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Contents lists available at ScienceDirect

Journal of Steroid Biochemistry and Molecular Biology

jo ur nal home page: www.elsev ier .com/ locate / j sbmb

egulation of the calcium-sensing receptor expression by,25-dihydroxyvitamin D3, interleukin-6, and tumor necrosis factorlpha in colon cancer cells�

rfete S. Fetahua, Doris M. Hummela, Teresa Manhardta,bhishek Aggarwala, Sabina Baumgartner-Parzerb, Eniko Kállaya,∗

Department of Pathophysiology and Allergy Research, Medical University of Vienna, Währinger Gürtel 18-20, Vienna, AustriaDepartment of Internal Medicine III, Medical University of Vienna, Währinger Gürtel 18-20, Vienna, Austria

r t i c l e i n f o

rticle history:eceived 1 August 2013eceived in revised form 3 October 2013ccepted 17 October 2013

eywords:alcium-sensing receptor

a b s t r a c t

Anti-proliferative effects of calcium in the colon are mediated, at least in part, via the calcium-sensingreceptor (CaSR), a vitamin D target gene. The expression of CaSR decreases during colorectal tumorprogression and the mechanisms regulating its expression are poorly understood. The CaSR promoterharbors vitamin D elements responsive to 1,25-dihydroxyvitamin D3 (1,25D3) and NF-�B, STAT, and SP1binding sites accounting for responsiveness to proinflammatory cytokines. Therefore, in the current studywe investigated the impact of 1,25D3, tumor necrosis factor alpha (TNF�), and interleukin (IL)-6 on CaSR

olon cancerumor necrosis factor alphanterleukin-6,25-dihydroxyvitamin D3

expression in a differentiated (Caco2/AQ) and in a moderately differentiated (Coga1A) colon cancer cellline. 1,25D3 induced CaSR expression in both cell lines. Treatment with TNF� was accompanied by a 134-fold induction of CaSR in Coga1A (p < 0.01). In Caco2/AQ cells the expression of CaSR was upregulatedalso by IL-6 (3.5-fold). Our data demonstrated transcriptional and translational activation of the CaSR by

nflammation 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.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Materials and methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.1. Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. RNA isolation, reverse transcription, and real time qRT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. Immunofluorescent staining of colon cancer cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.1. Impact of 1,25D3 on CaSR expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.2. Impact of TNF� and IL-6 on CaSR expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: I.S. Fetahu, et al., Reguldihydroxyvitamin D3, interleukin-6, and tumor necrosis factor alphhttp://dx.doi.org/10.1016/j.jsbmb.2013.10.015

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

� This is an open-access article distributed under the terms of the Creativeommons Attribution License, which permits unrestricted use, distribution, andeproduction in any medium, provided the original author and source are credited.∗ Corresponding author at: Department of Pathophysiology and Allergy Research,edical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria.

el.: +43 1 40400 5123; fax: +43 1 40400 5130.E-mail addresses: irfete.fetahu@meduniwien.ac.at (I.S. Fetahu),

oris.hummel@meduniwien.ac.at (D.M. Hummel),eresa.manhardt@meduniwien.ac.at (T. Manhardt),bhishek.aggarwal@meduniwien.ac.at (A. Aggarwal),abina.baumgartner-parzer@meduniwien.ac.at (S. Baumgartner-Parzer),nikoe.kallay@meduniwien.ac.at (E. Kállay).

960-0760/$ – see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights ttp://dx.doi.org/10.1016/j.jsbmb.2013.10.015

1. Introduction

Epidemiological studies demonstrate an inverse correlationbetween calcium and vitamin D intake and risk of tumordevelopment [1,2]. The calcium-sensing receptor (CaSR) is a puta-tive tumor suppressor gene in the colon, which partially mediatesthe anti-proliferative and pro-differentiating actions of calcium in

ation of the calcium-sensing receptor expression by 1,25-a in colon cancer cells, J. Steroid Biochem. Mol. Biol. (2013),

colonocytes (for review, see [3,4]). However, in colon cancer anti-proliferative effects of Ca2+ are lost [5,6], and this could be due toloss of CaSR expression during colorectal tumorigenesis [7]. Very

reserved.

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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 anda tor kappa B (NF-�B), specificity protein 1 (SP1)), which are critical for 1,25D3, TNF�, andI SS) 1 and 2 according to [12] were taken as point of reference for positioning the indicatedb

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ctivator of transcription (STAT), vitamin D response elements (VDRE), nuclear facL-6 responsiveness, as well as the CAAT and TATA boxes. Transcription start sites (Tinding sites in the corresponding promoters.

ittle is known about the factors that regulate the expression ofaSR in the colon. The CaSR gene contains 6 coding exons andwo 5′-untranslated exons (exons 1A and 1B), which are underhe control of promoter 1 and 2, respectively, yielding alternativeranscripts but coding for the same protein [8,9]. Several studieserformed in rat parathyroid, thyroid, and kidney have mappedinding sites of numerous transcription factors, including NF-�B,TAT, SP1, and vitamin D response elements in both CaSR promo-ers (Fig. 1) [9–12]. Currently, there is limited knowledge regardinghe role of 1,25D3 and of the proinflammatory cytokines TNF� andL-6 on CaSR expression in the colon. Therefore, in the presenttudy, we studied the impact of 1,25D3, TNF�, and IL-6 on trans-riptional and translational regulation of CaSR in two colon cancerell lines with different proliferation and differentiation properties,imicking different tumor stages.

. Materials and methods

.1. Cell culture

Caco2/AQ cells are a subclone of the Caco-2 cell line [13]. Thesearry a truncated APC and a missense mutation of �-catenin, and areble to differentiate spontaneously in culture. In the current studye used highly differentiated, 2 weeks post-confluent Caco2/AQ

ells. Coga1A is a cell line derived from a moderately differen-iated (G2) colon tumor [14]. These cells are heterozygous forruncated APC, without any known �-catenin mutations [15]. Con-uent Caco2/AQ and Coga1A cells were treated for 6, 12, 24, and8 h either with 10 nM 1,25D3, 50 ng/mL TNF� (Sigma Aldrich,SA), 100 ng/mL IL-6 (Immunotools, Germany), or the combinationf these compounds. Vehicle treated cells were used as controls.

.2. RNA isolation, reverse transcription, and real time qRT-PCR

RNA isolation and reverse transcription were performed asescribed previously [16]. Real time qRT-PCR analyses wereerformed in StepOne Plus system using POWER SYBR GREENastermix following the manufacturer’s recommendations (Life

echnologies, USA). Data were normalized to the expression ofhe reference genes: �2M or RPLP0 [17,18], and set relative tohe calibrator (Clontech, USA) to calculate the ��CT value. Primerequences for CaSR were: 5′-AGCCCAGATGCAAGCAGAAGG-3′ for-ard, 5′-TCTGGTGCGTAGAATTCCTGTGG-3′ reverse.

.3. Immunofluorescent staining of colon cancer cells

Cells were grown on sterile glass cover slips. After treatmentsells were fixed with 3.7% paraformaldehyde in PBS, permeabil-zed with 0.2% Triton-X (Sigma Aldrich, USA) for 20 min, and

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locked with 5% goat serum (Jackson ImmunoResearch, USA). Cellsere incubated either with rabbit polyclonal anti-CaSR antibody

1:100, Anaspec, USA) or mouse monoclonal anti-CaSR antibody1:200, Abcam, UK) for 1 h at room temperature. As negative

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 timepoints. Bars represent mean ± SEM of 2-3 independent experiments.

control we used rabbit or mouse IgG, respectively (Abcam, UK andLife Technologies, USA). As secondary antibody we used Dylightlabeled 549 goat-anti-rabbit or Alexa Fluor 647 goat-anti-mouseIgG (1:500, Vector Laboratories and Life Technologies, USA). Nucleiwere stained with DAPI (Roche, Switzerland). Images were acquiredusing TissueFAXS 2.04 (TissueGnostics, Austria).

2.4. Statistical analysis

All statistical analyses were performed with SPSS version 18 andgraphs were drawn with GraphPad Prism version 5. In case of non-normal distribution, data were log transformed to achieve normaldistribution and then subjected to one way ANOVA, followed byTukey’s multiple comparisons posttest. p-values smaller than 0.05were 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 andCoga1A cells with 1,25D3 for 6, 12, 24, and 48 h. In differentiatedCaco2/AQ cells treatment with 1,25D3 caused 2.4-fold inductionof CaSR expression after 6 h. The maximal effect of 1,25D3 onCaSR transcriptional activation in these cells was observed at 24 h(7.6-fold; Figs. 2A and 3C). In the less differentiated cells Coga1A1,25D3-induced CaSR transcription was 2.9-fold after 12 h and 4.2-fold after 24 h compared with the control group (Fig. 2B). 1,25D3increased CaSR translation as well. Immunofluorescence stainingdemonstrated upregulation of the CaSR protein in Caco2/AQ after24 h and Coga1A after 48 h (Fig. 3C and D).

3.2. Impact of TNF and IL-6 on CaSR expression

ation of the calcium-sensing receptor expression by 1,25-a in colon cancer cells, J. Steroid Biochem. Mol. Biol. (2013),

We treated Caco2/AQ and Coga1A cells with TNF� and IL-6 for6, 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 1,25D , 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 logt d corro ant chs

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ransformed to achieve normal distribution, then subjected to one way ANOVA anf 2-3 independent experiments, asterisks above bars indicate statistically significtaining of the CaSR protein (red) and nuclear staining (blue). Scale bar was 50 �m.

xpression. Treatment with IL-6 was accompanied by a 3.5-foldnduction after 6 h compared with control. Combined treatment

ith TNF� and IL-6 induced CaSR mRNA expression in Caco2/AQ0.3-fold (p < 0.05) after 24 h and 10.2-fold (p < 0.05) after 48 h.owever, the combination of all three compounds either had noffect or reduced CaSR expression (Fig. 3A).

In Coga1A cells, treatment with TNF� induced CaSR robustly,specially at 48 h (134-fold, p < 0.01). Treatment with IL-6 causednly marginal increases in CaSR mRNA expression. Furthermore,e observed upregulation of CaSR expression in the groups treatedith TNF�/IL-6 (68.5-fold) and TNF�/1,25D3 (121.2-fold, p < 0.05)

t 48 h. Similar results were observed in the groups that werereated with TNF�/IL-6/1,25D3 at 6 and 48 h (18.8-fold, p < 0.05 and7.7-fold, p < 0.05; Fig. 3B).

To address the question whether alterations on CaSR mRNA

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xpression were translated into protein, we performed immuno-uorescence staining. Fig. 3C and D demonstrates the upregulationf the CaSR protein upon treatments with the proinflammatoryytokines using the rabbit polyclonal anti-CaSR antibody. Protein

ected with Tukey’s posttest for multiple comparisons. Bars represent mean ± SEManges compared with control. *p < 0.05, **p < 0.01. (C and D) Immunofluorescence

expression data were confirmed using the mouse monoclonal anti-CaSR antibody (data not shown). Both antibodies gave the sameresults.

4. Discussion

Recent studies have demonstrated that murine CaSR activatesthe NLPR3 inflammasome, which in turn induces maturation andrelease of the inflammatory cytokine interleukin 1�, amplifying theinflammatory signal [19,20]. Inversely, mice double knockout forCaSR−/−/PTH−/− had increased inflammatory response after admin-istration of dextran sodium sulfate compared with control miceexpressing the receptor [21]. This suggests an important role forthe CaSR in inflammation. Therefore, it is essential to understandhow the expression of the CaSR is modulated in the colon.

ation of the calcium-sensing receptor expression by 1,25-a in colon cancer cells, J. Steroid Biochem. Mol. Biol. (2013),

It has been demonstrated previously that activation of VDREsby 1,25D3 and translocation of NF-�B to the nucleus after thetreatment with interleukin 1� led to induction of CaSR expressionin rat parathyroid, thyroid, and kidneys [9,10]. Furthermore, IL-6

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njection in rats caused induction of CaSR transcription via Stat1/3esponse elements in promoter 1 and Sp1/3 sites in promoter 2 [11],ut not much is known about the regulation of CaSR expression inhe colon.

Our study is the first to show that in colonocytes inflammatoryytokines are able to upregulate CaSR expression, and that thisffect is time- and cell line-specific. In the present study, we inves-igated the role of 1,25D3, TNF�, and IL-6 on the transcriptional andranslational activation of the CaSR in two cell lines representing

highly differentiated and a moderately differentiated colorectalumor.

1,25D3 is known for its anti-proliferative, pro-differentiatingffects (for review, see [22]), and its involvement in regulating epi-enetic mechanisms [23]. Inducing expression of CaSR, a putativeumor suppressor in the colon, might be one of the tumor preven-ive mechanisms of 1,25D3. In the differentiated Caco2/AQ cells,25D3 had more pronounced impact in inducing the expressionf CaSR than in the less differentiated Coga1A cells. In Caco2/AQells treatment with 1,25D3 reduced the expression of several pro-iferation markers also. This was much less evident in the Coga1Aells (data not shown), although the level of the vitamin D receptors similar [15].

In Caco2/AQ cells, both TNF� and IL-6 increased CaSR expres-ion to a lesser extent than 1,25D3. In combination, however, theyaused a strong upregulation at 6 h, which was lost at 12 h; at 24 hhe effect became additive and the CaSR level remained high alsofter 48 h. We hypothesized that two different mechanisms wereesponsible: first, direct upregulation of CaSR expression due to aransient activation of CaSR promoters by NF-�B upon treatmentith TNF� and Stat1/3 and Sp1/3 elements by IL-6. This was fol-

owed by a second induction of transcription that seems to bendirect. Some (still unknown) factors induced by TNF� and IL-6

ight be needed for this more stable induction of CaSR expression.nexpectedly, 1,25D3 counteracted this additive effect, suggesting

he existence of intricate feedback systems.In Coga1A cells, the CaSR was more sensitive to the proin-

ammatory cytokine TNF�, which was the main driver of CaSRxpression in these cells. The low effectiveness of IL-6 in upregulat-ng CaSR expression could be due to lower levels of the IL-6 receptoromplex (both the IL-6 binding � chain and the signal transducingnit gp130) in Coga1A cells compared with Caco2/AQ [24]. Inter-stingly, in these cells the CaSR protein levels remained enhancedn all combined treatments. The robust increase of CaSR expressiony TNF� treatment in Coga1A cells could be regarded as a defenseechanism against inflammation. Such protective mechanism was

hown in murine macrophages, where lipopolysaccharide-inducedNF� release upregulated CaSR expression leading to inhibition ofNF� synthesis, in a negative feedback manner [25].

In conclusion, our results demonstrate for the first time that inolon cancer cells not only 1,25D3, but also the proinflammatoryytokines TNF� and IL-6 were able to induce the expression ofaSR. How this observation can be translated in vivo and used

or the treatment of inflammation in the gut, still needs to bexplored.

cknowledgements

This study was funded by the Marie Curie ITN grant: FP7-64663, the Austrian Science Fund Project (FWF): P22200-B11, theroject Herzfelder’sche Familienstiftung: APP00422OFF.

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