Abstract
Cholesterol 7α-hydroxylase (CYP7a1) is the rate-limiting enzyme in the classic pathway of bile acid synthesis. Expression of CYP7a1 is regulated by a negative feedback pathway of bile acid signaling. Previous studies have suggested that bile acid signaling is also required for normal liver regeneration, and CYP7a1 expression is strongly repressed after 70% partial hepatectomy (PH). Both the effect of CYP7a1 suppression on liver regrowth and the mechanism by which 70% PH suppresses CYP7a1 expression are unknown. Here we show that liver-specific overexpression of an exogenous CYP7a1 gene impaired liver regeneration after 70% PH, which was accompanied by increased hepatocyte apoptosis and liver injury. CYP7a1 expression was initially suppressed after 70% PH in an farnesoid X receptor/ small heterodimer partner-independent manner; however, both farnesoid X receptor and small heterodimer partner were required to regulate CYP7a1 expression at the later stage of liver regeneration. c-Jun N-terminus kinase and hepatocyte growth factor signaling pathways are activated during the acute phase of liver regeneration. We determined that hepatocyte growth factor and c-Jun N-terminus kinase pathways were involved in the suppressing of the CYP7a1 expression in the acute phase of live regeneration. Taken together, our results provide the significance that CYP7a1 suppression is required for liver protection after 70% PH and there are two distinct phases of CYP7a1 gene regulation during liver regeneration.
Cholesterol 7α-hydroxylase (CYP7a1) is the rate-limiting enzyme in the classic pathway of bile acid synthesis. This enzyme is negatively regulated at the transcriptional level by bile acids returning to the liver via enterohepatic circulation (1). The CYP7a1 promoter contains two highly conserved bile acid response elements (BARE-I and BARE-II). The rodent (rat and mouse) CYP7a1 gene contains a BARE-I, to which the liver X receptor-α/retinoic X receptor heterodimer binds and activates transcription of CYP7a1. Oxysterols, metabolites from cholesterol, increase CYP7a1 transcription through the activation of liver X receptor-α (2). However, this binding element is not found in the human CYP7a1 promoter. The BARE-II contains overlapping direct repeat (DR)1 and DR5 motifs that bind hepatocyte nuclear factor (HNF)4α and the retinoic acid receptor α, respectively. Conditional knockout of the HNF4α gene in mouse liver reduces serum cholesterol and triglyceride levels, as well as CYP7a1 mRNA expression, suggesting that HNF4α plays a critical role in bile acid metabolism and lipid homeostasis (3–6).
The BARE-II also contains a binding site for the nuclear receptor (NR) 5A2 family of monomeric orphan receptors, including rat and human α-fetoprotein transcription factor (7), and in mouse the liver receptor homolog (8–10). It is now well known that bile acid-activated farnesoid X receptor (FXR) inhibits CYP7a1 transcription through an indirect FXR-short heterodimer partner (SHP) axis (7, 9). On the other hand, the CYP7a1 gene is also regulated by SHP-independent mechanisms (11–13). Activation of FXR by bile acids leads to the induction of expression of the fibroblast growth factor (FGF)15 and -19 genes. FGF is secreted from the intestine and signals to hepatocytes through its cell-surface receptor, FGF receptor 4, which consequently leads to the suppression of CYP7a1 expression through a c-Jun N-terminal kinase (JNK)-dependent signaling cascade (14). Bile acids can also activate protein kinase C (PKC) or induce the synthesis of inflammatory cytokines (TNFα, and IL-1β) and their release from Kupffer cells. Both PKC and these cytokines can suppress CYP7a1 expression in hepatocytes (14).
Recent studies showed that both cytokines and growth factors can activate the MAPK/JNK signaling pathway to inhibit CYP7a1 transcription. One of these growth factors, hepatocyte growth factor (HGF), and its receptor, c-met, have been shown to be required for efficient liver regeneration and repair. Mice with a conditional liver-specific mutation of the c-met gene showed lethal stress during the acute phase of liver regeneration (15). Recently, Song et al. (16) and Kakizaki et al. (17) reported that HGF could repress CYP7a1 gene expression in human hepatocytes.
Our previous studies indicate that CYP7a1 expression was strongly suppressed after 70% partial hepatectomy (PH), suggesting an increased bile acid influx in liver. This is also consistent with a strong activation of FXR by bile acids to promote liver regeneration (18). We hypothesize that CYP7a1 suppression is important to control the hepatic bile acid levels and is required for liver protection during liver regeneration. Indeed, our results showed that overexpression of exogenous CYP7a1 in mice impaired liver regeneration, which was accompanied by increased hepatocyte death. Moreover, we identified two distinct phases of CYP7a1 gene regulation during liver regeneration.
Results
Overexpression of an exogenous CYP7a1 impairs liver regeneration after 70% PH
Previously we showed that feeding the mice with a low dose of cholic acid (CA) (0.2%) promoted liver regeneration in an FXR-dependent manner (18). To better understand the effect of bile acid levels on liver regeneration, we fed mice a diet containing 1% CA for 4 d and then performed 70% PH. All of the mice died after the PH, suggesting that high levels of bile acids during liver regeneration are toxic and lethal (data not shown). Because CYP7a1 is the rate-limiting enzyme for bile acid synthesis in the liver, we investigated the effect of CYP7a1 gene expression on liver regeneration by infecting liver with recombinant adenoviruses that express CYP7a1 following a standard procedure. Three days after infection, the mice showed significantly increased levels of mRNA and protein of CYP7a1 expression in liver (Fig. 1, A and B). Considerably fewer 2-bromodeoxy- uridine (BrdU)-positive nuclei were found in the livers from infected mice on the second day after 70% PH than in those of control treated group (Fig. 1, C and D), indicating that CYP7a1 overexpression impaired normal liver regeneration. Terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining revealed that exogenous CYP7a1 expression also induced apoptosis after 70% PH (Fig. 2, A–D), which is accompanied by abnormal liver histology as shown by hematoxylin and eosin staining, including ballooning degeneration of hepatocytes (Fig. 2E) and fat accumulation in the liver (Fig. 2H). These results suggest that CYP7a1 suppression after 70% PH is required for preventing hepatocyte death and liver damage.
Exogenous CYP7a1 overexpression impaired liver regeneration. A, Mice (n = 3) were injected with adenovirus expressing the CYP7a1 gene, control adenovirus, or HBSS. Mice were subjected 3 d later to 70% PH. Livers were harvested 48 h after 70% PH. Total liver RNA was prepared, and CYP7a1 mRNA expression was measured by QRT-PCR. B, Total liver protein was extracted, and levels of CYP7a1 protein were measured by Western blottings. C, BrdU staining of liver sections from the mice described in panel A. D, Quantification of BrdU-positive hepatocytes in three groups of mice. *, P < 0.01.
Exogenous CYP7a1 overexpression increased liver injury after 70% PH. Mice (n = 4) were injected with adenovirus expressing the CYP7a1 gene, control adenovirus, or HBSS. Mice were subjected 3 d later to 70% PH. Livers were harvested 48 h later and sectioned. A–C, TUNEL staining. D, Quantification of TUNEL-positive hepatocytes in three groups of mice. E–H, Representative areas of hematoxylin and eosin staining. I, Serum ALT levels from the treated mice. J, Hepatic bile acid levels from the treated mice. K, Levels of serum total bile acids from the treated mice. **, P < 0.01; *, P < 0.05.
On d 5 after infection (2 d after 70% PH), serum alanine aminotransferase (ALT) levels in mice injected with adenovirus expressing CYP7a1 were significantly higher than those in mice that received control virus (Fig. 2I). These results further supported the idea that sustained expression of CYP7a1 after 70% PH will lead to liver cytotoxicity in mice. Mice infected with adenovirus expressing CYP7a1 also showed significantly greater levels of hepatic bile acids, when measured on the second day after PH (Fig. 2J). Although it did not reach statistic significance, the CYP7a1 groups had consistently higher levels of serum bile acids (Fig. 2K). These results suggest that CYP7a1 suppression may be critical for promptly lowering liver bile acids levels, and hence, preventing liver injury after 70% PH.
Suppression of CYP7a1 expression via different mechanisms during early and late stages of liver regeneration
As a rate-limiting enzyme for bile acid synthesis, CYP7a1 is crucial for maintaining bile acid homeostasis and protecting the liver from bile acid-induced cytotoxicity. We previously showed by Northern blots that CYP7a1 expression was strongly suppressed after 70% PH (18). To more clearly and quantitatively demonstrate the changes of CYP7a1 expression, we repeated the experiments and used quantitative RT-PCR (QRT-PCR) for measuring the mRNA levels of CYP7a1. Wild-type (WT) mice were fed standard (control), 0.2% CA, or 2% cholestyramine resin diets for 4 d before 70% PH. On d0, d1, d2, and d3 after PH, mouse livers were harvested, and CYP7a1 expression was assessed by QRT-PCR (Fig. 3, A–D). The 2% cholestyramine resin diet significantly reduced bile acid levels in mice (data not shown), which led to a much higher basal CYP7a1 expression. Starting from d 1 post-PH, CYP7a1 expression was strongly suppressed. In the 0.2% CA-fed mice, CYP7a1 was expressed at low levels at all time points, probably due to the sustained high level of bile acids. However, on the second day, the expression of CYP7a1 was increased in the cholestyramine feeding group. These results indicate that during liver regeneration, bile acid levels affected CYP7a1 expression through a negative feedback loop.
Bile acids suppressed CYP7a1 gene expression during liver regeneration. WT male mice (n = 3 for each time point) were fed standard (A), 0.2% CA (B), or 2% cholestyramine resin (C) diets before 70% PH. On 0 d, 1d , 2d, and 3 d post-PH, livers were harvested, and RNA was prepared and subjected to QRT-PCR analysis of CYP7a1 mRNA expression. D, Graph showing the changes in CYP7a1 gene expression over 3 d.
FXR has a central role in the regulation of bile acid synthesis, and it is well-established that the FXR-SHP pathway regulates the bile acid-mediated suppression of CYP7a1 expression. To investigate the roles of FXR and SHP in CYP7a1 gene regulation, we performed 70% PH on WT, FXR−/−, and SHP−/− mice. On d0, the expression of CYP7a1 in livers from WT mice was lower than in those from FXR−/− and SHP−/− mice (Fig. 4, A–D), indicating that the FXR-SHP pathway is required to maintain the levels of CYP7a1 gene expression under physiological conditions. On d 1 post-PH, qPCR showed a similar, dramatic decrease in CYP7a1 mRNA in all three groups of mice, suggesting that an FXR- and SHP-independent pathway might be involved in suppression of CYP7a1 gene expression. On d 2 (SHP−/−) and d 3 (FXR−/−), the CYP7a1 expression was higher than that in the WT group, suggesting that at later stages of post-PH, both FXR and SHP are required to regulate expression of CYP7a1. These results indicate that different mechanisms may be involved in regulating CYP7a1 expression during the early and late stages of liver regeneration.
FXR and SHP are required to regulate CYP7a1 gene expression at late stages of liver regeneration; 70% PH was performed on WT (A), FXR−/− (B), and SHP−/− (C) mice (n = 3 for each time point). On d 0, 1, 2, and 3 post-PH, livers were harvested, and RNA was prepared and subjected to QRT-PCR analysis of CYP7a1 expression. D, Changes in CYP7a1 gene expression over 3 d.
JNK1 is involved in CYP7a1 suppression in an FXR- and SHP-independent manner during the acute phase of liver regeneration
To further investigate the molecular mechanism of CYP7a1 repression during the early stages of liver regeneration, we determined the suppression of CYP7a1 expression at 1 and 6 h after 70% PH. CYP7a1 mRNA level decreased by approximately half 1 h after PH in all genotypes of mice. Six hours after PH, CYP7a1 mRNA expression was further suppressed in all three groups of mice (WT, FXR−/−, and SHP−/−) (Fig. 5A–D), although the WT mice had a greater fold of decrease in expression than the FXR−/− and SHP−/− mice. These results suggest that FXR- and SHP-independent pathways exist in CYP7a1 suppression during the acute phase of liver regeneration.
Expression of CYP7a1 was suppressed in an FXR- and SHP-independent manner during acute phase of liver regeneration. WT (A), FXR−/− (B), and SHP−/− (C) mice (n = 3 for each time point) were subjected to 70% PH. Livers were harvested 1 and 6 h after surgery. Total liver RNA was prepared and subjected to QRT-PCR analysis of CYP7a1 mRNA expression. D, Changes in CYP7a1 gene expression. *, P < 0.05.
Because JNK1 has been shown to be an important downstream pathway by different stimuli in suppressing CYP7a1 expression, we ask whether JNK1 is activated and is involved in CYP7a1 suppression during the acute phase of liver regeneration. FXR is known to activate FGF signaling in intestine and signals to liver, which also results in JNK activation. We also ask whether JNK is activated via an FXR-dependent pathway during acute phase of liver regeneration. We performed 70% PH on WT, FXR−/−, and JNK1−/− mice. Liver tissues were harvested for total RNA and total protein isolation at 1 and 6 h post-PH. Western blottings showed that both WT and FXR−/− mice had considerably increased levels of phosphorylated c-Jun protein at 1 and 6 h after 70% PH compared with the controls before surgery (Fig. 6A). Unlike in WT mice, CYP7a1 expression was not significantly repressed in JNK1 knockout mice 1 h after 70% PH (Fig. 6B). These results suggest that JNK1 may be involved in suppression of CYP7a1 expression during the acute phase of liver regeneration in an FXR-independent manner.
JNK1 contributes to CYP7a1 suppression in an FXR-independent manner. WT, FXR−/−, and JNK1−/− mice (n = 4 for each time point) were subjected to 70% PH. Livers were harvested at 1 h and 6 h, respectively, after surgery. A, Phosphorylated c-Jun (Phos-c-Jun), total c-Jun, and phosphorylated JNK (Phos-JNK) liver proteins were measured by Western blotting, as indicated. The same blot was reprobed with anti-β-actin antibody as control. B, QRT-PCR analysis of CYP7a1 mRNA expression. *, P < 0.05.
HGF/c-met inhibitor reduces CYP7a1 suppression during the acute phase of liver regeneration
We hypothesize that some early growth factors may play roles in CYP7a1 suppression during the acute phase of liver regeneration. As shown in Fig. 7, A and B, both circulating HGF and hepatic HGF were significantly increased after 70% PH. Previous reports indicate that the HGF/c-met pathway is required for efficient liver regeneration and repair (15), and HGF inhibits CYP7a1 gene expression in both SHP-dependent and -independent manner (16, 17). To determine whether HGF/Met plays a role in repressing CYP7a1 gene expression during liver regeneration, we injected the mice with a Met inhibitor, Su11274, before 70% PH. Significantly less repression of CYP7a1 expression was observed in the Su11274-treated group as compared with the vehicle-treated group 1 h after 70% PH (Fig. 7C). HGF activates both ERK and JNK1 pathways. Western blottings showed that MAPKs in the liver were activated in vehicle-treated control mice. However, mice pretreated with Su11274 exhibited reduced levels of both phosphorylated ERK and JNK 1 h after 70% PH (Fig. 7, D and E). These results suggest that HGF signaling is involved in suppressing CYP7a1 expression during the acute phases of liver regeneration.
Inhibition of HGF signaling reduced CYP7a1 suppression after 70% PH. Mice (n = 3 for each time point) were pretreated with Su11274 before 70% PH. RNA was prepared from livers that were harvested at 1 and 6 h after surgery. A and B, Serum and hepatic HGF levels were measured by ELISA. C, QRT-PCR analysis of CYP7a1 mRNA expression. *, P < 0.05. D, Western blotting analysis of liver levels of phosphorylated ERK1/2 (Phos-ERK1/2) and total ERK1/2 proteins from the mice treated with Su11274 (+) or vehicle control (−). E, Western blotting analysis of liver levels of phosphorylated JNK (Phos-JNK) and total JNK proteins from mice treated with Su11274 (+) or vehicle control (−).
Discussion
Liver regeneration after PH is a process of compensatory hyperplasia driven by the replication of existing hepatocytes. Many pathways are involved in this process such as cytokine, growth factor, and metabolic networks that link liver function with cell growth and proliferation (20). Bile acids are amphipathic products of cholesterol catabolism with well-established roles in dietary lipid absorption and cholesterol homeostasis. In addition, a large body of evidence demonstrates that bile acids are also signaling molecules with systemic endocrine functions. Bile acids can activate MAPK pathways (21, 22) and are ligands for the G-protein-coupled receptor TGR5 (23, 24). They also activate nuclear hormone receptors such as FXR (25–27), pregnane X receptor (28, 29), and vitamin D receptor (25). We recently showed that PH increases bile acid flux and generates a metabolic stress. On one hand, the bile acid receptor FXR is activated to promote liver regeneration. On the other hand, the expression of a key enzyme in bile acid synthesis, CYP7a1, is strongly repressed. These two parallel mechanisms may cooperate together to protect hepatocytes from bile acid-induced toxicity. After the liver has fully regenerated and the functional deficit is eliminated, the bile acids return to normal levels and the loss of proliferative stimuli leads to the cessation of liver regeneration and the reemergence of CYP7a1 expression (18). This fine tuning of bile acid homeostasis at the biosynthesis level is crucial for normal liver regeneration. Indeed, after 70% PH, the levels of hepatic CYP7a1 expression are sharply suppressed, but the functional importance of such regulation has not been clearly elucidated.
Our results indicate that exogenous overexpression of CYP7a1 significantly suppressed hepatocellular proliferation in WT mice subjected to 70% PH. We showed that bile acid levels are increased in mice that overexpress exogenous CYP7a1, which is consistent with a previous report (30). The impaired regenerative response seen in mice that overexpressed CYP7a1 may be due to increased levels of bile acids. Therefore, efficient suppression of CYP7a1 mRNA after 70% PH may be required for preventing bile acid-induced liver toxicity and accelerating the restoration of hepatic mass in the remaining liver. The mice that overexpress CYP7a1 eventually recovered and had full liver regeneration, probably due to the transient effect of virus infection. The more stable CYP7a1 transgenic mice may provide better models to investigate the long-term effects of CYP7a1 overexpression on liver regeneration.
CYP7a1 is under tight regulation by different pathways and is regulated mainly at the transcriptional level by a number of factors, including nuclear receptors, protein kinase C activator, cytokines, growth factors, and bile acids (12, 18, 31, 32). In our study, we found that a multifaceted molecular mechanism was involved in regulating CYP7a1 gene suppression during early and late stages after 70% PH.
As a bile acid sensor, the nuclear receptor FXR has been proposed to play a central role in the bile acid feedback inhibition of CYP7a1 expression. In addition, the FXR-SHP-regulatory cascade is involved in bile acid-mediated suppression of CYP7a1 (9). On d 1 post-PH, similar to the WT mice, FXR−/− and SHP−/− mice had a strong decrease in CYP7a1 mRNA levels. These results were in line with our previous report (18), which indicated that in the early phase of liver regeneration, FXR- and SHP-independent pathways might be involved in bile acid-mediated CYP7a1 gene suppression. However, on d 3 (FXR−/−) or d 2 (SHP−/−), CYP7a1 mRNA levels were significantly higher compared with the WT group, which demonstrated that in the relatively late stage of liver regeneration, FXR and SHP are required to regulate the transcription of CYP7a1 gene expression. Our data provide the first evidence that CYP7a1 suppression is achieved via different molecular mechanisms during the two distinct phases of liver regeneration. FXR- and SHP-independent mechanisms are involved in the inhibition of CYP7a1 expression during the acute stages, but both FXR and SHP are required for CYP7a1 suppression during the late stages.
JNK is a member of an evolutionarily conserved subfamily of MAPKs (33). The stimulation of JNK activity is one of the earliest events and most sensitive changes after PH. Bile acid signaling can induce the JNK activity via PKC and inflammatory cytokine-mediated pathways, or by FXR-induced expression of FGF-19 in intestine, a known upstream activator of the JNK signaling pathway (34). Phosphorylated c-Jun may form a transcriptional repressor complex with a positive transcription factor such as liver receptor homolog or HNF4α, and thereby preventing the transcription factors from activating CYP7a1. In addition, De Fabiani et al. (35) proposed that direct phosphorylation of HNF4α by JNK might also reduce HNF4α transactivation of CYP7a1. In our study, we found that in both WT and FXR−/− mice, the levels of phosphorylated c-Jun were significantly increased at 1 and 6 h after 70% PH. These results suggest that JNK is activated independently of FXR expression during the acute phase of liver regeneration. Repression of CYP7a1 gene expression in JNK1 knockout mice is significantly weaker at 1 h after 70% PH than in WT mice, suggesting that JNK1 may be involved in the suppression of CYP7a1 during the acute phase of liver regeneration. The JNK pathway may allow the hepatocytes to more rapidly adapt to the sudden increase in bile acid flux by suppressing CYP7a1 expression (36).
Our results also suggest a potentially important role for the HGF/c-met pathway in mediating suppression of CYP7a1 during the early stages after 70% PH. Previous reports indicate that HGF and its receptor, c-met, are required for efficient liver regeneration and repair (15–17). Mice with mutations in the c-met gene showed lethal stress during the acute phase of liver regeneration (15). Recently, in vitro work by Song et al. (16) and Kakizaki et al. (17) indicated that HGF inhibited CYP7a1 gene expression in both SHP-dependent and SHP-independent manners. Indeed, our results indicate that inhibition of Met by a chemical inhibitor significantly reduced the suppression of CYP7a1 gene expression at 1 h after 70% PH. Therefore, this growth factor-mediated regulation of CYP7a1 gene expression may represent one of the earliest pathways to suppress CYP7a1 expression. However, both JNK and HGF require more studies in term of their roles in suppressing CYP7a1 gene expression during liver regeneration. Important early events after 70% PH, i.e. the secretion of growth factors and cytokines such as TGF, produced in hepatocytes, and IL-6 and TNF, produced in nonparenchymal cells, have been shown to play important roles during liver regeneration. Further examination of the genes involved in the suppression of CYP7a1 gene expression during the acute phase of liver regeneration is likely to reveal novel regulatory mechanisms.
Liver regeneration has gained much interest in the medical field today, due to the increasing frequency of liver transplantation. The regrowth of transplanted livers appears to be regulated in the same manner as liver regeneration after PH in laboratory animals (37). Understanding the mechanisms of liver regeneration, such as the role of CYP7a1 and bile acid signaling, is crucial for the appropriate development of new therapies for many severe liver diseases, including acute liver failure and cirrhosis (20). However, human and rodents may have different regulatory mechanisms in CYP7a1 expression. Therefore, the roles of CYP7a1 suppression for normal liver regeneration in humans still need to be established.
Materials and Methods
Animal maintenance and treatments
FXR and SHP knockout mice were described previously (18). JNK1 knockout mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All procedures followed the NIH guidelines for the care and use of laboratory animals. Mice were housed in a pathogen-free animal facility under standard 12-h light, 12-h dark cycle and fed standard rodent chow and water ad libitum. Mice between 8 and 10 wk old were used in each group of experiments. For CA and cholestyramine feeding, mice were fed with a control diet, 0.2% CA diet, or 2% cholestyramine diet for 5 d before they were subjected to 70% hepatectomy, as described previously (18). After surgery, the mice were fed the same diet as before surgery until termination of experiments.
For c-met inhibition studies, wild-type mice were ip injected with Su11274([(3Z)-N-(3-chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-N-methyl-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide) (Calbiochem, CA) in 2% dimethylsulfoxide (40 μg/mice/d) for 3 d. A sc injection was given 2 h before 70% PH.
Propagation, purification, and injection of the adenovirus containing cytomegalovirus promotor (CMV) driven (AdCMV)-CYP7a1 adenovirus
The AdCMV-CYP7a1 viruses encoding the rat CYP7a1 were obtained from Dr. Philip B. Hylemon (Virginia Commonwealth University, VA). Recombinant virus was produced by infecting confluent monolayers of human embryonic kidney 293 cells grown in 10-cm tissue culture dishes with stock adenovirus at a multiplicity of 1 plaque-forming unit (pfu)/cell. After 3 h of infection, the media-containing virus was removed and replaced with DMEM with 10% fetal bovine serum. Adeno-X Maxi purification kits (CLONTECH, Mountainview, CA) were used to purify the adenoviruses encoding CYP7a1 and the control viruses, according to the manufacturer’s instructions. Briefly, the infected 293 cells were harvested when more than 90% of the cells showed cytopathic changes. A second round of amplification was carried out by infecting fresh human embryonic kidney 293 cells, and the viruses were purified via the adeno-X Maxi purification kits. The virus was aliquoted and stored at −80 C. The virus titer was determined by plaque assay, and viral particles were determined by optical density using spectrophotometry (=260 nm). C57BL/6 mice (8–10 wk old) were injected with one of the following: the rat CYP7a1 adenovirus, the control virus, or the saline vehicle (HBSS) via the tail vein (1010 pfu in a volume of 200–300 μl of HBSS). On the third day after adenovirus injection, 70% PH was performed.
Liver histology
After mice were euthanized, their livers were removed and small pieces from different lobes of the livers were fixed in 4% formaldehyde-PBS solution, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. For BrdU staining, mice were ip injected with BrdU solution (10 mg/kg body weight) 2 h before euthanasia. Liver sections were prepared and stained using a BrdU staining kit (Roche, Indianapolis, IN). The number of positively stained cells was counted in three randomly selected fields for each tissue section. The tissue sections were also subjected to TUNEL staining (kit from Roche) for detection of apoptotic cells.
RNA analysis
Total liver RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. QRT-PCR was performed using SYBR Green PCR Master Mix and an ABI prim 7300 Sequence Detection System (Applied Biosystems, Foster city, CA). Murine 36B4 and β-actin were used as internal controls. PCR primers specific for each gene are listed below (5′–3′): mouse CYP7a1-F (forward), CAA GAA CCT GTA CAT GAG GGA C; mouse CYP7a1-R (reverse), CAC TTC TTC AGA GGC TGC TTT C; 36B4-F, GCC CTG CAC TCT CGC TTT CT; 36B4-R, CAA CTG GGC ACC GAG GCA ACA GTT G; rat CYP7a1-F, ATG ACA CGC TCT CCA CCT TTG A; rat CYP7a1-R, AGC TCT TGG CCA GCA CTC TGT A; Quantum RNA β-actin (Ambion, Inc., Austin, TX).
Western blotting
Livers were homogenized in protein lysis buffer [50 mm Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonylfluoride, 1 mm Na3VO4, 1 mm NaF, and proteinase inhibitor cocktail (Roche)]. Proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane and detected by chemiluminescence (Supersignal; Pierce Chemical Co., Rockford, IL). Western blotting was performed using antibodies from Signaling Technology: anti-ERK1/2 MAPK, anti-phospho-ERK1/2 (Thr202/Tyr204), JNK (56G8) rabbit monoclonal antibody, phospho-JNK (Thr183/Tyr185), phospho-c-Jun (Ser63) II, c-Jun (60A8) rabbit monoclonal antibody. Anti-CYP7a1 and anti-β-actin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Serum and hepatic bile acids
Serum and regenerating livers were collected after 70% PH, and the bile acids were measured as we described previously (18) using a kit from Diagnostic Chemicals Ltd. (Charlottetown, Prince Edward’s Island, Canada).
Serum ALT levels
ALT levels were measured at the clinical laboratory at City of Hope Helford Hospital.
Serum and hepatic HGF levels
Mice serum and liver were collected at indicated time points after 70% PH. Serum HGF were measured directly by using HGF Duoset ELISA Development kit (R&D Systems, Inc., Minneapolis, MN), and hepatic HGF was measured according to a previous report (19).
Statistical analyses
Data are expressed as means ± sem. Two-tailed Student’s t test was used to determine differences between data groups. All analyses were performed using one-way ANOVA. P < 0.05 was considered statistically significant unless otherwise stated.
Acknowledgments
We thank Dr. Keely Walker for her help in manuscript editing and Dr. Philip B. Hylemon for his generous gift of CYP7a1 adenoviruses.
W.H. is supported by the Sidney Kimmel Foundation for Cancer Research, Margaret E. Early Trust Medical Research Award, and developmental funds from City of Hope. D.D.M. is supported by National Institutes of Health Grant RO1 DK053366.
Disclosure Statement: The authors have nothing to disclose.
L.Z. and X.H. contributed equally to this work.
Abbreviations:
- ALT,
Alanine aminotransferase;
- BARE,
bile acid response element;
- BrdU,
2-bromodeoxy-uridine;
- CA,
cholic acid;
- CYP7a1,
cholesterol 7α hydroxylase;
- FGF,
fibroblast growth factor;
- FXR,
farnesoid X receptor;
- HGF,
hepatocyte growth factor;
- HNF,
hepatocyte nuclear factor;
- JNK,
c-Jun N-terminus kinase;
- PH,
partial hepatectomy;
- PKC,
protein kinase C;
- QRT-PCR,
quantitative real-time PCR;
- SHP,
small heterodimer partner;
- TUNEL,
terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling;
- WT,
wild type.
