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Journal of Steroid Biochemistry & Molecular Biology xxx (2013) xxx–xxx

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Journal of Steroid Biochemistry and Molecular Biology

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Regulation of the calcium-sensing receptor expression by

1,25-dihydroxyvitamin D3 , interleukin-6, and tumor necrosis factor
alpha in colon cancer cells夽
Irfete S. Fetahu a , Doris M. Hummel a , Teresa Manhardt a ,
Abhishek Aggarwal a , Sabina Baumgartner-Parzer b , Enikő Kállay a,∗
a
Department of Pathophysiology and Allergy Research, Medical University of Vienna, Währinger Gürtel 18-20, Vienna, Austria
b
Department of Internal Medicine III, Medical University of Vienna, Währinger Gürtel 18-20, Vienna, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Anti-proliferative effects of calcium in the colon are mediated, at least in part, via the calcium-sensing
Received 1 August 2013 receptor (CaSR), a vitamin D target gene. The expression of CaSR decreases during colorectal tumor
Received in revised form 3 October 2013 progression and the mechanisms regulating its expression are poorly understood. The CaSR promoter
Accepted 17 October 2013
harbors vitamin D elements responsive to 1,25-dihydroxyvitamin D3 (1,25D3 ) and NF-␬B, STAT, and SP1
binding sites accounting for responsiveness to proinflammatory cytokines. Therefore, in the current study
Keywords:
we investigated the impact of 1,25D3 , tumor necrosis factor alpha (TNF␣), and interleukin (IL)-6 on CaSR
Calcium-sensing receptor
expression in a differentiated (Caco2/AQ) and in a moderately differentiated (Coga1A) colon cancer cell
Colon cancer
Tumor necrosis factor alpha
line. 1,25D3 induced CaSR expression in both cell lines. Treatment with TNF␣ was accompanied by a 134-
Interleukin-6 fold induction of CaSR in Coga1A (p < 0.01). In Caco2/AQ cells the expression of CaSR was upregulated
1,25-dihydroxyvitamin D3 also by IL-6 (3.5-fold). Our data demonstrated transcriptional and translational activation of the CaSR by
Inflammation 1,25D3 , TNF␣, and IL-6 in a time- and cell line-dependent manner.
This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’.
© 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Materials and methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. RNA isolation, reverse transcription, and real time qRT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3. Immunofluorescent staining of colon cancer cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.4. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.1. Impact of 1,25D3 on CaSR expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.2. Impact of TNF␣ and IL-6 on CaSR expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

夽 This is an open-access article distributed under the terms of the Creative 1. Introduction
Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.
∗ Corresponding author at: Department of Pathophysiology and Allergy Research, Epidemiological studies demonstrate an inverse correlation
Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria. between calcium and vitamin D intake and risk of tumor
Tel.: +43 1 40400 5123; fax: +43 1 40400 5130. development [1,2]. The calcium-sensing receptor (CaSR) is a puta-
E-mail addresses: irfete.fetahu@meduniwien.ac.at (I.S. Fetahu), tive tumor suppressor gene in the colon, which partially mediates
doris.hummel@meduniwien.ac.at (D.M. Hummel), the anti-proliferative and pro-differentiating actions of calcium in
teresa.manhardt@meduniwien.ac.at (T. Manhardt),
abhishek.aggarwal@meduniwien.ac.at (A. Aggarwal),
colonocytes (for review, see [3,4]). However, in colon cancer anti-
sabina.baumgartner-parzer@meduniwien.ac.at (S. Baumgartner-Parzer), proliferative effects of Ca2+ are lost [5,6], and this could be due to
enikoe.kallay@meduniwien.ac.at (E. Kállay). loss of CaSR expression during colorectal tumorigenesis [7]. Very

0960-0760/$ – see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jsbmb.2013.10.015

Please cite this article in press as: I.S. Fetahu, et al., Regulation of the calcium-sensing receptor expression by 1,25-
dihydroxyvitamin D3 , interleukin-6, and tumor necrosis factor alpha in colon cancer cells, J. Steroid Biochem. Mol. Biol. (2013),
http://dx.doi.org/10.1016/j.jsbmb.2013.10.015
 ARTICLE IN PRESS
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Fig. 1. Schematic illustration of the CaSR promoter region including exon 1A and exon 1B. Position of binding sites for regulatory elements is shown (signal transducer and
activator of transcription (STAT), vitamin D response elements (VDRE), nuclear factor kappa B (NF-␬B), specificity protein 1 (SP1)), which are critical for 1,25D3 , TNF␣, and
IL-6 responsiveness, as well as the CAAT and TATA boxes. Transcription start sites (TSS) 1 and 2 according to [12] were taken as point of reference for positioning the indicated
binding sites in the corresponding promoters.
little is known about the factors that regulate the expression of
CaSR in the colon. The CaSR gene contains 6 coding exons and
two 5 -untranslated exons (exons 1A and 1B), which are under
the control of promoter 1 and 2, respectively, yielding alternative
transcripts but coding for the same protein [8,9]. Several studies
performed in rat parathyroid, thyroid, and kidney have mapped
binding sites of numerous transcription factors, including NF-␬B,
STAT, SP1, and vitamin D response elements in both CaSR promo-
ters (Fig. 1) [9–12]. Currently, there is limited knowledge regarding
the role of 1,25D3 and of the proinflammatory cytokines TNF␣ and
IL-6 on CaSR expression in the colon. Therefore, in the present
study, we studied the impact of 1,25D3 , TNF␣, and IL-6 on trans-
criptional and translational regulation of CaSR in two colon cancer
cell lines with different proliferation and differentiation properties,
mimicking different tumor stages.
2. Materials and methods
2.1. Cell culture
Caco2/AQ cells are a subclone of the Caco-2 cell line [13]. These
carry a truncated APC and a missense mutation of ␤-catenin, and are
able to differentiate spontaneously in culture. In the current study
we used highly differentiated, 2 weeks post-confluent Caco2/AQ
cells. Coga1A is a cell line derived from a moderately differen-
tiated (G2) colon tumor [14]. These cells are heterozygous for
truncated APC, without any known ␤-catenin mutations [15]. Con-
fluent Caco2/AQ and Coga1A cells were treated for 6, 12, 24, and
48 h either with 10 nM 1,25D3 , 50 ng/mL TNF␣ (Sigma Aldrich,
USA), 100 ng/mL IL-6 (Immunotools, Germany), or the combination
of these compounds. Vehicle treated cells were used as controls.
2.2. RNA isolation, reverse transcription, and real time qRT-PCR
RNA isolation and reverse transcription were performed as
described previously [16]. Real time qRT-PCR analyses were
performed in StepOne Plus system using POWER SYBR GREEN
Mastermix following the manufacturer’s recommendations (Life
Technologies, USA). Data were normalized to the expression of
the reference genes: ␤2M or RPLP0 [17,18], and set relative to
the calibrator (Clontech, USA) to calculate the CT value. Primer
sequences for CaSR were: 5 -AGCCCAGATGCAAGCAGAAGG-3 for-
ward, 5 -TCTGGTGCGTAGAATTCCTGTGG-3 reverse.
2.3. Immunofluorescent staining of colon cancer cells
Cells were grown on sterile glass cover slips. After treatments
cells were fixed with 3.7% paraformaldehyde in PBS, permeabil-
ized with 0.2% Triton-X (Sigma Aldrich, USA) for 20 min, and
blocked with 5% goat serum (Jackson ImmunoResearch, USA). Cells
were incubated either with rabbit polyclonal anti-CaSR antibody
(1:100, Anaspec, USA) or mouse monoclonal anti-CaSR antibody
(1:200, Abcam, UK) for 1 h at room temperature. As negative
Please cite this article in press as: I.S. Fetahu, et al., Regulation of the calcium-sensing receptor expression by 1,25-
dihydroxyvitamin D3 , interleukin-6, and tumor necrosis factor alpha in colon cancer cells, J. Steroid Biochem. Mol. Biol. (2013),
http://dx.doi.org/10.1016/j.jsbmb.2013.10.015
A Caco2/AQ B Coga1A
16 16
control
8 8 1,25D 3
Relative to control
4 4
2 2
1 1
0.5 0.5
6 12 24 48 6 12 24 48
time of treatment (h) time of treatment (h)
Fig. 2. Transcriptional regulation of CaSR by 1,25D3 in colon cancer cell lines.
Caco2/AQ and Coga1A cells were treated with 10 nM 1,25D3 for the indicated time
points. Bars represent mean ± SEM of 2-3 independent experiments.
control we used rabbit or mouse IgG, respectively (Abcam, UK and
Life Technologies, USA). As secondary antibody we used Dylight
labeled 549 goat-anti-rabbit or Alexa Fluor 647 goat-anti-mouse
IgG (1:500, Vector Laboratories and Life Technologies, USA). Nuclei
were stained with DAPI (Roche, Switzerland). Images were acquired
using TissueFAXS 2.04 (TissueGnostics, Austria).
2.4. Statistical analysis
All statistical analyses were performed with SPSS version 18 and
graphs were drawn with GraphPad Prism version 5. In case of non-
normal distribution, data were log transformed to achieve normal
distribution and then subjected to one way ANOVA, followed by
Tukey’s multiple comparisons posttest. p-values smaller than 0.05
were regarded as statistically significant.
3. Results
3.1. Impact of 1,25D3 on CaSR expression
To study the role of vitamin D response elements on trans-
criptional regulation of CaSR expression we treated Caco2/AQ and
Coga1A cells with 1,25D3 for 6, 12, 24, and 48 h. In differentiated
Caco2/AQ cells treatment with 1,25D3 caused 2.4-fold induction
of CaSR expression after 6 h. The maximal effect of 1,25D3 on
CaSR transcriptional activation in these cells was observed at 24 h
(7.6-fold; Figs. 2A and 3C). In the less differentiated cells Coga1A
1,25D3 -induced CaSR transcription was 2.9-fold after 12 h and 4.2-
fold after 24 h compared with the control group (Fig. 2B). 1,25D3
increased CaSR translation as well. Immunofluorescence staining
demonstrated upregulation of the CaSR protein in Caco2/AQ after
24 h and Coga1A after 48 h (Fig. 3C and D).
3.2. Impact of TNF˛ and IL-6 on CaSR expression
We treated Caco2/AQ and Coga1A cells with TNF␣ and IL-6 for
6, 12, 24, and 48 h. In Caco2/AQ treatment with the proinflam-
matory cytokine TNF␣ caused only modest upregulation of CaSR
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Fig. 3. Effect of 1,25D3 , TNF␣, and IL-6 on CaSR expression. (A and B) mRNA expression of Caco2/AQ and Coga1A cells assessed by real time qRT-PCR. Data were log
transformed to achieve normal distribution, then subjected to one way ANOVA and corrected with Tukey’s posttest for multiple comparisons. Bars represent mean ± SEM
of 2-3 independent experiments, asterisks above bars indicate statistically significant changes compared with control. *p < 0.05, **p < 0.01. (C and D) Immunofluorescence
staining of the CaSR protein (red) and nuclear staining (blue). Scale bar was 50 ␮m.

expression. Treatment with IL-6 was accompanied by a 3.5-fold
induction after 6 h compared with control. Combined treatment
with TNF␣ and IL-6 induced CaSR mRNA expression in Caco2/AQ
10.3-fold (p < 0.05) after 24 h and 10.2-fold (p < 0.05) after 48 h.
However, the combination of all three compounds either had no
effect or reduced CaSR expression (Fig. 3A).
In Coga1A cells, treatment with TNF␣ induced CaSR robustly,
especially at 48 h (134-fold, p < 0.01). Treatment with IL-6 caused
only marginal increases in CaSR mRNA expression. Furthermore,
we observed upregulation of CaSR expression in the groups treated
with TNF␣/IL-6 (68.5-fold) and TNF␣/1,25D3 (121.2-fold, p < 0.05)
at 48 h. Similar results were observed in the groups that were
treated with TNF␣/IL-6/1,25D3 at 6 and 48 h (18.8-fold, p < 0.05 and
47.7-fold, p < 0.05; Fig. 3B).
To address the question whether alterations on CaSR mRNA
expression were translated into protein, we performed immuno-
fluorescence staining. Fig. 3C and D demonstrates the upregulation
of the CaSR protein upon treatments with the proinflammatory
cytokines using the rabbit polyclonal anti-CaSR antibody. Protein
expression data were confirmed using the mouse monoclonal anti-
CaSR antibody (data not shown). Both antibodies gave the same
results.
4. Discussion
Recent studies have demonstrated that murine CaSR activates
the NLPR3 inflammasome, which in turn induces maturation and
release of the inflammatory cytokine interleukin 1␤, amplifying the
inflammatory signal [19,20]. Inversely, mice double knockout for
CaSR−/− /PTH−/− had increased inflammatory response after admin-
istration of dextran sodium sulfate compared with control mice
expressing the receptor [21]. This suggests an important role for
the CaSR in inflammation. Therefore, it is essential to understand
how the expression of the CaSR is modulated in the colon.
It has been demonstrated previously that activation of VDREs
by 1,25D3 and translocation of NF-␬B to the nucleus after the
treatment with interleukin 1␤ led to induction of CaSR expression
in rat parathyroid, thyroid, and kidneys [9,10]. Furthermore, IL-6

Please cite this article in press as: I.S. Fetahu, et al., Regulation of the calcium-sensing receptor expression by 1,25-
dihydroxyvitamin D3 , interleukin-6, and tumor necrosis factor alpha in colon cancer cells, J. Steroid Biochem. Mol. Biol. (2013),
http://dx.doi.org/10.1016/j.jsbmb.2013.10.015
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injection in rats caused induction of CaSR transcription via Stat1/3
response elements in promoter 1 and Sp1/3 sites in promoter 2 [11],
but not much is known about the regulation of CaSR expression in
the colon.
Our study is the first to show that in colonocytes inflammatory
cytokines are able to upregulate CaSR expression, and that this
effect is time- and cell line-specific. In the present study, we inves-
tigated the role of 1,25D3 , TNF␣, and IL-6 on the transcriptional and
translational activation of the CaSR in two cell lines representing
a highly differentiated and a moderately differentiated colorectal
tumor.
1,25D3 is known for its anti-proliferative, pro-differentiating
effects (for review, see [22]), and its involvement in regulating epi-
genetic mechanisms [23]. Inducing expression of CaSR, a putative
tumor suppressor in the colon, might be one of the tumor preven-
tive mechanisms of 1,25D3 . In the differentiated Caco2/AQ cells
1,25D3 had more pronounced impact in inducing the expression
of CaSR than in the less differentiated Coga1A cells. In Caco2/AQ
cells treatment with 1,25D3 reduced the expression of several pro-
liferation markers also. This was much less evident in the Coga1A
cells (data not shown), although the level of the vitamin D receptor
is similar [15].
In Caco2/AQ cells, both TNF␣ and IL-6 increased CaSR expres-
sion to a lesser extent than 1,25D3 . In combination, however, they
caused a strong upregulation at 6 h, which was lost at 12 h; at 24 h
the effect became additive and the CaSR level remained high also
after 48 h. We hypothesized that two different mechanisms were
responsible: first, direct upregulation of CaSR expression due to a
transient activation of CaSR promoters by NF-␬B upon treatment
with TNF␣ and Stat1/3 and Sp1/3 elements by IL-6. This was fol-
lowed by a second induction of transcription that seems to be
indirect. Some (still unknown) factors induced by TNF␣ and IL-6
might be needed for this more stable induction of CaSR expression.
Unexpectedly, 1,25D3 counteracted this additive effect, suggesting
the existence of intricate feedback systems.
In Coga1A cells, the CaSR was more sensitive to the proin-
flammatory cytokine TNF␣, which was the main driver of CaSR
expression in these cells. The low effectiveness of IL-6 in upregulat-
ing CaSR expression could be due to lower levels of the IL-6 receptor
complex (both the IL-6 binding ␣ chain and the signal transducing
unit gp130) in Coga1A cells compared with Caco2/AQ [24]. Inter-
estingly, in these cells the CaSR protein levels remained enhanced
in all combined treatments. The robust increase of CaSR expression
by TNF␣ treatment in Coga1A cells could be regarded as a defense
mechanism against inflammation. Such protective mechanism was
shown in murine macrophages, where lipopolysaccharide-induced
TNF␣ release upregulated CaSR expression leading to inhibition of
TNF␣ synthesis, in a negative feedback manner [25].
In conclusion, our results demonstrate for the first time that in
colon cancer cells not only 1,25D3 , but also the proinflammatory
cytokines TNF␣ and IL-6 were able to induce the expression of
CaSR. How this observation can be translated in vivo and used
for the treatment of inflammation in the gut, still needs to be
explored.
Acknowledgements
This study was funded by the Marie Curie ITN grant: FP7-
264663, the Austrian Science Fund Project (FWF): P22200-B11, the
Project Herzfelder’sche Familienstiftung: APP00422OFF.
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