BrdU labeling in decompensated cirrhosis (Fig. 1A) confirmed the inhibition of PCNA by p21.
Absence of p21 Improves Ability to Compensate and Delays Architectural Distortion After BDL.
If p21 expression contributes to hepatocyte mito-inhibition after BDL and during decompensation, we hypothesized that p21−/− mice subjected to BDL for 12 weeks should show increased hepatocyte proliferation, a larger liver mass, and less architectural distortion. The reason for expecting less architectural distortion is that hepatocyte mito-inhibition combined with a strong regenerative stimulus results in an exaggerated ductular response,25 which in turn distorts the liver architecture. In contrast, maintenance of hepatocyte proliferation in the presence of a regenerative stimulus maintains the liver architecture.26, 27
The liver mass from age-matched normal (untreated) p21−/− mice was significantly smaller than wild-type controls (data not shown). However, by 12 weeks after BDL, livers of p21−/− mice showed significantly higher hepatocyte BrdU labeling and a significantly larger liver mass than similarly treated wild-type controls (Fig. 4A-B). Interestingly, the magnitude of the difference in liver mass index between p21−/− and p21+/+ mice (∼15%) was substantially less than might be expected given the threefold difference in hepatocyte BrdU incorporation, but very similar to the differences in hepatocyte proliferation and liver mass between livers with compensated and decompensated cirrhosis. This suggests that proliferating p21−/− hepatocytes can proceed through the G1 checkpoint and complete the S phase, but may be unable to successfully negotiate the G2-M transition in a toxic environment.28, 29 Indeed, Ki67 labeling, whose expression peaks during the G2-M phase of the cell cycle, revealed a difference of only 15% between p21−/− and p21+/+ mice 12 weeks after BDL (Fig. 4C). This was similar to the difference observed in liver mass.
There was also a clear difference in the severity of architectural distortion, or stage of biliary fibrosis, between p21−/− and p21+/+. The average stage of fibrosis and architecture distortion was significantly less in the p21−/− mice at 12 weeks after BDL (1.3 ± 0.4 vs. 2.6 ± 0.7; P < .01) (Fig. 4D-E).
Possible Mechanisms of Increased Hepatocyte p21 Expression in Decompensated Cirrhosis.
As an explanation for the upregulation of hepatocyte p21, we hypothesized that chronic oxidative stress30 associated with chronic cholestasis31 leads to stress-induced, p21-mediated mito-inhibition of hepatocytes.32 Indicators of oxidative stress include reactive nitrogen species (RNS), which were measured in normal liver and livers with compensated and decompensated cirrhosis at 12 weeks after BDL (Fig. 5). Consistent with previous studies, RNS levels were significantly increased in livers at 12 weeks after BDL in both groups compared with normal livers31 (Fig. 5). More importantly, livers with decompensated cirrhosis showed significantly higher RNS levels than livers with compensated cirrhosis at 12 weeks after BDL.
Hepatocyte p21 and Ki67 in Human Liver Disease.
Based on the above-mentioned results in experimental animals, we tested the hypothesis that the p21/Ki67 ratio should increase along with the histopathological severity, stage of disease, and MELD score in patients with biliary (primary biliary cirrhosis and primary sclerosing cholangitis) and nonbiliary (autoimmune hepatitis and chronic hepatitis C) liver disease. Immunohistochemical staining for p21 and Ki67 (Fig. 6A-B) was used to generate p21 and Ki67 ratios (see Materials and Methods). Similar to the mouse model, there was a direct correlation in candidates with biliary cirrhosis between p21/Ki67 ratio and histopathology stage of disease alone (correlation coefficient: 0.378; P < .05) (Fig. 6C, left), and histopathological stage of disease plus cholestasis score (correlation coefficient: 0.398; P < .05) (Fig. 6C, right). The latter combination was included because it provides more information about the decompensation process beyond the final histopathology stage. There was also a positive correlation between p21/Ki67 ratio and histopathology stage of disease plus cholestasis score in patients with nonbiliary disease, but this did not achieve statistical significance (data not shown).
The p21+ hepatocyte nuclei in decompensated cirrhosis were concentrated along the fibrous septae (Fig. 6A,E), colocalizing with or immediately adjacent to periportal hepatocyte copper deposits in patients with biliary cirrhosis (Fig. 6D,F), which is typical of chronic cholestatic disorders. Copper deposition in the liver is known to be hepatotoxic and a stimulator of oxidative stress.33, 34 Other generators of oxidative stress were also increased, including cholestasis, inflammation, and iron accumulation in patients with nonbiliary liver disease (data not shown).
MELD risk scores are used to prioritize candidates for transplantation and predict decompensation. Patients with MELD scores of 15 or lower usually do not require immediate liver transplantation, whereas those with MELD scores of 20 or higher definitely show inferior survival without liver replacement.4, 5, 35, 36 We used these guidelines to define two groups of patients: (1) compensated cirrhosis with relatively low MELD scores under 16; and (2) decompensated cirrhosis with relatively high MELD scores greater than 20 (Table 1). Consistent with the mouse model,9 the p21/Ki67 ratio in these two groups differed significantly in patients with both biliary and nonbiliary disease (Table 1).
Table 1. Comparison of the Liver Architecture, Liver Mass Index, and p21 and Ki67 Labeling in Patients With Chronic Biliary and Nonbiliary Liver Disease
MELD Score < 15
MELD Score > 20
Primary biliary cirrhosis and primary sclerosing cholangitis.
Experimental animal models show that hepatocyte nuclear p21 expression induced by the noxious stimuli of 2-acetaminofluorene treatment,16 fatty liver disease,17 ethanol,18 copper sequestration,19 old age,20 or an overexpressing transgene can acutely impair hepatocyte proliferation after partial hepatectomy.21 In contrast, Albrecht et al.8 showed that increased hepatocyte nuclear p21 protein expression also occurs during and immediately after hepatocyte proliferation following partial hepatectomy. In diseased human livers, hepatocyte p21 expression has been associated with histological markers of hepatocyte proliferation and disease activity.37–40 Upregulation of hepatocyte p21 after proliferative stimuli might serve to prevent premature hepatocyte re-entry into cell cycling. This study is unique because it shows that p21 can be associated with either hepatocyte proliferation or mito-inhibition, and the latter significantly contributes to the evolution of cirrhosis. Therefore, simultaneous analyses of p21 and proliferation markers are needed to determine the functional significance of hepatocyte p21 expression in specific situations.
Livers with compensated cirrhosis show a relatively low hepatocyte p21/BrdU ratio, coexpression of p21 and Ki67 in the same nuclei, and significantly lower p21/PCNA ratio in the insoluble DNA-bound protein fraction.6, 7, 23 Conversely, in livers with decompensated cirrhosis and impaired regeneration, the hepatocyte p21/BrdU ratio is relatively high, there is mostly exclusive hepatocyte labeling of p21 and Ki67, and there is a significantly higher p21/PCNA ratio in the insoluble DNA-bound protein fraction.6, 7, 23 Because nuclear localization is required for the proproliferative, DNA excision/repair, and mito-inhibition functions of p21,7, 41 it is important to include immunocytochemical staining and quantitative morphometry methods of analysis, which may ultimately have clinical use.
The cell cycle regulatory role of p21 is complex and not completely understood.41 In some cell types, p21 is required for G1→S phase progression, and it can mediate formation of cyclin D1/CDK4 and other cyclin/cdk complexes.24, 41 This could explain increased p21 levels soon after partial hepatectomy8 and in other situations where p21 might facilitate cell cycle progression.24, 41, 42 However, p21 is most commonly known for its ability to inhibit cell cycle progression.7, 41 It does so by blocking cyclin/cdk complex activity and the replicative function of PCNA, which is required for DNA polymerase δ activity.7, 41 Although this study focuses on the role of p21 in cell cycle regulation, virtually all of the other known functions of p21, such as DNA excision and repair, protection from apoptosis, and cellular senescence,7, 41 are also of particular interest in the study of liver disease.
Despite a three-fold decrease in hepatocyte BrdU labeling in decompensated cirrhosis, liver mass is only 15% smaller than in compensated cirrhosis.9 In turn, absence of p21 in p21−/− mice caused nearly a threefold increase in hepatocyte BrdU labeling but only a 15% increase in liver mass compared with wild-type controls at 12 weeks after BDL. This observation suggested that proliferating hepatocytes appear to be able to undergo S phase, but then have problems negotiating the G2-M transition. This finding was confirmed by a comparison with Ki67 labeling, which showed that a significant proportion of cycling hepatocytes initiate a cell death program when exposed to a cholestatic environment, similar to events that occur after exposure of other p21−/− cells to DNA-damaging agents such as irradiation and anticancer drugs.28
This study confirms previous studies showing that oxidative stress is induced by cholestasis after BDL31; it also extends this line of inquiry by showing that oxidative stress is significantly greater in livers with decompensated compared with compensated cirrhosis. 3-Nitrotyrosine is a molecular footprint of oxidative stress left by the interaction between free radical nitric oxide (·NO) or RNS, and oxidants such as superoxide radicals (O2·), hydrogen peroxide (H2O2), and transition metal centers (e.g., iron).43 Reactive oxygen species induce cell cycle arrest through elevated p21 in H2O2-treated fibroblasts44 and in hepatocytes of ethanol-fed mice.18 Oxidative stress is also a common feature of chronic cholestatic liver diseases in humans, such as primary biliary cirrhosis.45
Chronic cholestatic injury preferentially targets periseptal hepatocytes for mito-inhibition. Periseptal hepatocytes are preferentially targeted in cholestatic liver diseases because they: (1) produce and secrete bile and are the major site of bile stasis and copper accumulation; (2) far outnumber biliary epithelial cells; and (3) contain many more mitochondria than biliary epithelial cells or cholangiocytes, and mitochondria are the major site of superoxide production.46 Thus, in a cholestatic and cytokine and growth factor–rich environment, mature biliary epithelial cells and cholangiocytes enjoy a relative growth advantage over hepatocytes, particularly in the periseptal regions. This leads to an exaggerated ductular reaction and more rapid architectural distortion compared with similar situations when hepatocyte proliferation is maintained.
Previous studies show that oval cell proliferation, or a type IV ductular reaction, occurs when p21-mediated mito-inhibition of hepatocytes is combined with the strong acute regenerative stimulus of partial hepatectomy.16, 47 However, BDL in adult mice does not cause oval cell expansion,48 a process that is usually transient and one that does not generally stimulate fibrosis. Moreover, no previous studies have examined the effect of chronic differences in hepatocyte p21 expression on other types of ductular reactions and on the evolution of cirrhosis, including architectural distortion and decompensation. Thus, it is not unreasonable to propose the following model (Fig. 7).
The important concept illustrated in this study is that when a persistent low-grade regenerative stimulus of chronic injury is combined with chronic preferential hepatocyte mito-inhibition and time, there is a selective growth advantage for biliary epithelial cells or cholangioles and myofibroblasts (or ductular reaction). These conditions result in more rapid distortion of the liver architecture and decompensation compared with situations where hepatocyte proliferation is maintained.9, 49 This report focuses primarily on the development of biliary cirrhosis, but data in patients with nonbiliary diseases and a collaborative study with Clouston et al.50 show that this concept is applicable to the evolution of HCV-induced cirrhosis and other causes of liver disease where oxidative stress contributes to hepatocyte injury. A shortcoming of many experimental models of liver injury is that they focus solely on short-term in vivo manipulations. The results are then extrapolated to human liver disease without examining the same processes under chronic conditions of naturally occurring human liver diseases. The time factor is important, because human liver diseases usually evolve over years to decades when many adaptive and counterregulatory influences develop to compensate for disease processes.
In conclusion, this study has directly linked processes in an experimental animal model where pathogenic mechanisms could be vigorously explored with human liver diseases under very similar conditions.