Contributed equally
Fibroblast growth factor 21 (FGF21) as a member of the FGFs serves a key role in glucose homeostasis and protection of the liver, heart, kidney and skin from damage as well as cancer cell development. In addition, transcription of
Fibroblast growth factors (FGFs) include 23 members, which serve important roles in metabolism regulation and development (
Fasting-mediated induction of
In the present study, the appearance of the putative TCF/LEF binding motifs in the promoter of
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
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
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 (
ORF regions of
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.
After β-catenin accumulates in the nucleus, it interacts with TCF/LEF, and activates the transcription of Wnt signaling genes (
As TCF4 and β-catenin bound to the F1 region of the
As overexpression of
FGF21 is sensitive to diverse physiological changes (e.g., fasting and injury) and serves an important role in development and metabolism, including glucose metabolism (
TCF and β-catenin are the master transcriptional regulators of the Wnt signaling pathway, and translocation of β-catenin from the cytosol to the nucleus resulted in formation of a TCF complex, including TCF and β-catenin (
Previous reports identified that Wnt/β-catenin acts upstream of
The present study was funded by Wenzhou Medical University (grant no. 025gt8972). This work was also supported by the Doctoral Scientific Research Foundation of Xingxiang Medical University (grant no. XYBSKYZZ201512&201513) and grants from The Education Department of Henan Province (grant nos. 201610472040, 172102310584). The vectors and yeast strain used in the yeast two hybrid assay were kindly donated by the Yuan lab at Wenzhou Medical University.
TCF4 bound directly to the promoter of
β-catenin physically interacts with TCF4 and binds to the promoter of
Overexpression of
Suppression of
Primer sequences.
Primer | Sequences (5′-3′) |
---|---|
FGF21 | F: GATGACGACCAAGACACTG |
R: CGGCCCTGTAAAGGCTCT | |
TCF4 | F: AGAGCGACAAGCCCCAGAC |
R: ATTCGCTGCGTCTCCCATC | |
β-catenin | F: TCGCCAGGATGATCCCAGC |
R: GCCCATCCATGAGGTCCTG | |
GAPDH | F: GCCAAGGTCATCCATGACAACT |
R: GAGGGGCCATCCACAGTCTT | |
pFGF21 | F: GAATTCCAGAGTTCCAGGGCCA CATCA |
R: GAGCTCCAGGGCTGCGCTCCGTTCGGGAG | |
FGF21 F1 | F: CTAAGCAGGGGTTGGTGAGG |
R: GCGTGTCTGAGGCTTTCTTTC | |
FGF21 F2 | F: ACACCAGCTCAGTTGCTTACAC |
ACTGAAGTCTACACTCCTGGGTCT | |
β-catenin ORF | F: AAGCTTATGGCTACTCAAGCTGACCTGATG |
R: CTCGAGTTACAGGTCAGTATCAAACCAGGC | |
TCF4 ORF | F: AAGCTTATGCATCACCAACAGCGAATG |
R: CTCGAGTCACATCTGTCCCATGTGATTC |
FGF21, fibroblast growth factor 21; F, forward; R, reverse; TCF4, transcription factor 4; ORF, open reading frame.