Inhibiting post-translational core fucosylation prevents vascular calcification in the model of uremia

The International Journal of Biochemistry & Cell Biology 79 (2016) 69–79

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Inhibiting post-translational core fucosylation prevents vascular calcification in the model of uremia Xinyu Wen a,b,1 , Anqi Liu a,b,1 , Changqing Yu b , Lingyu Wang b , Mengying Zhou b , Nan Wang b , Ming Fang b , Weidong Wang b , Hongli Lin b,∗ a b

Graduate School of Dalian Medical University, Dalian, China Department of Nephrology, Liaoning Translational Medicine Center of Nephrology, The First Affiliated Hospital of Dalian Medical University, Dalian, China

a r t i c l e

i n f o

Article history: Received 1 December 2015 Received in revised form 20 June 2016 Accepted 9 August 2016 Available online 10 August 2016 Keywords: ␣-1,6-fucosyltransferase Vascular calcification TGF␤-R

a b s t r a c t Vascular calcification (VC) is an independent risk factor for cardiovascular disease and mortality in uremia. Post-translational core fucosylation is implicated in a number of pathological processes. First, we investigated the role of core fucosylation and key TGF-␤1 pathway receptors in calcified arteries in vivo. To determine whether blocking core fucosylation effectively inhibited VC and TGF-␤/Smad signaling pathway, we established an in vitro model of phosphate-induced calcification in rat vascular smooth muscle cells (VSMCs) to assess the role of core fucosylation in VC. Core fucose could be detected at markedly higher levels in calcified VSMCs than control cells. Fut8 (␣-1,6 fucosyltransferase), the only enzyme responsible for core fucosylation in humans, was significantly upregulated by high phosphate. Exposed to high phosphate media and blocking core fucosylation in VSMCs by knocking down Fut8 using a siRNA markedly reduced calcium and phosphorus deposition and Cbf␣1 expression (osteoblast-specific transcription factor), and increased ␣-Sma expression (smooth muscle cell marker). Fut8 siRNA significantly inhibited TGF-␤/Smad2/3 signaling activation in VSMCs cultured in high phosphate media. In conclusion, this study provides evidence to suggest core fucosylation plays a major role in the process of VC and appropriate blockade of core fucosylation may represent a potential therapeutic strategy for treating VC in end-stage renal disease. © 2016 Published by Elsevier Ltd.

1. Introduction Vascular calcification (VC) is a common complication and the major cause of cardiovascular disease in patients with end-stage renal disease (Karohl et al., 2011). The prevalence of VC increases during the progression of chronic kidney disease (CKD), ranging from 40% among patients with stage 3 CKD to 80–99% in patients with end-stage renal disease on dialysis (Garland et al., 2008; Sigrist et al., 2006; Adeney et al., 2009; Chertow et al., 2002). However, the precise molecular mechanisms underlying VC still need to be clarified. VC is no longer regarded as a passive process and is considered an actively-regulated and complex process that is not yet

Abbreviation: VSMCs, vascular smooth muscle cells; CKD, chronic kidney disease; Fut8, ␣-1,6 fucosyltransferase; cbf␣1, core-binding factor subunit 1 ␣; ␣-SMA, ␣-smooth muscle actin; TGF␤1, transforming growth factor-␤1; LCA-FITC, fluorescent L. culinaris agglutinin–fluorescein complex. ∗ Corresponding author at: Department of Nephrology, The First Affiliated Hospital of Dalian Medical University, No. 222 Zhongshan Road, Dalian 116011, China. E-mail address: [email protected] (H. Lin). 1 These authors contributed equally to this work and share the first authorship. http://dx.doi.org/10.1016/j.biocel.2016.08.015 1357-2725/© 2016 Published by Elsevier Ltd.

completely understood. During VC, the specific, indispensable transcriptional regulator of osteoblastic differentiation core-binding factor subunit 1␣ (Cbf␣1) is upregulated, while expression of the VSMC marker ␣-smooth muscle actin (␣-SMA) decreases (Giachelli et al., 2005; Hruska et al., 2005). A number of cytokines and signaling pathways have been demonstrated to stimulate the occurrence and development of VC, including TNF-␣, osteonectin, osteocalcin, the BMP-2 signaling pathway, transforming growth factor-␤ (TGF-␤) signaling pathway (Yetkin and Waltenberger, 2009; Wang et al., 2013) and the Wnt/␤-catenin/OPG/RANKL/RANK axis (Evrard et al., 2015). These studies have provided important data regarding the mechanisms that underlie VC and helped to infer the process that occur during the progression of VC. Most of the above-mentioned studies have explored the roles of key proteins in VC by altering the expression of these genes or proteins. In fact, in addition to gene and protein expression levels, post-translational modifications of proteins can also have a major effect on protein function. Data increasingly indicates that post-translational modifications directly and definitively regulate protein function in a range of pathophysiological processes, and in some cases, this regulation is independent of the

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expression levels of the modified proteins (Kumar and Klein, 2004; Hao et al., 2011). Therefore, post-translational modifications may be critical regulators of protein function. Glycosylation is a key type of post-translational modification and has significant effects on the regulation of various physiological processes, including cell growth, differentiation and migration (Takahashi et al., 2009; Zhao et al., 2008). Core fucosylation, which consists of an ␣-1,6 fucose substitution on the innermost N-acetylglucosamine (GlcNAc) of the pentasaccharide core of Nlinked glycans, is a special protein glycosylation pattern associated with a number of biological and pathological functions (Ferrara et al., 2011; Wang et al., 2006a,b; Akasaka-Manya et al., 2008). Recent investigations reported that core fucosylated proteins are associated with various cancers, such as pancreatic cancer, lung cancer, ovarian cancer and prostate cancer (Noda et al., 1998a,b, 2003; Geng et al., 2004; Chen et al., 2013; Saldova et al., 2007; Tabarés et al., 2006; Okuyama et al., 2006; Ahn et al., 2014). Additionally, the core fucosylated ␣-fetoprotein (AFP-L3) has been approved by the Food and Drug Administration (FDA) for the early diagnosis of hepatocellular carcinoma (HCC). Our previous studies showed that TGF-␤RII and ALK5 are modified by core fucosylation via a process dependent on Fut8 (Shen et al., 2013; Lin et al., 2011), which is the only enzyme that catalyzes ␣1,6-linked core fucosylation in humans (Uozumi et al., 1996; Yanagidani et al., 1997). This data indicates the absence of core fucose on TGF-␤ receptors markedly dysregulated downstream TGF-␤/Smad2/3 signaling and induces pathological changes in renal tubular cells, and more importantly, these effects were independent of the expression of TGF-␤RII and ALK5. However, it remains unclear whether core fucosylation plays crucial role in VC. The aim of the present study was to determine whether blocking core fucosylation effectively inhibits VC and over activation of the TGF-␤/Smad signaling pathway in CKD. To test this, we investigated the role of core fucosylation and key receptors of the TGF-␤1 pathway that participate in VC in the calcified arteries of rats with adenine-induced CKD and human patients with CKD. As phosphate loading is known to induce VC in animal models of CKD, and hyperphosphatemia is a significant risk factor for VC and cardiovascular mortality in patients with preexisting CKD (Adeney et al., 2009; El-Abbadi et al., 2009; Kestenbaum et al., 2005), we established an in vitro model of CKD using high phosphate culture medium. Fut8 was knocked down in rat aortic vascular smooth muscle cells (VSMCs) exposed to high phosphate media. This study suggests that inhibition of core fucosylation attenuates VC, and indicates that the ability to target post-translational modifications of key proteins may provide a novel and effective strategy for the treatment of VC.

(LCA-FITC) were purchased from Vector (Burlingame, CA, USA). Anti-Fut8 antibody (sc-34629; Yanagidani et al., 1997), anti-TGF␤1 antibody (sc-146; Li et al., 2013), anti-TGF-␤RII antibody (sc-17791; Recouvreux et al., 2011), anti-ALK5 antibody (sc-399; Velasco et al., 2008), anti-Smad 2/3 antibody (sc-8332; Zhou et al., 2010), antip-Smad2/3 antibody (sc-11769; Wang et al., 2012), anti-Cbf␣1 antibody (sc-10758; Xiao et al., 2005), anti-␤-actin antibody (sc47778) and Protein G PLUS-Agarose (sc-2002; Ma et al., 2001) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-␣-smooth muscle actin (SMA) antibody (ab5694; Ramani et al., 2010) was purchased from Abcam (Cambridge, UK). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). DMEM, FBS and pancreatic enzyme were purchased from Hyclone (UT, USA). RIPA was purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). SYBR PrimerScript RT-PCR Kit and ISOPLUS were purchased from Takara (Takara, Otsu, Shiga, Japan). Horseradish peroxidase (HRP)-goat anti-rabbit antibody (ZB-2301), HRP-goat anti-mouse antibody (ZB-2305) and HRP-rabbit antigoat antibody (ZB-2306) were purchased from Beijing Zhong Shan Golden Bridge Biological Technology (Beijing, China). FITC- conjugated goat anti-rabbit antibody (SA00003-2), FITC-conjugated goat anti-mouse antibody (SA00003-1) and TRITC-conjugated goat anti-rabbit antibody (SA00007-2) were purchased from Proteintech Group (Chicago, IL, USA) and the BCA protein assay kit from Pierce (Madison, WI, USA).

2.3. Human radial artery samples Part of this study assessed two patients with upper limb trauma with normal renal function who had no signs of cardiovascular disease or diabetes (control group) and four patients with CKD who exhibited clinical calcification (CKD group). Radial arterial samples in control group with upper limb trauma operation and CKD group during arterial venous fistula plasty were obtained after the patients provided informed consent.

2.4. Rat model of CKD Following acclimatization to a standard diet for 7 days, 10week-old male SD rats were randomly subdivided into two groups and treated as follows for 6 weeks: (1) normal group (n = 8), rats were given 0.8% saline once a day by gavage; and (2) CKD group (n = 8), given 2% adenine by gavage once a day at 250–300 mg/kg/d. Six weeks later, the rats were humanely scarified and the abdominal aortas were collected and processed for protein analyses and to determine the calcium content.

2. Materials and methods 2.1. Ethical considerations and animal care

2.5. Histology and immunohistochemistry

All experimental protocols were reviewed and approved by the Committee on Ethics of Animal Experimentation of Dalian Medical University. Animal experiments were conducted in accordance with the regulations set by the institutional committee for the care and use of laboratory animals, and approved by local authorities. Male Sprague-Dawley (SD) rats (250–300 g) were housed under a 12 h light/dark cycle with free access to food and water.

For histological analysis, artery tissues were fixed in 4% buffered paraformaldehyde and embedded in paraffin. Sections (2 um thick) were prepared and deparaffinized, endogenous peroxidase activity was quenched in 3% H2 O2 for 15 min, then the sections were washed in PBS, incubated with anti-Cbf␣1, ␣-SMA, Fut8, TGF␤1, TGF␤RII, ALK5 or p-Smad2/3 antibodies at 37◦ C for 1 h, then incubated with the appropriate biotinylated secondary antibodies, followed by treatment with 3,3 -diaminobenzidine (DAB) as a chromogen. Slides were counterstained with Mayer’s hematoxylin and mounted in glycerol jelly. Quantification of the area of immunostaining (brown color) in the artery regions of each tissue section was conducted using computer-based morphometric analysis (Name software; Olympus, Tokyo, Japan).

2.2. Reagents and antibodies Fut8 small interfering (si) RNA fragments were purchased from Genepharma (Shanghai, China). Biotinylated Lens culinaris agglutinin (LCA-Biotin), fluorescein-labeled L. culinaris agglutinin

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Fig. 1. Calcification and upregulation of Cbf␣1 and the TGF␤/smad2/3 signaling pathway in CKD arteries in vivo. (A–B) Alizarin red staining of the aortas of the rats in the control group and CKD group (A) and arteries of human controls and patients with CKD (B). (C-D) Expression of Cbfa1 (1:100), ␣-Sma (1:500), TGF␤1 (1:100), TGF␤RII (1:100), ALK5 (1:100), Smad2/3 (1:100) and p-Smad2/3 (1:200) were assessed using immunohistochemistry in the aortas of the rats in the control group and CKD group (C) and arteries of human controls and patients with CKD (D). (E) Protein expression of Cbfa1 (1:100), ␣-Sma (1:200), TGF␤1 (1:100), TGF␤RII (1:200), ALK5 (1:200), Smad2/3 (1:500) and p-Smad2/3 (1:500) were determined by Western blotting in the aortas of the rats in the control group and CKD group. Data are mean ± SD of three independent experiments. ␤-Actin (1:10000) was used as an internal control; ## P < 0.01, # P < 0.05 versus normal. Scale bar = 50 um.

2.6. Cell culture and in vitro calcification model A7r5 rat aortic vascular smooth muscle cells (VSMCs) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and confirmed free of mycoplasma contamination. The cells were routinely cultured in DMEM high glucose medium supplemented with 10% FBS and plated at a density of 2 × 105 cells/well in six-well plates. After reaching confluence, VSMCs were switched to calcification medium (DMEM containing

10% FBS and 2.5 mM/L Pi supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin) for up to 14 days.

2.7. Detection of core fucosylation LCA-FITC was used to detect the expression of core fucose in rat VSMCs. Cells were fixed in 4% paraformaldehyde, blocked by incubation in 1% (wt/vol) goat serum for 30 min, incubated in LCAFITC (1:1, 000) for 1 h at 37 ◦ C, washed three times with 0.01 M

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Fig. 2. Fut8 is upregulated in CKD arteries. Representative Western blot assessing Fut8 protein expression in the aortas of the rats in the control group and CKD group. (B-C) Immunohistochemistry for Fut8 in the aortas of the rats in the control group and CKD group. (B) and radial arteries of human controls and patients with CKD (C). Data are mean ± SD of three independent experiments. ␤-Actin (1:10000) was used as an internal control; # P < 0.05 versus normal. Scale bar = 50 um.

Fig. 3. High phosphate media induces calcification and upregulates the expression of core fucose in VSMCs. Alizarin red and von Kossa staining indicated that deposits could not be detected in control cells, whereas a punctate pattern of mineral accumulation was observed after treatment of VSMCs with high phosphate media (2.5 mM Pi) 14 d (×400 magnification). (B) Representative Western blotting analysis and (C) quantification of Cbfa1 and ␣-Sma protein expression in VSMCs cultured in control media and high phosphate media (2.5 mM Pi). (D) Representative images and (E) densitometric quantification of Lens culinaris agglutinin–fluorescein complex (LCA-FITC) staining for core fucose in VSMCs cultured in control media and high phosphate media (2.5 mM Pi); original magnification, × 400. (F) Representative Western blot analysis and quantification of Fut8 protein expression in VSMCs cultured in control media and high phosphate media (2.5 mM Pi). Results are expressed as the mean ± SD of three independent experiments. ␤-Actin (1:10000) was used as an internal control; ## P < 0.01 versus control cells.

PBS, and then mounted in an anti-fade medium and examined by fluorescence microscopy. 2.8. Design, preparation, and transfection of siRNAs SiRNA targeting rat Fut8 (5 -CCAGCGGAGAAUAACUUAUTTA UAAGUUAUUCUCCGCUGGTT-3 ) and a scrambled siRNA were synthesized by Genepharma (Shanghai, China). The siRNA sequences

were validated using BLAST and the rat genome database to assess possible cross-reactivity. Cells cultured in 6-well plates were transfected with siRNA using the Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, and then incubated for 8 h at 37 ◦ C in antibiotic-free medium before the addition of 10% fetal calf serum and culture for 48 h. All cells were cultured for 14 days and transient transfection was performed once every 3 days.

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Fig. 4. Relative protein expression levels of the TGF␤/Smad2/3 pathway in calcified VSMCs. (A-D) Representative Western blots and quantification of TGF␤1(1:100), TGF␤RII (1:200), ALK5 (1:200), Smad2/3 (1:500), and p-Smad2/3 (1:500) expression in VSMCs cultured in control media and high phosphate media (2.5 mM Pi). Results are mean ± SD of three independent experiments. ␤-Actin (1:10000) was used as an internal control; # P < 0.05, ## P < 0.01 versus control cells.

2.9. Real-time RT-PCR analysis

2.12. Western blotting

Total RNA was extracted from the VSMCs using ISOPLUS according to the manufacturer’s instructions. Total RNA was reverse transcribed using PrimeScriptTM RT Master Mix (Takara, Otsu, Shiga, Japan). The cDNA samples were added to a PCR array containing SYBR PrimerScript RT-PCR Kit to simultaneously examine mRNA expression levels according to the manufacturer’s instructions using the following primers: Fut8, 5 - GCTACCGATGACCCTGCTTTG-3 (forward) and 5 -CCGATTGTGTAATCCAGCTGACC-3 (reverse), and Gapdh, 5 -GCACCGTCAAGGCTGAGAAC-3 (forward) and TGGTGAAGACGCCAGTGGA-3 (reverse). PCR was performed at 30 s at 95 ◦ C, followed by 40 cycles of 5 s at 95 ◦ C and 30 s at 60 ◦ C. The specificity of the PCR products was assessed by melting curve analysis. To control for variations in the amount of DNA available for PCR in different samples, the gene expression levels of the target sequence were normalized to that of the housekeeping gene Gapdh using the Ct method. All samples were analyzed in triplicate.

Cultured cells and aortas were harvested, lysed in RIPA buffer, centrifuged at 15,000g for 10 min at 4 ◦ C, and the supernatants were collected and the protein concentrations were determined using a BCA protein assay kit. Protein samples were heated to 100 ◦ C for 5 min, and 20 ␮l aliquots were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Blots were probed with the appropriate primary antibodies diluted in 1 ml dilution buffer with agitation at 4 ◦ C overnight to detect Fut8, Cbf␣1, ␣SMA, TGF␤1, TGF-␤RII, ALK5, Smad2/3 and p-Smad2/3. Then, the blots were incubated with horseradish peroxidase–conjugated secondary antibody (1:10000) in dilution buffer with agitation for 1 h at 37 ◦ C, followed by ECL reagent (Amersham-Pharmacia Biotech, Little Chalfont, UK) and quantified using LabworksTM Image Analysis software (UVP, Upland, USA).

2.10. Immunoprecipitation assays Cells were washed three times with ice-cold PBS, lysed in cold RIPA lysis buffer, incubated on ice for 30 min, the lysates were centrifuged (12,000 rpm at 20 min, 4 ◦ C) and the supernatants were transferred to clean tubes, pre-cleared using Protein G PLUS-Agarose, and then incubated with anti-TGFRII or anti-ALK5 antibody (1:50) coupled with 20 ␮l of Protein G PLUS Agarose at 4◦ C with continual shaking for 2 h. Protein-antibody complexes were collected using 20 ␮l of Protein G PLUS-Agarose after rotary agitation overnight at 4 ◦ C. As a negative control, the primary antibody was omitted. The immunoprecipitate was washed three times with lysis buffer. Equal amounts of proteins were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes for lectin blotting (as described below).

2.13. Immunofluorescent analysis of p-Smad2/3, Cbf˛1 and ˛-SMA Rat VSMCs were incubated at 4 ◦ C overnight with rabbit anti-pSmad2/3 antibody (l:150), anti-Cbf␣1 antibody (l:100) and anti-␣SMA antibody (l:100), followed by incubation with FITC-goat antirabbit antibody (1:100) and TRITC-mouse anti-rabbit antibody for 1 h at 37 ◦ C in the dark. After washing three times in 0.01 M PBS for 5 min, the sites of immunoreactivity were observed by fluorescence microscopy. 2.14. Von Kossa staining Cells in culture were incubated with 5% silver nitrate solution and placed under a UV light source for 30 min. After several washes with distilled water, unreacted silver was removed by incubation in 5% sodium thiosulfate for 5 min, then the cells were rinsed in distilled water, counter-stained with hematoxylin and stored in distilled water. The presence of black staining confirmed the presence of calcium phosphate deposits.

2.11. Lectin blotting 2.15. Alizarin red staining Polyvinylidene difluoride membranes were blocked with 5% BSA (wt/vol) in Tris-buffered saline containing 0.05% Tween 20 (TBST) at 4 ◦ C overnight, and then incubated for 1 h at 37 ◦ C in TBST containing LCA-Biotin (1:200), which preferentially recognizes Fuc1,6GlcNAc. After washing with TBST four times for 10 min each, lectin-reactive proteins were detected using an ECL kit.

Two-micrometer sections of paraffin-embedded aorta were deparaffinized and processed for Alizarin red staining. Cells cultured in 6-well plates were washed three times with PBS, fixed with 10% formaldehyde for 10 min, and washed three with PBS. The cells and aorta were exposed to 0.5% Alizarin red for 40 min then washed

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Fig. 5. Fut8 siRNA effectively reverses the expression of endogenous a-1,6 fucosyltransferase (Fut8) and core fucosylation induced by high phosphate. Representative Western blot analysis and (B) quantification of Fut8 (1:200) protein expression in calcified VSMCs transfected with the scrambled siRNA or Fut8 siRNA. Results are mean ± SD of three independent experiments; ␤-Actin (1:10000) was used as an internal control; **P < 0.01 versus 2. 5 mM Pi group; # P < 0.05, ## P < 0.01 versus control cells. (C) Representative photomicrographs of rhodamine-labeled Lens culinaris agglutinin (LCA-FITC) staining for core fucosylation in VSMCs cultured in control media and high phosphate media (2.5 mM Pi) transfected with the Fut8 siRNA or scrambled siRNA; original magnification × 400. (D) Densitometric quantification of core fucosylation levels. Data are mean ± SD of three independent experiments; # P < 0.05 versus control cells; *P < 0.05 versus 2.5 mM Pi. (E) Representative real-time reverse transcriptase–PCR (RT–PCR) analysis of Fut8 mRNA expression in the VSMCs cultured in control media or high phosphate media and transfected with the scrambled siRNA or Fut8 siRNA; *P < 0.05 versus 2.5 mM Pi; # P < 0.05 versus control cells.

with distilled water. Positively-stained sections displayed a reddish color.

3. Results 3.1. Adenine-based CKD induces arterial medial calcification and upregulates expression of the TGFˇ/Smad pathway in rat arteries in vivo

2.16. Statistical analysis Data are expressed as the mean ± SD values from three independent experiments. Statistical analysis of data was performed using the Student’s t-test with SPSS 13.0 software (SPSS, Chicago, USA); statistical significance was defined as P < 0.05.

Rats were treated with 2% adenine for 6 weeks and then sacrificed. Alizarin red staining revealed the presence of obvious calcified areas in the arteries of CKD group compared with the normal group (Fig. 1A). To determine whether the TGF␤/Smad2/3 pathway is associated with VC, we performed immunohistochemistry and Western blotting for Cbf␣1, ␣-SMA, and members of the

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TGF␤/Smad2/3 pathway using the aorta samples from each group (Fig. 1C,E). Positive staining for both Cbf␣1 and the TGF␤/Smad2/3 pathway members was evident in and around the calcified area of the aorta of the CKD group while the expression of ␣-SMA was reduced compared to the control group. The expression patterns of the members of the TGF␤/Smad2/3 pathway were closely associated with the staining for Cbf␣1 in the aortas of the CKD group. 3.2. The TGFˇ/Smad pathway is upregulated in the radial arteries of patients with CKD and VC To confirm whether VC is associated with upregulation of the TGF␤/Smad pathway in the arteries of patients with CKD, radial artery samples were obtained from patients with upper limb trauma and patients with CKD during arterial venous fistula plasty. VC of the radial arteries of the patients with CKD was confirmed by Alizarin red staining and upregulation of Cbf␣1 was also observed (Fig. 1B,D). Moreover, the TGF␤/Smad 2/3pathway was upregulated in the arteries obtained from the patients with CKD compared to the arteries obtained from non-CKD donors (Fig. 1D). 3.3. Fut8 is upregulated in calcified arteries in vivo To determine whether core fucosylation is associated with VC, we assessed the expression of Fut8, the enzyme uniquely responsible for core fucosylation in vivo. Immunohistochemistry and Western blotting revealed Fut8 was significantly upregulated in the calcified arteries of the rats with CKD and patients with CKD (Fig. 2). 3.4. High phosphate media promotes calcification in VSMCs Hyperphosphatemia is considered the major cause of VC in patients with end-stage renal disease (Lilien and Groothoff, 2009; Kendrick and Chonchol, 2011), and phosphate loading is known to cause VC in animal models of CKD. Therefore, we established an in vitro model of high phosphate-induced calcification in rat vascular smooth muscle cells (VSMCs). Compared to the control media, precipitation of calcium and phosphate dramatically increased in the VSMCs cultured in the high phosphate media, predominantly in the extracellular regions (Fig. 3A). Additionally, expression of Cbfa1, the earliest marker of osteogenesis, obviously increased in VSMCs cultured in the high phosphate media, while expression of the smooth muscle cell protein ␣-Sma reduced (Fig. 3B,C). 3.5. Core fucosylation increases in VSMCs during calcification As core fucosylation has not yet been investigated in VSMCs, we firstly detected the characteristics of core fucosylation in calcified VSMCs using fluorescent L. culinaris agglutinin–fluorescein complex (LCA-FITC). We observed a low level of core fucosylation in the control cells; however, the level of core fucosylation markedly increased in the VSMCs cultured in media containing 2.5 mM Pi (Fig. 3D,E). Fut8 was also significantly upregulated in the VSMCs cultured in the high phosphate media compared to control cells (Fig. 3F). 3.6. Activation of the TGFˇ/Smad pathway in calcified VSMCs We next investigated the activity of the TGF-␤1 pathway, and the downstream Smad2/3 signaling pathway in VMSCs. Western blotting demonstrated activated TGF-␤1 protein expression significantly increased in cells cultured in the high phosphate media. Additionally, TGF-␤RII, ALK5 and p-Smad2/3 were also specifically upregulated in the VSMCs cultured in 2.5 mM Pi compared to the control cells (Fig. 4).

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3.7. Knockdown of Fut8 in VSMCs As described above, core fucosylation increased in calcified arteries and VSMCs cultured in media containing 2.5 mM Pi. Nevertheless, it remained unclear whether core fucosylation plays an important role in VC or is only a concomitant response. In order to study the role of core fucosylation in VC, RNA interference was used to knockdown the expression of Fut8, the enzyme uniquely responsible for core fucosylation in humans. Western blotting (Fig. 5A, B) and real-time RT-PCR (Fig. 5E) confirmed that the Fut8 siRNA effectively decreased Fut8 protein and mRNA expression in both control VSMCs and VSMCs cultured in 2.5 mM Pi. Furthermore, staining with rhodamine-labeled L. culinaris agglutinin (LCA-FITC) demonstrated that the Fut8 siRNA effectively reversed the increased level of core fucosylation observed in VSMCs cultured in 2.5 mM Pi in comparison with VSMCs cultured in the presence of 2.5 mM Pi and transfected with the scrambled siRNA (Fig. 5C, D). 3.8. Blocking core fucosylation inhibits calcification in VSMCs To address the role of core fucosylation in VC, we examined whether silencing Fut8 could inhibit calcification in VSMCs. Western blotting and immunofluorescent staining analysis revealed that the expression of Cbf␣1 significantly increased and the expression of ␣-Sma significantly decreased in VSMCs cultured in 2.5 mM Pi, while transfection of Fut8 siRNA markedly attenuated these changes (Fig. 6A-D). Finally, we analyzed the effect of Fut8 siRNA on calcification in VSMCs using von Kossa and Alizarin red staining. Fut8 siRNA markedly decreased calcium and phosphorus deposition in VSMCs cultured in 2.5 mM Pi (Fig. 6E). 3.9. Fut8 siRNA suppresses core fucosylation of TGF-ˇRII and ALK5, but does not affect their protein expression levels in calcified VSMCs Our previous study demonstrated that TGF-␤RII and ALK5 are modified by core fucosylation and play crucial roles in renal fibrosis (Shen et al., 2013; Lin et al., 2011). In this study, we observed positive core fucose bands were present in TGF-␤RII and ALK5 immunoprecipitates, indicating that TGF-␤RII and ALK5 are modified by core fucosylation in VSMCs (Fig. 7A–D). Furthermore, TGF-␤RII and ALK5 were upregulated in parallel with increased core fucosylation in VSMCs cultured in 2. 5 mM Pi. Additionally, while the Fut8 siRNA significantly reduced core fucosylation of TGF-␤RII and ALK5, it did not reverse the overexpression of these proteins induced by culture in media containing 2. 5 mM Pi (Fig. 7A–D). 3.10. Fut8 siRNA inhibits Smad2/3 phosphorylation by suppressing core fucosylation of TGF-ˇRII and ALK5 As ALK5 and TGF-␤RII are two key receptors of the TGF-␤ and Smad2/3 pathway, we explored whether inhibiting core fucosylation of ALK5 and TGF-␤RII using the Fut8 siRNA could abolish activation of the TGF-␤/Smad2/3 pathway in VSMCs. Western blotting analysis revealed that p-Smad2/3 was significantly upregulated in VSMCs cultured in 2.5 mM Pi; however, the Fut8 siRNA markedly inhibited high phosphate-induced upregulation of pSmad2/3 (Fig. 7E,F). 4. Discussion The present study provides novel insight into the pathogenesis of VC in end-stage renal disease. The role of core fucosylation, an important post-translational modification, was assessed in vascular disease for the first time.

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Fig. 6. Blocking core fucosylation affects expression of ␣-smooth muscle actin (SMA) and the osteoblast transcription factor Cbfa1 and suppresses VSMC calcification. Representative Western blot analysis and (B) quantification of ␣-Sma and Cbf␣1 protein expression in VSMCs cultured in control media or high phosphate media (2.5 mM Pi) and transfected with the scrambled siRNA or Fut8 siRNA. ␤-Actin was used as an internal control; *P < 0.05, **P < 0.01 versus 2.5 mM P; ## P < 0.01 versus control cells. (C) Representative images of immunofluorescent analysis and (D) densitometric quantification of ␣-Sma (l:100) and Cbf␣1 (l:100) protein expression in VSMCs cultured in control media or high phosphate media (2.5 mM Pi) and transfected with the scrambled siRNA or Fut8 siRNA; original magnification × 400. *P < 0.05, **P < 0.01 versus 2.5 mM Pi; ## P < 0.01 versus control cells. (E) Representative images of Alizarin red and von Kossa staining analysis to detect calcium and phosphate deposition in VSMCs cultured in control media or high phosphorus media (2.5 mM Pi) and transfected with the scrambled siRNA or Fut8 siRNA; original magnification × 400. All values are mean ± SD of three independent experiments.

Emerging evidence indicates that aberrant protein glycosylation plays an important role in tumor development, often independently of protein expression levels (Pinho et al., 2009). Post-translational glycosylation is considered an attractive treatment target for cancer (Matsumoto et al., 2009; Jin and Zangar, 2009; Goldman et al., 2009). Our previous studies confirmed that blocking core fucosylation of TGF-␤RII and ALK5 using a Fut8 siRNA could inactivate TGF-␤/Smad2/3 signaling pathway and reverse the renal tubular epithelial–mesenchymal transition in cultured human renal proximal tubular epithelial cells (Lin et al., 2011) and

attenuate renal interstitial fibrosis in a rat model of with unilateral ureteral obstruction (Shen et al., 2013). The fucosyltransferase FUT8 specifically catalyzes the introduction of fucose to position 6 of the initial N-acetyl glucosamine residue of the N-glycan core to produce ‘core fucose’ (Ihara et al., 2006; Imai-Nishiya et al., 2007). In our experiments, expression of Fut8 was markedly increased in calcified arteries and VSMCs (Figs. 2, 3), suggesting core fucosylation may play a role in VC. To further elucidate the role of core fucosylation in VC, we synthesized fluorescently-labeled Fut8 siRNA, and confirmed that it significantly knocked down the expression of endogenous Fut8 in rat

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Fig. 7. Blocking core fucosylation of TGF␤II and ALK5 inhibits TGF-␤/Smad2/3 pathway activation. (A, C) Representative Western blot and lectin blot analyses and (B, D) quantification of the expression and core fucosylation levels of TGF-␤RII and ALK5 in VSMCs cultured in control media or high phosphate media (2.5 mM Pi) and transfected with the scrambled siRNA or Fut8 siRNA. TGF-␤RII and ALK5 were immunoprecipitated from cell lysates and then subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed using Lens culinaris agglutinin (LCA)–Biotin (1:200). Results are mean ± SD of three independent experiments; ␤-Actin was used as an internal control; *P < 0.05 versus 2.5 mM Pi; # P < 0.05, ## P < 0.01 versus control cells. (E) Representative Western blot analysis and (F) quantification of Smad2/3 and p-Smad2/3 protein expression in VSMCs cultured in control media or high phosphate media (2.5 mM Pi) and transfected with the scrambled siRNA or Fut8 siRNA. Results are mean ± SD of three independent experiments. ␤-actin was used as an internal control; *P < 0.05 versus 2.5 mM Pi group; # P < 0.05 versus control cells.

VSMCs (Fig. 5). Inhibition of core fucosylation mediated by transfection of the Fut8 siRNA markedly reversed the alterations in the expression of Cbf␣1 and ␣-SMA induced by high phosphate media (Fig. 6A–D). Additionally, VSMCs transfected with Fut8 siRNA exhibited lower levels of calcium and phosphate deposition (Fig. 6E). These findings indicate inhibition of core fucosylation using the Fut8 siRNA prevented calcification in VSMCs cultured in high phosphate media. Next, we explored the mechanisms by which core fucosylation affects calcification in VSMCs. TGF-␤1 is present in calcified aortic valves and regulates VC and osteoblastic transition of VSMCs (Clark-Greuel et al., 2007). Expression of TGF-␤1 and TGF-␤RII increase during VC (Wang et al., 2010; Christian and Uwe, 2014) and upregulation of active TGF-␤1/T␤RII within the aged aortic wall results in activation of the Smad signaling pathways (Wang et al., 2006a,b). In accordance with previous research, we con-

firmed that TGF-␤RII and ALK5 are critical receptors associated with VC. The protein expression levels of both TGF-␤RII and ALK5 were obviously upregulated in the calcified arteries and VSMCs (Figs. 1 and 4). Intriguingly, the Fut8 siRNA inhibited core fucosylation of TGF-␤RII and ALK5, but had no effect on the expression of these proteins (Fig. 7A–D). Additionally, blocking core fucosylation of TGF-␤RII and ALK5 effectively reduced the levels of phosphorylated Smad2/3 in calcified VSMCs (Fig. 7E,F). These results suggest that the roles of fucosylated TGF-␤RII and ALK5 in VC are independent of their protein expression levels, and core fucosylation regulates the TGF-␤/Smad2/3 signaling pathway during VC. Collectively, these results indicate that when hyperphosphatemia occurs in patients with uremia, activation of TGF-␤/smad2/3 signaling − due to fucosylatation of the extracellular domains of TGF-␤ receptors by the fucosyltransferase Fut8 − subsequently induces VSMCs calcification, upregulates Cbf␣1 and downregulates ␣-Sma. Con-

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Fig. 8. General scheme for the fucosylation inhibition on phosphate-induced calcification on VSMCs. Hyperphosphatemia upregulates core fucosylation in VSMCs, promoting the fucosyltransferase Fut8 to fucosylate the extracellular domains of TGF-␤ receptors, activate TGF␤/Smad2/3 signaling, and subsequently inducing VSMC calcification, upregulating the osteogenic differentiation maker protein Cbf␣1, and decreasing the expression of ␣-Sma. Fut8 siRNA significantly diminished VSMC calcification and Cbf␣1 expression in the model of high-phosphate-induced VSMC calcification via abrogating core fucosylation of the TGF-␤ receptors. Collectively, this evidence suggests core fucosylation plays a major role in VSMC calcification and appropriate blockade of core fucosylation may be a potential therapeutic strategy for treating VC in end-stage renal disease.

versely, blocking core fucosylation by knocking down Fut8 using a siRNA markedly abrogated core fucosylation of the TGF-␤ receptors and attenuated VC after hyperphosphatemia (Fig. 8). While our experimental conditions were not suitable for assessing the effect of fucosylation on TGF-␤ receptors and ligand binding occurs via direct or indirect mechanisms, this study provides a basis for further investigations to elucidate the mechanisms of this process. Likewise, further investigations are required to gain additional mechanistic insights into core fucosylation in order to prevent VC. We also recognize other glycoprotein receptors relevant to VC may also be fucosylated, such as type-2 BMP receptors, a type of N-linked glycoprotein. Moreover, the effects of the Fut8 siRNA may not be specific to the TGF-␤ receptor; therefore, we plan to investigate whether Fut8 also modifies other target receptors in future studies. 5. Conclusions This study provides the first data to show that inhibition of core fucosylation effectively suppresses VC in CKD. These results suggest the ability to target post-translational core fucosylation modifications of key proteins may represent an attractive treatment for VC in patients with CKD. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This research was supported by a grant (201502023) from the Special Scientific Research Fund of Public Welfare Profession of China; a grant (2014225018) from the Renal Translational Medicine Center of Liaoning Province. We thank Department of Central Laboratory (The First Affiliated Hospital of Dalian Medical University, Dalian, China) for reagents and initial guidance on the project. References Adeney, K.L., Siscovick, D.S., Ix, J.H., Seliger, S.L., Shlipak, M.G., Jenny, N.S., Kestenbaum, B.R., 2009. Association of serum phosphate with vascular and valvular calcification in moderate CKD. J. Am. Soc. Nephrol. 20, 381–387.

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