In vascular smooth muscle cells paricalcitol prevents phosphate-induced
Wnt/-catenin activation
Julio M. Martínez-Moreno,
1
Juan R. Muñoz-Castañeda,
1
Carmen Herencia,
1
Addy Montes de Oca,
2
Jose C. Estepa,
2
Rocio Canalejo,
1
Maria E. Rodríguez-Ortiz,
1
Pablo Perez-Martinez,
3
Escolástico Aguilera-Tejero,
2
Antonio Canalejo,
4
Mariano Rodríguez,
1
and Yolanda Almadén
3
1
Servicio de Nefrologia, Red in Ren, Instituto Maimónides de Investigación Biomédica de Córdoba, (IMIBIC) Hospital
Universitario Reina Sofia, Cordoba, Spain;
2
Department of Medicina y Cirugia Animal, Universidad de Cordoba, Cordoba,
Spain;
3
Lipid and Atherosclerosis Unit, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Reina Sofia
University Hospital/University of Cordoba, and Centro de Investigación Biomédica en Red de la Fisiopatología de la
Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III, Cordoba, Spain; and
4
Department of Biologia Ambiental y
Salud Publica, Universidad de Huelva, Huelva, Spain
Submitted 20 December 2011; accepted in final form 2 August 2012
Martínez-Moreno JM, Muñoz-Castañeda JR, Herencia C,
Montes de Oca A, Estepa JC, Canalejo R, Rodríguez-Ortiz ME,
Perez-Martinez P, Aguilera-Tejero E, Canalejo A, Rodríguez M,
Almadén Y. In vascular smooth muscle cells paricalcitol prevents
phosphate-induced Wnt/-catenin activation. Am J Physiol Renal
Physiol 303: F1136 –F1144, 2012. First published August 8, 2012;
doi:10.1152/ajprenal.00684.2011.—The present study investigates the
differential effect of two vitamin D receptor agonists, calcitriol and
paricalcitol, on human aortic smooth muscle cells calcification in
vitro. Human vascular smooth muscle cells were incubated in a high
phosphate (HP) medium alone or supplemented with either calcitriol
10
⫺8
M (HP ⫹ CTR) or paricalcitol 3·10
⫺8
M (HP ⫹ PC). HP
medium induced calcification, which was associated with the upregu-
lation of mRNA expression of osteogenic factors such as bone
morphogenetic protein 2 (BMP2), Runx2/Cbfa1, Msx2, and osteocal-
cin. In these cells, activation of Wnt/-catenin signaling was evi-
denced by the translocation of -catenin into the nucleus and the
increase in the expression of direct target genes as cyclin D1, axin 2,
and VCAN/versican. Addition of calcitriol to HP medium (HP ⫹
CTR) further increased calcification and also enhanced the expression
of osteogenic factors together with a significant elevation of nuclear
-catenin levels and the expression of cyclin D1, axin 2, and VCAN.
By contrast, the addition of paricalcitol (HP ⫹ PC) not only reduced
calcification but also downregulated the expression of BMP2 and
other osteoblastic phenotype markers as well as the levels of nuclear
-catenin and the expression of its target genes. The role of Wnt/-
catenin on phosphate- and calcitriol-induced calcification was further
demonstrated by the inhibition of calcification after addition of Dick-
kopf-related protein 1 (DKK-1), a specific natural antagonist of the
Wnt/-catenin signaling pathway. In conclusion, the differential ef-
fect of calcitriol and paricalcitol on vascular calcification appears to
be mediated by a distinct regulation of the BMP and Wnt/-catenin
signaling pathways.
vascular calcification; calcitriol; paricalcitol; VSMCs; Wnt/-catenin
UREMIC PATIENTS FREQUENTLY present vascular calcifications
(VC), which contributes to the high rate of cardiovascular
morbidity and mortality observed in these patients (61). The
generation of VC in uremic patients is multifactorial, and the
mechanisms are partially understood (35). Abnormal mineral
metabolism, and particularly the accumulation of phosphate,
plays a central role in the generation of VC (3, 25). Chronic
inflammation has also been proposed as an important factor in
the process of VC (34, 50).
Calcitriol [1,25(OH)
2
D
3
], the most active metabolite of
vitamin D, is a key regulator of mineral metabolism through its
direct effect on intestine, kidney, bone, and parathyroid glands.
Chronic kidney disease (CKD) patients, often develop second-
ary hyperparathyroidism (2°HPT) because of the retention of
phosphate and the deficiency of calcitriol synthesis, both due to
the reduction of functional renal mass, which eventually lead to
hypocalcemia. Thus, in the last decades, therapeutic strategies
to control 2°HPT incorporated high doses of vitamin D, mainly
as calcitriol, that may contribute to the development of VCs
(13). The vitamin D analog 19-nor-1,25(OH)
2
D
2
(paricalcitol)
is now commonly used to treat 2°HPT in CKD patients
because it effectively suppress parathyroid hormone but is less
calcemic and phosphatemic than calcitriol (29, 53).
The administration of calcitriol to CKD patients may cause
VC by increasing the serum levels of calcium and phosphate.
Other studies (45) have suggested that calcitriol and other
vitamin D receptor (VDR) activators such as paricalcitol may
have a direct effect on VC that is independent of calcium and
phosphorus. Paricalcitol seems to produce less calcification
than calcitriol (4, 28, 33). However, the cellular mechanisms
driving the differential effects of calcitriol and paricalcitol on
VC have not been elucidated. We hypothesized that factors
regulating osteogenic differentiation of vascular smooth mus-
cle cells (VSMCs) may respond differently to calcitriol and
paricalcitol.
Bone morphogenetic proteins (BMPs) and proteins of the
Wnt family are extremely potent anabolic regulators of bone
formation, and both have been implicated in the regulation of
VC (7, 17, 31, 49, 51, 57). BMP2 upregulates transcription
factors as the runt-related transcript factor 2 (Runx2/Cbfa1)
and the Msh homeobox 2 (Msx2), master regulators of osteo-
genesis (30). Furthermore, Wnt signaling, which is essential
for the commitment of pluripotent mesenchymal cells, has also
been shown to be activated during the development of VC in
vivo and in vitro (5, 8, 23, 31, 51). Wnt proteins (revised in 26)
are a large family of secreted signaling molecules that signal
through binding to a coreceptor complex formed by the pro-
teins of the frizzled (Fzd) family and the lipoprotein receptor-
related 5/6 proteins (Lrp5/6). The activation of the canonical
Address for reprint requests and other correspondence: Y. A. Peña, Unidad
de Investigacion, IMIBIC, Hospital Reina Sofía, Avda. Menéndez Pidal s/n,
14004 Córdoba, Spain (e-mail: yolandaalmaden@yahoo.es).
Am J Physiol Renal Physiol 303: F1136–F1144, 2012.
First published August 8, 2012; doi:10.1152/ajprenal.00684.2011.
1931-857X/12 Copyright
©
2012 the American Physiological Society http://www.ajprenal.orgF1136
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Wnt pathway results in the inactivation of a destruction com-
plex that phosphorylates -catenin and targets it for ubiquitin-
proteasome-mediated degradation; thus -catenin is able to
translocate to the nucleus and regulates the expression of target
genes.
The aim of the present study was to investigate possible
differences between the effect of calcitriol and paricalcitol on
osteogenic signals, mainly the Wnt/-catenin signaling, in
human aortic smooth muscle cells (HASMCs) in vitro.
MATERIALS AND METHODS
Cell culture. HASMCs were obtained from Clonetics (Lonza,
Walkersville, MD). Cells were cultured in DMEM supplemented with
15% heat-inactivated FBS (BioWhittaker), Na piruvate (1 mmol/l),
glutamine (4.5 g/l), penicillin (100 U/ml), streptomycin (100 mg/ml),
and HEPES (20 mmol/l) at 37°C in a humidified atmosphere with 5%
CO
2
. HASMCs of passage 5– 8 were used in the experiments. To
induce calcification, after reaching 80% confluence, HASMCs were
incubated for 9 days in a high phosphate medium (calcification
medium) that contained Na
2
HPO
4
3⫺
and NaH
2
PO
4
3⫺
salts in 1:2
proportion (Sigma Aldrich) to obtain a final phosphate concentration
of 3.3 mmol/l. The medium was replaced with fresh medium every
2–3 days. Depending on the experiments, calcification medium was
supplemented with calcitriol 10
⫺8
M, paricalcitol 3·10
⫺8
M, or other
drugs such as Dickkopf-related protein 1 (DKK-1; ref 5439-DK-010;
R&D Systems; 100 ng/ml; a secreted, endogenous extracellular Wnt/
-catenin inhibitory gene product that is commercially available).
Cells that were incubated in normal phosphate (0.9 mmol/l) medium
were used as controls.
Assessment of calcium deposition. After 9 days of incubation,
calcification was quantified. Cells were decalcified with HCl (0.6
mol/l) for 24 h. The calcium content of the supernatants was deter-
mined by spectrophotometer at 612 nm by a kit containing phenol-
sulphonephthalein dye (no. DICA008, QuantiChrom calcium assay
kit; BioAssay Systems). Then, the cells were washed three times with
PBS (Sigma Aldrich) and solubilized in 0.1 mol/l NaOH/0.1% SDS.
The protein content was measured using the Bio-Rad protein assay
(Bio-Rad Laboratories, Munich, Germany), and the calcium content
was normalized for total protein.
Real-time RT-PCR. Total RNA was isolated from each sample of
HASMCs using 500 l Trizol (Sigma) by processing according to the
manufacturer’s recommendation. Real-time RT-PCR was performed
in duplicate with QuantiTect SYBR Green one-step RT-PCR Kit (ref.
no. 204243; Qiagen) in a final volume of 20 ul from 30 ng of total
RNA. All PCR amplifications were carried out using Lightcycler 480
(Roche Molecular Biochemicals, Indianapolis, IN). The expression of
target genes was normalized to the expression of GAPDH. The
primers for PCR amplification are indicated in Table 1.
Protein extracts and Western blot. Proteins were isolated from
HASMCs using lysis buffer containing HEPES (10 mmol/l), KCl (10
mmol/), EDTA (0.1 mmol/l), EGTA (0.1 mmol/l), dithiothreitol (1
mmol/l), PMSF (0.5 mmol/l), protease inhibitor cocktail (70 mg/ml;
Sigma Aldrich), and Igepal CA-630 (0.6%) at pH 7.9. The suspension
was centrifuged, and the supernatant (cytosolic extract) was stored.
Nuclear extracts were obtained by incubating the pellet separated
from the cytosolic extract in a lysis buffer containing HEPES (20
mmol/l), NaCl (0.4 mmol/l), EDTA (1 mmol/l), EGTA (1 mmol/l),
dithiothreitol (1 mmol/l), PMSF (1 mmol/l), and protease inhibitor
cocktail (46 mg/ml) at pH 7.9. Protein concentration was determined
with the Bradford method (Bio-Rad Laboratories, Munich, Germany).
For Western blot, equal amounts of protein were electrophoresed in
10% SDS-polyacrylamide gel (Invitrogen, Carlsbad, CA) and subse-
quently transferred to a nitrocellulose membrane (Invitrogen). The
membranes were blocked with 5% nonfat dried milk for1hatroom
temperature and then incubated with primary antibody for2hatroom
temperature. Primary antibodies used included rabbit polyclonal
-catenin antibody (ref. no. L9562; Cell Signaling) and rabbit poly-
clonal TFIIB antibody (ref. no. SC-225; Santa Cruz Biotechnology,
Santa Cruz, CA). Blots were immunolabeled using a horseradish
peroxidase-conjugated secondary antibody and developed on autora-
diographic film using the ECL Plus Western blotting detection system
(Amersham Biosciences, Little Chalfont, England). Specific bands
were quantified by densitometric analyses with Quantity One 4.4.0
software (Bio-Rad Laboratories) and were normalized to TFIIB levels.
Confocal microscopy. Cells were seeded on coverslips, and after
reaching 90% confluence, they received the different treatments for 24
h. Then, they were rinsed in PBS and fixed and permeated in cold 50%
methanol for 2 min, cold 100% methanol for 20 min, and cold 50%
methanol for 2 min. The specimens were subsequently washed once in
PBS (3 ⫻ 5 min) and incubated for 2 h with anti--catenin antibody
(1:50; BD Pharmigen, Franklin Lakes, NJ) in blocking solution (1%
BSA) at room temperature. After being washed with PBS (3 ⫻ 5 min),
specimens were incubated for 1 h with Alexa Fluor 488 F(ab=)
2
fragment of rabbit anti-mouse IgG (1:500; ref. no. A-21204; Invitro-
gen) in PBS containing 1% BSA. After a final wash with PBS (3 ⫻
5 min), the specimens were counterstained with DAPI for nuclear
stain. Cells were mounted on slides to examine fluorescence using a
LSM 5 Exciter Carl Zeiss confocal microscope. Pictures were ob-
tained at ⫻40 in Axio Observer Z1 inverted confocal microscope
(LSM5 Exciter Zeiss). ImageJ software (National Institutes of Health)
was used to analyze confocal immunofluorescence staining. Mander’s
coefficient M2 plugin (DAPI vs. green) was used to analyze nuclear
translocation of -catenin. Mander’s coefficient M2 is the percentage
of above-background pixels in blue color (DAPI) that overlap above-
background pixels in green color (-catenin).
Statistical analysis. Results are expressed as means ⫾ SE. The
difference between means for three or more groups was assessed by
one-way ANOVA followed by post hoc Duncan analysis. The difference
between means for two different groups was determined by t-test. A P
value ⬍0.05 was considered significant. These analyses were performed
with the assistance of a computer program (SPSS 15.0, Chicago, IL).
RESULTS
Effect of calcitriol and paricalcitol on VSMC calcification.
Incubation of HASMCs in a high phosphate (3.3 mmol/l)
medium for 9 days (HP) induced calcification compared with
Table 1. Primers used for quantitative real-time RT-PCR
Gene Sense Primer Antisense Primer
GAPDH 5=-TGATGACATCAAGAAGGTGGTGAAG-3= 5=-TCCTTGGAGGCCATGTGGGCCAT-3=
Cyclin D1 5=-CCGAGGAGCTGCTGCAAATGGA-3= 5=-ATGGAGGGCGGATTGGAAATGAAC-3=
BMP2 5=-AGGAGGCAAAGAAAAGGAACGGAC-3= 5=-GGAAGCAGCAACGCTAGAAGACAG-3=
Msx2 5=-AAATTCAGAAGATGGAGCGGCGTG-3= 5=-CTGGGATGTGGTAAAGGGCGTGCG-3=
Runx2 5=-CCGGAGTGGACGAGGCAAGAGTT-3= 5=-AGCTTCTGTCTGTGCCTTCTGGG-3=
Osteocalcin 5=-GCAGAGTCCAGCAAAGGTGCAGCC-3= 5=-GCCTCCTGAAAGCCGATGTGGTCA-3=
BMP2, bone morphogenetic protein 2.
F1137PARICALCITOL AND Wnt/-CATENIN
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cells maintained in a medium with 0.9 mmol/l phosphate
(control; P ⬍ 0.05; Fig. 1). The addition of calcitriol (10
⫺8
M)
to cells in HP medium (HP ⫹ CTR) further increased the
degree of calcification observed with HP alone (P ⬍ 0.05).
Conversely, the addition of paricalcitol (3 ⫻ 10
⫺8
M; HP ⫹
PC) produced a significant reduction in calcification relative to
the observed in cells in HP medium (P ⬍ 0.05). Nevertheless,
in the HP ⫹ PC cells the calcium content remained higher than
in control cells cultured in 0.9 mmol/L P (P ⬍ 0.05).
Osteoblast phenotype. The levels of BMP2 mRNA were
greater in cells incubated in HP medium than in controls (P ⬍
0.05; Fig. 2A). The addition of calcitriol further increased the
expression of BMP2 mRNA compared with the HP cells (P ⬍
0.05). In cells treated with paricalcitol, the levels of BMP2
mRNA were not different from control cells.
The gene expression of the osteoblastic-specific marker
Runx2 was significantly increased in cells on HP compared
with controls (P ⬍ 0.05; Fig. 2B). Again calcitriol caused
additional increase of Runx2 expression (P ⬍ 0.05 vs. HP
cells), whereas paricalcitol failed to increase the expression of
Runx2 mRNA beyond the values observed in HP cells.
Similarly to that observed with Runx2, the expression of
Msx2 mRNA was increased in HP and further increased by
addition of calcitriol (Fig. 2C). The addition of paricalcitol
decreased the Msx2 mRNA levels to control values. The
expression of Bglap/osteocalcin (OC) was not increased by HP
but it increased when calcitriol was added to the HP medium
(Fig. 2D). By contrast, paricalcitol did not modify the OC
expression.
Role of the canonical Wnt/

-catenin signaling pathway.
Activation of the Wnt/-catenin signaling pathway results in
nuclear translocation of -catenin. The presence of -catenin
in the nucleus was assessed by Western blotting of nuclear
extracts. The incubation of cells in HP induced a significant
increase of the expression of nuclear -catenin compared with
controls (P ⬍ 0.05). The addition of calcitriol to HP medium
increased the nuclear content of -catenin; however, the addi-
tion of paricalcitol caused a reduction in the levels of nuclear
-catenin to a level similar to that observed in control cells
(Fig. 3A). Intracellular localization of -catenin was visualized
by immunofluorescence using confocal microscopy (Fig. 3B).
Control cells showed immunofluorescence staining of -catenin
only in the cytoplasm, whereas cells cultured in HP showed
marked expression of -catenin at the nuclear level. HP ⫹ CTR
cells also exhibited marked staining for nuclear -catenin. By
contrast, in HP ⫹ PC, -catenin expression was mainly re-
stricted to the cytoplasm. Quantification by the Mander’s
3
4
5
6
on
(µg/mg protein)
*
*
#
*
0
1
2
Ca
depositio
Control HP HP+CTR HP+PC
*
#
Fig. 1. Effect of calcitriol and paricalcitol on human aortic smooth muscle cell
(HASMC) calcification. HASMCs are incubated for 9 days in a high (3.3
mmol/l) phosphate (HP) medium (calcification medium) alone or supple-
mented with either calcitriol 10
⫺8
M (HP ⫹ CTR) or paricalcitol 3·10
⫺8
M
(HP ⫹ PC). Cells incubated in normal phosphate (0.9 mmol/l) medium are
used as controls. Calcium content is determined with the phenolsulphoneph-
thalein dye. Bars are means ⫾ SE (5 independent experiments; 6 repetitions in
each experiment). *P ⬍ 0.05 vs. control. #P ⬍ 0.05 vs. HP.
3
ntrol)
*
#
8
ntrol)
*
#
BA
0
0.5
1
1.5
2
2.5
BMP2 mRNA levels (vs. Con
Control HP HP+CTR HP+PC
*
#
0
2
4
6
Runx 2 mRNA levels (vs. Con
Control HP HP+CTR HP+PC
**
*
0.5
1
1.5
2
2.5
Msx 2 mRNAlevels (vs. Control)
*
*
#
0.5
1
1.5
2
OC mRNA levels (vs. Control)
*
CD
0
Control HP HP+CTR HP+PC
0
Control HP HP+CTR HP+PC
Fig. 2. Effect of calcitriol and paricalcitol on the expression of markers of osteoblastic phenotype during HASMC calcification. HASMCs are incubated for 9
days in a high (3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supplemented with either calcitriol 10
⫺8
M (HP ⫹ CTR) or paricalcitol
3·10
⫺8
M (HP ⫹ PC). Cells incubated in normal phosphate (0.9 mmol/l) medium are used as controls. mRNA levels are analyzed by real-time RT-PCR technique.
Expression of target genes are normalized to the expression of GAPDH. A: bone morphogenetic protein 2 (BMP2) mRNA expression. B: Runx2/Cbfa1 mRNA
expression. C: Msx2 mRNA expression. D: osteocalcin/Bglap mRNA expression. Bars are means ⫾ SE (3 independent experiments; 6 repetitions in each
experiment). OC, osteocalcin. *P ⬍ 0.05 vs. control. #P ⬍ 0.05 vs. HP.
F1138 PARICALCITOL AND Wnt/-CATENIN
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coefficient M2 plugin (DAPI vs. green), confirmed the signif-
icance of differences in the levels of -catenin fluorescence in
HP and HP ⫹ CTR compared with control cells (P ⬍ 0.05 and
P ⬍ 0.05, respectively; Fig. 3C).
The relevance of Wnt/-catenin signaling activation in cal-
cification was tested by addition to culture medium (100 ng/ml)
of DKK-1, a commercially available specific endogenous ex-
tracellular antagonist of the Wnt signaling (1, 26). As shown in
Fig. 4, the addition of DKK-1 reduced the level of calcification
induced by high phosphate (P ⬍ 0.05 vs. HP cells) and
prevented the increase in calcification induced by calcitriol
(P ⬍ 0.05 vs. HP ⫹ CTR cells); a trend toward a decreased
calcification, although not significant, was also observed after
addition of DKK-1 to HP ⫹ PC cells. The effect of DKK-1 on
-catenin translocation was also evaluated by confocal immu-
nofluorescence (Fig. 3, B and C). The increase of nuclear
-catenin expression in HP and HP ⫹ CTR cells was abolished
by coincubation with DKK-1 (P ⬍ 0.05 vs. controls).
Additional direct Wnt/-catenin transcriptional targets were
examined, including cyclin D1 (an early marker of cells enter-
ing the cell cycle), VCAN/versican (a large chondroitin sulfate
proteoglycan; a member of the aggrecan/versican proteoglycan
family), and axin 2 (axis inhibition protein 2 or conductin; a
member of the Wnt/-catenin signaling pathway that regulates
the stability of -catenin). Effects of cotreatment with the
inhibitor DKK-1 on the expression of genes were also tested
(Fig. 5). The mRNA expression of cyclin D1 was similarly
elevated in cells in HP and HP ⫹ CTR compared with control
cells (P ⬍ 0.05). In cells treated with HP ⫹ PC, the cyclin D1
mRNA level was greater than in control (P ⬍ 0.05) but lower
than in HP and HP ⫹ CTR cells (P ⬍ 0.05). Addition of
DKK-1 dramatically reduced cyclin D1 mRNA to a level not
different from that of control. VCAN expression was also
increased in HP, HP ⫹ CTR, and HP ⫹ PC cells over control
(P ⬍ 0.05). Cotreatment with DKK-1 significantly reduced
VCAN mRNA levels to control values in all the groups.
Finally, compared with control, axin 2 mRNA expression was
elevated in HP and HP ⫹ CTR (P ⬍ 0.05) but not in HP ⫹ PC
group; this increase was abolished by DKK-1 (P ⬍ 0.05).
DISCUSSION
In the present study, we have explored the differential effect
of two VDR agonists, calcitriol and paricalcitol, on VSMC
A
C
HP HP+CTR
HP+PC
Nuclear
beta-catenin
TFIIB
0
40
80
120
160
200
Nuclear beta-catenin
(integrated OD vs
. Control)
Control HP HP+CTR HP+PC
*
*
#
B
C
-catenin
3
4
5
Nuclear beta
(Mander’s coefficient vs. Control)
0
1
2
#
#
Fig. 3. Effect of calcitriol and paricalcitol on the canonical Wnt/-catenin signaling pathway during HASMC calcification. HASMCs are incubated for 24 h in
a high (3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supplemented with either calcitriol 10
⫺8
M (HP ⫹ CTR) or paricalcitol 3·10
⫺8
M
(HP ⫹ PC). Cells incubated in normal phosphate (0.9 mmol/l) medium are used as controls. A: levels of nuclear -catenin is assessed by western blotting of
nuclear extracts. Image represents 3 different experiments. Quantification is performed by measurement of the integrated optical density (OD) and normalized
to TFIIB levels. Data are means ⫾ SE of the 3 independent experiments. *P ⬍ 0.05 vs. control. #P ⬍ 0.05 vs. HP. B: HASMCs are incubated for 24 h in a
high (3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supplemented with either calcitriol 10
⫺8
M (HP ⫹ CTR) or paricalcitol 3·10
⫺8
M
(HP ⫹ PC) alone or with the addition to culture medium (100 ng/ml) of commercially available Dickkopf-related protein 1 (DKK-1), a specific endogenous
extracellular antagonist of the Wnt signaling. Intracellular localization of -catenin is visualized by immunofluorescence using confocal microscopy. For each
treatment, -catenin staining (green immunofluorescence) is shown at left;atmiddle, the same sample is counterstained with DAPI (blue) for nuclear stain; the
merged image is shown at right. Images represent 3 different experiments. C: quantification of nuclear -catenin staining is performed by the Mander’s coefficient
(M2 plugin: DAPI vs. green). *P ⬍ 0.05 vs. control. #P ⬍ 0.05 vs. the same treatment without DKK-1.
F1139PARICALCITOL AND Wnt/-CATENIN
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calcification in vitro. We found that calcitriol increased calci-
fication, which appeared to be associated with the activation of
the Wnt/-catenin and BMP2 signaling pathways. By contrast,
paricalcitol decreased calcification, which was accompanied by
an inhibition of the Wnt/-catenin pathway and downregula-
tion of osteoblastic gene expression.
As previously demonstrated by us and other researchers (6,
36, 40, 44, 47, 49, 58), VSMCs cultured in high phosphate
undergo osteogenic transformation and calcification. BMPs
and Wnt ligands have been implicated in the regulation of both
osteoblastic transdifferentiation of aortic VSMCs in vitro and
VC (7, 17, 31, 49, 51, 57). In our study, we found that the
calcification process was associated with the upregulation of
osteogenic factors such as BMP2, the transcription factors
Runx2/Cbfa1 and Msx2, and the procalcificant protein OC.
This is in agreement with previous results (56) showing that the
expression of all these markers was significantly induced in
calcified vessels. BMP2 is a key factor for osteogenic differ-
entiation of mesenchymal cells that upregulates Runx2 and
Msx2 (30). OC is a downstream target gene of Runx2 and
Msx2 (16).
In the present study, we also show that calcification of the
HASMCs is also associated with a concomitant activation of
the canonical Wnt/-catenin signaling pathway. This was
quantitatively demonstrated both immunocytochemically by
the translocation of -catenin into the nucleus and by the
significant increase of the expression of nuclear -catenin by
Western blotting. Furthermore, the addition of DKK-1, a se-
creted, endogenous extracellular Wnt/-catenin inhibitory
gene product (1, 26) that is commercially available, inhibited
the calcification and the concomitant cellular changes induced
by high phosphate.
The activation of -catenin signaling modulates osteoblast
proliferation and differentiation (32, 41). Other authors (22)
observed VSMC proliferation in vessels developing calcifica-
tion. Notably, we demonstrated that the calcification observed
in the high phosphate-treated cells was accompanied by a
*
*
e
ls
*
*
e
ls
2
e
ls
#
Cyclin D mRNA lev
e
(vs. Control)
*
$
#
#
Cyclin D mRNA lev
e
(vs. Control)
0
0.5
1.5
1
Cyclin D mRNA lev
e
(vs. Control)
*
$
#
levels
ol)
*
#
*
#
1.5
2
levels
ol)
*
#
*
#
Control HP HP+CTR HP+PC HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
Control HP HP+CTR HP+PC HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
Control HP HP+CTR HP+PC HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
Control HP HP+CTR HP+PC
Axin 2 mRNA
(vs. Contro
HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
#
0
0.5
1
Control HP HP+CTR HP+PC
Axin 2 mRNA
(vs. Contro
HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
#
A
N
mRNA
levels
(vs. Control)
#
#
*
*
AN
mRNA
levels
(vs. Control)
1
2
1.5
AN
mRNA
levels
(vs. Control)
*
#
#
*
*
Control HP HP+CTR HP+PC
VCA
HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
Control HP HP+CTR HP+PC
VC
A
HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
0
0.5
Control HP HP+CTR HP+PC
VC
A
HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
Fig. 5. Effect of calcitriol and paricalcitol on the expression of -catenin direct
transcriptional target genes, cyclin D1, axin 2, and VCAN/versican, during
HASMC. HASMCs are incubated for 24 h in a high (3.3 mmol/l) phosphate
(HP) medium (calcification medium) alone or supplemented with either cal-
citriol 10
⫺8
M (HP ⫹ CTR) or paricalcitol 3·10
⫺8
M (HP ⫹ PC) alone or with
the addition to culture medium (100 ng/ml) of commercially available DKK-1,
a specific endogenous extracellular antagonist of the Wnt signaling. Cells
incubated in normal phosphate (0.9 mmol/l) medium are used as controls.
mRNA levels are analyzed by real-time RT-PCR technique. Expression of
target gene mRNA is normalized to the expression of GAPDH. Bars are
means ⫾ SE (3 independent experiments; 6 repetitions in each experiment).
*P ⬍ 0.05 vs. control. $P ⬍ 0.05 vs. HP. #P ⬍ 0.05 vs. the same treatment
without DKK-1.
5
* $
*
#
2
3
4
* $
#
$
Calcium deposition
g
/mg protein vs. Control)
0
1
Control HP HP+CTR HP+PC HP
+
DKK1
HP+CTR
+
DKK1
HP+PC
+
DKK1
(µ
g
Fig. 4. Effect of the inhibition of the canonical Wnt/-catenin signaling
pathway by DKK-1 on HASMC. HASMCs are incubated for 24 h in a high
(3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supple-
mented with either calcitriol 10
⫺8
M (HP ⫹ CTR) or paricalcitol 3·10
⫺8
M
(HP ⫹ PC) alone or with the addition to culture medium (100 ng/ml) of
commercially available DKK-1, a specific endogenous extracellular antagonist
of the Wnt signaling. Cells incubated in normal phosphate (0.9 mmol/l)
medium are used as controls. Calcium content is determined with the phenol-
sulphonephthalein dye. Bars are means ⫾ SE (3 independent experiments; 6
repetitions in each experiment). *P ⬍ 0.05 vs. control. $P ⬍ 0.05 vs. HP.
#P ⬍ 0.05 vs. the same treatment without DKK-1.
F1140 PARICALCITOL AND Wnt/-CATENIN
AJP-Renal Physiol • doi:10.1152/ajprenal.00684.2011 • www.ajprenal.org
by 10.220.32.246 on November 21, 2017http://ajprenal.physiology.org/Downloaded from
