Orphan nuclear receptor NR4A2 inhibits hepatic stellate cell proliferation through MAPK pathway in liver fibrosis

Antibodies and reagents

Antibodies against glyceral-dehydephosphate dehydrogenase (GAPDH) (SC-25778), NR4A1 (SC-5569), NR4A2 (SC-991), NR4A3 (SC-30154) and α-SMA (SC-32251) were purchased from Santa Cruz (CA, USA). Alexa Fluor^® 546 IgG antibody (A10040) and Alexa Fluor^® 488 IgG (H + L) antibody (A21202) were obtained from Life Technologies (Carlsbad, CA, USA). IRDye680 anti-rabbit antibody (926-32221) and IRDye680 anti-mouse antibody (926-32220) were from LI-COR Biosciences (Lincoln, NE, USA). Antibody against Erk1/2 (4695P), P-erk1/2 (4370P), JNK (9258P), P-JNK (4668P), P-P38 (4511P) and P38 (8690P) were purchased from Cell Signaling (Danvers, MA, USA). Nycodenz (QK1002424-1) was from Nycomed (Zurich, Switzerland). NC nitrocellulose was from Pall (Port Washington, NY, USA). Deoxyribonuclease I was obtained from Roche (Basel, Switzerland). D-HanK’s and 30% acrylamide solution were from Bio-Light (Shanghai, China). Diaminobenzidine (DAB) Kit (GK50705) was from GeneTech (Shanghai, China). Cell cycle and Apoptosis Analysis Kit were purchased from R&S (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit was from Keygen (NJ, China). TGF-β1 was obtained from Peprotech (Rocky Hill, NJ, USA). Tween-20 and ammonium persulfate were obtained from Sinopharm (Beijing, China). Lipofect-amineTM 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Collagenase IV, PDGF BB and Pronase were purchased from Sigma (St. Louis, MO, USA). Fecal calf serum (FBS) was purchased from Gibco (Waltham, MA, USA). RIPA buffer, phenylmethylsulfonyl fluoride, TEMED, 5× SDS loading buffer and BCA kit were from Beyotime (Shanghai, China).

Animals and liver samples

Male Sprague-Dawley rats (SLAC, Shanghai, China), weighing approximately 250–270 g were maintained in a 12-h dark/12-h light cycle. All animal experiments were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (License No. SYXK2011-0128). Human liver samples were offered by hepatobiliary surgery department of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. All samples were obtained in accordance with the ethics committee of Shanghai Jiao Tong University.

Cell culture

The HSC-T6 cells were kindly provided by Dr. Friedman of Mount Sinai School of Medicine of New York University (MSSM). Cells were cultured in DMEM medium supplemented with 10% heat-inactivated fetal calf serum (FBS). All cells were maintained in a humidified incubator at 37 °C with 5% CO₂. Platelet-derived growth factor (PDGF) BB or TGF-β was used for stimulation.

RNA isolation and quantitative real time-polymerase chain reaction

Total RNA was extracted from HSC-T6 cell using Trizol (Takara, Dalian, China) and reverse transcribed using PrimeScript RT Master Mix (Takara). Real time polymerase chain reaction (RT-PCR) was performed using SYBR Green PCR Kit (Takara) and Applied Biosystems 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Primer sequences (Sangon Biotech, Shanghai, China) were as follows: NR4A1 (Fw: 5′-ACACCGGAGAGTTTGACACC-3′; Rev: 5′-GGGTAGCAGCCATACACCTG-3′); NR4A2 (Fw: 5′-AGATTCCTGGCTTTGCTGAC-3′; Rev: 5′-CTGGGTTGGACCTGTATGCT-3′); NR4A3 (Fw: 5′-GGCTGCAAGGGCTTCTTCA-3′; Rev: 5′-CACCATCCCGACACTGAGACA); α-SMA (Fw: 5′-CCAGGGAGTGATGGTTGGA-3′; Rev: 5′-CCGTTAGCAAGGTCGGATG-3′); Col1 (Fw: 5′-AGGCATAAAGGGTCATCGTG-3′; Rev: 5′-ACCGTTGAGTCCATCTTTGC-3′); β-actin (Fw: 5′-ACCCACACTGTGCCCATCTATG-3′; Rev: 5′-AGAGTACTTGCGCTCAGGAGGA-3′). The cycle conditions were 95 °C for 30 s, 40 cycles at 95 °C for 5 s and 60 °C for 34 s, one cycle at 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. The resulting sequences were normalized to the β-actin expression levels, and relative gene expression was measured by the 2^−ΔΔCT method.

Immunohistochemistry and immunofluorescence

Hepatic tissues were embedded in paraffin and sectioned. The sections were incubated in 3% H₂O₂ in methanol and nonspecific binding was blocked with 10% normal goat serum. The sections were incubated with primary antibody at 4 °C overnight, washed and incubated with secondary antibody for 60 min at room temperature. Antigen–antibody complexes were visualized using DAB kits.

Immunofluorescence was performed as described previously (García-Pérez et al., 2013; García-Pérez et al., 2015). For detecting NR4A2 and α-smooth muscle actin (α-SMA) expression in liver tissue, the sections were incubated for 48 h at 4 °C with the following primary antibodies: NR4A2 (1:50), Alexa Fluor^® 546 IgG (1:500), Alexa Fluor^® 488 IgG (H + L) (1:500). The sections were incubated in 4, 6-diamino-2-phenylindole (DAPI, 1:100) for 1 min and mounted in antifade reagent.

NR4A2 siRNA transfection

For RNA interference assay, the cells were seeded into 6-well cell culture plates at a density of 5 × 10⁵ cells. The cells were transfected with siRNAs and Lipofectamine 2000 according to the manufacturer’s instructions. The double strands of small interfering RNAs (siRNAs) targeting NR4A2 (GenePharma, Shanghai, China) were as follows: siRNA (1) (Fw: 5′-CGCGAAAUAUGUGUGUUUATT-3′; Rev: 5′-UAAACACACAUAUUUCGCGTT-3′); siRNA (2) (Fw: 5′-GACCAUGUGACUUUCAAUATT-3′; Rev: 5′-UAUUGAAAGUCACAUGGUCTT-3′); siRNA (3) (Fw: 5′-GACCUCACCAACACUGAAATT-3′; Rev: 5′-UUUCAGUGUUGGUGAGGUCTT-3′); Negative control (Fw: 5′-UUCUCCGAACGUGUCACG UTT-3′; Rev: 5′-ACGUGACACGUUCGGAGAATT-3′). Briefly, 100 nmol siRNAs and 5 µl Lipofectamine 2000 were diluted in serum and antibiotic free opti-DMEM (Invitrogenn, Carlsbad, CA, USA) at a final volume of 500 µl. After mixing for 20 min at room temperature, the siRNA/Lipofectamine 2000 mixture was added to the cells and incubated at 37 °C in a humidified CO₂ incubator. Nonsilencing siRNA with no known homology to rat genes was synthesized as a negative control.

Primary HSC isolation

Sprague-Dawley rats (SLAC, Shanghai, China) for primary HSCs were prepared. After anesthesia, the rat abdominal wall was opened and the intestines were moved aside so as to expose the vena cava and portal vein. A fine cannula ligated and fixed to a proper location was inserted into the vein after placing a thread around it. Perfusion solutions were incubated at 37 °C before usage. An adjustable peristaltic pump was then used to control the in situ liver perfusion process. After the liver swelled, the cannula in the inferior vena cava was opened to allow draining. Subsequently, the liver was perfused in turn with D-HanK’s solution, Collagenase IV solution, Pronase solution for 15 min at 37 °C. The cell fraction was collected, washed and put between a top layer of buffer and a bottom cushion of 18% Nycodenz. After centrifugation at 1,400 g for 22 min, the HSCs fraction at the interface between the top and intermediate layer was obtained. Finally, the cells were cultured in DMEM medium supplemented with 10% FBS.

Western blotting

Western blot analysis was performed according to standard protocols as described previously (Ogawa et al., 2012). Western blots were performed using antibodies against α-SMA (1:500), GAPDH (1:1,000), NR4A1 (1:500), NR4A2 (1:500), NR4A3 (1:500), IRDye680 anti-rabbit (1:5,000), IRDye680 anti-mouse (1:5,000), Erk1/2 (1:5,000), P-erk1/2 (1:1,000), JNK (1:1,000), P-JNK (1:1,000), P-P38 (1:1,000) and P38 (1:1,000).

Protein samples were subjected to SDS-PAGE and then transferred onto a polyvinylidenefluoride membrane. After blocking, the membranes were incubated with primary antibodies followed by peroxidase-conjugated secondary antibodies. The protein samples were scanned through Odyssey Infrared Imaging System.

CCK-8 assay

Cell viability was assessed using the Cell Counting Kit-8 (Dojindo, KMJ, Japan) according to the manufacturer’s instructions. Briefly, HSC-T6 cells transfected with NR4A2 siRNA were seeded into 96-well plates (3 × 10³ cells per well). Next, 10 µl of CCK8 was added to each well and the cells were incubated for 2 h at 37 °C. Absorbance was assessed at 450 nm with a microplate reader (Bio-Tek, Winooski, VT, USA). The mean optical density (OD) of the wells in each group was used to draw cell growth curve.

Cell cycle analysis

Cells were seeded into 6-well cell culture plates at a density of 5 × 10⁵ cells. The cells were transfected with siRNA NR4A2 and 48 h later the cells were harvested, stained and fixed with propidium iodide (PI) according to the manufacturer’s protocol and subject to cell cycle analysis using a flow cytometer (BD, Franklin Lakes, NJ, USA).

Cell apoptotic assay

Cells were seeded into 6-well cell culture plates at a density of 5 × 10⁵ cells. The cells were transfected with siRNA NR4A2 and 48 h later the cells were harvested. Apoptosis assay was measured using FITC AnnexinV Apoptosis Detection Kit according to the manufacturer’s protocol and subject to cell cycle analysis using flow cytometer.

Statistical analysis

The results were expressed as mean ± SD and were analyzed by analysis of variance (ANOVA) or Student’s t-test followed by paired comparison as appropriate. P < 0.05 was taken as the minimum level of significance.

NR4A2 expression was reduced in activated HSCs

HSCs play an important role in liver fibrosis. Quiet HSCs could be activated by numerous factors and produce extracellular matrix. α-SMA is a well-established marker for liver fibrosis. PDGF and TGF-β are strong stimulating factors. After HSC-T6 cells was stimulated by PDGF BB (10 ng/ml or 20 ng/ml), NR4A2 mRNA level decreased significantly whereas α-SMA level increased significantly (Fig. 1A). The level of NR4A2 protein has significantly reduced after treatment with PDGF BB (10 ng/ml, 20 ng/ml respectively) in HSC-T6 cells, yet inconspicuous changes occurred in NR4A1 and NR4A3 expression (Fig. 1B). Similar observation has been made after treatment with TGF-β (2 ng/ml) (Fig. 1C). We also screened NR4A2 expression on short time upon stimulation by PDGF BB (Fig. 1D) or TGF-β (Fig. 1E). It was revealed NR4A2 mRNA level for 12 h increased significantly compared to control group meanwhile NR4A2 protein levels elevated slightly after stimulated by PDGF BB (Fig. 1F) or TGF-β (Fig. 1G).

Further, we isolated primary HSCs from SD rats. Primary hepatic stellate cells can be spontaneously stimulated and proliferate several days after isolation (Davis & Vucic, 1988). We observed that NR4A2 mRNA expression decreased significantly while α-SMA level was increased 2–7–folds in primary hepatic stellate cells after isolation (Fig. 2).

NR4A2 expression in fibrotic liver tissue decreased

SSkip[0.25em plus .1em minus .1em] α-SMA expressed mainly in fibrotic area where extracellular matrix accumulated whilst NR4A2 expressed in both fibrotic and non-fibrotic area of liver tissue. However, fibrotic area was dominant in fibrotic liver compared with normal liver. Immunohistochemistry analysis illustrated lower expression of NR4A2 in fibrotic area than non-fibrotic area (Figs. 3A and 3B). The same result was observed by immunofluorescence analysis (Fig. 3C).

NR4A2 inhibited α-SMA and Col1 expression in HSCs

To clarify the correlation between NR4A2 and liver fibrosis, we knocked down NR4A2 via three siRNAs and examined the expression of extracellular matrix markers in HSC-T6 cells. RT-PCR analysis showed that the mRNA level of NR4A2 decreased by more than 50% (Fig. 4A) while α-SMA and Col1 increased by more than50% (Figs. 4B and 4C).

NR4A2 suppressed cell proliferation, modulated cell cycle andpromoted apoptosis

After NR4A2 knocked down in HSC-T6 cells, we analyzed cell cycle and apoptosis by flow cytometry and examined optical density by CCK8 assay. Flow cytometry showed decreased percentage of cells in G1 phase and increased percentage in S stage compared with control group after NR4A2 knockout (Figs. 5A and 5B). NR4A2 knockout also led to reduced apoptosis (Figs. 5C and 5D) and promoted proliferation (Fig. 5E) in HSC-T6 cells.

NR4A2 upregulation of MAPK pathway in HSC proliferation

In liver fibrogenesis, MAPK is an important signaling pathway which consists of ERK1/2, P38 and JNK. Western blotting analysis indicated reduced phosphorylation of ERK1/2, P38 and JNK with NR4A2 knockout in HSC-T6 cells (Figs. 6A–6C).