Dynamic expression of extracellular signal-regulated kinase in rat liver tissue during hepatic fibrogenesis

Abstract

AIM: To investigate whether extracellular signal-regulated kinase 1 (ERK₁) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue.

METHODS: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were evaluated by hematoxylin and eosin staining, and Masson’s trichrome method. ERK₁ mRNA in rat liver tissue was determined by reverse transcription-polymerase chain reaction, while the distribution of ERK₁ was assessed by immunohistochemistry. ERK₁ protein was detected by Western blotting analysis. The number of activated HSCs was quantified after alpha smooth muscle actin (α-SMA) staining.

RESULTS: With the development of hepatic fibrosis, the positive staining cells of α-SMA increased obviously, and mainly resided in the portal ducts. Fiber septa and perisinuses were accompanied with proliferating bile ducts. The positive staining areas of the rat livers in model groups 1-4 wk after ligation of common bile duct (12.88% ± 2.63%, 22.65% ± 2.16%, 27.45% ± 1.86%, 35.25% ± 2.34%, respectively) were significantly larger than those in the control group (5.88% ± 1.46%, P < 0.01). With the development of hepatic fibrosis, the positive cells of ERK₁ increased a lot, and were mainly distributed in portal ducts, fiber septa around the bile ducts, vascular endothelial cells and perisinusoidal cells. Western blotting analysis displayed that the expression of ERK₁ and ERK₂ protein was up-regulated during the model course, and its level was the highest 4 wk after operation, being 3.9-fold and 7.2-fold higher in fibrotic rat liver than in controls. ERK₁ mRNA was expressed in normal rat livers as well, which was up-regulated two days after BDL and reached the highest 4 wk after BDL. The expression of ERK₁ was positively correlated with α-SMA expression (r = 0.958，P < 0.05).

CONCLUSION: The expression of ERK₁ protein and mRNA is greatly increased in fibrotic rat liver tissues, which may play a key role in HSC proliferation and hepatic fibrogenesis.

Key Words: Extracellular signal-regulated kinase; Hepatic fibrosis; Hepatic stellate cells; Proliferation

INTRODUCTION

Mitogen-activated protein kinase (MAPK) pathway is an important intracellular signal transduction system[1], and extracellular signal-regulated kinase 1 (ERK₁) is the critical and classical pathway of MAPK and plays an important role in several physiological phenomena, including cell local adhesion, migration, proliferation, differentiation, apoptosis and cell cycle[2]. Hepatic stellate cells (HSCs) play a pivotal role in hepatic fibrogenesis[3-6]. In normal liver, HSCs are perisinusoidal mesenchymal elements characterized by intracytoplasmic lipid droplets rich in retinyl esters. On the contrary, in chronic liver injury, HSCs undergo a response known as “activation”, which is the transition of quiescent cells into proliferative, fibrogenic and contractile myofibroblasts[7-9].

Up till now, many studies have focused on the role of MAPK in various cultured cells[10,11], little is known about its regulation in vivo[12]. No report on dynamic expression of ERK₁ mRNA in fibrotic liver tissue is available. To probe into the molecule mechanism of fibrogenesis by ERK₁, the changes in distribution and contents of ERK₁ protein and mRNA in rat hepatic fibrogenesis were observed by immunohistochemistry, Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The dynamic expression of α-smooth muscle (α-SMA) as a marker of activated HSCs was also examined by immunohistochemistry in this study.

MATERIALS AND METHODS

Reagents

The monoclonal antibodies against ERK₁ and α-SMA were products of Santa Cruz Biotech Inc. Streptavidin-peroxidase immunohistochemical kit was purchased from Zhongshan Biological Technology Co (Beijing). Trizol reagent was obtained from Life Technologies Inc (USA). One tube RT-PCR kit was from Promega Co (USA). Primers for rat ERK₁ and β-actin were designed by ourselves in accordance with gene sequence in Genebank, synthesized and purified by Bao Biological Engineering Co (Dalian). All other reagents were analytically pure.

Animal model and experimental protocol

A total of 80 adult male Sprague-Dawley rats weighing 350-400 g were purchased from the Experimental Animal Center of Hebei Medical University (Clearing Grade, Certificate No. 04057). All rats were housed in plastic cages with free access to food and water. The rats were randomly divided into 8 groups (10 rats in each group). The rats were subjected to laparotomy with complete ligation of the common bile duct and received ketamine hydrochloride at a dose of 100 mg/kg by intraperitoneal injection[13]. Under deep anaesthesia, the peritoneal cavity was opened and the common bile duct was double-ligated with 3-0 silk and cut between the ligatures. Control animals underwent a sham operation that consisted of exposure but not ligation of the common bile duct. At various intervals postoperatively, the animals were anaesthetised and the livers were harvested. Liver tissue specimens were routinely fixed in 10% phosphate-buffered formaldehyde and embedded in paraffin. Some liver tissue specimens were used for light microscopy and immunohistochemistry by using anti-α-SMA and ERK₁, while others were snap-frozen in liquid nitrogen and stored at -80°C for RNA analysis. In addition, control livers were harvested 4 wk after sham operation.

Histopathology

Liver specimens were routinely fixed overnight in 10% phosphate-buffered formaldehyde, embedded in paraffin for light microscopic examination. Tissue sections (5-μm thick) were stained with haematoxylin and eosin (H&E) for morphological evaluation and Masson’s trichrome for assessment of fibrosis.

Immunohistochemical detection of α-SMA and ERK₁

All immunohistochemical studies using the streptavidin-peroxidase technique were performed on 5-μm thick paraformaldehyde-fixed and paraffin-embedded liver tissue sections mounted on APES-coated slides. Slides were deparaffined in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was quenched with a 3% hydrogen peroxide solution in methanol at room temperature for 30 min, followed by rinsing in pH 6.0 phosphate-buffered saline (PBS). After antigen retrieval in a water bath set in a 10 mmol/L citrate buffer (pH 6.0) at 94°C for 8 and 10 min, respectively, the slides were immediately cooled for 20 min at room temperature. Non-specific binding sites were blocked by incubation with wash buffer containing 10% normal goat serum at 37°C for 30 min. The sections were then incubated overnight at 4°C with a mouse monoclonal antibody directed against α-SMA or a rabbit monoclonal antibody directed against ERK₁ at a dilution of 1:100. The secondary antibody bindings were localized using a biotin-conjugated rabbit anti-mouse IgG for α-SMA and goat anti-rabbit IgG for ERK₁ (1:100 dilution), followed by incubation with streptavidin-peroxidase complex (1:200 dilution). Peroxidase conjugates were subsequently visualised by utilizing diaminobenzidine (DAB) solution in hydrogen peroxide as a chromogen yielding a brown reaction product. The sections were then counterstained with Mayer’s hematoxylin and mounted on a cover slip. All incubations were performed in a moist chamber. Furthermore, between each incubation step, the slides were washed 3 times with PBS for 5 min. To ensure the specificity of antibody, negative control samples were processed in parallel under the same conditions but with omission of the first antibody, which was replaced by an equal volume of PBS. The α-SMA and ERK₁ positive parenchymas were measured by a video-image analysis system and expressed as a percentage of area occupied by the signal.

Preparation of cell lysates and Western blotting analysis

Liver tissues from control and bile duct ligation (BDL) animals were quickly removed and washed twice with ice-cold PBS, and then homogenized in modified radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L Tris-HCl, pH 7.5; 150 mmol/L NaCl; 1% Nonident P-40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate; 1 mmol/L EDTA; 1 mmol/L PMSF; 2 μg/mL l × leupeptin) for 30 min at 4°C, followed by rotating the tubes at 12 000 × g at 4°C for 10 min. After centrifugation, cleared tissue lysates were collected and stored at -80°C and protein concentration of each sample was determined by Coomassie brilliant blue protein assay. Each sample was adjusted up to a desired protein content of 150 μg, an equal volume of 5 × SDS loading buffer was added and the sample was incubated at 100°C for 3 min. Lysate containing 150 μg of protein was separated by electrophoresis on 10% acrylamide sodium dodecyl sulfate (SDS) gels and transferred onto nitrocellulose membranes. After blocked in a buffer (pH 7.2) containing 1% bovine serum albumin and 5% skim milk powder, the membranes were incubated with rabbit anti-ERK₁ antibodies diluted at 1:600 overnight at 4°C, then incubated for 2 h at 37°C with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody diluted 1:5000 in a blocking buffer. After washed 3 times with PBS containing 0.1% Tween 20, immunoreactive proteins were visualized with an enhanced chemiluminescence detection system on radiograph film. The intensity of the bands was determined by scanning video densitometry. Experiments were performed at least 3 times with similar results.

RNA extraction and RT-PCR assay

Expression of ERK₁ mRNA was evaluated by RT-PCR. Total RNA from liver specimens (100 mg) was isolated using a monophasic solution of phenol and guanidine thiocyanate (Trizol), precipitated in ethanol and resuspended in sterile RNAase-free water for storage at -80°C until use, as recommended by the suppliers. Total RNA was quantified spectrometrically at 260 nm, and the quality of isolated RNA was analysed on agarose gels under standard conditions. One-step RT-PCR was performed according to the manufacturer’s instructions. Two-microgram RNA was added to each reaction and RT-PCR was routinely performed utilizing 5 units of AMV reverse transcriptase, 5 units of Tfl DNA polymerase, 10 pmol of each oligonucleotide primer, 10 pmol of dNTP mix and 25 mmol/L MgSO₄ in a final reaction volume of 50 μL. Primer sequences were as follows: ERK₁ : forward 5’- GCT GAC CCT GAG CAC GAC CA -3’ and reverse 5’- CTG GTT CAT CTG TCG GAT CA -3’, fragment length 451 bp; β-actin: forward 5’- AGC TGA GAG GGA AAT CGT GCG -3’ and reverse 5’- GTG CCA CCA GAC AGC ACT GTG -3’, fragment length 300 bp. RT-PCR was performed in the following steps: reverse transcription at 41°C for 45 min, pre-denaturation at 94°C for 2 min. Then amplification was performed in a thermal controller for 35 cycles (denaturation at 94°C for 40 s, annealing at 52°C for 1 min, extension at 72°C for 1.5 min, and a final extension at 72°C for 10 min). Ten μL of the PCR products was analyzed by 1.5% agarose gel electrophoresis with TAE buffer at 80 V for 40 min, visualized with ethidium bromide staining and photographed under UV illumination. The band intensities were quantified by densitometry. ERK₁/β-actin quotient was the indication of ERK₁. Experiments were performed at least 3 times with similar results.

Statistical analysis

The data were expressed as mean ± SD. Group means were compared by using analysis of variance followed by the Student-Newman-Keuls test if the former was significant. The correlation between the expressions of ERK₁ and α-SMA was analyzed for statistical significance by the simple linear regression analysis. P < 0.05 was considered statistically significant.

RESULTS

Histology of progressive fibrotic liver injury

In the present study, spotted (or scattered) perivenular degeneration of hepatocytes, increased inflammatory infiltrate in the necrotic areas and bile ductular proliferation in the portal triads were observed after 1 wk of BDL. After 2 wk of BDL, all rats showed expanded portal tracts with fibrous tissue, portal-to-portal fibrous bridging, nodular transformation and widespread proliferating bile ductules which extended into the parenchyma in places, without clear-cut cirrhosis. After 3-4 wk of BDL, the animals developed severe fibrosis associated with proliferating bile ducts which formed a continuous meshwork of connective tissue with complete distortion of lobular architecture, whereas there was no histological abnormality or evidence of stainable collagen in any of the sham-operated control livers.

Identification of proliferating and activated HSCs

Immunostaining for α-SMA was used to detect and quantify the number of activated HSCs in this study. The α-SMA positive cells in the sham-operated control livers were observed in vascular smooth muscle cells and sinusoids with a weak staining. With the development of hepatic fibrosis，the number of positive α-SMA cells was greatly elevated and mainly resided in the cells of portal ducts, fiber septa, and perisinuses accompanied with proliferating bile ducts. The positive areas of the rat livers were larger in model groups at 1 to 4 wk (12.88 % ± 2.63%, 22.65% ± 2.16%, 27.45% ± 1.86%, 35.25% ± 2.34%) than in control group (5.88% ± 1.46%) (P < 0.01, Figure 1A-1C).

Figure 1 α-SMA protein expression in immunohistochemically-stained liver tissue (SP × 200). A: Few α-SMA expressions in sham operation group; B: Positive α-SMA cells residing in the cells of portal ducts, fiber septa, perisinuses and around the proliferated bile ducts 2 wk after BDL; C: Positive areas of α-SMA expression in model groups at wk 1 to 4 after common bile duct ligation.

Distribution of ERK₁ protein in bile duct-ligated rat liver tissues

To explore the distribution of ERK₁, the normal and fibrotic rat liver tissue sections were immunostained using specific monoclonal anti-ERK₁ antibody. ERK₁ was found in vascular endothelial and perisinusoidal cells of normal rat liver tissue sections. With the development of hepatic fibrosis, the positive ERK₁ cells increased a lot and were mainly distributed in portal ducts, fiber septa, around the bile ducts, as well as in vascular endothelial and perisinusoidal cells and hepatocytes. ERK₁ protein was expressed not only in cytoplasm mentioned above, but also in nuclear membrane, indicating its activation. The positive areas of rat livers in model groups at 1 to 4 wk (9.58% ± 1.01%, 17.43% ± 1.78%, 23.88% ± 2.97%, 28.63% ± 2.72%) were larger than those in control group (5.03% ±1.10%) (P < 0.01, Figure 2A-2C).

Figure 2 ERK₁ distribution in immunohistochemically-stained liver tissue (SP × 200). A: ERK₁ distribution in sham operation group; B: ERK₁ distribution 2 wk after BDL; C: The time course of ERK₁ distribution in hepatic fibrogenesis.

Expression of ERK₁ protein in common bile duct-ligated rat livers

Western blot analysis showed that ERK₁ was expressed in normal rats with a prolonged model-making period. The expression of ERK₁ increased 3.07-, 4.51-, 4.36-, 4.36-, 4.36- and 4.71-fold at 2, 6 h; 2 d; 1, 2 and 3 wk; respectively, and reached the peak at 4 wk, which was 4.93 times of the expression in sham-operated group (P < 0.01, Figure 3A and 3B).

Figure 3 Time course of ERK₁ protein expression (A) and quantitative results (B) in fibrotic and normal rat liver tissues by Western blotting analysis. 1: 4 wk after BDL; 2: 3 wk after BDL; 3: 2 wk after BDL; 4: 1 wk after BDL; 5: 2 d after BDL; 6: 6 h after BDL; 7: 2 h after BDL; 8: Sham operation group.

Increased ERK₁ mRNA expression in bile duct-ligated rat livers

Although ERK₁ protein is produced by liver tissue in vivo, it is not clear whether the ERK₁ mRNA level under fibrogenic response is increased in vivo. Therefore, we investigated the production of ERK₁ mRNA in liver. Total liver RNA was analyzed by RT-PCR. The results revealed faint transcript of ERK₁ mRNA in sham-operated rat livers. However, specific banding to ERK₁ mRNA was detected in fibrotic liver sections. Moreover, ERK₁ mRNA expression was initially up-regulated at 6 h and reached its peak level 4 wk after BDL, which increased 2.4-fold. Levels of the housekeeping gene, β-actin, did not show any significant differences between control and BDL rat liver tissue (Figure 4A and 4B).

Figure 4 Electrophoresis of RT-PCR for ERK₁ mRNA expression in hepatic fibrogenesis at different time points (A) and percentage of optical density of ERK₁ mRNA electrophoretic strip (B). 1: marker; 2: sham operation group; 3: 2 h after BDL; 4: 6 h after BDL; 5: 2 d after BDL; 6: 1 wk after BDL; 7: 2 wk after BDL; 8: 3 wk after BDL; 9: 4 wk after BDL.

Analysis of relation between ERK₁ and α-SMA

Immunohistochemistry experiments were performed to analyze whether the above described ERK₁ protein distribution was correlated with α-SMA in sham operated livers compared with BDL rats. The results indicated that ERK₁ was positively correlated with α-SMA (r = 0.958, P < 0.05).

DISCUSSION

HSCs, the principal cellular source of extracellular matrix during chronic liver injury, undergo a transition into α-SMA-expressing myofibroblastlike cells in response to various liver injuries such as ECM, cytokine, inflammation mediator, ethanal, oxygen radical and lactic acid. Furthermore, HSC activation is associated with stellate cell proliferation, increased contractility and enhanced matrix production. HSCs express a number of fibrogenic and proliferative cytokines and their cognate receptors. Therefore, HSCs play a crucial role in cellular and molecular events that lead to hepatic fibrosis[4]. Cassiman et al[13] and Ramm et al[14] have demonstrated that positive α-SMA cells mainly reside in the portal ducts and fiber septa, accompanied with proliferating tubercle, corresponding to the distribution of collagen. This study displayed that with the development of hepatic fibrosis, the positive α-SMA cells were greatly elevated and mainly situated in cells of the portal ducts, fiber septa, perisinuses accompanied with proliferating bile ducts. The positive areas of α-SMA in rat livers of model groups at 1 to 4 wk increased in turn and were larger than those in control group (P < 0.01). The result is in accordance with the above reports and indicates that the level of liver fibrosis can respond to α-SMA.

The activation and proliferation of HSCs may be regulated by various factors and signal transduction pathways[7,15]. MAPK is a group of cytoplasmic serine/threonine kinases distributed extensively in cytoplasm, as a point of convergence of extracelluar signal causing nuclear reaction[1]. It becomes activated after threonine and tyrosine are phosphorylated altogether. In the MAPK family, ERK has been identified. ERK subset includes 2 subtypes: ERK₁ (P44^MAPK) and ERK₂ (P42^MAPK). The activation pathway is RAS-RAF-MEK-ERK. The combination of extracellular stimulation signal and receptors in cell membrane provokes dimerization of receptors and then phosphorylates self residue-Tyr. Some adapter proteins such as (growth factor receptor bound₂ (Grb₂) are recruited. Grb₂ is composed of one SH₂ region and two SH₃ regions. The SH₃ region of Grb₂ can combine with son of sevenless, which provocates the change of Ras’s GDP and GTP. RAS activates RAF kinases further and phosphorylated RAF activates mitogen-activated protein kinase kinase (MEK), which can phosphorylate Thr and Tyr residues of ERK_1/2. At last, activated ERK enters nuclear membrane to regulate gene transcription and provokes cell biologic effects.

The role of ERK in promoting HSC proliferation in vitro has been increasingly acquainted in recent years. As mentioned earlier, it has been suggested that many cytokines, such as platelet derived growth factor (PDGF), endothelin (ET), epidermal growth factor (EGF), insulin, insulin-like growth factor-1 (IGF-1) and tumor necrosis factor (TNF), as well as endotoxin, ethanol, acetaldehyde, ROS and ECM, can all activate HSCs and promote the prodution of ERK[10,16-19]. PD98059, an inhibitor of MEK kinase which is a upstream signal molecule of ERK, can restrain the transformation of HSCs into myofibroblasts, HSC proliferation and collagen synthesis[14,20-22]. The study on HSC-deleting MAPK gene in vitro discovered that the collagen gene expression of HSCs decreases to one third of the primary[23]. On the contrary, little is known about the role and regulation mechanism of ERK in vivo. In addition, the mechanism of ERK mRNA is unknown. Nguyen et al[24] have demonstrated that the expression of ERK₁ and ERK₂ in patients with alcoholic liver disease increases 3.9- and 3.2-fold respectively, compared with that in normal people. By using immunohistochemistry, Alvaro et al[25] have confirmed that the activated ERK_1/2 in rat liver enters nuclear membrane from cytoplasm, and the expression coincides with liver pathology, suggesting that ERK₁ protein and ERK₁ mRNA are scarcely expressed in normal rat livers. In addition, ERK₁ protein and ERK₁ mRNA expressions are elevated with the progression of hepatic fibrosis. The position distribution demonstrates that ERK₁ protein is scarcely expressed in vascular endothelial and perisinusoidal cells in normal rat liver. With the development of hepatic fibrosis, the positive ERK₁ cells are mainly resided along with HSCs, in portal ducts, fibrotic septa, and epithelium of bile ducts. ERK₁ protein is expressed not only in plasma of cells, but also in nuclearmembrane, indicating its activation. Based on these results, we presume that hepatocytes, vascular endothelial cells, epithelium of bile ducts and HSCs are all cellular sources of ERK₁. However, the major cellular source is HSCs during hepatic fibrosis. ERK₁ in the MAPK family probably participates in the development of hepatic fibrogenesis.

In conclusion, ERK₁ protein distribution is positively correlated with α-SMA. ERK₁ is associated with HSC proliferation. Activation of ERK₁ in rat liver tissue can activate the downstream signal molecule which modulates gene expression of HSCs and gives rise to hepatic fibrosis.