Wnt3a‑regulated TCF4/β‑catenin complex directly activates the key Hedgehog signalling genes Smo and Gli1
- Authors:
- Published online on: June 29, 2018 https://doi.org/10.3892/etm.2018.6379
- Pages: 2101-2107
Abstract
Introduction
The canonical Wnt signalling pathway, also known as the Wnt/β-catenin or the β-catenin/T-cell factor (TCF) pathway (1), modulates diverse biological processes via signal transduction (2–4). The key step in Wnt/β-catenin signalling pathway is the stabilization and accumulation of cytosolic β-catenin. Under normal conditions without stimulation, β-catenin is constantly phosphorylated by the glycogen synthase kinase-3β (GSK3β) complex (5). Phosphorylated β-catenin is subsequently ubiquitinated and targeted for degradation by the 26s proteasome (5,6). Wnt signalling activation leads to the inhibition of GSK3β activity and β-catenin accumulation in the cytosol and nucleus. In the nucleus, β-catenin forms a complex with T-cell factor/lymphoid enhancer binding factor (TCF/LEF) and induces the expression of downstream genes involved in Wnt signalling (5). A genome-wide scan of TCF/LEF binding sites revealed that TCF/LEF binds to the putative cis-elements (T/AC/GAAAG) in the target gene promoters (7).
Hedgehog signalling plays important roles in both vertebrate and invertebrate development (8). Hedgehog was first identified as a secreted signalling protein whose expression is induced in the Drosophila melanogaster embryonic segment (9). The three mammalian Hedgehog (hh) genes, namely, Sonic hedgehog, Indian hedgehog, and Desert hedgehog, are important in the patterning of many tissues and biological structures (9). In addition, abnormal activation of Hedgehog signalling is required for nearly all basal cell carcinomas, medulloblastomas, and rhabdomyosarcomas; however, overactivation of Hedgehog signalling has also been observed in some tumours (8,10–12). In the absence of Hedgehog stimulation, the transmembrane protein Patched1 (Patch1) interacts with Smoothened (Smo), another transmembrane protein that maintains inactivity of the Hedgehog pathway. Sonic hedgehog binds to and inactivates Patch1 after secretion, leading to Smo activation (11,13) and subsequent transcription of downstream genes via the GLI-Kruppel family transcription factors (13). Among the three GLI-Kruppel family members (Glis), Gli1 and Gli2 are positive regulators, while Gli3 is a negative regulator (14). Gli1 overexpression was associated with promoting gastric initiation and progression in patients (14). A previous study identified the novel function of Gli1 in modulating E-cadherin/β-catenin-regulated cancer cell properties. Gli1 was demonstrated to interfere with membrane localization of E-cadherin by upregulating MUC5AC, a gel-forming mucin, which in turn weakens E-cadherin-dependent cell-cell adhesion and promotes cell migration and invasiveness in pancreatic ductal adenocarcinoma (15). In addition, Gli3 deficient showed prevention of premature fusion of calvarial suture in mice via eliminate one allele of a key transcription factor Runx2 (16).
The extracellular matrix (ECM) is the largest component of the dermal skin layer (17). Fibroblasts are known to participate in ECM assembly and remodelling (18,19), demonstrating their important roles in wound repair. More recently, Wnt signalling was found to be involved in human fibroblast repair (20). In our transcriptome study using Wnt3a-stimulated cells, Wnt signalling activation was observed to alter the expression patterns of a large number of genes, including Hedgehog signalling genes (21). In addition, Hedgehog signalling genes have been demonstrated to regulate fibroblast repair and are controlled by β-catenin, a Wnt signalling regulator (22). However, the association between Wnt and Hedgehog signalling pathways remain unclear.
In the present study, we further examined regulation of Hedgehog signalling genes by Wnt signalling by examining the gene expression profiles of Hedgehog signalling genes under Wnt3a stimulation. In addition, promoter sequence analysis combined with ChIP and yeast-one hybrid assays were performed to explore the potential mechanisms that mediate the activation of Smo and Gli1 transcription by β-catenin/TCF4 complex. Next, TCF4 and β-catenin were transiently overexpressed in fibroblasts, after which Smo and Gli1 expression levels were determined. In addition, β-catenin expression was suppressed via siRNA transfection, and Smo and Gli1 levels were monitored. In conclusion, our analyses indicated that the β-catenin/TCF4 complex directly regulates Smo and Gli1 transcription, which provided direct evidence for the link between the Wnt and Hedgehog signalling pathways.
Materials and methods
Human foreskin fibroblast cell culture
Human foreskin samples were isolated from 4 patients in the Department of Dermatology, the First Affiliated Hospital, Wenzhou Medical University (Wenzhou, China). The present study was approved by the Ethics Committee of Wenzhou Medical University (Wenzhou, Zhejiang, China) and written informed consent was obtained from all of the patients involved (21). All the procedures followed for purification and culture of human fibroblasts were described by Xuan et al (23).
Cell culture
Human foreskin fibroblast cells were cultured for 12 h at 37°C in an incubator with 5% CO2 in Petri dishes and subsequently cultured into a monolayer until reaching confluence (23). Afterwards, cells were cultured in DMEM containing 0.5% FBS and treated with Wnt3a (100 ng/ml). Cells were harvested after 0, 1, and 3 h of Wnt3a treatment.
Yeast-one hybrid assay
For the yeast one-hybrid assay, the 1.5-kb promoters of Smo and Gli1 were synthesized by Sangon Biotech (www.sangon.com/) and subsequently cloned into the pHISi vector. The TCF4 ORF sequences were cloned into the pGAD424 yeast expression vector. The pGAD424-TCF4 and pSmo-His, mpSmo-His, pGli1-His, or mpGli1-His were transformed into the yeast one-hybrid bait strain (YM4271). The growth of yeast cells was monitored on synthetic dropout Leu or His.
ChIP assay
Chromatin immunoprecipitation (ChIP) assay was performed using a chromatin immunoprecipitation assay kit (cat no. 17-295, Millipore, Billerica, MA) according to the manufacturer's instructions. Immunoglobulin (1 µg; IgG; Abcam, ab172730), anti-TCF4 antibody (Cell Signaling Technology, 2566), and anti-β-catenin antibody (Abcam, ab32572) were used for immunoprecipitation (24). The immunoprecipitated DNA fragments were analysed via quantitative PCR. Primer sequences used for ChIP-PCR are listed in Table I.
Western blot analysis
Cells were lysed in an ice-cold lysis solution [7 M urea, 2 M thiourea, 2% CHAPS, 40 mM Trizma base, 40 mM dithiothreitol (DTT), and 1% protease inhibitor]. After complete lysis of the cells and centrifugation at 15,000 × g for 15 min, the total protein concentration in the supernatant was measured using a Bradford protein assay kit (Bio-Rad, Richmond, CA, USA). Proteins were separated via SDS-PAGE and electrotransferred onto Immobilon-P Transfer Membranes (Millipore, Tokyo, Japan). Membranes were incubated in TBS containing 5% skim milk and 0.05% Tween-20 for 1 h and subsequently blotted with primary antibodies at 4°C overnight. Anti-Smo antibody (1:1,000, Abcam, ab72130), anti-Gli1 antibody (1:2,000, Abcam, ab49314), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam, ab8245) were used as the primary antibodies. Membranes were incubated for 1 h with an anti-mouse or anti-rabbit HRP-linked secondary antibody (1:2,000; Cell Signaling Technology).
Total RNA extraction, cDNA synthesis, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA (1 µg) was extracted from human foreskin fibroblast cells and reverse-transcribed using a GoScript Reverse Transcription Kit (Reverse Transcription System, Promega) according to the manufacturer's instructions. Gene expression was quantified via RT-qPCR as previously described (25). A SYBR Green Master Mix kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for performing qPCR on an Illumina Eco 3.0 (Illumina, Inc., San Diego, CA, USA). A typical reaction consisted of 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 55°C and extension at 72°C for 30 sec, followed by a final extension at 72°C for 5 min. The transcription levels were normalized against those of GAPDH using the 2−ΔΔCq method (26). Primer sequences used for RT-qPCR are listed in Table I.
Overexpression (OX) and RNA interference
Open reading frame (ORF) regions of TCF4 (NM_013685.2, NCBI) and β-catenin (NM_001165902.1, NCBI) were synthesized and cloned into the pcDNA3.1 (+) vector to generate the TCF4 OX and β-catenin OX constructs. siRNA targeting β-catenin (ON-TARGET plus SMART pool, L-004018), siRNA targeting Gli1 (ON-TARGETplus SMART pool, J-041026-05), and negative control siRNA (ON-TARGETplus Non-targeting Control Pool, D-001810) were purchased from Dharmacon RNA Technologies (Chicago, IL, USA). Fibroblast cells were seeded for 12 h before transfection and allowed to reach 30 to 50% confluence at the time of transfection. Afterwards, 30 nM siRNA duplex and 2 µg of TCF4 OX or β-catenin OX plasmids were transfected on day 0 using Lipofectamine 2000 (Invitrogen) and Opti-MEM®I Reduced Serum Medium (Gibco) according to the manufacturer's instructions. Cells reached confluence at 24 h after transfection, and the OX or siRNA solutions were replaced with full growth medium. The transfected cells were used for two experiments, namely, cell migration assay (60–80% confluence at the time of cell migration assay on day 2) and RT-qPCR (up to a density of 80–90% confluence at the time of harvest for RNA preparation on day 3).
Cell proliferation assay
Cell proliferation was examined after siRNA treatment. Proliferation ability was measured using a CCK-8 Kit (Dojindo Bio., Japan) according to the manufacturer's instructions. The cell densities of the β-catenin and Gli1 siRNA-transfected cells were analysed relative to those of the control group as previously described (23).
Statistical analysis
Statistical calculations were performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Data are expressed as the mean ± standard deviation. Significant differences between groups were analysed by one-way analysis of variance, followed by Bonferroni's multiple comparison post hoc tests. P<0.05 was considered to indicate a statistically significant difference.
Results
Wnt3a induced the expression of Hedgehog signalling genes
To verify the effect of Wnt3a on the expression of hedgehog signalling genes, human fibroblast cells were treated with Wnt3a (100 ng/ml) for 1 and 3 h. RT-qPCR assay was performed to monitor the expression of Smo, PTCH, Gli1, Gli2, and Gli3. The results indicated that expression of all the above mentioned genes was induced by Wnt3a treatment, and peak expression levels for all genes tested were detected after 3 h of Wnt3a treatment (Fig. 1A). Western blot analysis was performed to verify the results of RT-qPCR analysis. Consistent with the results of RT-qPCR, western blotting results revealed that Smo and Gli1 expression was induced after 3 h of Wnt3a treatment (Fig. 1B).
The β-catenin/TCF4 complex directly activates Smo and Gli1 transcription
Smo and Gli1 are induced by Wnt3a treatment. Therefore, we next determined whether the key Wnt signalling transcription factor complex β-catenin/TCF4 can directly induce the transcription of Smo and Gli1. Promoter sequence analysis revealed that two and one putative TCF/LEF binding motifs (AGAAAG) (7) was located within 1.5 kb of the Smo and Gli1 promoters, respectively (Fig. 2A). To investigate whether β-catenin/TCF4 directly binds to the putative motifs located in the Smo and Gli1 promoters, ChIP assays were performed using antibodies targeting TCF4 or β-catenin, with IgG as the control. Immunoprecipitated DNA fragments were amplified using the primer pairs targeting the F1, F2, and F3 regions of the Smo and Gli1 promoters (Fig. 2B). Data were normalized using the input DNA as template. ChIP-PCR results showed that β-catenin and TCF4 can bind to the F1, F2, and F3 regions (Fig. 2B). To verify the ChIP findings, yeast one-hybrid assay was performed by co-expressing AD (activation domain)-TCF4, and pSmo-His and mpSmo-His or pGli1-His and mpGli1-His (Fig. 2C). The results indicated that cells transfected with AD-TCF4 co-expressing pSmo-His or pGli1-His were able to grow in the SD medium without histidine, whereas cells transfected with AD-TCF4 co-expressing mpSmo-His or mpGli1-His did not grow. All transformants were normally grown in SD medium without leucine (Fig. 2D). The above findings indicated that TCF4 activates the transcription of Smo and Gli1 by binding to their promoters in yeast cells.
Overexpression of TCF4 and β-catenin activated Smo and Gli1
Given that TCF4 and β-catenin bind to the Smo and Gli1 promoters, their expression levels were further analysed in the TCF4- and β-catenin-overexpressing cells. TCF4 and β-catenin were transiently overexpressed (TCF4 OX and β-catenin OX) in human fibroblasts by transformation of the plasmids pcDNA3.1-TCF4 or pcDNA3.1-β-catenin using Lipofectamine 2000 reagent. Gene expression analysis via RT-qPCR revealed that TCF4 and β-catenin were significantly upregulated after 24 h of transfection (Fig. 3). In addition, Smo and Gli1 expression levels showed 2.2- and 2.8-fold upregulation in TCF4 OX cells relative to control cells, respectively (Fig. 3A). In addition, the results showed that Smo and Gli1 expression levels were upregulated by 2.2- and 2.6-fold in β-catenin OX cells relative to control cells, respectively (Fig. 3B).
Suppression of β-catenin reduced Smo and Gli1 levels with or without Wnt3a stimulation
Considering that overexpression of TCF4 and β-catenin induced Smo and Gli1 expression, TCF4 and β-catenin levels were further analysed in the β-catenin siRNA-transfected human fibroblast cells with or without Wnt3a treatment. RT-qPCR results indicated that transfection with β-catenin-specific siRNA significantly downregulated the expression of β-catenin independent of Wnt3a treatment (about 60–70%), and Wnt3a treatment induced β-catenin levels (Fig. 4). Wnt3a treatment also upregulated Smo and Gli1 expression in cells with or without Wnt3a stimuli; however, the relative increases in expression were lower in β-catenin siRNA-transfected cells compared to those of control cells. Furthermore, siRNA suppression of β-catenin expression significantly reduced Smo and Gli1 levels in human fibroblasts (Fig. 4).
Effects of β-catenin and Gli1 suppression on cell proliferation in fibroblasts
Given that the Wnt and Hedgehog signalling pathways have been demonstrated to be involved in cell proliferation, we examined the effects of β-catenin and Gli1 suppression on fibroblast proliferation. Before evaluating cell proliferation ability, fibroblasts were transfected with Gli1 siRNA and control siRNA, after which Gli1 expression levels were measured by RT-qPCR. Gli1 was significantly downregulated in Gli1-siRNA transfected cells relative to those of control cells (Fig. 5A). In addition, cell proliferation rates were determined in β-catenin and Gli1 siRNA-transfected cells relative to those of the control group. Cell density measurements evidently showed that suppression of β-catenin and Gli1 inhibited cell proliferation (Fig. 5B).
Discussion
The skin is the tissue layer that protects the body from external damage. Fibroblasts are a cell type that play a major role in wound repair. Extensive studies have been conducted to elucidate the regulatory mechanisms underlying fibroblast cell migration and proliferation (20–23). TCF and β-catenin are master transcriptional regulators of Wnt signalling, and nuclear translocation of β-catenin leads to the formation of a TCF/β-catenin transcription factor complex (2–4). GSK3β-β-catenin signalling was reported to regulate cell migration in human fibroblasts via feedback regulation of basic fibroblast signalling (20). Another study demonstrated that key regulators of Hedgehog signalling, including Smo and Gli1, are controlled by β-catenin to modulate fibroblast cell migration (22). In addition, connections between Wnt and hedgehog signalling was reported (27). In colon cancer, GSK3β and CK1α phosphorylate full-length Gli3 (28), leading to the degradation of C-terminal peptides of Gli3 to produce truncated form, Gli3R. Gli3R further inhibits Gli1 activity (29). A negative regulator (kinase) of both Wnt/β-catenin and Hedgehog/Gli signaling pathways, Sufu interacts with β-catenin and Gli1 to modulate their nuclear-cytoplasmic distributions (30,31). However, the relationship between β-catenin and Gli as well as Wnt and hedgehog signalling remains unclear in fibroblasts. Our previous transcriptome study revealed that Wnt3a stimulation in fibroblasts induced the expression of Hedgehog signalling genes, such as Smo, PTCH, and Gli (21). To further confirm the relationship between Wnt and Hedgehog signalling, fibroblast cells were subjected to Wnt3a treatment, and expression patterns of key hedgehog signalling genes were analysed. The results of RT-qPCR and western blot analyses demonstrated that Wnt3a-induced Wnt signalling activation upregulated the expression of Smo, PTCH, Gli1, Gli2, and Gli3 (Fig. 1).
A previous study demonstrated that the TCF/β-catenin complex activates various downstream genes by binding to specific sequences in the promoters of the target genes (7). Interestingly, promoter sequence analysis revealed that the putative TCF/LEF binding motifs (7) to which the TCF/β-catenin complex binds were located within 1.5 kb of the Smo and Gli1 promoters. We next performed ChIP and yeast-one hybrid assays to determine whether the TCF/β-catenin complex directly binds to the Smo and Gli1 promoters. Our findings indicated that the TCF4/β-catenin complex directly binds to two and one TCF/LEF motifs in the Smo and Gli1 promoters, respectively (Fig. 2B).
Given that the TCF4/β-catenin complex binds to the Smo and Gli1 promoters, the expression levels of Smo and Gli1 were examined in the cells overexpressing TCF4 or β-catenin. TCF4 or β-catenin was highly expressed in the cells transfected with pCDNA3.1-TCF4 or pCDNA3.1-β-catenin (Fig. 3). In addition, the results indicated that TCF4 or β-catenin overexpression activated Smo and Gli1 transcription. Moreover, Smo and Gli1 expression levels were examined in β-catenin siRNA-transfected cells. siRNA transfection evidently suppressed β-catenin levels. In addition, Smo and Gli1 levels were lower in β-catenin siRNA-transfected cells compared to those of control cells (Fig. 4), which suggested that β-catenin is located upstream of Smo and Gli1. To confirm whether Wnt3a stimuli-mediated induction of Smo and Gli1 are mediate by β-catenin, control siRNA- and β-catenin siRNA-transfected cells were treated with Wnt3a. The results indicated that Wnt3a induced β-catenin, Smo, and Gli1 expression in both the control siRNA- and β-catenin siRNA-transfected cells (Fig. 4). However, the fold inductions in β-catenin siRNA-transfected cells were lower than those in control cells, suggesting that β-catenin mediates Wnt3a-induced activation of Hedgehog signalling.
We further analysed the biological function of β-catenin as a key regulator of Hedgehog signalling genes. The Wnt signalling pathway is known to regulate cell proliferation (2). Thus, β-catenin and Gli1 expression was suppressed via siRNA transfection of human fibroblasts, and cell proliferation rates were determined. The results suggested that β-catenin and Gli1 participate in the same signalling pathway and act as positive regulators of cell proliferation.
Wnt and Hedgehog signalling play crucial roles in diverse processes involved in mammalian development. In the present study, our findings revealed that the Wnt and Hedgehog signalling pathways directly share a common molecular mechanism, which is the binding of the TCF4/β-catenin complex to Smo and Gli1 promoters in human fibroblasts. Furthermore, β-catenin and Gli1 were demonstrated to positively regulate cell migration and proliferation in human fibroblasts (2; Fig. 5B), thereby demonstrating the strong link between gene regulation and biological function. Previous study showed the post-transcription regulation of Gli family proteins by Wnt signalling regulators GSK3β and CK1α (26–28), but transcriptional regulation between two pathways has not been observed. In this study, we identified a transcriptional regulation between β-catenin and Smo or Gli1, which extended knowledge of Wnt and hedgehog relationship, which could be important for understanding regulatory networks not only in fibroblasts, but also in cancer cells.
Acknowledgements
The authors would like to thank Mr. Rui Wen (Department of Basic Medical Science, Wenzhou Medical University, Zhejiang, China) for their assistance.
Funding
The present study was supported by Wenzhou City Public Welfare Technology Projects (grant no. Y20170033).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
YPW, PPL and QW performed the experiments. YPW, MQZ and LXP analyzed and interpreted the data. YPW, MQZ and LXP were the major contributors in writing the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Ethics Committee of Wenzhou Medical University (Wenzhou, Zhejiang, China) and written informed consent was obtained from all of the patients involved.
Patient consent for publication
The patients provided written informed consent for the publication of any associated data.
Competing interests
The authors declare that they have no competing interests.
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