The mouse forestomach carcinoma (MFC) fibroblast cell line, purchased from Bena Culture Collection (Suzhou, China; cat. no. BNCC100581), was plated at a density sufficient to create a confluent monolayer following 12 h of culture at 37°C in an incubator with 5% CO₂. Cells were cultured in Dulbecco's modified Eagle's medium (cat. no. 11965-092) containing 0.5% fetal bovine serum (cat. no. 10437-028; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

For a yeast one-hybrid assay, the 1.5-kb FGF21 was amplified from genomic DNA of MFC fibroblast cells by polymerase chain reaction (PCR) under the following conditions: Initial denaturation at 98°C for 30 sec, followed by 30 cycles at 98°C for 10 sec, 65°C for 20 sec and 72°C for 2 min, with a final extension step at 72°C for 10 min. PCR was performed using Q5^® High-Fidelity DNA Polymerase (cat. no. M0491; New England Biolabs, Inc., Ipswich, MA, USA) and the primers listed in Table I. Then, FGF21 promoter sequences were cloned into the pHISi vector (cat. no. 102239; BioVector NTCC Inc., Beijing, China), in which FGF21 promoter drives histidine synthase coding sequences. The open reading frame (ORF) sequences of TCF4 were cloned into the pGAD424 vector (BioVector NTCC Inc., Beijing, China). The constructed pGAD424-TCF4 or empty vector pGAD424 was transformed into the yeast one hybrid bait strain (YM4271) using a transformation solution (polyethylene glycol, lithium acetate, Tris, and EDTA; STZ; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) under the following conditions: 28°C for 30 min, 42°C for 15 min and 28°C for 5 min. After transformation, the yeast cells were harvested at 10,000 × g for 1 min, and the supernatant was removed. The cells were suspended in the dH₂O and plated on synthetic dropout (SD)-Leu or -His media (Takara Biotechnology Co., Ltd., Dalian, China) with subsequent growth at 30°C for 3 days.

ChIP assay was performed using a ChIP assay kit (cat no. 17-295; EMD Millipore, Billerica, MA, USA) according to the manufacturer's instructions. A total of 1 µg anti-TCF4 antibody (cat. no. 2566; Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-β-catenin antibody (cat. no. ab32572; Abcam, Cambridge, UK) were used for immunoprecipitation. The immunoprecipitated (IP) DNA by the antibodies were compared with the DNA precipitated without addition of antibodies using quantitative PCR (qPCR) with SYBR-Green mixture (cat. no. 4472908; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. A reaction of ChIP-PCR was performed with an initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation for 30 sec at 95°C, annealing for 30 sec at 58°C, and extension at 72°C for 30 sec, followed by a final extension at 72°C for 5 min. GAPDH was used as a reference gene to normalize data. The primer sequences for qPCR are listed in Table I. The 2^−ΔΔCq method was used for the fold change calculation (27).

A Nuclear-Cytosolic Protein Extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used for protein extraction of MFC cells. A total of 500 µg (in 500 µl) extracted protein was incubated at 4°C with 1 µg anti-TCF4 antibody (cat. no. 2566; Cell Signaling Technology, Inc.), anti-β-catenin antibody (cat. no. ab32572; Abcam), and horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) H&L (ab6789; Abcam) for 1 h. Subsequently, 20 µl MagnaBind Protein A Beads (cat no. 21348; Thermo Fisher Scientific, Inc.) were added and incubated for 2 h. Following centrifugation at 10,000 × g at 4°C for 5 min, the beads were washed four times with extraction buffer containing 0.1% Triton X-100 and eluted with SDS sample buffer.

Cells were lysed in an ice-cold lysis solution containing 7 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 40 mM Trizma base, 40 mM dithiothreitol and 1% protease inhibitor. Following complete lysis of the cells and centrifugation at 15,000 × g for 15 min at 4°C, the total protein concentration in the supernatant was measured using a Bradford protein assay kit (Bio-Rad Laboratories, Inc., Richmond, CA, USA). The proteins (40 µg/lane) were separated on 12% SDS-PAGE and electrotransferred onto Immobilon-P transfer membranes (EMD Millipore). The membranes were incubated in TBS containing 5% skimmed milk and 0.05% Tween-20 for 1 h and blotted with primary antibodies at 4°C overnight. Anti-TCF4 (1:1,000; cat. no. 2566; Cell Signaling Technology, Inc.), anti-β-catenin (1:2,000; cat. no. ab32572; Abcam) and anti-GAPDH (1:2,000; cat. no. ab8245; Abcam) antibodies were used as the primary antibodies. GAPDH was used as the internal control. The membranes were incubated for 1 h at 37°C with an anti-mouse or anti-rabbit horseradish peroxidase-linked secondary antibody (1:2,000; cat. nos. 7076 and 7074, Cell Signaling Technology, Inc.), and the signal was visualized using an electrochemiluminescence kit (GE Healthcare, Chicago, IL, USA).

In total, 2 µg total RNA, extracted with RNeasy Mini kit (cat. no. 74104; Qiagen China Co., Ltd., Shanghai, China) from MFC cells, was reverse-transcribed using a GoScript reverse transcription kit (Promega Corporation, Madison, WI, USA) following the manufacturer's instructions. RT-qPCR was performed with SYBR Green mixture (cat. no. 4472908; Thermo Fisher Scientific, Inc.), and gene expression was quantified as described previously (27). GAPDH was used as an internal control. The sequences of primers used in RT-qPCR are listed in Table I.

ORF regions of TCF4 (NM_013685.2, NCBI) and β-catenin (NM_001165902.1, NCBI) were amplified from cDNA of MFC cells by the gene specific primers with Q5^® High-Fidelity DNA Polymerase (cat. no. M0491; New England Biolabs, Inc.) and cloned into the pcDNA3.1 (+) (cat. no. V79020; Thermo Fisher Scientific, Inc.) expression vector to contract TCF4 overexpression and β-catenin overexpressed plasmids. The primers used for TCF4 and β-catenin are listed in Table I. siRNA for β-catenin (ON-TARGET plus SMART pool, L-004018) and negative control siRNA (ON-TARGETplus si CONTROL non-targeting pool, D-001810) were purchased from Dharmacon RNA Technologies (Chicago, IL, USA). The MFC cells were seeded 12 h prior to transfection at 37°C with 5% CO₂ and reached a density of 30–50% confluence at the time of transfection. Subsequently, 30 nM siRNA duplex and 2 µg TCF4 overexpression or β-catenin overexpression plasmids were transfected on day 0 using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and Opti-MEM I Reduced Serum Medium (Gibco; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. On day 1 (24 h after transfection), the cells were confluent and the overexpression or siRNA solutions were exchanged with full growth medium. These transfected cells were subsequently used for RT-qPCR (as described above), for which the density reached 80–90% confluence at the time of harvest for RNA preparation on day 3 (72 h after transfection).

Data are presented as the mean ± standard error of the mean. Each experiment was performed in triplicate. Statistical calculations were performed with Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Data were analyzed using the Student's t-test for two groups and one-way analysis of variance followed by Tukey's test for more than two groups. P<0.05 was considered to indicate a statistically significant difference.

FGF21 promoter sequences analysis demonstrated that two putative TCF/LEF binding motifs (AGAAAG) (26) appeared within 1.5 kb of the promoter. The two putative binding motifs are located 1,163 and 876 bp upstream of the start codon, respectively (Fig. 1A). In order to examine whether TCF4 directly binds to the putative motifs, ChIP assays were performed using the TCF4 antibody and the precipitation without antibody as the control. FGF21 exhibited a higher expression level in the MFC cells (data not shown); therefore, the MFC cells were used in the experiments (14,15). The IP DNA fragments were amplified by two primer pairs, which spanned the F1 and F2 regions in the FGF21 promoter (Fig. 1B). The ChIP-PCR data was normalized to input DNA and the results revealed that TCF4 bound to the F1 but not F2 region in the promoter of the FGF21 gene (Fig. 1B). To further verify the ChIP results, a yeast-one hybrid assay was performed by co-expressing activation domain (AD)-TCF4 and pFGF21-His or mpFGF21-His (Fig. 1C). The results indicate that AD-TCF4 co-expressing pFGF21-His were able to grow in SD media missing histidine, whereas the empty vector transforming or AD-TCF4 co-expressing mpFGF21-His cells failed to grow (Fig. 1D). These data indicated that TCF4 activates the FGF21 promoter in yeast.

After β-catenin accumulates in the nucleus, it interacts with TCF/LEF, and activates the transcription of Wnt signaling genes (24). To test whether TCF4 and β-catenin interact in MFC cells, Co-IP assay was performed. The cell lysate was incubated with TCF4 or IgG antibodies, and β-catenin and TCF4 were detected using β-catenin and TCF4 antibodies prior to and following immunoprecipitation (Fig. 2A). GAPDH levels in IgG and TCF4 incubated samples were similar (Fig. 2A). The results indicate that β-catenin and TCF4 physically interact with the MFC cells. To further test the binding of the β-catenin complex to the promoter of FGF21, ChIP assays were performed using β-catenin. ChIP-PCR results indicate that similar to the TCF4, β-catenin IP DNA was enriched in the F1 region but not in the F2 region (Fig. 2B and C).

As TCF4 and β-catenin bound to the F1 region of the FGF21 promoter, effects of TCF4 and β-catenin on FGF21 expression were tested. TCF4 and β-catenin modulated transcription of FGF21 was confirmed by plasmid (pcDNA3.1-TCF4 or pcDNA3.1-β-catenin)-mediated transfection experiments using Lipofectamine 2000. At 24 h following transfection, TCF4 was ~22-fold and β-catenin was ~20-fold higher in the TCF4 overexpression and β-catenin overexpressed cells compared with the control in which empty vector was transformed (Fig. 3A and B). Furthermore, FGF21 expression levels were monitored by RT-qPCR. The results revealed that the FGF21 transcript was 2.7 and 2.6 fold higher in TCF4 overexpression and β-catenin overexpressed cells compared with the control, respectively (Fig. 3C).

As overexpression of β-catenin induced FGF21 expression, FGF21 levels in the β-catenin knockdown MFC cells was analyzed further. RT-qPCR results indicated that two independent transfection of β-catenin specific siRNAs significantly reduced the expression of β-catenin (~70–80%; Fig. 4A). In addition, siRNA-mediated knockdown of β-catenin largely contributed to the reduction in the expression of FGF21 (~50%; Fig. 4B).