LX2 cells, immortalized human HSCs, were kindly provided by Professor S.L. Friedman (Liver Disease Research Center of San Francisco General Hospital, San Francisco, CA, USA) and were maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% fetal bovine serum (FBS) and 1% streptomycin/penicillin. For western blot analysis, LX2 cells were seeded at ~70% confluence, on 60-mm dishes (2×10⁵ cells) or 100-mm dishes (7×10⁵ cells) and cultured for 24 h. After starvation for 12 h (reduction of FBS to 0.5%), LX2 cells were treated with AIMP1 peptide as indicated. For the ERK inhibition assay, LX2 cells, starved for 12 h as above, were treated with AIMP1 peptide in the presence or absence of the selective inhibitor of MAP kinases, U0126 (10 μM) for 30 min, before TGFβ (2 ng/ml) was added.

For protein extraction, LX2 cells were washed with cold 1X PBS, and lysed on ice for 30 min in cold RIPA buffer (Sigma, 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10% Glycerol, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 12 mM β-glycerophosphate, 10 mM NaF, 1 mM NaOV₃, 1 mM PMSF, 2 mM β-mercaptoethanol) containing a protease inhibitor cocktail (Cell Signaling Technology), followed by centrifugation at 15,000 rpm for 20 min. The cells were fractionated into nucleus and cytoplasm using the anuclear/cytosol fractionation kit (BioVision) according to the manufacturer's instructions, and confirmed by western blot analysis using α-tubulin and lamin A/C for cytoplasm and nucleus, respectively. Approximately 20 μg of total proteins was loaded and separated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes using the wet transfer kit (Bio-Rad Laboratories, Hercules, CA, USA), followed by blocking for 30 min with 5% skim milk in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBS-T), and then incubated overnight at 4°C in TBS containing 5% skim milk with the indicated primary antibodies; anti-pSMAD2 (Ser465/467, 1:1,000; Cell Signaling Technology), anti-pSMAD3 (Ser423/425, 1:1,000; Cell Signaling Technology), anti-SMAD2/3 (1:1,000; Cell Signaling Technology), anti-SMAD4 (1:1,000; Santa Cruz Biotechnology), anti-collagen type I (1:1,000; Millipore), anti-Lamin A/C (1:1,000; Abcam) anti-α-Tubulin (1:1,000; Sigma). The membranes were then washed four times for 10 min in TBS-T solution and incubated with horseradish peroxidase-conjugated secondary antibodies (0.1 μg/ml; Santa Cruz Biotechnology). Immunoreactivity bands were detected using the western blot detection kit (Abc-3001; AbClon), according to the manufacturer's instructions.

LX2 cells (2×10⁴ cells) were cultured for 24 h on round glass coverslips (VWR LabShop, Batavia, IL, USA) in 24-well plates. After 0.5% FBS starvation for 12 h, the cells were treated with 5 μg/ml of AIMP1 peptide for 30 min, followed by 2 ng/ml of TGFβ for 1 h. The cells were fixed with 4% formaldehyde freshly prepared from paraformaldehyde for 10 min and then permeabilized with PBS supplemented with 0.1% Triton X-100 (PBST) for 5 min. After washing with PBST three times for 5 min, fixed cells were incubated for an additional 30 min in PBST containing 5% BSA to prevent non-specific binding of the antibodies, followed by an overnight incubation at 4°C with the indicated primary antibodies. Cells were washed three times with PBST solution, and then incubated with Alexa 488 and 594-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Molecular Probes) for 1 h at room temperature. Nuclear DNA was counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) and the fluorescence was captured with the Leica confocal microscope TCS SP5 (Leica Microsystems, Wetzlar, Germany).

After serum starvation for 4 h, LX2 cells were treated with AIMP1 peptide (5 μg/ml) in the presence or absence of U0126 (10 μM). Cells were harvested, lysed with RIPA buffer, and incubated on ice for 30 min. Whole cell lysates were prepared by centrifugation at 25,000 × g for 10 min at 4°C. The protein extracts (300 μg) were incubated with anti-SMAD2/3 antibody (1.5 μg; Cell Signaling Technology) for 4 h at 4°C and then with protein A agarose for 4 h, also at 4°C. The beads were washed three times with RIPA buffer without β-mercaptoethanol, and resuspended in 1X SDS sample buffer. The samples were boiled and loaded into 9% SDS-PAGE. Phosphorylation of SMAD2/3 was confirmed by pSer antibody (Abcam).

LX2 cells (5×10⁴ cells) were seeded on a 12-well plate and cultured in DMEM supplemented with 2% fetal bovine serum (FBS) and 1% streptomycin/penicillin. The cells were then co-transfected with a SBE4-Luc vector containing the SMAD binding element (SBE) and Renilla luciferase vector using Lipofectamine 2000 (Invitrogen) for 12 h. After serum starvation with 0.5% FBS for 4 h, LX2 cells were treated with AIMP1 peptide for 10 min and then TGFβ was added for an additional 20 h. Luciferase activity was determined using the Dual-Luciferase Reporter Assay system (Promega) and quantified using GloMax (Promega) according to the manufacturer's instructions. Luciferase activity was normalized to Renilla luciferase activity.

Total RNA was extracted using an RNA isolation kit (iNtRON Biotechnology, Inc., Seoul, Korea) according to the manufacturer's instructions. cDNA was prepared by reverse transcription with 0.5 μg of total RNA. Type I collagen mRNA (NM_000088.3) (forward, 5′-CCCCTGGAAAGAATGGAGATG-3′ and reverse, 5′-TCCAAACCACTGAAACCTCTG-3′); GAPDH (forward, 5′-CGAGATCCCTCCAAAATCAA-3′ and reverse, 5′-TGTGGTCATGAGTCCTTCCA-3′) were amplified by real-time PCR, which was performed with the AccuPower^® GreenStar qPCR PreMix (SYBR-Green PreMix; Bioneer Corp., Daejeon, Korea) and StepOne Real-Time PCR system (Applied Biosystems). GAPDH was used as an endogenous control.

Male BALB/c mice (6 weeks old, weighing 20–22 g) were obtained from Orient Bio. Animal Inc. (Seoul, Republic of Korea). Animal care and all experimental procedures were conducted in accordance with the approval and guidelines of the Inha Institutional Animal Care and Use Committee (Inha IACUC) of the Medical School of Inha University (approval ID: 111024-1). The animals were fed standard chow and tap water ad libitum, and were maintained with a 12-h dark/light cycle at 21°C. Acute liver damage was induced by intraperitoneal injection of 40% CCl₄ in corn oil at a single dose of (150 μl) twice a week for one month. Control animals were treated with the same volume of corn oil alone. Mice in the scrambled AIMP1 and AIMP1 peptide groups were treated twice-weekly with intraperitoneal injections for 2 weeks during the CCl₄ administration. All mice were sacrificed by ether anesthesia after 4 weeks of treatment. The livers were excised and weighed and the specimens were immediately fixed in 10% neutral buffered formalin for histochemical studies. Blood samples for biochemical analyses were obtained by cardiac puncture.

Liver samples fixed in a 10% buffered formaldehyde solution were processed using a paraffin slice technique. Sections ~4-μm thick were stained with hematoxylin and eosin (H&E) for routine histological examination. The sections were first stained with hematoxylin for 3 min, washed, and then stained with 0.5% eosin for an additional 3 min. For staining of liver fibrosis, the fixed liver tissue samples were stained with Masson's trichrome according to standard protocol. After an additional wash with water, the slides were sequentially dehydrated in 70, 95 and 100% ethanol and cleared in xylene. The degree of liver damage was examined in a blinded manner by a pathologist using a light microscope (Olympus).

Serum Total-Bilirubin, D-Bilirubin, aspartate transaminase (AST), and alanine transaminase (ALT) levels were measured at the Green Cross Reference Lab (Seoul, Republic of Korea).

Immunohistochemical staining was performed using formalin-fixed and deparaffinized tissue sections as previously described (34,35). After blocking with normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h, primary antibodies specific for TGFβ, collagen I, and α-SMA (Sigma-Aldrich) were used. After removing the unbound primary antibody, the sections were incubated with secondary antibody in 1.5% horse serum/PBS at room temperature for 1 h. The sections were visualized by an avidin-biotin peroxidase complex solution using an ABC kit (Vector Laboratories). The sections were washed in PBS, developed with a diaminobenzidine tetrahydrochloride substrate for 15 min and then the nucleus was counterstained with hematoxylin. Terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) was performed using the TUNEL kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions.

Data are expressed as the mean ± SD, and analyzed with an ANOVA and unpaired Student's t-test. A P-value ≤0.05 was considered to indicate a statistically significant result. Statistical calculations were performed using SPSS software for the Windows operating system (version 10.0; SPSS, Inc., Chicago, IL, USA).

We have previously reported that the AIMP1 peptide activates MAPKs including ERK (33). In the present study, we used LX2 cells for human HSCs that were spontaneously immortalized in low serum condition, which express α-smooth muscle actin, vimentin, glial fibrillary acid protein and other HSC biomarkers upon activation (36). We first examined whether the AIMP1 peptide could induce ERK phosphorylation in LX2 cells. AIMP1 peptide increased ERK phosphorylation in a time-dependent manner (Fig. 1A). Since the activation of ERK induces TGFβ-independent phosphorylation of SMAD and inhibits nuclear translocation (15,18,37), we examined whether AIMP1-mediated ERK phosphorylation induced SMAD2/3 phosphorylation. After treatment of LX2 cells with the AIMP1 peptide as indicated, cell lysates were prepared and the SMAD2/3 protein was immunoprecipitated using a specific antibody as described in Materials and methods. The western blot analysis using anti-phospho-serine antibody, showed that the AIMP1 peptide induced phosphorylation of SMAD2 in a time-dependent manner (Fig. 1B). To verify whether the phosphorylation at the serine residue of SMAD2/3 was induced by AIMP1 peptide-mediated ERK phosphorylation, we treated the cells with U0126, an ERK inhibitor, and then analyzed the SMAD2/3 phosphorylation. The suppression of ERK phosphorylation by U0126 indeed reduced the AIMP1 peptide-mediated SMAD2 phosphorylation (Fig. 1C and D).

In order to transduce TGFβ signaling, the TGFβ receptor, activated by the ligand binding, phosphorylates SMAD2/3. The pSMAD2/3 then forms a complex with SMAD4, resulting in translocation to the nucleus, and regulation of a variety of TGFβ target genes including type I collagen (38). To investigate whether the AIMP1 peptide-mediated ERK activation could inhibit the TGFβ-induced nuclear translocation of pSMAD2/3, we separated whole cell lysates into the cytoplasmic and nuclear fractions after treatment with AIMP1 peptide. Analysis of the separate fractions clearly showed that pre-treatment of LX2 cells with AIMP1 peptide reduced the nuclear translocation of pSMAD2, but not of pSMAD3 (Fig. 2A). We confirmed this result further by immunofluorescence staining. TGFβ stimulation of LX2 cells induced the nuclear translocation of pSMAD2/3 and SMAD4, whereas pretreatment with AIMP1 peptide inhibited the nuclear translocation of pSMAD2/3 (Fig. 2B and C). These results suggest that the AIMP1 peptide inhibits the nuclear translocation of pSMAD2/3 without affecting the phosphorylation of SMAD2/3 by TGFβ.

TGFβ has been reported to induce the deposition of type I collagen at sites of injury in the liver, leading to liver fibrosis (39–41). Thus, we examined whether the reduction in the nuclear translocation of pSMAD2/3 seen after treatment with the AIMP1 peptide could inhibit the TGFβ-mediated synthesis of type I collagen. Pretreatment with the AIMP1 peptide decreased type I collagen synthesis in a dose-dependent manner at the level of both mRNA and protein (Fig. 3A), suggesting that the decreased expression level of type I collagen was due to the reduced transcriptional activity of the SMAD complex in the nucleus. This is a result of the fact that the AIMP1 peptide inhibited the nuclear translocation of the pSMAD induced by TGFβ. To further confirm the transcriptional activity of the SMAD complex, we carried out a luciferase activity assay using the SBE4-Luc vector containing SMAD binding element (SBE). The AIMP1 peptide attenuated the TGFβ-mediated increase of luciferase activity in a dose-dependent manner (Fig. 3B). In addition, we investigated whether the AIMP1 peptide-mediated decrease of type I collagen expression was dependent on ERK activation. As shown in Fig. 3C, pre-treatment with U0126 abolished the AIMP1 peptide-mediated reduction of type I collagen expression. The luciferase activity assay further confirmed that the AIMP1 peptide-mediated decrease of type I collagen expression is ERK-dependent (Fig. 3D).

We next assessed whether the AIMP1 peptide could have a therapeutic effect in the CCl₄-induced mouse model of liver, by inhibiting the expression of type I collagen induced by TGFβ. The AIMP1 peptide was administered intraperitoneally twice weekly as described in Materials and methods. Histological analysis showed that treatment with the AIMP1 peptide decreased the CCl₄-induced liver damage in a dose-dependent manner (Fig. 4A). In addition, Masson-Trichrome staining further clearly showed that collagen deposition was reduced by AIMP1 peptide treatment (Fig. 4A). The level of liver toxicity was examined by analysis of blood chemistry parameters. Treatment with the AIMP1 peptide significantly decreased the levels of total (T) and indirect (D) bilirubin, which were increased by CCl₄. In addition, the AIMP1 peptide significantly decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), suggesting that it could have a therapeutic effect on liver fibrosis in this model (Fig. 4B and C). To further confirm whether the therapeutic effect of the AIMP1 peptide is specific, we used scrambled AIMP1 peptide (sAIMP1) as a negative control, as previously described for a defective mutant Y24T (42). H&E and Masson-Trichrome staining showed that the wild-type AIMP1 peptide significantly attenuated CCl₄-induced liver fibrosis, whereas the sAIMP1 peptide did not, thereby confirming the specificity of the therapeutic effect (Fig. 4D and E). In addition, immunohistochemical staining clearly showed that the AIMP1, but not the sAIMP1, peptide reduced the levels of expression of α-SMA, TGFβ and collagen (Fig. 4D and E). Furthermore, the AIMP1 peptide decreased hepatic apoptosis induced by CCl₄ as measured by TUNEL (Fig. 4D and E). Serum markers for liver fibrosis including AST, ALT, albumin and BUN showed that the AIMP1 peptide significantly improved liver fibrosis, while the sAIMP1 peptide had no effect (Table I). In conclusion, these results suggest that the AIMP1 peptide attenuates liver fibrosis by inhibiting apoptosis induced by CCl₄, which, in turn, leads to decreased collagen deposition, α-SMA activation and TGFβ accumulation.