Fucosylation regulates various pathological events in cells. We reported that different levels of CRT (calreticulin) affect the cell adhesion and metastasis of bladder cancer. However, the precise mechanism of tumour metastasis regulated by CRT remains unclear. Using a DNA array, we identified FUT1 (fucosyltransferase 1) as a gene regulated by CRT expression levels. CRT regulated cell adhesion through α1,2-linked fucosylation of β1 integrin and this modification was catalysed by FUT1. To clarify the roles for FUT1 in bladder cancer, we transfected the human FUT1 gene into CRT-RNAi stable cell lines. FUT1 overexpression in CRT-RNAi cells resulted in increased levels of β1 integrin fucosylation and rescued cell adhesion to type-I collagen. Treatment with UEA-1 (Ulex europaeus agglutinin-1), a lectin that recognizes FUT1-modified glycosylation structures, did not affect cell adhesion. In contrast, a FUT1-specific fucosidase diminished the activation of β1 integrin. These results indicated that α1,2-fucosylation of β1 integrin was not involved in integrin–collagen interaction, but promoted β1 integrin activation. Moreover, we demonstrated that CRT regulated FUT1 mRNA degradation at the 3′-UTR. In conclusion, the results of the present study suggest that CRT stabilized FUT1 mRNA, thereby leading to an increase in fucosylation of β1 integrin. Furthermore, increased fucosylation levels activate β1 integrin, rather than directly modifying the integrin-binding sites.

INTRODUCTION

Fucosylation is a common type of post-translational protein glycosylation, which regulates various physiological and pathological events in cells. This process is catalysed by at least 11 types of FUTs (fucosyltransferases), which transfer a fucose residue from GDP-fucose to oligosaccharides through α1,2-, α1,3/4- or α1,6-linkages [1]. Aberrant expression of fucosylated haptoglobin has been reported in various tumour tissues, particularly during the advanced stages of cancers [24]. In papillary carcinoma, higher FUT8 levels are significantly correlated with tumour size and lymph node metastasis [5]. In addition, fucosylated haptoglobin is considered to be a novel biomarker for pancreatic cancer detection [6]. These results suggest that fucosylation may be a key event in regulating cancer development and progression.

FUT1 is a common type of FUT and is involved in catalysing the addition of fucose via α1,2-linkage to galactose. It has been reported that higher levels of Lewis Y antigen, catalysed by FUT1 and FUT4, are found in over 60% of human epithelial carcinomas [79]. Moreover, studies suggest that in certain cancer cells, tumour growth and metastasis are regulated by changes in FUT1 levels [10,11]. These results indicated that FUT1-regulated fucosylation is closely associated with tumour progression.

Integrins are a family of receptors comprising α- and β-subunits. They are involved in cell–cell and cell–matrix interactions and regulate key physiological processes such as cell migration and adhesion and cancer metastasis. Accumulated evidence indicates that integrin function is regulated by glycosylation [12,13]. Overexpression of GnT (N-acetylglucosaminyltransferase)-V, which increases β1,6-branching of N-linked glycan, enhanced α5β1 integrin-mediated cell migration and invasion [14], whereas an increase in the GlcNAc (N-acetylglucosamine) bisecting linkage of N-glycan by GnT-III inhibited α5β1 integrin-mediated cell spreading and migration [15]. Furthermore, FUT8-catalysed core fucosylation (α1,6-fucosylation) of α5β1 integrins stimulated cell migration via laminin 5 [16]. However, the consequences of FUT1 expression and its effect on integrins have not been surveyed so far.

CRT (calreticulin) is a multifunctional chaperon protein majorly localized to the ER (endoplasmic reticulum). It has been reported that CRT participates in a variety of important biological processes. Previously, CRT has been identified as an RNA-binding protein that regulates mRNA stability [17]. Our previous studies have shown that suppression of CRT levels in human bladder cancer cell line diminishes cell adhesion and migration, and subsequently inhibits cancer metastasis [18]. However, the precise mechanism by which CRT modulates the above-mentioned processes remains unclear.

In order to evaluate the metastatic mechanisms of bladder cancer, we generated stable CRT-knockdown cell lines and used DNA microarrays to identify CRT-regulated genes. Compared with control cells, FUT1 expression was significantly lower in CRT-knockdown (CRT-RNAi) cells. Since several studies have highlighted that CRT is essential for integrin-mediated cell adhesion [19,20], we hypothesized that CRT may affect FUT1 and regulate integrin functions as well as cell adhesion. In the present study, we have demonstrated that CRT-RNAi lead to significantly lower glycosylation of β1 integrins. Our results further indicated that changes in β1 integrin fucosylation affected integrin activity. Furthermore, we demonstrated that CRT regulated FUT1 expression levels by regulating its mRNA stability. In conclusion, we suggested that FUT1-modified fucosylation play an important role in β1 integrin activation and this process can enhance cancer metastasis of bladder cancer.

EXPERIMENTAL

Cell culture and generation of stably transfected cell lines

Details of the CRT-knockdown human bladder cancer cell line J82 (J82 CRT-RNAi) have been described previously [18]. J82 CRT-RNAi cells were infected with pLKO_AS2.zeo-EGFP and selected with 250 μg/ml zeocin to generate J82 CRT-RNAi–GFP cells. J82 CRT-RNAi–GFP cells were then infected with pLKO_AS2.puro or pLKO_AS2.puro-FUT1 to generate the J82 CRT-RNAi–GFP Control (#R-EGFP) and J82 CRT-RNAi–GFP FUT1 (#R-FUT1) cell lines. The stably infected cells were selected using 500 μg/ml G418, 250 μg/ml zeocin and 2 μg/ml puromycin. The J82 human bladder cancer cell line was cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS. For subcultures, cells were trypsinized with 0.05% EDTA/trypsin.

Plasmid construction

The coding region sequence of the FUT1 (NCBI Reference Sequence NM_000148) gene was purchased from OriGene. The lentival vectors for cDNA expression, including pLKO_AS2.puro and pLKO_AS2.zeo were obtained from the National RNAi Core Facility Platform, Academia Sinica, Taipei, Taiwan. EGFP template was purchased from pEGFP-C1 (Clontech). PCR was performed using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific). FUT1 was ligated into pLKO_AS2.puro between the NheI and BsrGI sites to build pLKO_AS2.puro-FUT1. EGFP was subcloned into pLKO_AS2.zeo between the NheI and EcoRI sites to generate pLKO_AS2.zeo-EGFP. The following primers were used for FUT1 PCR: Fut1-forward-NheI, 5′-AAGCTAGCATGT-GGCTCCGGAGCCATCGTCAG-3′ and Fut1-reverse-BsrGI, 5′-GGTGTACATCAAGGCTTAGCCAATGTCCAGAG-3′.

Cell adhesion assay

The 96-well culture plates were coated with 10 μg/ml type I collagen (Sigma) and incubated at 37°C for 30 min followed by washing with PBS. The adhesion assay procedure was described previously [18]. To determine the effects of various functional blocking antibodies or lectin on cell adhesion, suspensions of J82 cells suspension was pre-incubated with an anti-integrin antibody (1 μg/ml for α1, α2, α3, αV, β3 and αVβ3 and 0.25 μg/ml for β1; Millipore) for 1 h or lectin (Vector Laboratories) for 30 min. Adhesion then was determined by using the adhesion assay as described previously [18].

Western blot analyses

The whole cell lysate were lysed with 1% NP40 (Nonidet P40) lysis buffer and then incubated with the primary antibodies anti-β1 integrin (1:2000 dilution; BD Transduction Laboratories and GeneTex), anti-CRT (1:5000 dilution; Millipore), anti-(biotin-labelled UEA-1) (where UEA-1 is Ulex europaeus agglutinin-1; 1:5000 dilution; Vector Laboratories), and anti-FUT1 and anti-FUBP1 (far-upstream-binding protein 1; 1:2000 dilution; Santa Cruz Biotechnology) for 4°C overnight. Membranes were washed and then incubated with HRP (horseradish peroxidase)-conjugated secondary antibodies or HRP-conjugated streptavidin (1:5000 dilution) for 1 h. Immunoreactive bands were quantified using the Total Lab V2.01 software.

Lectin pull-down assay

Detection of glycoproteins decorated with terminal galactose was achieved by lectin pull-down assays using biotinylated UEA-1 or LTL (Lotus tetragonolobus lectin; Vector Laboratories). Briefly, 500 mg of total cell lysates were incubated with biotinylated UEA-1. Following a 16 h incubation at 4°C, streptavidin–agarose beads were added and incubated for an additional 6 h. The pulled-down proteins were then subjected to Western blot analysis.

RNA isolation and real-time PCR

Total RNA was isolated using the TRIzol® reagent (Life Technologies) following the manufacturer's instructions. RT (reverse transcription)–PCR was carried out using ReverTra Ace reverse transcriptase (Toyobo). Real-time PCR was performed using the iCycle iQ real-time detection system with the dsDNA-specific SYBR Green I dye for detection (Bio-Rad Laboratories). For quantification, the target gene was normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase), an internal control gene. The primer sequences used were: GAPDH, 5′-GGTGGTCTCCTCTGACTTCAAC-3′ (forward) and 5′-TCTCTCTTCCTCTTGTGCTCTTG-3′ (reverse); FUT1-CDS (coding sequence), 5′-AAGTTCTACGGTGA-CGAGGAG-3′ (forward) and 5′-GTCGATGTTCTGCTCATGT-TTC-3′ (reverse); FUT1-3′UTR, 5′-CGTGCTCATTG-CTAACCACTGTC-3′ (forward) and 5′-TCGTGCTCCTG-CCTGGATCT-3′ (reverse); EGFP, 5′-GTGAGCAAGGGCGA-GGAG-3′ (forward) and 5′-CGTAGGTCAGGGTGGTCA-3′ (reverse); DsRed 5′-GAGGGCTTCAAGTGGGAG-3′ (forward) and 5′-CATAGTCTTCTTCTGCATTACGG-3′ (reverse); and FBP1 5′- TGGGACCATACAACCCTGCACCT-3′ (forward) and 5′-AGCTGGATCAGGAGCCTG-3′ (reverse).

Microarray analysis

Total RNA from control and CRT-RNAi cells were isolated using the TRIzol® reagent and quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). The cRNA was fragmented and then hybridized to a Human OneArray™ microarray (HOA 4.3; Phalanx Biotech) containing 30968 human gene probes. Arrays were scanned by Microarray Scanner GenePix 4000B (Axon Instruments), and fluorescence intensities were measured by GenePix Pro version 6.0 (Molecular Devices). The raw data were preprocessed by log2 transformation and global LOWESS (locally weighted scatterplot smoothing) normalization.

Determination of integrin activation

Cells were trypsinized and re-suspended in 100 μl of PBS containing α1,2-fucosidase (1:100 dilution; BioLabs) at 37°C. After 30 min, cells were stained with PE (phycoerythrin)-conjugated anti-CD29 antibody (HUTS-21; BD Pharmingen) and analysed by flow cytometry.

Determination of mRNA stability

J82 cells were treated with 5 μg/ml Act-D (actinomycin D; Sigma) at the indicated time points. FUT1 mRNA was detected by real-time PCR.

REMSA (RNA EMSA)

The synthesized biotinylated FUT1-ARE (AU-rich element) and mARE (mutant ARE) probes (MDBio) were incubated with recombinant CRT protein (Abnova) for 10 min at 4°C in REMSA buffer (100 mM Hepes, 200 mM KCl, 10 mM MgCl2 and 10 mM DTT) containing 2 μg of tRNA to a final volume of 20 μl. Samples were analysed by native PAGE [4% polyacrylamide/bisacrylamide (37.5:1)]. Following electrophoresis, the gel was transferred on to a nylon membrane and the RNA–protein complexes were detected by chemiluminescence following the manufacturer's instructions (Thermo Scientific).

RNA immunoprecipitation

The synthetic biotinylated FUT1-ARE and mARE probes were incubated with whole cell lysate (500 μg) containing Streptavidin Mag Sepharose (GE Healthcare) at 4°C overnight. The pulled-down RNA–protein complexes were analysed by Western blotting using the anti-CRT antibody.

Statistical analysis

Data were statistically analysed using one-way ANOVA followed by Fisher's protected LSD (least-significant difference) test (StatView; Abacus Concept). Each result was obtained from at least three independent experiments and P<0.05 was considered statistically significant.

RESULTS

β1 Integrin participated in CRT-mediated cell adhesion in J82 human bladder cancer cells

It has been reported previously that down-regulation of CRT in J82 cells suppresses cell adhesion and metastatic behaviour [18]. Since integrins play a crucial role in cell adhesion, we used a collagen-specific integrins investigator kit to identify the type of integrins involved in CRT-mediated changes in cell adhesion. As shown in Figure 1(A), functional blocking antibodies for α2 or β1 integrin inhibited the adhesion in vector control cells, but not in CRT-RNAi cells. Blocking other integrins did not result in any significant differences in cell adhesion between control and CRT-RNAi cells. These suggested that α2 and β1 integrin might be involved in CRT-mediated adhesion on type I collagen. However, the level of total β1 integrin showed no difference between the control and CRT-RNAi cells (Figure 1B). Subsequently, we used DNA arrays to compare the gene expression profiles between J82 control and CRT-RNAi cells and identified many candidate genes whose expression was likely to be CRT-regulated (Supplementary Tables S1 and S2 at http://www.biochemj.org/bj/460/bj4600069add.htm). Among these, FUT1 was significantly suppressed in J82 CRT-RNAi cells. Because previous studies have shown that changes in glycosylation of β1 integrin modulates its function [21], we performed a lectin pull-down assay using either LTL or UEA-1, which are known to bind to α1,2-linkage oligosaccharides through a reaction catalysed by FUT1. The pull-down samples were then subjected to immunoblotting against antibodies specific for β1 integrin. The fucosylation levels of β1 integrin were decreased profoundly in CRT-RNAi cells (Figure 1C). In addition, we also performed glycomics analysis in our stable cell lines. J82 cells had different UEA-1-positive glycoprotein expression patterns, as shown by Western blotting, when we knocked down CRT (Figure 1D). These results suggested that CRT mediates cell adhesion through post-translational modification of β1 integrin in bladder cancer.

Integrin α2β1 was involved in CRT-mediated cell adhesion in J82 bladder cancer cells

Figure 1
Integrin α2β1 was involved in CRT-mediated cell adhesion in J82 bladder cancer cells

(A) To analyse the role of integrins in J82 cell adhesion, cells were pre-incubated with normal mouse IgG or functional blocking antibodies at 37°C for 1 h. Cells (5×104) were seeded in 96-well collagen-coated plates (10 μg/ml) for 20 min. NS, not significant. (B) Western blot analysis showing no changes in the total β1 integrin expression levels between the control and CRT-knockdown cells. Human β-actin was used as a loading control. (C) FUT1-modified glycosylation on β1 integrin in J82 stable cell lines. Cell lysates were pulled down with UEA-1 or LTL followed by Western blotting with an anti-(β1 integrin) antibody. (D) Western blot analysis showing the glycomic differences between the control and CRT-knockdown cells. Cell lysates were separated by SDS/PAGE (10% gel) and probed with biotin-labelled UEA-1. Representative data from three independent experiments are shown. (A and B) Results are means±S.D. **P<0.01 and ***P<0.001 compared with the control cells.

Figure 1
Integrin α2β1 was involved in CRT-mediated cell adhesion in J82 bladder cancer cells

(A) To analyse the role of integrins in J82 cell adhesion, cells were pre-incubated with normal mouse IgG or functional blocking antibodies at 37°C for 1 h. Cells (5×104) were seeded in 96-well collagen-coated plates (10 μg/ml) for 20 min. NS, not significant. (B) Western blot analysis showing no changes in the total β1 integrin expression levels between the control and CRT-knockdown cells. Human β-actin was used as a loading control. (C) FUT1-modified glycosylation on β1 integrin in J82 stable cell lines. Cell lysates were pulled down with UEA-1 or LTL followed by Western blotting with an anti-(β1 integrin) antibody. (D) Western blot analysis showing the glycomic differences between the control and CRT-knockdown cells. Cell lysates were separated by SDS/PAGE (10% gel) and probed with biotin-labelled UEA-1. Representative data from three independent experiments are shown. (A and B) Results are means±S.D. **P<0.01 and ***P<0.001 compared with the control cells.

FUT1 overexpression in CRT-knockdown cells enhanced cell adhesion

Fucosylation is one of the common post-translational protein modifications. It has been shown that transfection of FUT1 into RMG-1 cells enhanced cell adhesion [22]. To further examine the role of FUT1 on CRT-mediated changes in cell adhesion, the FUT1 gene was stably transfected into CRT-RNAi cells to generate J82 #R-FUT1 cells. FUT1 overexpression in J82 #R-FUT1 cells was confirmed by real-time PCR (Figure 2A). To investigate the effect of FUT1 on the changes in β1 integrin fucosylation, we pulled down the lectin UEA-1 and probed for β1 integrin, as described above. Binding of β1 integrin to UEA-1 lectin was increased significantly in J82 #R-FUT1 cells compared with the control vector-transfected cells (Figure 2B). Furthermore, as shown in Figure 2(C), reintroduction of FUT1 restored cell adhesion in CRT-RNAi stable cells. These results suggested that CRT modulated cell adhesion in bladder cancer by regulating β1 integrin fucosylation through FUT1.

FUT1 overexpression rescued cell adhesion in CRT-knockdown cells

Figure 2
FUT1 overexpression rescued cell adhesion in CRT-knockdown cells

Reintroduction of FUT1 in J82 CRT-RNAi cells. CRT-knockdown stable cells were infected with the control (EGFP) or FUT1 plasmid to generate FUT1-overexpressed stable cell lines. (A) mRNA expression was confirmed by real-time PCR in FUT1-overexpressing stable cell lines. GAPDH was used as an internal control. (B) A lectin pull-down assay was performed to assess whether overexpressed FUT1 could enhance glycosylation of β1 integrin. Cell lysates were pulled down with UEA-1 followed by Western blotting with the anti-(β1 integrin) antibody. Representative data from three independent experiments are shown. (C) Effects of FUT1 on CRT-mediated cell adhesion were performed by adhesion experiment. Cells (5×104 cells/100 μl) were seeded in 96-well collagen-coated (10 μg/ml) plates for 20 min. (A and C) Results are means±S.D. *P< 0.05 and **P<0.01 compared with the vector control level. #C, control cells; #R, CRT-RNAi cells; #R-EGFP, CRT-knockdown cells infected with EGFP; #R-FUT1, CRT-knockdown cells infected with FUT1–EGFP.

Figure 2
FUT1 overexpression rescued cell adhesion in CRT-knockdown cells

Reintroduction of FUT1 in J82 CRT-RNAi cells. CRT-knockdown stable cells were infected with the control (EGFP) or FUT1 plasmid to generate FUT1-overexpressed stable cell lines. (A) mRNA expression was confirmed by real-time PCR in FUT1-overexpressing stable cell lines. GAPDH was used as an internal control. (B) A lectin pull-down assay was performed to assess whether overexpressed FUT1 could enhance glycosylation of β1 integrin. Cell lysates were pulled down with UEA-1 followed by Western blotting with the anti-(β1 integrin) antibody. Representative data from three independent experiments are shown. (C) Effects of FUT1 on CRT-mediated cell adhesion were performed by adhesion experiment. Cells (5×104 cells/100 μl) were seeded in 96-well collagen-coated (10 μg/ml) plates for 20 min. (A and C) Results are means±S.D. *P< 0.05 and **P<0.01 compared with the vector control level. #C, control cells; #R, CRT-RNAi cells; #R-EGFP, CRT-knockdown cells infected with EGFP; #R-FUT1, CRT-knockdown cells infected with FUT1–EGFP.

Fucosylation status of β1 integrin affected integrin activation, but not association between integrin and collagen

Since the fucosylation status of β1 integrin could influence cell adhesion, we hypothesized that α1,2-linked fucosylated glycan on β1 integrin might participate in β1 integrin and type I collagen interaction. To assess the potential inhibitory effects of UEA-1, we tested J82 cells in a cell adhesion assay in the presence of UEA-1 lectin. As shown in Figure 3(A), treatment with UEA-1 did not show any effect on adhesion even at higher dosages. These results prompted us to inspect the activation status of β1 integrin. Several studies have revealed that variation in the glycosylation patterns of β1 integrin has an influence on its functions [15,23,24]. To verify the effect of fucosylation levels on β1 integrin activity, the active forms of β1 integrin were detected by flow cytometry. We found that active integrins were significantly lower in CRT-RNAi cells (13.0±2.6) than in control cells (22.9±3.9) (Figure 3B). These results suggested that β1 integrin fucosylation modified integrin activity. To further confirm this hypothesis, cells were treated with a FUT1-specific α1,2-fucosidase. Both the control and CRT-RNAi cells showed reduced β1 integrin activity upon fucosidase treatment (Figure 3B). Taken together, these findings suggested that β1 integrin fucosylation affected the J82 cell adhesion ability by activating β1 integrin, rather than directly modifying the integrin-binding sites. The data for down-regulation of α1,2-fucosylation after fucosidase treatment are shown in Figure 3(C).

The activation of β1 integrin decreased by reduction in α1,2-linked fucosylation

Figure 3
The activation of β1 integrin decreased by reduction in α1,2-linked fucosylation

(A) To evaluate the involvement of α1,2-linked glycan in integrin–collagen interaction, cells were pre-incubated with various concentrations of UEA-1 for 30 min. Cells (5×104) were seeded in 96-well collagen-coated plates (10 μg/ml) for 20 min. Results are means±S.D. (B) The activity of β1 integrin was analysed by flow cytometry. Cells were trypsinized and incubated with PE-conjugated anti-CD29 antibody (HUTS21; active form of β1 integrin) for 20 min. Cells were also pre-treated with α1,2-fucosidase for 30 min before staining with the anti-CD29 antibody to further analyse the effect of α1,2-linked fucosylation on β1 integrin activity. Representative data from three independent experiments are shown. Results are means±S.D. *P<0.05 compared with the no-fucosidase group. #C, control cells; Fs, cells were treated with α1,2-fucosidase; NS, not significant; #R, CRT-RNAi cells. (C) FUT1-modified glycosylation of β1 integrin after treatment with α1,2-fucosidase. J82 control cells were pre-treated with α1,2-fucosidase before being lysed with NP40 lysis buffer. Cell lysates were pulled down with UEA-1 or LTL followed by Western blotting with an anti-(β1 integrin) antibody.

Figure 3
The activation of β1 integrin decreased by reduction in α1,2-linked fucosylation

(A) To evaluate the involvement of α1,2-linked glycan in integrin–collagen interaction, cells were pre-incubated with various concentrations of UEA-1 for 30 min. Cells (5×104) were seeded in 96-well collagen-coated plates (10 μg/ml) for 20 min. Results are means±S.D. (B) The activity of β1 integrin was analysed by flow cytometry. Cells were trypsinized and incubated with PE-conjugated anti-CD29 antibody (HUTS21; active form of β1 integrin) for 20 min. Cells were also pre-treated with α1,2-fucosidase for 30 min before staining with the anti-CD29 antibody to further analyse the effect of α1,2-linked fucosylation on β1 integrin activity. Representative data from three independent experiments are shown. Results are means±S.D. *P<0.05 compared with the no-fucosidase group. #C, control cells; Fs, cells were treated with α1,2-fucosidase; NS, not significant; #R, CRT-RNAi cells. (C) FUT1-modified glycosylation of β1 integrin after treatment with α1,2-fucosidase. J82 control cells were pre-treated with α1,2-fucosidase before being lysed with NP40 lysis buffer. Cell lysates were pulled down with UEA-1 or LTL followed by Western blotting with an anti-(β1 integrin) antibody.

CRT knockdown suppressed FUT1 expression by regulating its mRNA stability through an ARE

In order to verify how CRT affected FUT1 expression, real-time PCR and Western blot analyses were performed in our stable cell lines. The results showed that CRT down-regulation attenuated the expression of FUT1 protein and mRNA levels of the 3′-UTR, but not the mRNA level of the CDS (Figure 4). It is known that certain specific sequences in 3′-UTR of mRNA can influence the RNA stability and protein expression [25]. These reports prompted us to investigate whether CRT regulates FUT1 expression levels by affecting mRNA stability rather than transcription. An Act-D time course experiment was performed to determine the degradation rate of FUT1 mRNA. We found that FUT1 mRNA was significantly less stable following CRT knockdown in J82 cells (Figure 5A). These results strongly suggested that CRT down-regulation affected the mRNA stability of FUT1.

CRT knockdown inhibited the FUT1 protein and 3′-UTR mRNA expression levels, but not the mRNA of the CDS

Figure 4
CRT knockdown inhibited the FUT1 protein and 3′-UTR mRNA expression levels, but not the mRNA of the CDS

(A) FUT1 mRNA levels were detected by real-time PCR in J82 control and CRT-knockdown cells. mRNA expression was normalized to the internal control GAPDH. (B) Western blot analysis demonstrates protein expression in J82 stable cell lines. The human β-actin level was used as a loading control. Results are means±S.D. ***P<0.001 compared with the control level. NS, no significant.

Figure 4
CRT knockdown inhibited the FUT1 protein and 3′-UTR mRNA expression levels, but not the mRNA of the CDS

(A) FUT1 mRNA levels were detected by real-time PCR in J82 control and CRT-knockdown cells. mRNA expression was normalized to the internal control GAPDH. (B) Western blot analysis demonstrates protein expression in J82 stable cell lines. The human β-actin level was used as a loading control. Results are means±S.D. ***P<0.001 compared with the control level. NS, no significant.

CRT-knockdown destabilized FUT1 mRNA through the ARE

Figure 5
CRT-knockdown destabilized FUT1 mRNA through the ARE

(A) Cells were treated with 5 μg/ml Act-D for the indicated time. FUT1 mRNA was assessed by real-time PCR and normalized to the internal control GAPDH. (B) Schematic diagram of reporter constructs containing EF1α-driven DsRed and CMV-driven EGFP fused to various ARE sequences. (C) Cells were transfected with the plasmids shown in (B). Total RNA was harvested after 24 h of transfection. Results are the ratio of EGFP to the internal transfected control DsRed. Results are means±S.D. **P<0.01 and ***P<0.001 compared with the control level. dARE, deleted ARE; wtARE, wild-type ARE.

Figure 5
CRT-knockdown destabilized FUT1 mRNA through the ARE

(A) Cells were treated with 5 μg/ml Act-D for the indicated time. FUT1 mRNA was assessed by real-time PCR and normalized to the internal control GAPDH. (B) Schematic diagram of reporter constructs containing EF1α-driven DsRed and CMV-driven EGFP fused to various ARE sequences. (C) Cells were transfected with the plasmids shown in (B). Total RNA was harvested after 24 h of transfection. Results are the ratio of EGFP to the internal transfected control DsRed. Results are means±S.D. **P<0.01 and ***P<0.001 compared with the control level. dARE, deleted ARE; wtARE, wild-type ARE.

Numerous studies have shown that in sequences rich in adenosine and uridine nucleotides, called AREs, the 3′-UTRs are important sequences for mRNA decay [2628]. The FUT1 gene contains an ARE sequence at its 3′-UTR. J82 control and CRT-RNAi cells were transfected with a CMV (cytomegalovirus)-driven EGFP reporter fused to wild-type, mutated or ARE-deleted FUT1 3′-UTR as shown in Figure 5(B). The EF1α (elongation factor 1α)-driven DsRed was used as an internal control. EGFP mRNA expression was measured by real-time PCR after 48 h of transfection. Control cells showed a lower EGFP/DsRed ratio when the ARE sequences from the FUT1 3′-UTR were mutated or deleted (Figure 5C, left-hand panel). However, no significant differences were observed between the wild-type, mutated or ARE-deleted FUT1 3′-UTR constructs in CRT-knockdown cells (Figure 5C, right-hand panel). Consequently, these results indicate that CRT influenced FUT1 mRNA stability through the ARE sequence.

Identification of FBP1 as an FUT1 mRNA-binding protein

The proteins which bind to AREs, called ARE-BPs (ARE-binding proteins), play an important role in the regulation of mRNA stability [26]. CRT was reported previously as an ARE-BP that regulates mRNA degradation in various cell types [29,30]. We carried out REMSAs using a biotin-labelled probe and recombinant CRT to assess CRT–FUT1 ARE interaction in vitro. No specific bands with a higher molecular mass than the biotin-labelled probes were observed (Figure 6A). We next incubated whole cell extracts with the biotin-labelled probes and the bound proteins were pulled down with streptavidin. As shown in Figure 6(B), CRT did not co-precipitate with the ARE or mARE probes. These results indicate that CRT did not bind to the ARE in the FUT1 3′-UTR.

CRT knockdown suppressed FBP1 that binds to the ARE at the FUT1 3′-UTR

Figure 6
CRT knockdown suppressed FBP1 that binds to the ARE at the FUT1 3′-UTR

(A) Biotinylated RNA probes (ARE or mARE) were incubated with recombinant CRT. The RNA–protein complex was separated by native PAGE and detected using chemiluminescence. (B) Whole cell lysates were incubated with biotinylated RNA probes (ARE or mARE). The RNA–protein complex was pulled down by streptavidin beads and analysed using Western blotting with an anti-CRT antibody. IP, immunoprecipitation. (C) Identification of FBP1 as the FUT1 ARE-BP. Biotinylated RNA probes (ARE or mARE) were incubated with whole cell lysate and the RNA–protein complex was pulled down by streptavidin beads. Arrow indicates the putative binding protein. (D) Real-time PCR was performed to detect FBP1 expression levels in J82 control and CRT-RNAi cells. mRNA expression was normalized to the internal control GAPDH. Results are means±S.D. **P<0.01 compared with the control level. (E) Western blot analysis demonstrating protein expression in J82 stable cell lines. Human β-actin was used as a loading control.

Figure 6
CRT knockdown suppressed FBP1 that binds to the ARE at the FUT1 3′-UTR

(A) Biotinylated RNA probes (ARE or mARE) were incubated with recombinant CRT. The RNA–protein complex was separated by native PAGE and detected using chemiluminescence. (B) Whole cell lysates were incubated with biotinylated RNA probes (ARE or mARE). The RNA–protein complex was pulled down by streptavidin beads and analysed using Western blotting with an anti-CRT antibody. IP, immunoprecipitation. (C) Identification of FBP1 as the FUT1 ARE-BP. Biotinylated RNA probes (ARE or mARE) were incubated with whole cell lysate and the RNA–protein complex was pulled down by streptavidin beads. Arrow indicates the putative binding protein. (D) Real-time PCR was performed to detect FBP1 expression levels in J82 control and CRT-RNAi cells. mRNA expression was normalized to the internal control GAPDH. Results are means±S.D. **P<0.01 compared with the control level. (E) Western blot analysis demonstrating protein expression in J82 stable cell lines. Human β-actin was used as a loading control.

To identify the proteins that did bind to the FUT1 ARE, the synthetic probe was incubated with whole cell lysate. We performed an RNA pull-down assay and the RNA–protein complex was separated by SDS/PAGE (8% gel). In all three independent experiments, we consistently noticed a band near 75 kDa, which was suppressed in the mARE samples (Figure 6C). Using MS we identified this ARE-binding protein as FBP1. Our results confirmed that both the protein and mRNA levels of FBP1 were reduced in CRT-knockdown cells (Figures 6D and 6E). Consequently, these results suggest that CRT affected FUT1 mRNA stability through FBP1 regulation, which binds to the ARE sequence in the FUT1 3′-UTR.

DISCUSSION

RNA stability is an important control point for gene expression. It has been reported that interaction between specific sequences (cis-acting elements) in the 3′-UTR of mRNA and specialized RNA-binding proteins (trans-acting factor) regulate mRNA degradation [31]. On the basis of the characteristics of the cis-acting elements, there are three classes of mRNA, and the most common type identified in the 3′-UTR is the ARE [25,28,32,33]. A number of studies have shown that certain metastasis-associated genes with AREs are overexpressed in cancers [3436]. Previously, we have reported that CRT levels regulate the metastasis of bladder cancer cells [18]. In the present study, we showed that CRT knockdown suppressed FUT1 mRNA degradation in J82 cells. Screening using the ARE database (http://brp.kfshrc.edu.sa/ARED/) classified FUT1 as class II cluster I of the ARE mRNA. In our fluorescence reporter experiments, mutations or deletions in the FUT1 3′-UTR ARE sequence resulted in a significant decrease in EGFP levels in control cells. These results are consistent with previous reports that the ARE sequence is important for regulating mRNA stability [32,37]. Consequently, we suggested that CRT may modulate the metastasis of bladder cancer by influencing FUT1 mRNA stability in the ARE region.

Protein factors that bind to AREs are key regulators for mRNA stability. Many ARE-BPs, including AUF1 (ARE RNA-binding protein 1), HuR and TTP (tristetraprolin) have been well studied [26]. Numerous studies have reported that these ARE-BPs bind to AU-rich regions and influence the stability of certain inflammatory and tumour-associated genes, including c-Myc, c-Fos, GMCSF (granulocyte/macrophage colony-stimulating factor), TNFA (tumour necrosis factor α) and VEGF (vascular endothelial growth factor) [3840]. CRT is a multifunctional protein that participates in various cell processes. In 2002, Nickenig et al. [29] first indicated CRT as a novel mRNA-binding protein that destabilizes type I angiotensin II receptor mRNA by binding to the AU-rich region at the 3′-UTR. Moreover, Totary-Jain et al. [30] reported that CRT also binds to a specific element in the 3′-UTR of glucose transporter-1 mRNA and destabilizes the mRNA under high-glucose conditions. In the present study, the fluorescence signal of mARE or ARE-deleted FUT1 was suppressed in the control cells, but not in CRT-RNAi cells, suggesting that CRT levels influence FUT1 mRNA stability. However, we did not notice any direct association between CRT and the FUT1 ARE directly in J82 cells. On the other hand, we identified FBP1 as the protein that interacts with an FUT1 ARE probe. Previous studies have indicated that FBP1 is an RNA-binding protein that participates in mRNA translation and/or stabilization [4143]. In the present study, FBP1 levels were significantly decreased in CRT-knockdown cells. Taken together, our results suggested that CRT can stabilize FUT1 mRNA through FBP1, which binds to the FUT1 ARE at the 3′-UTR.

Fucosylation-regulated cancer cell tumorigenesis and metastasis has been studied extensively [1]. It has been reported that FUT1/FUT4-knockdown by siRNA significantly diminishes cell proliferation and tumour growth both in vitro and in vivo [10]. FUT1 overexpression restored cell adhesion in CRT-RNAi cells, suggesting that FUT1-related fucosylation affects cell adhesion and subsequent metastasis. Nevertheless, a deficiency in GDP-mannose-4,6-dehydratase, resulting in a loss of fucosylation, has been shown to cause colon cancer cell metastasis by escaping from tumour immune surveillance [44,45]. Although those studies contradicts the results of the present study, cumulative evidence suggests that tumour tissues express higher levels of fucosylation compared with normal tissues [4648]. Furthermore, Numahata et al. [49] showed that sialyl Lewis X, a blood carbohydrate catalysed by fucosyltransferase, is a predictor of an invasive and metastatic outcome of bladder cancer. These findings suggested a positive correlation between fucosylation and bladder cancer progression. Our results provided further evidence that FUT1 plays an important role in the metastasis of bladder cancer.

Cell adhesion is one of the common features of cancer metastasis. It is known that integrins are the main cell surface molecules that regulate cell–matrix interaction. Many studies have revealed that changes in glycan modification of integrins affect its biological function [50,51]. A previous study showed that deletion of core fucosylation (FUT8) on α3β1 integrin suppressed cell migration on laminin 5 in MEFs (mouse embryonic fibroblasts) [16]. In the present study, we found that FUT1 increased fucosylation of β1 integrin and promoted the cell adhesion of bladder cancer cells with type I collagen. Overexpression of the FUT1 gene enhanced cell adhesion in CRT-knockdown cells. However, higher levels of FUT1 only partially restored cell adhesion in CRT-knockdown cells. Consequently, our results provide evidence that FUT1-mediated fucosylation of β1 integrin is one of the important mechanisms for cell adhesion in J82 bladder cancer cells. In another study FUT1 has been shown to increase the total expression of α5β1 integrin and promote RMG-1 cell adhesion on fibronectin [22]. Nevertheless, on the basis of the results of the present study, we suggest that these observations might due to increased activity of β1 integrin. We also noticed a clear reduction in the activity of β1 integrin with reduction in fucosylation in our stable cell lines. Most importantly, cells treated with FUT1-specific α1,2-fucosidase effectively diminished integrin activity. Taken together, our results suggest that CRT regulates cell adhesion in bladder cancer by regulating β1 integrin activity through FUT1-dependent fucosylation. Since previous studies only indicated that changes in the glycosylation of integrins may affect its cell adhesion and migration function, the results of the present study provide further evidence showing the role of fucosylation on the activity of β1 integrin. It is known that there are 12 potential N-glycosylation sites on the β1 integrin subunit [12]. Our stable cell lines can be excellent tools to identify the functional fucosylation sites on β1 integrin.

We have shown previously that alterations in CRT expression levels affect cell adhesion and the metastasis of bladder cancer. In the present study we further analysed the mechanistic details of CRT-dependent fucosylation and bladder cancer progression. Our findings demonstrate that CRT stabilizes FUT1 mRNA through affecting the level of the FBP1 ARE-BP. Therefore FUT1 activates β1 integrin by fucosylation, thereby enhancing bladder cancer adhesion and subsequent metastasis. The present study provides new insights into the biological function of α1,2-linked fucosylation on β1 integrin. Studies are underway to address the specific fucosylation modification site of β1 integrin. The results of the present study can provide a new strategy towards therapeutic development for bladder cancer.

Abbreviations

     
  • Act-D

    actinomycin D

  •  
  • ARE

    AU-rich element

  •  
  • ARE-BP

    ARE-binding protein

  •  
  • CDS

    coding sequence

  •  
  • CMV

    cytomegalovirus

  •  
  • CRT

    calreticulin

  •  
  • EF1α

    elongation factor 1α

  •  
  • FBP1/FUBP1

    far-upstream-binding protein 1

  •  
  • FUT

    fucosyltransferase

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • GnT

    N-acetylglucosaminyltransferase

  •  
  • HRP

    horseradish peroxidase

  •  
  • LTL

    Lotus tetragonolobus lectin

  •  
  • mARE

    mutant ARE

  •  
  • NP40

    Nonidet P40

  •  
  • PE

    phycoerythrin

  •  
  • REMSA

    RNA EMSA

  •  
  • UEA-1

    Ulex europaeus agglutinin-1

AUTHOR CONTRIBUTION

Hsinyu Lee and Cheng-Chi Chang designed the experiments. Yi-Chen Lu performed experiments, analysed the data and wrote the paper. All other authors performed experiments.

We thank Enago (http://www.enago.tw) for a review of the English used in the present paper.

FUNDING

This work was supported by the Cutting-Edge Steering Research Project of the National Taiwan University [grant number NTU-CESRP-102R76263A], the National Science Council of Taiwan [grant number NSC 97-2311-B-002 -002 -MY3] and the National Health Research Institutes of Taiwan [grant number NHRI-EX101-10130BI (to H.L.)].

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Author notes

1

These authors contributed equally to this work.

Supplementary data