Stanniocalcin 2 is associated with ectopic calcification in α-klotho mutant mice and inhibits hyperphosphatemia-induced calcification in aortic vascular smooth muscle cells

Bone 50 (2012) 998–1005

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Stanniocalcin 2 is associated with ectopic calcification in α-klotho mutant mice and inhibits hyperphosphatemia-induced calcification in aortic vascular smooth muscle cells Yuichiro Takei a, 1, 2, Hironori Yamamoto a,⁎, 1, Tadatoshi Sato b, Ayako Otani a, Mina Kozai a, Masashi Masuda a, 3, Yutaka Taketani a, Kazusa Muto-Sato a, 4, Beate Lanske b, Eiji Takeda a a b

Department of Clinical Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Kuramoto-cho 3, Tokushima 770-8503, Japan Department of Developmental Biology, Harvard School of Dental Medicine, Boston, MA 02115, USA

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Article history: Received 7 September 2011 Revised 4 January 2012 Accepted 11 January 2012 Available online 21 January 2012 Edited by: Toshio Matsumoto Keywords: Stanniocalcin 2 (STC2) α-Klotho mutant mice (kl/kl) Fgf-23 Ectopic calcification Hyperphosphatemia

a b s t r a c t Ectopic calcification of soft tissues can have severe clinical consequences especially when localized to vital organs such as heart, arteries and kidneys. Mammalian stanniocalcin (STC) 1 and 2 are glycoprotein hormones identified as calcium/phosphate-regulating hormones. The mRNA of STCs is upregulated in the kidney of α-klotho mutant (kl/kl) mice, which have hypercalcemia, hyperphosphatemia and hypervitaminosis D and exhibit a short life span, osteopenia and ectopic calcification. In the present study, we investigated the distribution and localization of STCs in kl/kl mice. Quantitative RT-PCR revealed that renal mRNA expression of STC2 was increased in both kl/kl mice and fibroblast growth factor 23 (Fgf23)-null mice compared with wild type mice. Interestingly, STC2 protein was focally localized with the calcified lesions of renal arterioles, renal tubular cells, heart and aorta in kl/kl mice. In vitro analysis of rat aortic vascular smooth muscle (A-10) cells showed that inorganic phosphate (Pi) stimulation significantly increased STC2 mRNA levels as well as that of osteocalcin, osteopontin and the type III sodium-dependent phosphate co-transporter (PiT-1), and induced STC2 secretion. Interestingly, the knockdown with a small interfering RNA or the over-expression of STC2 showed acceleration and inhibition of Pi-induced calcification in A-10 cells, respectively. These results suggest that the up-regulation of STC2 gene expression resulting from abnormal α-klotho-Fgf23 signaling may contribute to limitation of ectopic calcification and thus STC2 represents a novel target gene for cardio-renal syndrome. © 2012 Elsevier Inc. All rights reserved.

Introduction Patients with chronic kidney disease and osteoporosis and as well as the elderly people are at greater risk of heterotopic ossification. The pathogenesis of vascular mineralization involves both passive and active processes [1,2]. Passive mineralization is a deposition of supersaturated mineral on the region of cellular damage and degeneration. The active mechanism of calcification has many similarities with ossification via expression of osteogenic differentiation markers, such as Runx2, osteocalcin (OCN) and osteopontin (OPN). Recently, in vivo and in

⁎ Corresponding author at: Department of Clinical Nutrition, Institute of Health Biosciences, University of Tokushima, Kuramoto-cho 3-18-15, Tokushima City, 770-8503, Japan. E-mail address: [email protected] (H. Yamamoto). 1 Authors contributed equally to this work. 2 Present address: Aab Cardiovascular Research Institute (CVRI), University of Rochester, Rochester, NY 14642, USA. 3 Present address: Dept. of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Denver, CO 80045, USA. 4 Present address: Dept. of Developmental Biology, Harvard School of Dental Medicine, Boston, MA 02115, USA. 8756-3282/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2012.01.006

vitro studies have demonstrated that hyperphosphatemia is a crucial factor for vascular calcification [3,4]. Elevated inorganic phosphate (Pi) leads to transformation of vascular smooth muscle cells (VSMC) into osteoblast-like cells by mediating upregulation of osteogenic markers and initiation of extracellular matrix calcification [4–6]. Meanwhile, it has also been reported that Pi-induced calcification is caused by apoptosis of VSMC [7], although vesicles derived from apoptotic VSMC may perform similar functions to bone-specific matrix vesicles [8]. The serum level of Pi is regulated by dietary phosphorus, 1,25dihydroxyvitamin D3 [1,25(OH)2D3], parathyroid hormone (PTH), fibroblast growth factor-23 (Fgf23), and other factors [9,10]. Recently, it has been reported that α-klotho acts as an essential cofactor of Fgf23 [11,12]. α-Klotho mutant (kl/kl) mice exhibited hypercalcemia, hyperphosphatemia and hypervitaminosis D [13]. After weaning, kl/kl mice display various phenotypes resembling human aging syndromes, including short life span, poor growth, cognitive deficit, osteopenia and ectopic calcification of various soft tissues, especially the arteries and kidneys [13,14]. Indeed, Fgf23-null mice exhibit the similar phenotype to kl/kl mice [14,15]. Various age-related disorders shown in kl/kl mice and Fgf23-null mice are ameliorated by feeding a low-Pi diet [16,17]. Fgf-23 and type IIa sodium-dependent phosphate cotransporter

Y. Takei et al. / Bone 50 (2012) 998–1005

Real-time RT-PCR and western blot analysis Total RNA was extracted with RNA iso Plus reagent (Takara Bio Inc., Shiga, Japan) and then dissolved in RNase-free water. First-strand cDNA was synthesized from 2.5 μg of total RNA using a first-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Two microliters of the cDNA was then used for quantitative PCR, with 20 to 30 cycles of amplification, and the PCR products were then separated by electrophoresis using 2% agarose gels. The primer sequences used for PCR amplification are described in Table 1. Real-time quantitative RT-PCR analysis was performed using the LightCycler™ (Roche Diagnostics, Tokyo, Japan) as previously described [26]. For western blot analysis, the conditioned medium from A-10 cells (see below) was centrifuged at 1500 ×g for 10 min at 4 °C. The

A 5

WT

4

kl/kl

3 2 1 0

Name

GenBank accession no.

Primer sequence

Mouse STC1

NM_009285

Mouse STC2

NM_011491

Mouse β-actin

NM_007393

Rat STC1

NM_031123

Rat STC2

NM_022230

Rat PiT1

NM_031148

Rat OCN

NM_013414

Rat OPN

NM_012881

Rat β-actin

NM_031144

5′-AAGCGCAACCCGGAAGCCATCACTG-3′ 5′-GGCTGTCTCTGATTGTACTGACCGTG-3′ 5′-AGCCCAGGAGAACGTCGGTGTGATTG-3′ 5′-CAGCAGCAGGTTCACAAGGTCCACATAG-3′ 5′-CTGACCCTGAAGTACCCCATTGAACA-3′ 5′-CTGGGGTGTTGAAGGTCTCAAACATG-3′ 5′-CCAAGGTCTTCCTTGCCATT-3′ 5′-TGCTGCAAACATTGAGCTTG-3′ 5′-TACCAGTTGCAGAGGGAATG-3′ 5′-TTACAAGGTCCACGTAGGGT-3′ 5′-CCCATCAGCACAACACATTG-3′ 5′-TAGGGACGGTGACAAACCAG-3′ 5′-CTGCATTCTGCCTCTCTGAC-3′ 5′-CCGGAGTCTATTCACCACCT-3′ 5′-GATAGCTTGGCTTACGGACTG-3′ 5′-CTTGATAGCCTCATCGGACTC-3′ 5′-CTAAGGCCAACCGTGAAAAGA-3′ 5′-TGGTACGACCAGAGGCATACA-3′

25 20 15 10 5

*

* en

is

le

ia

st

sp

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ar

ur m

lv ca

um

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fe

t

ey

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B Table 1 Sequence of oligonucleotide primers for quantitative RT-PCR analysis.

*

30

Relative mRNA levels (FGF23-/-/WT)

Heterozygous α-klotho mutant mice were purchased from CLEA Japan (Osaka, Japan) and mated to produce wild-type (WT) mice and homozygous kl/kl mice. Mice were maintained with 12 h:12 h light–dark cycles with free access to regular mouse chow and water under pathogen-free conditions. Mice genotypes were confirmed using genomic DNA extracted from tail clippings and amplified by PCR using specific primers: 5′-TGGAGATTGGAAGTGGACG-3′, 5′CAAGGACCAGTTCATCATCG-3′ and 5′-TTAAGGACTCCTGCATCTGC-3′ [28]. Mice were weaned at 3 week (wk) of age and were given free access to water and regular mouse chow. Male and female

STC2 mRNA levels (vs. WT brain)

35

Experimental animals

en

Materials and methods

heterozygous-Fgf23 +/− mice were bred to attain WT and Fgf23 −/− at 6 wk. Routine PCR was used to identify the genotypes of various mice as described previously [29]. The breeding and handling of αklotho mutant mice and Fgf23 −/− mice in experiments were approved by the Animal Experimentation Committee of the University of Tokushima and the institutional animal care and use committee at the Harvard Medical School, respectively.

STC1 mRNA levels (vs. WT brain)

(NaPi-2a) double-knockout mice, which do not have hyperphosphatemia, are viable and rescued from some diseases [18]. Together, these data suggest that high serum levels of Pi play a key role in these disorders. The stanniocalcin (STC) family of glycoproteins consists of STC1 and STC2 which have 35% conserved sequence identity [19]. STC was first identified from the corpuscles of Stannius in bony fish, as a regulator of mineral homeostasis, having effects on calcium influx [20,21]. Like fish STC, it is thought that mammalian STC1 and STC2 exhibit similar roles in regulating calcium (Ca) and Pi homeostasis [19]. Indeed, it has been shown that STC1 stimulates phosphate reabsorption in the small intestine and proximal tubules of the kidney [22,23], and renal STC1 mRNA expression is increased by 1,25(OH)2D3 in rat [24]. We have previously characterized the human STC2 gene and shown that human STC2 decreases Pi uptake activity and human NaPi-2a gene promoter activity in opossum renal proximal tubular cell line (OK cells). We also observed that the STC2 gene is widely expressed in mice; however, its expression in hypophosphatemic (Hyp) mice, which is a model of human X-linked hypophosphatemic vitamin D-resistant rickets (XLH), was down-regulated in many organs [25]. Our more recent work identified the opossum STC2 gene and observed that its expression and secretion in OK cells were positively and negatively controlled by 1,25(OH)2D3 and PTH [26]. Interestingly, Yahata et al. indicated the up-regulation of STC1 and STC2 gene expressions in the kidney of kl/kl mice [27]. However, the role of STCs in kl/kl mice has been unclear. In the present study, to clarify the role of increased STC2 in kl/kl mice, we analyzed the gene expression and localization of STC2. We found that STC2 localized to ectopic calcification sites in kidney, heart and aorta of kl/kl mice. Furthermore, we investigated the role of STC2 in ectopic calcification by using rat aortic smooth muscle cells.

999

15

*

WT FGF23 -/-

10

5

0 STC1

STC2

Fig. 1. The mRNA expressions of STC1 and STC2 in various tissues of kl/kl mice and kidney of Fgf23−/− mice.The mRNA levels of STC1 and STC2 were determined by real-time quantitative RT-PCR analysis using (A) brain, heart, kidney, duodenum, muscle, femur, calvaria, testis and spleen from 6 to 7-wk-old WT and kl/kl mice, and (B) kidney of 6-wk-old Fgf23−/− mice. Results were normalized to the mRNA level of β-actin. The data in A are represented as mean fold increases ± SD (n = 3–5) above the levels of WT mice brain, and the data in B are represented as mean fold increases ± SD (n = 3–5) above the levels of WT mice *p b 0.05 compared with WT mice.

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supernatant was concentrated by acetone precipitation. Whole kidney was homogenized in lysis buffer containing 1% Triton X-100, 1% Na deoxycholate, 0.1% SDS, 10 mM Tris · HCl, pH 8.0, 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, and 5 mM iodoacetamide. Proteins were electrophoresed on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. After blocking, membranes were incubated with affinity-purified anti-STC2 antibody [26] or anti-His antibody (MBL: Medical and Biological Laboratories, Nagoya, Japan) and horseradish peroxidase (HRP)-labeled anti-rabbit IgG antibody, and then, the membranes were developed using an ECL plus western blotting system (GE Healthcare, Buckinghamshire, UK).

Immunohistochemical analysis The harvested tissues were fixed with 4% PFA in PBS, and then dehydrated in ascending ethanol series, embedded in paraffin and sliced at 2 μm thickness. After blocking with 0.8% BSA in PBS for 1 h, the tissue sections were incubated with affinity-purified anti-STC2 rabbit antibody, and subsequently immersed in 3,3′-diaminobenzidine (DAB) solution. Then, the sections were counterstained by hematoxylin. The specificity of anit-STC2 antibody was confirmed by using antigenic peptide corresponding to a 12-amino acid residues (291–302: EDEQSEYSDIRR) of STC2 protein [26].

Von Kossa staining Von Kossa stain was performed to detect ectopic calcification. The tissue sections were treated with 5% silver nitrate solution under ultraviolet light for 1 h. The sections were then washed with distilled water and immersed in 5% hypo solution (sodium thiosulfate). Sections were counterstained with hematoxylin–eosin (HE). In situ hybridization A fragment of mouse STC2 cDNA (1–862) was subcloned into the pGEM-T easy plasmid (Promega), and complementary RNA probes were prepared using a DIG RNA Labeling kit (Boehringer Mannheim Biochemica, Mannheim, Germany) with RNA polymerases (T7 RNA polymerase for the antisense probe and T3 RNA polymerase for the sense probe) according to the manufacturer's instructions. Hybridization was performed at 50 °C for 16 h, and signals were visualized with a Dako Gen Point® kit using the horseradish peroxidase-conjugated anti-digoxigenin antibody. Cell culture and treatment Rat aortic smooth muscle cell-line, A-10 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin.

kl/kl

B

kl/kl

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kl/kl

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WT

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A b c

Fig. 2. Focal localization of STC2 in kidney of kl/kl mice.Kidney sections from WT and kl/kl male mice at 6 wk were incubated with affinity-purified STC2 antibody and stained by DAB staining (brown stain; arrows), followed by counterstaining with hematoxylin (blue stain). (A) STC2 localization in kidney of kl/kl mice (original magnification, × 40). Arrowed areas (b and c) are magnified in (B) and (C), respectively. STC2 accumulation in tubules (B) and arterioles (C) of renal cortex of kl/kl mice (original magnification, × 200). (D) STC2 in renal tubules of WT mice (original magnification, × 200). (E) The specificity of anit-STC2 antibody in serial sections from kidney of WT mice was confirmed by using of STC2 specific antigenic peptide (5 μg/section).

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Subcultures were obtained every three days by using 0.25% trypsin in Ca2 +- and Mg2 +-free Eagle's salt solution that contained 0.02% ethylenediaminetetraacetic acid. The cultures were maintained at 37 °C in humidified atmosphere of 5% CO2 and 95% air. For Pi-induced calcification, the cells were seeded at 25,000 cells/cm2 on collagen-coated plates. Pi (a mixed solution of Na2HPO4 and NaH2PO4 whose pH was adjusted to 7.4) was added to 10% FBS supplemented DMEM to final Pi concentrations of 2 to 4 mM (termed as calcification medium). After reaching 100% confluency (day 3), A-10 cells were cultured in calcification medium or control medium (Pi concentrations of 0.9 mM) for 3 days. Ca depositions of the cultures were visualized by von Kossa staining and determined by using the Calcium-E test (Wako, Osaka, Japan), as will hereinafter be described in detail. Passage number tested in all experiments was from 33 to 38. Construction of human STC2 (hSTC2) expression vector and short interfering RNA (siRNA) targeting rat STC2 Full-length human STC2 cDNA was cloned from human renal cancer cell line (ACHN cells) by RT-PCR, using the forward primer 5′GCTCTAGAACCATGTGTG CCGAGCGGCTGG-3′ and the reverse primer 5′-GCTCTAGACTCCGGATATCAGAATACTCAG-3′. The PCR product was cloned into the Xba I site of pcDNA™ 3.1/myc-His(−) B vector (Invitrogen), such that the myc-epitope tag and 6× his tag were in frame for subsequent translation. The siRNA sequences for rat STC2, were as follows: No, 1 STC2 siRNA (forward, 5′-UACAAGGUCCACGUAGGGUUCAUGC-3′; reverse, 5′-GCAUGAACCCUACGUGGACCUUGUA-3′), No, 2 STC2 siRNA (forward, 5′-UUGUGCAGAAACGUCAUGCAAAUCC-3′; reverse, 5′-GGAUUUGCAUGAC GUUUCUGCACAA-3′), No, 3 STC2 siRNA (forward, 5′-UGAUGAAUGACUUGC CCUGGGCAUC-3′; reverse, 5′-GAUGCCCAGGGCAAGUCAUUC AUCA-3′). In RNA interference experiments, we used a mix of these three annealed double-stranded oligonucleotides. AllStars Negative Control siRNA (Qiagen, Tokyo, Japan) was used as a negative control. When A-10 cells reached 80–90% confluency, the cells were washed and exchanged in DMEM containing 5% FBS without antibiotics.

A

1001

Transfection of the expression vector or siRNA duplexes into cells was achieved using Lipofectamine 2000 reagent (Invitrogen). After 4 h of transfection, FBS was added to the cells to achieve a final concentration of 10% in DMEM and incubated for 48 h. Quantification of Ca deposition Cells were decalcified with 0.6 N HCl for 24 h. The Ca contents of HCl supernatants were colorimetrically determined by the Calcium-E test. After decalcification, the cells were washed three times with PBS and solubilized with 0.1 N NaOH/0.1% SDS. The protein content was measured with a Bio-Rad protein assay kit (Bio-Rad Laboratories, CA, USA). Statistical analysis Data are expressed as means ± SD. Statistical significance between groups was determined using ANOVA followed by post hoc testing using Fisher's protected least significant difference (PLSD). A p value b0.05 was considered to be significant. Results The mRNA expressions of STC1 and STC2 in various tissues of kl/kl mice and in kidney of Fgf23 −/− mice Previous reports show that STC1 and STC2 mRNA are expressed in various tissues, e.g. kidney, heart, skeletal muscle, neurocyte and reproductive organs [30,31], and renal STC mRNA expression is up-regulated in kl/kl mice [27]. However, it is not clear whether the mRNA expressions of STC1 and STC2 were also changed in extra-renal tissues of kl/kl mice and in kidney of Fgf23 −/− mice compared with WT mice. As shown in Fig. 1A, the distributions of STC1 and STC2 mRNA were notable in kidney of both WT and kl/kl mice. Although renal STC1 mRNA expression of kl/kl mice tended to be up-regulated (p value b0.06) compared with WT mice, the levels of renal STC2 mRNA were significantly (220%) increased in kidney of kl/kl mice,

B

C

D

Fig. 3. Co-localization of STC2 with renal calcified area in kl/kl mice.Serial sections from kidney of kl/kl mice were analyzed by von Kossa staining and immunostaining of STC2, respectively. Calcification of renal arterioles (A; original magnification, × 100) and tubular cells (C; original magnification, × 200) is indicated by arrows. The sections were counterstained by hematoxylin–eosin. (B, D) STC2 co-localization with renal calcified area was detected on serial sections (brown stain; arrows).

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consistent with a previous report [27]. In addition, although STC2 mRNA levels of kl/kl mice did not show significant changes in extra-renal tissues including brain, heart, duodenum, skeletal muscle and spleen, STC2 mRNA expression was decreased in the femur and calvaria of kl/kl mice by 30% and 40%, respectively. Furthermore, we analyzed the renal STC2 mRNA expression in Fgf23−/− mice because of the strikingly similar biochemical and morphological phenotypes of kl/kl mice and Fgf23−/− mice [14,15]. Importantly, we found that up-regulation of renal mRNA expression of the STC2 gene, not the STC1 gene was also observed in Fgf23−/− mice (Fig. 1B). These results suggest that renal STC2 mRNA expression is increased as a result of abnormal Fgf23-α-klotho signaling.

kidney of kl/kl mice [13,15,16], we next examined the association with renal calcified lesions in kl/kl mice. Renal calcification was particularly shown in arterioles of cortical layer, and frequently detected in tubular cells (Figs. 3A and C). We then assessed calcification sites and localization sites of STC2 by using these serial sections. Interestingly, focal STC2 expressions co-localized with calcified areas in arterioles and tubules (Figs. 3A–D). On the other hand, we observed that STC1 localization did not alter between WT and kl/kl mouse kidney and did not correlate with calcified lesions, e.g. arterioles of renal cortex (data not shown). Demonstration of STC2 mRNA in kidney of WT and kl/kl mice by in situ hybridization

STC2 protein localization and ectopic calcification in kl/kl mice Next, we assessed localization of STC2 protein in kl/kl mouse kidney. Immunohistochemical analysis demonstrated that STC2 was localized in tubular cells of WT mice and the specificity of anit-STC2 antibody was confirmed by using of antigenic peptide (Figs. 2D and E). On the other hand, kl/kl mice exhibited the focal expression of STC2 in renal cortex (Fig. 2A). In particular, STC2 was strongly expressed in distinct areas part of arterioles and tubular cells (Figs. 2B and C). Because the ectopic calcification is observed in the

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In order to elucidate the STC2 mRNA distribution in kidney of kl/kl mice, in situ hybridization analysis was performed. As shown in Figs. 4A and B, STC2 mRNA staining was mainly observed in proximal and distal tubular cells, but not expressed in glomeruli of both mice, and its expression was highly induced in kl/kl mice compared with WT mice. In contrast, no staining was seen when a sense probe of STC2 was used as the negative control (Fig. 4E). Intriguingly, its mRNA distribution was quite different to the focal localization patterns of STC2 protein in kidney of kl/kl mice (Figs. 4C and D).

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100μm

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100μm Fig. 4. Distribution of STC2 mRNA and protein in the kidney of WT and kl/kl mice.The kidneys were obtained from WT and kl/kl male mice at the 6 wk of age after perfusion fixation with 4% paraformaldehyde, and then embedded in paraffin. The tissue sections were cut at a 2 μm thickness. The antisense RNA probe of STC2 was hybridized with the kidney sections of WT (A) and kl/kl (B, D) mice by in situ hybridization. (C) The kidney sections of kl/kl mice were incubated with affinity-purified STC2 antibody and stained by DAB staining (brown stain; arrows), followed by counterstaining with hematoxylin (blue stain). (E) A sense RNA STC2 probe was used as negative control.

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Localization of STC2 in the calcified lesions in heart and aorta of kl/kl mice It was previously documented that kl/kl mice exhibit cardiac- and vascular calcification as well as renal calcification [13]. Therefore, we next analyzed the STC2 localization and calcified lesions in cardiacand vascular tissues of kl/kl mice. Interestingly, as shown in Figs. 5A–D, STC2 proteins were also detected in calcified sites of heart and aorta as well as kidney.

Contribution of STC2 on Pi-induced calcification in aortic vascular smooth muscle cells To understand the contribution to STC2 on hyperphosphatemiainduced vascular calcification, we next assessed whether STC2 expression was altered by Pi stimulation. It has been previously reported that the calcification of aortic smooth muscle cells can be induced by an elevated extracellular Pi concentration [38,40]. We first confirmed that treatment of rat aortic smooth muscle (A-10) cells with 2.9 to 4.9 mM of Pi for 3 to 6 days promoted mineralization, as detected using von Kossa staining or alizarin red S staining (data not shown). Fig. 6A shows the significant increase of STC2 mRNA levels by Pi stimulation after 6 days, as well as the up-regulation of osteoblast-related genes including osteocalcin (OCN), osteopontin (OPN) and type III sodium-dependent Pi cotransporter (PiT1) in a time-dependent manner. Interestingly, 12 h treatment with 2 mM of Pi strongly induced STC2 protein secretion (Fig. 6B). To address the role of STC2 on ectopic calcification, A-10 cells with either knockdown or over-expression of STC2 were established. The endogenous STC2 mRNA level was significantly suppressed (5%) in A-10 cells using RNAi (Fig. 6C). As shown in Fig. 6D, after Pi stimulation for 3 days, STC2 RNAi-treated (siSTC2) cells showed significantly more mineral deposition than the negative control RNAi-treated (siControl) cells. His-tagged human STC2 was transiently overexpressed in A-10 cells (Fig. 6E). In contrast to knockdown, the over-expression of STC2 gene in A-10 cells significantly prevented Pi-induced Ca deposition (Fig. 6F).

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Discussion In this study, we revealed the localization of STC2 with ectopic calcification in kidney, heart and aorta of kl/kl mice and showed that renal STC2 mRNA transcripts were significantly increased in kl/kl mice compared with WT mice. It is known that kl/kl mice exhibit hypervitaminosis D and low serum PTH levels [32], and, we have previously reported that STC2 mRNA expression was increased by 1,25(OH)2D3 but decreased by PTH in renal tubules [26]. Hence, induction of renal STC2 mRNA in kl/kl mice might be regulated by these factors. And, the α-klotho gene high-expresses especially in kidney [13]. Importantly, STC2 mRNA expression was also significantly increased in the kidney of Fgf23-null mice (Fig. 1B). These data suggest that renal tissue-specific up-regulation of STC2 mRNA is associated with a direct and/or indirect effect of Fgf23 signaling, and that the Fgf23-α-klotho axis is a key factor in regulatory mechanism governing STC2. Interestingly, kl/kl mice displayed a decrease in STC2 mRNA levels in calvaria and femur. It has been reported that the STC1 mRNA expression in mouse bone and its developmental expression pattern are similar to that of kidney, and STC1 stimulates osteoblast differentiation in rat calvaria cells [41,42], however, STC2 exhibited potent growth-suppressive properties in transgenic mice independently of growth hormone and IGFs [43]. Because kl/kl mice exhibit osteoporosis and abnormal bone formation [13], a detailed examination of STC2 expression in bone may provide important insight into the bone abnormalities in kl/kl mice. We next histologically analyzed the localization of STC2 in kl/kl mouse kidney. Surprisingly, STC2 was focally localized in renal arterioles and to areas of tubular cells that were calcified in kl/kl mice (Figs. 3A–D). Moreover, focal STC2 localization was also observed in areas of ectopic calcification in cardiac and vascular tissues of kl/kl mice (Fig. 5). However, the mRNA expression of STC2 gene is not enhanced in the heart of kl/kl mice while STC2 protein localized in the region of ectopic calcification (Figs. 1A and 5B). We noticed that the cardiac calcification was not severe compared with that of kidney in kl/kl mice. Therefore, there is a possibility that any difference in heart STC2 mRNA levels between the kl/kl mice and WT mice was not observed. As another possibility, one of the intriguing facets of

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Fig. 5. Cardiac and vascular calcification and STC2 localization in kl/kl mice.Serial sections from heart and aorta of kl/kl mice were analyzed by von Kossa staining and immunostaining of STC2, respectively. (A) Calcification of cardiac muscle and (B) cardiac STC2 localization (original magnification, × 400) in serial sections of the heart and aorta from 6wk-old kl/kl mice. (C) Aortic calcification and (D) localization of STC2 (original magnification, × 100).

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Relative mRNA levels

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Ca content (μg/mg protein)

Relative mRNA levels

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STC2 (His) β-actin

Ca content (μg/mg protein)

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Fig. 6. Up-regulation of STC2 expression and its contribution on Pi-induced calcification in aorta smooth muscle cells.(A) A-10 cells were treated with high Pi medium (4.9 mM of Pi) for different time periods. The mRNA expression levels of STC2, PiT-1, OCN and OPN were determined by real-time RT-PCR analysis. *p b 0.05 compared with 0 day. (B) At 12 h after treatment with 2.9 mM of Pi, STC2 protein levels were measured by western blot analysis. (C) Endogenous STC2 mRNA detected by real-time quantitative PCR analysis 48 h after transfection with control or STC2 siRNA. *p b 0.05 compared with control. (E) STC2-His tagged protein expressions 48 h after transfection were detected by western blot analysis. The transfected A-10 cells were treated with high Pi medium (4.9 mM of Pi) for 3 days. (D, F) Ca depositions were visualized by von Kossa staining and quantified by the Calcium-E test. *p b 0.05 compared with control or Mock.

STC physiology is considered as the role of the circulating hormone. Indeed, it has been reported that high affinity receptor of STC1 was identified on red blood cells from all species [33]. McCudden et al. also identified recombinant STC1 protein binding sites, suggesting the presence of a putative STC1 receptor in plasma membranes and mitochondria of nephron cells and liver hepatocytes, but STC2 did not displace the binding of STC1 [34]. These reports suggest that the high affinity receptor of STC2 is different from that of STC1. Future studies are needed to identify a STC2 receptor. Hyperphosphatemia causes ectopic calcification, particularly vascular calcification, in vivo and in vitro [3,4]. Indeed, it was reported that renal calcification of kl/kl mice is ameliorated by restriction of dietary Pi [35]. RT-PCR analysis using aortic smooth muscle A-10 cells showed that the mRNA levels of the STC2 gene were significantly increased by Pi treatment in a time-dependent manner (Fig. 6A). More interestingly, we found that the STC2 secretion is quite strongly enhanced by Pi treatment while mRNA increased after 6 days of treatment (Fig. 6B). These data suggest that the STC2 gene expression is up-regulated by extracellular Pi on transcriptional and post-translational levels and could help to explain the regional STC2 expression in heart or aorta of kl/kl mice. We also demonstrate that STC2 inhibits Pi-induced calcification

(Figs. 6D and F). Our previous report documented that STC2 inhibits the Pi uptake and suppresses the promoter activity of NaPi-2a in OK cells [25]. Another Pi transporter, PiT-1, is predominantly expressed in VSMC and essential for Pi-induced calcification [4,39]. However, the Pi uptake activity in A-10 cells was not changed by knockdown of STC2 (data not shown). These results suggest that STC2 had no effect on Pi uptake in A-10 cells and so attenuated calcification via an alternative mechanism. We have previously reported that elevated extracellular Pi levels can promote reactive oxygen species, a marker of oxidative stress, in endothelial cells [36,37]. Furthermore, it has been reported that hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, statins protect against Pi-induced calcification of human aortic smooth muscle cells by inhibiting apoptosis [7]. The gene expression and secretion of STC2 in neurocyte were induced by oxidative stress and hypoxia, and STC2 protected against cell damage [38]. Therefore, the upregulation of STC2 expression in ectopic calcification area of kl/kl mice and/or in A-10 cells stimulated by Pi is proposed to protect against aggravation of calcified lesion. It still remains to be determined whether STC2 up-regulation could regulate the ectopic calcifications of kl/kl and Fgf23-null mice. Further studies are needed to determine the mechanisms by which STC2 inhibits ectopic calcification.

Y. Takei et al. / Bone 50 (2012) 998–1005

In conclusion, we firstly revealed that STC2 is associated with ectopic calcification in kidney, heart and aorta of kl/kl mice, and inhibits Pi-induced calcification in aortic vascular smooth muscle cells. The up-regulation of STC2 gene expression by abnormal Fgf23-α-klotho signaling may represent a novel target in the treatment of vascular calcification and cardio-renal syndrome.

Acknowledgments We thank Dr. K-I. Miyamoto (University of Tokushima, Tokushima, Japan) and Dr. Y. Yoshiko (University of Hiroshima, Hiroshima, Japan) for helpful discussions and comments. This work was supported by Grants 16790526 (to H. Yamamoto) and 13470013 (to E. Takeda) from the Ministry of Education, Science, Sports and Culture of Japan and the Human Nutritional Science on Stress Control 21st Century Center of Excellence Program (COE), and the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References [1] Moe SM, Chen NX. Pathophysiology of vascular calcification in chronic kidney disease. Circ Res 2004;95:560–7. [2] Hofbauer LC, Brueck CC, Shanahan CM, Schoppet M, Dobnig H. Vascular calcification and osteoporosis—from clinical observation towards molecular understanding. Osteoporos Int 2007;18:251–9. [3] El-Abbadi MM, Pai AS, Leaf EM, Yang HY, Bartley BA, Quan KK, Ingalls CM, Liao HW, Giachelli CM. Phosphate feeding induces arterial medial calcification in uremic mice: role of serum phosphorus, fibroblast growth factor-23, and osteopontin. Kidney Int 2009;75:1297–307. [4] Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res 2000;87:E10–7. [5] Chen NX, O'Neill KD, Duan D, Moe SM. Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells. Kidney Int 2002;62: 1724–31. [6] Giachelli CM, Jono S, Shioi A, Nishizawa Y, Mori K, Morii H. Vascular calcification and inorganic phosphate. Am J Kidney Dis 2001;38:S34–7. [7] Son BK, Kozaki K, Iijima K, Eto M, Kojima T, Ota H, Senda Y, Maemura K, Nakano T, Akishita M, Ouchi Y. Statins protect human aortic smooth muscle cells from inorganic phosphate-induced calcification by restoring Gas6-Axl survival pathway. Circ Res 2006;98:1024–31. [8] Proudfoot D, Skepper JN, Hegyi L, Bennett MR, Shanahan CM, Weissberg PL. Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies. Circ Res 2000;87:1055–62. [9] Nemere I. The ins and outs of phosphate homeostasis. Kidney Int 2007;72:140–2. [10] Shimada T, Kakitani M, Yamazaki Y, Hasegawa H, Takeuchi Y, Fujita T, Fukumoto S, Tomizuka K, Yamashita T. Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J Clin Invest 2004;113:561–8. [11] Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, Fujita T, Fukumoto S, Yamashita T. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 2006;444:770–4. [12] Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, Rosenblatt KP, Baum MG, Schiavi S, Hu MC, Moe OW, Kuro-o M. Regulation of fibroblast growth factor-23 signaling by klotho. J Biol Chem 2006;281:6120–3. [13] Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45–51. [14] Nagai T, Yamada K, Kim HC, Kim YS, Noda Y, Imura A, Nabeshima Y, Nabeshima T. Cognition impairment in the genetic model of aging klotho gene mutant mice: a role of oxidative stress. FASEB J 2003;17:50–2. [15] Razzaque MS, Lanske B. The emerging role of the fibroblast growth factor-23-klotho axis in renal regulation of phosphate homeostasis. J Endocrinol 2007;194:1–10. [16] Tsujikawa H, Kurotaki Y, Fujimori T, Fukuda K, Nabeshima Y. Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system. Mol Endocrinol 2003;17:2393–403.

1005

[17] Stubbs JR, Liu S, Tang W, Zhou J, Wang Y, Yao X, Quarles LD. Role of hyperphosphatemia and 1,25-dihydroxyvitamin D in vascular calcification and mortality in fibroblastic growth factor 23 null mice. J Am Soc Nephrol 2007;18:2116–24. [18] Sitara D, Kim S, Razzaque MS, Bergwitz C, Taguchi T, Schüler C, Erben RG, Lanske B. Genetic evidence of serum phosphate-independent functions of FGF-23 on bone. PLoS Genet 2008;4:e1000154. [19] Yoshiko Y, Aubin JE. Stanniocalcin 1 as a pleiotropic factor in mammals. Peptides 2004;25:1663–9. [20] Wagner GF, Hampong M, Park CM, Copp DH. Purification, characterization, and bioassay of teleocalcin, a glycoprotein from salmon corpuscles of Stannius. Gen Comp Endocrinol 1986;63:481–91. [21] Lafeber FP, Hanssen RG, Choy YM, Flik G, Herrmann-Erlee MP, Pang PK, Bonga SE. Identification of hypocalcin (teleocalcin) isolated from trout Stannius corpuscles. Gen Comp Endocrinol 1988;69:19–30. [22] Madsen KL, Tavernini MM, Yachimec C, Mendrick DL, Alfonso PJ, Buergin M, Olsen HS, Antonaccio MJ, Thomson AB, Fedorak RN. Stanniocalcin: a novel protein regulating calcium and phosphate transport across mammalian intestine. Am J Physiol 1998;274:G96–G102. [23] Wagner GF, Vozzolo BL, Jaworski E, Haddad M, Kline RL, Olsen HS, Rosen CA, Davidson MB, Renfro JL. Human stanniocalcin inhibits renal phosphate excretion in the rat. J Bone Miner Res 1997;12:165–71. [24] Honda S, Kashiwagi M, Ookata K, Tojo A, Hirose S. Regulation by 1alpha,25dihydroxyvitamin D(3) of expression of stanniocalcin messages in the rat kidney and ovary. FEBS Lett 1999;459:119–22. [25] Ishibashi K, Miyamoto K, Taketani Y, Morita K, Takeda E, Sasaki S, Imai M. Molecular cloning of a second human stanniocalcin homologue (STC2). Biochem Biophys Res Commun 1998;250:252–8. [26] Takei Y, Yamamoto H, Masuda M, Sato T, Taketani Y, Takeda E. Stanniocalcin 2 is positively and negatively controlled by 1,25(OH)(2)D(3) and PTH in renal proximal tubular cells. J Mol Endocrinol 2009;42:261–8. [27] Yahata K, Mori K, Mukoyama M, Sugawara A, Suganami T, Makino H, Nagae T, Fujinaga Y, Nabeshima Y, Nakao K. Regulation of stanniocalcin 1 and 2 expression in the kidney by klotho gene. Biochem Biophys Res Commun 2003;310:128–34. [28] Segawa H, Yamanaka S, Ohno Y, Onitsuka A, Shiozawa K, Aranami F, Furutani J, Tomoe Y, Ito M, Kuwahata M, Imura A, Nabeshima Y, Miyamoto K. Correlation between hyperphosphatemia and type II Na-Pi cotransporter activity in klotho mice. Am J Physiol Renal Physiol 2007;292:F769–79. [29] Sitara D, Razzaque MS, Hesse M, Yoganathan S, Taguchi T, Erben RG, Jüppner H, Lanske B. Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol 2004;23:421–32. [30] Chang AC, Jellinek DA, Reddel RR. Mammalian stanniocalcins and cancer. Endocr Relat Cancer 2003;10:359–73. [31] Wagner GF, Dimattia GE. The stanniocalcin family of proteins. J Exp Zool A Comp Exp Biol 2006;305:769–80. [32] Yoshida T, Fujimori T, Nabeshima Y. Mediation of unusually high concentrations of 1,25-dihydroxyvitamin D in homozygous klotho mutant mice by increased expression of renal 1alpha-hydroxylase gene. Endocrinology 2002;143:683–9. [33] James K, Seitelbach M, McCudden CR, Wagner GF. Evidence for stanniocalcin binding activity in mammalian blood and glomerular filtrate. Kidney Int 2005;67:477–82. [34] McCudden CR, James KA, Hasilo C, Wagner GF. Characterization of mammalian stanniocalcin receptors. Mitochondrial targeting of ligand and receptor for regulation of cellular metabolism. J Biol Chem 2002;277:45249–58. [35] Morishita K, Shirai A, Kubota M, Katakura Y, Nabeshima Y, Takeshige K, Kamiya T. The progression of aging in klotho mutant mice can be modified by dietary phosphorus and zinc. J Nutr 2001;131:3182–8. [36] Takeda E, Yamamoto H, Nashiki K, Sato T, Arai H, Taketani Y. Inorganic phosphate homeostasis and the role of dietary phosphorus. J Cell Mol Med 2004;8:191–200. [37] Shuto E, Taketani Y, Tanaka R, Harada N, Isshiki M, Sato M, Nashiki K, Amo K, Yamamoto H, Higashi Y, Nakaya Y, Takeda E. Dietary phosphorus acutely impairs endothelial function. J Am Soc Nephrol 2009;20:1504–12. [38] Nishiwaki-Yasuda K, Suzuki A, Kakita A, Sekiguchi S, Asano S, Nishii K, Nagao S, Oiso Y, Itoh M. Vasopressin stimulates Na-dependent phosphate transport and calcification in rat aortic smooth muscle cells. Endocr J 2007;54:103–12. [39] Li X, Yang HY, Giachelli CM. Role of the sodium-dependent phosphate cotransporter, Pit-1, in vascular smooth muscle cell calcification. Circ Res 2006;98:905–12. [40] Villa-Bellosta R, Bogaert YE, Levi M, Sorribas V. Characterization of phosphate transport in rat vascular smooth muscle cells: implications for vascular calcification. Arterioscler Thromb Vasc Biol 2007;27:1030–6. [41] Yoshiko Y, Son A, Maeda S, Igarashi A, Takano S, Hu J, Maeda N. Evidence for stanniocalcin gene expression in mammalian bone. Endocrinology 1999;140: 1869–74. [42] Yoshiko Y, Maeda N, Aubin JE. Stanniocalcin 1 stimulates osteoblast differentiation in rat calvaria cell cultures. Endocrinology 2003;144:4134–43. [43] Gagliardi AD, Kuo EY, Raulic S, Wagner GF, DiMattia GE. Human stanniocalcin-2 exhibits potent growth-suppressive properties in transgenic mice independently of growth hormone and IGFs. Am J Physiol Endocrinol Metab 2005;288:E92–105.