Rapid effects of 17β-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells

Cell Calcium (2008) 44, 441—452

journal homepage: www.elsevier.com/locate/ceca

Rapid effects of 17␤-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells Mustapha Irnaten ∗,1, Nicolas Blanchard-Gutton 1, Brian J. Harvey Molecular Medicine Laboratories, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland Received 22 May 2007; received in revised form 18 January 2008; accepted 12 February 2008 Available online 18 April 2008

KEYWORDS Non-genomic; 17␤-Estradiol; TRPV6; Calcium; T84 colonic cells

Summary The control of calcium homeostasis is essential for cell survival and is of crucial importance for several physiological functions. The discovery of the epithelial calcium channel Transient Receptor Potential Vaniloid (TRPV6) in intestine has uncovered important Ca2+ absorptive pathways involved in the regulation of whole body Ca2+ homeostasis. The role of steroid hormone 17␤-estradiol (E2 ), in [Ca2+ ]i regulation involving TRPV6 has been only limited at the protein expression levels in over-expressing heterologus systems. In the present study, using a combination of calcium-imaging, whole-cell patch-clamp techniques and siRNA technology to specifically knockdown TRPV6 protein expression, we were able to (i) show that TRPV6 is natively, rather than exogenously, expressed at mRNA and protein levels in human T84 colonic cells, (ii) characterize functional TRPV6 channels and (iii) demonstrate, for the first time, the rapid effects of E2 in [Ca2+ ]i regulation involving directly TRPV6 channels in T84 cells. Treatment with E2 rapidly (<5 min) enhanced [Ca2+ ]i and this increase was partially but significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV6 protein expression. These results indicate that when cells are stimulated by E2 , Ca2+ enters the cell through TRPV6 channels. TRPV6 channels in T84 cells contribute to the Ca2+ entry/signalling pathway that is sensitive to 17␤-estradiol. © 2008 Elsevier Ltd. All rights reserved.

Introduction Calcium is one of the most abundant cation in the human body and it is required for many important physiological processes. The Ca2+ concentration in blood and extracel-

∗ Corresponding author. Department of Molecular Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, PO Box 9063, Dublin 9, Ireland. Tel.: +353 1 809 3825; fax: +353 1 809 3778. E-mail address: [email protected] (M. Irnaten). 1 Both authors contributed equally to this work.

lular fluids is dependent upon the balance of movement of Ca2+ via intestinal absorption, bone formation or breakdown, and kidney reabsorption. As all of the Ca2+ in the body is obtained from the diet through gastrointestinal absorption, the regulation of intestinal Ca2+ absorption is crucial for maintaining whole body calcium homeostasis [1]. The human T84 colonic epithelial cell line is a well-characterized tissue culture model of intestinal ion transport and is responsive to hormones involved in the regulation of Ca2+ -dependent absorptive processes. Many hormones, neurotransmitters and physical stimuli elicit cellular responses through changes in the levels of the

0143-4160/$ — see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2008.02.007

442 ubiquitous second messenger Ca2+ . An increase in cytosolic Ca2+ concentration is used as a signal where it activates a large number of key processes including neurotransmitter release, muscle contraction, gene transcription, cell growth and proliferation [2]. Eukaryotic cells can increase cytosolic Ca2+ concentration either by Ca2+ influx into the cytosol across the plasma membrane or by release of Ca2+ from internal stores. However, Ca2+ stores have only a limited capacity and therefore Ca2+ entry is essential for driving most Ca2+ -dependent responses. Adequate with the importance of Ca2+ entry, several distinct Ca2+ -permeable ion channels exist in the plasma membrane. Of these, the known and, perhaps, primordial, is the TRPV6 channel [3]. The basic mechanisms used to signal through [Ca2+ ]i are determined by the fact that the resting level of cytosolic Ca2+ is very low (∼100 nM), while that in intracellular stores and in the surrounding extracellular milieu is around 2 mM (∼10,000-fold higher). As a result, [Ca2+ ]i is set by the balance of passive influx into the cytoplasm and active extrusion of Ca2+ from the cytoplasmic compartment. Calcium ions cross the cell barrier from the apical to the basolateral side by two routes [4]; (i) a passive paracellular pathway in which Ca2+ diffuses down its electrochemical gradient through tight junctions and (ii) transcellular absorption in which Ca2+ enters across the apical membrane of the cell down an electrochemical gradient via apical Ca2+ entry channels [5], crosses the cytosol from the apical side to the basal side by diffusion facilitated by Ca2+ -binding proteins (calbindin-D9k in the intestine) [6] and finally Ca2+ is actively extruded across the basolateral membrane by a plasma membrane Ca2+ -ATPase and a Na+ /Ca2+ -exchanger [7,8]. The molecular identity of the epithelial Ca2+ entry channels remained elusive until the identification of a novel family of Ca2+ -permeable cation channels, the TRPV channels. The epithelial calcium channel TRPV6 is a member of the TRPV subfamily [3]. TRPV6 channels exhibit currents profiles characterized by steep inward rectification at negative membrane potentials, are highly Ca2+ -selective (PCa/PNa > 100), and exhibit robust constitutive activity when over-expressed [9]. TRPV6 channel protein is expressed in kidney, intestine, brain and testis [7,10,11]. TRPV6 is also highly expressed in exocrine tissues such as pancreas, prostate, mammary gland, salivary gland and sweet gland [5,27]. Tissue distribution and electrophysiological properties of TRPV6 channel are consistent with its role in apical Ca2+ uptake during transcellular transport in the intestine. Immuno-histochemical studies have shown that TRPV6 channels are mainly located on the apical membrane small intestine and colon [12]. Functional studies have shown that TRPV6 has a role as an apical Ca2+ entry channel in the transcellular Ca2+ transport pathway [11] and likely controls the apical entry of Ca2+ in gastrointestinal tract and appears to function as gatekeepers for transepithelial Ca2+ movement in the body by intestinal route [13]. TRPV6 form a channel through which Ca2+ enters cells in response to depletion of internal Ca2+ stores, which is known as capacitative Ca2+ entry or storeoperated Ca2+ entry [14]. Active transcellular Ca2+ transport is under the control of calcitrophic hormones including 1␣,25-dihydroxyvitamin D3 (1␣,25(OH)2 D3 ) [15], which presumably acts by transcriptional regulation through the classical vitamin D receptor

M. Irnaten et al. [1,16,17]. Estrogen is considered as a calciotropic hormone although it does not produce immediate detectable effects on extracellular Ca2+ homeostasis. However, there is evidence that E2 plays a key role in long-term whole body Ca2+ homeostasis [18]. Clinical studies demonstrated that E2 deficiency is coupled with a negative Ca2+ balance associated with osteoporosis in postmenopausal women [18,19] and E2 deficiency is also associated with Ca2+ mal-absorption in the intestine which can be corrected by E2 replacement therapy [18,20]. This data suggests a key role of E2 in the maintenance of Ca2+ balance by controlling absorption of Ca2+ through the regulation of TRPV6 Ca2+ channels. However, in spite of the efforts of several laboratories to describe the regulation of TRPV protein expression so far no reports of a rapid modulation of TRPV6 by E2 or other steroid hormones are available. The majority of studies are limited to TRPV6 over-expression in heterologous systems. Here we test the hypothesis that endogenous TRPV6 could be a target for estrogen in the regulation of Ca2+ entry. Previously, we reported the involvement of cytosolic Ca2+ mediating rapid responses to steroid hormone E2 in colonic epithelial cells [21]. On the basis of that initial finding, we suggested that the possible link between E2 and calcium uptake might involve TRPV6 channels. We therefore tested the [Ca2+ ]i responses evoked by E2 in T84 colonic epithelial cells, using siRNA targeting TRPV6. This is the first report to investigate the acute effects of E2 in T84 cells with a particular regard to its eventual effect on calcium influx occurring via TRPV6 channels. Our data support the concept of an implication of TRPV6 channels in [Ca2+ ]i increase evoked by 17␤-estradiol in T84 cells. TRPV6 may be the molecular target for E2 effects on the whole body Ca2+ homeostasis regulation via transepithelial absorption.

Materials and methods Cell culture T84 colonic epithelial cells were initially grown (5—8 days) on T-25 tissue culture flasks in a 1:1 mixture of phenol-red free DMEM/F-12 medium (Sigma Chemical Co., Ireland), supplemented with 10% (v/v) foetal calf serum (Gibco, Paisley, UK), 2 mM L-glutamine (Gibco), 2 U/ml penicillin, 2 mg/ml streptomycin (Gibco) and 1% (v/v) non-essential amino acid at 37 ◦ C in a humidity controlled incubator with 5% CO2 . When confluent, cells were subcultured onto glass coverslips or plastic tissue culture dishes (SARSTEDT, NC, USA). For calcium-imaging measurements, confluent cells grown on glass coverslips were used 3—4 days after plating. Cells were kept for 45 min in Krebs solution to recover prior the experiment. For patch-clamp recordings, after trypsinisation, cells were also kept in Krebs solution for recovering period of minimum 30 min prior to recording. No significant difference in TRPV6 currents in response to E2 in either, cells were kept at 4 ◦ C or at room temperature.

Solutions and chemicals For calcium-imaging and whole-cell patch-clamp experiments, cells were continuously superfused with a standard ‘‘Krebs’’ solution containing (in mM): 145 NaCl; 6 CsCl;

Rapid effects of 17␤-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells 1 MgCl2 ; 10 CaCl2 10 HEPES and 10 glucose, pH 7.4 with CsOH. Na+ -free conditions were obtained by using NMDG-Cl instead of NaCl. When the extracellular Ca2+ concentration was increased to 10, 20 and 100 mM, extracellular NaCl was equimolarly decreased to 130, 115 and 0 mM, respectively, to keep the osmolarity constant. In ‘‘nominal divalent-free solutions’’, Ca2+ and Mg2+ were omitted from the bathing solution. The patch pipette solution contained in all experiments (in mM): 20 CsCl; 100 Cs—Aspartate; 1 MgCl2 ; 10 BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid); Na2 ATP 4, 10 HEPES (pH 7.2). Chemicals were purchased from Sigma Chemical Co. (Ireland) or Tocris Cookson Ltd. (Avonmouth, UK). In all experiments, stock solutions of E2 were prepared in methanol. No effect of the methanol vehicle on TRPV6 currents was observed at the concentrations used to study E2 effects.

Patch-clamp measurements Patch-clamp experiments were performed in the standard whole-cell recording configuration [22]. Initially, we tried to record currents of T84 cells attached to glass cover slips. However, electrophysiological analysis was not feasible under these conditions. We therefore detached subconfluent cells from 6 well plates using trypsin—EDTA. Cells were transferred to a small chamber (Warner Instruments, Hamden, CT, USA) containing appropriate Krebs solution (see Materials and Methods section) and allowed to attach to the glass bottom and recover for 30 min prior the experiment began. Isolated cells were patch-clamped on an inverted microscope (TE 2000-S, Nikon Ltd., Japan). Patch pipettes were prepared from capillary glass (GC150F-10, Harvard Apparatus Ltd., UK) using a programmable puller (DMZ-Universal Puller, Zeitz-Instruments GmbH, Germany) and had an electrical resistance of 2—5 M when filled with the pipette solution. The reference electrode was an Ag—AgCl plug submerged in the bath. The whole-cell configuration was obtained from cell-attached mode after breaking the patch membrane. Whole-cell currents were amplified (Axopatch 200B, Axon instrument, CA), digitised at 5 kHz and low pass-filtered at 1 kHz through an eightpole Bessel filter. Membrane currents were recorded in response to voltage steps (from −120 mV to +100 mV with steps of 20 mV). In some of experiments cells were voltageclamped at a series of holding potentials (+20 mV, −50 mV, −80 mV and −110 mV). Average capacitance of the cell was approximately 14.5 ± 2.2 pF. Experimental protocols and data acquisition were controlled by Axon Instruments pClamp 9.2 software via a Digidata 1322A acquisition system. Drug actions were measured only after steady-state conditions were reached.

RNA isolation and RT-PCR Total RNA was isolated by using Tri-Reagent kit (Molecular Research Center, USA) and reverse-transcribed to the first-strand cDNA with ImProm IITM reverse-transcriptase (Promega, USA). The resulting cDNA was used as template for PCR amplification. Primers for TRPV6 channels were designed by GeneFisher-Software [23], synthesized and purchased (MWG, UK). The following primers were synthe-

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sized: forward 5 -GGTTCCAGACAT-CTTCAGA-3 and reverse 5 -CAATGAGGAGGTTGAGCA-3 for human TRPV6 (accession no. NM 018646). PCR amplification was performed with initial heating for 2 min at 94 ◦ C, followed by 35 cycles of 1 min denaturation at 94 ◦ C, annealing for 1 min at 50.3 ◦ C and extension for 2 min at 72 ◦ C. BLASTN search was performed on primers to confirm that the sequences were not shared with other known genes. The PCR products were electrophoresed through a 2% tris-Borate-EDTA (TBE) agarose gel and amplified cDNA bands were visualized by ethidium bromide staining. The bands were analyzed via Gene Tools software (Syngene, UK).

Western Blot analysis Detection of TRPV6 in T84 cells was carried out using standard immunoblotting protocol and commercially available antibodies. T84 cells were gently washed twice with cold PBS and then scraped into homogenization buffer containing in (mM): 20 Tris—HCl pH 7.6, 250 NaCl, 3 EGTA, 3 EDTA, protease inhibitors and 2 mM DTT. The crude cell lysate was homogenized, sonicated and subsequently centrifuged for 15 min at 14,000 rpm. Protein concentration was determined with the Bio-Rad Protein Assay (Bio-Rad, UK). Equal amounts of extracted total proteins were separated on an 8% SDS-PAGE gels and transferred to PVDF membranes using standard semi-dry technique. Membranes were blocked with 5% non-fat dry milk in Tris Buffered Saline with Tween-20 (TBST) overnight at 4 ◦ C. Rabbit anti-human TRPV6 primary antibody (ACC-036, Alamone Laboratories, Israel) was used at a dilution of 1:500 and an incubation time of 2 h in blocking solution. After washing, membranes were incubated with HRP-Rabbit secondary antibody (1/10,000; Sigma, Ireland) for 1hr. Antibody—protein complexes were detected using an enhanced ECL-plus and chemiluminescent reagent (Amersham, Biosciences, UK). Anti-␤-actin monoclonal antibody (1/4000; Sigma, Ireland) was used as a Loading control. Isolated human colonic crypts and Chinese Hamster Ovary (CHO) cells were used as positive and negative controls, respectively. Densitometric analysis was performed using a Bio-Rad image acquisition system (Bio-Rad Laboratories).

Imaging and cytosolic calcium measurements Experiments were performed on sub-confluent T84 cells cultured on a glass cover slip Petri dishes. [Ca2+ ]i was measured using the Ca2+ -sensitive dye Fura 2-AM. In brief, cells were preloaded in DMEM-F12 containing Fura 2-AM (5 ␮M) for 45 min at room temperature and kept for additional 45 min in appropriate Krebs solution to recover prior the experiment. Cells were then rinsed twice in a solution containing (in mM): NaCl 145, CsCl 6, MgCl2 1, CaCl2 10, HEPES 10, glucose 10 (pH 7.4). We used cesium chloride in the Fura 2-AM experiments to use the same experimental conditions as in the patch-clamp recordings, where CsCl was used instead of KCl to prevent K+ currents. After rinsing, cells were mounted on the stage of an inverted epi-fluorescence microscope (Diaphot 200, Nikon) and excited alternately at wavelengths of 340 and 380 nm. The resultant fluorescence at each excitation wavelength was measured at 510 nm collected using an intensified CCD camera system (Hammatsu, Japan).

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Figure 1 Extracellular Ca2+ -dependence of whole-cell currents in T84 cells. (A) Cells were incubated in Krebs solution with no divalent cations (0 mM Ca2+ , 0 mM Mg2+ ). Under these conditions, data showed a substantial increase of inward current amplitudes in the presence of NaCl in the Krebs solution. This increase was completely abolished when NaCl was replaced by NMDG. Addition of 1 and 10 mM of extracellular Ca2+ induced rise of inward current amplitudes in [Ca2+ ]e -dependent manner. (B) Cells were perfused with five increasing concentrations of extracellular Ca2+ . Typical whole-cell inward current traces measured at the first voltage step of −120 mV at five extracellular Ca2+ concentrations (in mM: 0.5, 1.5, 10, 20 and 100). (C) Current/voltage relationships recorded by measuring the amplitude of currents at: 0.5 mM (䊉), 1.5 mM Ca2+ (), 10 mM Ca2+ (), 20 mM Ca2+ (䊉) and 100 mM Ca2+ (). Data are presented as mean ± S.E.M., n = 9 cells obtained from 9 separate experiments.

Images were digitised and analyzed using Openlab2 software (Improvision, UK). Drug actions were measured only after steady-state conditions were reached. As an internal control of cell Ca2+ responsiveness, 1 ␮M Thapsigargin was added at the end of each experiment. All calcium-imaging experiments were performed in dark at room temperature (20—22 ◦ C) to minimise dye leakage.

Synthesis and transfection of siRNA for human TRPV6 The 21-nucleotide siRNA sequences specifically targeting human TRPV6 were designed and synthesized using the Silencer Pre-designed siRNA construction kit (Ambion Inc., UK). The three specific TRPV6 target sequences used in this study are listed in table one. Cells (0.5 × 106 well−1 ) were seeded into 6-well plates in 2 ml of serum-free medium OptiMEM® 12 h prior to transfection by the calcium phosphate co-precipitation technique. During transfection, cells were incubated at 37 ◦ C in Opti-MEM® serum and antibiotic-free medium for 18 h. Full fresh growth medium was then added. All experimental measurements were performed 72—96 h post-transfection. For functional studies and to monitor transfection efficiencies cells were transfected with a FAM tagged siRNA and 72 h post-transfection FAM positive (fluorescent) cells were counted and compared to the total cell

number. Functional non-coding siRNA#1 (Ambion Research Inc.) was used as control. The estimated percentage of transfection was >90%. The optimal concentration of siRNA used to transfect cells was 25 nM.

Data analysis All data are presented as mean ± S.E.M. for a series of the indicated number of experiments. Patch-clamp data analysis was performed using the clampfit software of the p-clamp suite version 9.2 and Origin 7.5 (OriginLab Corp, MA, USA). Statistical analysis was performed using t-tests and ANOVA followed by hoc test to compare multiple groups, with a P value of 0.05 being considered significant. The use of a paired test reflected that control and experimental measurements were obtained in the same cell.

Results Electrophysiological characterization of TRPV6 channels in T84 cells It has been previously shown that TRPV6 is an inwardly rectifying Ca2+ -permeable channel with a prominent monovalent permeability in divalent cation-free solution [24]. Under these conditions in the presence of sodium as a

Rapid effects of 17␤-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells charge carrier in the extracellular medium, cells show an increase to large inward currents that are completely abolished by substituting extracellular Na+ by NMDG+ (Fig. 1A). Re-application of NaCl to the bathing solution re-established the inward currents indicating that calcium channels lose their selectivity for Ca2+ ions upon removal of extracellular Ca2+ . Na+ currents are greater than when Ca2+ is the charge carrier. When Ca2+ is added to the Krebs solution, the inward current amplitudes increased in calcium-dependent manner (Fig. 1A). Fig. 1B shows typical traces of whole-cell inward Ca2+ currents measured at the first voltage step of −120 mV, witch increase in extracellular Ca2+ concentration-dependent manner. In this protocol, cells were perfused with increasing extracellular Ca2+ concentrations (in mM: 0.5; 1.5, 10, 20 and 100) and clamped at a holding potential of +20 mV. Note that the normal luminal [Ca2+ ] fluctuates between 10 mM and 30 mM in intestine lumen. Fig. 1C shows current—voltage relationships recorded under different extracellular Ca2+ concentrations as indicated in the graph (n = 9 cells). Note that at low [Ca2+ ]e (0.5 mM CaCl2 ), equimolar (0.5 mM) MgCl2 was added to the bath solution to keep divalent cation concentration constant. At this concentration (0.5 mM) the current amplitudes were nearly linear, the membrane potential reverses close to zero and show no inward rectification. Increasing the [Ca2+ ]e to 1.5, 10, 20 and 100 mM, respectively, significantly increased whole-cell inward currents indicating that the current is mainly carried by Ca2+ ions. At [Ca2+ ]e of 100 mM, the mean maximal current density measured at the first voltage step (Vp = −120 mV) was larger than that at lower [Ca2+ ]e (65 ± 6 pA/pF at 100 mM [Ca2+ ]e compared with 51.8 ± 8 pA/pF at 20 mM; 16.2 ± 4 pA/pF at 10 mM; 7.2 ± 3.3 pA/pF at 1.5 mM and 4.7 ± 2.8 pA/pF at 0.5 mM, n = 9). Characteristically, the current profiles displayed a steep inward rectification at negative potentials. The current profiles observed here were similar to those reported by Clapham [25]. Based on these studies and our results, we therefore considered that wholecell inward Ca2+ currents could be mediated by TRPV6 channels. The voltage-dependence of whole-cell Ca2+ currents was also investigated in T84 cells. To address this, we tested if membrane voltage influences the activity of the channels by measuring current activation when cells were clamped at different holding potentials. Whole-cell current density measurements showed that the current amplitude increased in a voltage-dependant manner. Fig. 2A shows superimposed typical traces of whole-cell calcium current recorded at different holding potentials. From a holding potential of +20 mV the membrane potential was changed to −50 mV, to −80 mV and to −110 mV. Currents represented in the graph were measured at the first voltage step of −120 mV. Fig. 2B illustrates the average current amplitude recorded from 7 cells clamped at different holding potentials over the range +20, −50, −80 and −110 mV. At a holding potential of −110 mV, the current measured at the first voltage step (−120 mV) was larger than that at more depolarised membrane potentials (−59.0 ± 6.6 pA/pF at −110 mV compared with −45.8 ± 5.6 pA/pF at −80 mV; −24.2 ± 3.7 pA/pF, at −50 mV; −4.9 ± 3.8 pA/pF at +20 mV, n = 7). The sensitivity of the channel to voltage suggests that a hyperpolarizing potential favors calcium influx. Taken together, these results

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Figure 2 Voltage-dependence of whole-cell currents in T84 cells. T84 Cells were incubated in Krebs solution containing 10 mM Ca2+ in the perfusion medium and 10 mM BAPTA was added to the pipette solution. From a holding potential of +20 mV the membrane potential was changed to −50 mV, to −80 mV and to −110 mV. Note the currents were measured at the first voltage step of −120 mV. (A) Superimposed typical current traces recorded with corresponding holding potentials as indicated in the graph, in response to 2.5 s voltage steps to −120 mV. (B) Average of current amplitudes recorded from 7 cells at the corresponding holding potentials of +20, −50, −80 and −110 mV, respectively.

indicate that the calcium influx into cell may occur via TRPV6 channels in T84 cells.

TRPV6 mRNA is expressed in T84 colonic epithelial cells The whole-cell current profiles recorded resembled the activity of TRPV6 channels; therefore, we explored the presence of the TRPV6 mRNA in T84 cells. For identification of TRPV6 mRNA, RT-PCR was performed with one set of primer pair as described in Materials and Methods section. In all PCR experiments single band at the expected molecular weight was obtained. Ethidium bromide staining of RT-PCR product showed a single band at ∼465 bp corresponding to TRPV6 (Fig. 3, lane 1, upper panel). Positive control has been performed with mRNA extracted from human colon (Fig. 3, lane1, lower panel). Negative controls (RT-PCR run without sample or Reverse-Transcriptase) have been performed and no band was expressed (Lanes 2 and 3) indicating that TRPV6 mRNA is specifically expressed in human T84 colonic cells. Lane 4 represents molecular marker.

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Figure 3 TRPV6 mRNA is expressed in T84 cells. RT-PCR experiments were performed on mRNA isolated from T84 cells and human colon tissue. Lanes 1 represents human colonic T84 cell line (top) and human colon tissue (bottom) as positive control. Lanes 2 and 3 are negative controls (RT-PCR run with no cDNA template or Reverse-Transcriptase. Lane 4 represents DNA ladder. A single band of the predicted size of ∼500 bp was obtained in both T84 cells and human control colon tissue. Data are representative of four similar experiments.

TRPV6 protein is expressed in T84 colonic epithelial cells Western blot experiments were performed on lysates obtained from T84 cells. A single band was detected by rabbit anti-human TRPV6 antibody (Alomone Labs, Israel) and estimated to be of size ∼88 kDa (Fig. 4A, lane 1). To confirm that the proteins detected resulted from specific immunoreaction with the primary antibody, two control blots were performed. (i) Positive control blots were loaded with samples prepared from human colon (Fig. 4, lane 3); (ii) negative control blots run in parallel with samples prepared from CHO cells (Fig. 4, lane 2) compared to blots loaded with samples prepared from T84 cells. No significant expression of TRPV6 (∼4% of control) was observed in CHO cells. This result is in agreement with previously reported studies showing that TRPV6 was not endogenously expressed in CHO cells [26].

E2 elevates [Ca2+ ]i in T84 cells Functional studies were performed in Fura 2-AM loaded T84 cells to determine the modulation of the activity of TRPV6 channel by E2 . Data from spectrofluorescence measurements of [Ca2+ ]i demonstrated that E2 , at a concentration of 50 nM, rapidly (<5 min) induced an increase in [Ca2+ ]i in 50 ± 11% of cells examined (n = 35). Fig. 5A illustrates an original representative experiment. The responding cells showed an E2 -induced sharp and transient rise in [Ca2+ ]i which returned rapidly (within 1—2 min) to basal levels. The [Ca2+ ]i increase was specific to E2 , as vehicle controls, methanol and Krebs solutions, alone did not affect intracellular Ca2+ levels.

Figure 4 TRPV6 protein is expressed in T84 cells. (A) Representative original blot performed with whole-cell protein loaded with samples from T84 cells (lane 1), CHO cells (lane 2, negative control) and human colon tissue (lane 3, positive control). Blots were stripped and reprobed with an anti-␤actin antibody. Single bands are indicated by arrows and the corresponding molecular weight (kDa). (B) The graphs represent densitometric analysis of the blots plotted as percentage of control. Values are displayed as mean ± S.E.M. (n = 3) [*** Significance (p < 0.01) between T84 cells, CHO cells, and human colon tissue.

E2 effect on intracellular Ca2+ and whole-cell currents in T84 cells Whole-cell currents were recorded over 5 min before addition of E2 to ensure stability of the current recording. Exposure of T84 cells to E2 evoked a rapid and transient rise of Ca2+ currents through TRPV6 channels (Fig. 5B). The basal TRPV6 current measured at the first voltage step (−120 mV) was 4.6 ± 3 pA/pF and E2 application stimulated a mean maximal increase in TRPV6 currents of 63 ± 14. pA/pF (at Vp = −120 mV) (n = 9). The involvement of TRPV6 channels in the E2 -induced [Ca2+ ]i increase was tested using ruthenium red, a wellknown effective inhibitor of TRPV6 channels, in Fura 2-AM loaded T84 cells (Fig. 6). Under the absence of ruthenium red conditions E2 induced a significant increase in [Ca2+ ]i (130 ± 4% compared to normalized basal levels 100%, n = 35 cells, P < 0.05). However, when cells were pre-incubated with ruthenium red (50 ␮M), the [Ca2+ ]i increase in response to E2 was partially but significantly reduced (130 ± 4% for E2 alone versus 116 ± 3% for E2 in the presence of ruthe-

Rapid effects of 17␤-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells

Figure 5 Effect of E2 on intracellular Ca2+ and whole-cell currents through TRPV6 in T84 cells. (A) Calcium-imaging performed in Fura 2 loaded T84 cells. Addition of E2 induced a rapid (<5 min) increase in [Ca2+ ]i . E2 -evoked [Ca2+ ]i increase was sharp and transient and Ca2+ returned to basal level within 1—2 min. No increase in [Ca2+ ]i levels was observed in control (untreated) cells (Krebs and Vehicle). The results shown are tracings of a representative cell with similar results observed in separate experiments from 35 cells. (B) Effect of E2 on whole-cell TRPV6 current amplitudes in T84 cells. Time dependence of change in mean maximal current (measured at Vp −120 mV). Cells were allowed to stabilize and dialyze in whole-cell patch-clamp configuration for at least 5 min before exposure to E2 (20 nM). The results shown are mean ± S.E.M. (n = 9) obtained at −120 mV). Arrow indicates time of addition of E2 .

nium red, n = 38 cells, P < 0.05) (Fig. 6). These results suggest that the E2 -induced [Ca2+ ]i increase occurs in part via TRPV6 channels. In addition, the number of cells responding with a rise in intracellular Ca2+ to E2 exposure was reduced in the presence of ruthenium red [50 ± 11% of cells for E2 alone (n = 35 cells) versus 31 ± 3% of cells for E2 in the presence of ruthenium red (n = 38 cells)]. To examine whether the source of [Ca2+ ]i increase induced by E2 is due to external Ca2+ entry, calcium-imaging experiments were performed in nominally extracellular Ca2+ -free Krebs solution (0 mM Ca2+ ). Under these conditions, treatment of T84 cells with E2 did not evoke any increase in [Ca2+ ]i (Fig. 6). These data support the conclusion that the rise of [Ca2+ ]i induced by E2 requires the presence and entry of Ca2+ from the external milieu rather than release from intracellular stores.

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Figure 6 The effect of E2 on [Ca2+ ]i is sensitive to ruthenium red in T84 cells. (A) Data were normalized and presented as % of [Ca2+ ]i changes form control cells. Experiments were performed in different conditions: (i) control Krebs, non-E2 stimulated cells, (ii) cells stimulated with E2 (20 nM), (iii) cells pre-treated with ruthenium red (50 ␮M) and stimulated with E2 and (iv) T84 cells stimulated with E2 in Ca2+ -free conditions. The number of cells is shown inside bar. Data are expressed as mean ± S.E.M. (* Significant differences between cells treated with E2 in absence or presence of ruthenium red. * P < 0.05; *** P < 0.001).

Rapid effect of E2 on TRPV6 channels activity in T84 cells We tested whether the E2 -induced whole-cell Ca2+ current response is mediated by TRPV6 channels in T84 cells. The experiments consisted to expose cells to E2 (20 nM) in the absence or presence of 50 ␮M ruthenium red in the perfusion solution. Patch pipettes were filled with a Cs—Asp solution containing 10 mM BAPTA and the external solution contained 10 mM CaCl2 . Note that the normal luminal Ca2+ concentration fluctuates between 10 mM and 30 mM in the intestine. Fig. 7a—c illustrate representative traces of whole-cell currents obtained in control (untreated) and E2 treated cells in the absence or presence of ruthenium red, respectively. Fig. 7d shows the I—V relationship of E2 effect on TRPV6 whole-cell currents in the absence or presence of ruthenium red. E2 application stimulated a mean maximal increase in TRPV6 currents of 50 ± 6 pA/pF (at Vp = −120 mV) (n = 9; P < 0.001). Ruthenium red addition to the cells significantly reduced the E2 -activated mean maximal increase in TRPV6 currents by 62 ± 4%, corresponding to a reduction of 19.5 ± 2.1 pA/pF (at Vp = −120 mV) (n = 8, P < 0.02). These results further support the conclusion that E2 modulates TRPV6 channels activity in T84 cells. The E2 -induced increase in Ca2+ current is due to Ca2+ influx from the extracellular space as nearly no Ca2+ current increase occurred when T84 cells were incubated in Kerbs containing low (1.5 mM) extracellular Ca2+ (Fig. 7d). The whole-cell conductance (Gc) over the Vp range −120 to −40 mV was increased to 516 ± 35 pS (n = 9) by E2 at 10 mM extracellular Ca2+ compared to

448 Table 1

M. Irnaten et al. Sense and anti-sense siRNA primers designed for knockdown human TRPV6

Target sequences of human TRPV6 siRNA siRNA

Target sequences

siRNA#1 sense Antisense

GCUCUAUGAGGGUCAGACUtt AGUCUGACCCUCAUAGAGCtc

555—573

48 52

siRNA#2 sense Antisense

CCUGCGUGGGAUAAUCAACtt GUUGAUUAUCCCACGCAGGtc

2334—2352

33 33

siRNA#3 sense Antisense

GCACUUUAAAAACAGGCCAtt UGGCCUGUUUUUAAAGUGCtc

2679—2697

38 43

102 ± 1.17 pS (n = 7) at 1.5 mM extracellular Ca2+ . Ruthenium red reduced the E2 change in Gc to 121 ± 8 pS (n = 8), a values close to the Gc recorded under low [Ca2+ ]e conditions. Other reports have established the possibility that ruthenium red could be used as an inhibitor of TRPV6 channels activity [9]. However, this compound has limited suitability for functional studies because of its lack of specificity.

Figure 7 The effect of E2 on currents through TRPV6 is sensitive to ruthenium red in T84 cells. T84 cells were stimulated with E2 (20 nM) in absence and presence of 50 ␮M of ruthenium red (RR) with 10 mM or 1.5 mM [Ca2+ ]e as indicated in the graph. The pipette solution was a Cs—Cl solution supplemented with 10 mM of BAPTA and the holding potential was +20 mV. Typical whole-cell current traces recorded in (a) untreated cells, (b) cells treated with E2 in the absence or (c) in the presence of ruthenium red. (d) Current/voltage relationships of TRPV6 currents measured at: 1.5 mM Ca2+ + 20 nM E2 (n = 7) (), 10 mM Ca2+ + 20 nM E2 (n = 9) () and 10 mM Ca2+ + 50 ␮M RuR + 20 nM E2 (n = 8) (䊉). Data represent the mean ± S.E.M. * Significant differences between cells stimulated by E2 in the absence and presence of RuR, P < 0.05.

Position in gene sequence

GC content (%)

Rapid response of TRPV6 channel activity to E2 in T84 cells Faced with the lack of specific inhibitor of TRPV6 channels, siRNA was employed as an alternative approach to specifically knockdown TRPV6 channel activity in T84 cells. To assess the importance of TRPV6 expression, we determined whether TRPV6 activity induced by E2 is suppressed in TRPV6 siRNA-transfected cells. As different siRNAs targeting the same gene are often differentially effective in silencing, three different siRNAs (siRNA#1, siRNA#2 and siRNA#3) recognizing distinct target sequences in TRPV6 were tested in T84 cells (Table 1). Fig. 8 illustrates the expression of TRPV6 protein in transfected T84 cells in control siRNA (Functional non-coding siRNA#1 (Ambion, Research Inc.)) and three TRPV6-siRNAs. Western blot analysis showed that the expression of TRPV6 protein was reduced by 74 ± 8% and 78 ± 6% when cells were transfected with TRPV6-siRNA#2 and siRNA#3, respectively, compared to control (cells transfected with control functional non-coding siRNA) (100%). In contrast, Western blotting for TRPV6-siRNA#1 revealed no significant reductions in TRPV6 protein expression compared with control cells (n = 3, P > 0.05). Based these results, for subsequent functional expression studies, siRNA#3 was selected for knock down TRPV6 and the control functional non-coding siRNA#1 (Ambion, Research Inc.) was used as control-siRNA. Calcium-imaging and whole-cell patch-clamp techniques were performed in the same experimental conditions. T84 cells were loaded in Ca2+ -sensitive dye Fura2-AM. Fig. 9 shows a (A) typical experiment and (B) average data from spectrofluorescence measurements of [Ca2+ ]i demonstrating that E2 (50 nM) induced [Ca2+ ]i rise in cells transfected with control functional non-coding siRNA, but not in cells treated with TRPV6-siRNA#3 (Fig. 9A and B). The effect of silencing TRPV6 expression in whole-cell TRPV6 mediated currents has been also investigated. Fig. 9C shows a comparison of current amplitudes recorded using the same voltage protocol as in Fig. 7. In cells transfected with TRPV6-siRNA#3, the mean maximal currents induced by E2 treatment was substantially inhibited compared to control cells (transfected with functional non-coding siRNA), corresponding to E2 control of 82.6 ± 11 pA/pF (at Vp = −120 mV) (n = 12, P < 0.005) compared to E2 responses in TRPV6-siRNA#3 cells of 17 ± 4.8 pA/pF (at Vp = −120 mV) (n = 6, P < 0.005). Taken together these results show that the siRNA#3 specifically decreased TRPV6 protein expression

Rapid effects of 17␤-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells

Figure 8 Western blot analysis in cells expressing siRNAtargeting TRPV6. Western blot analysis was performed as described in Materials and Methods section. Blots were stripped and re-probed with an anti-␤-actin antibody. (A) Representative blot for TRPV6 protein expression in T84 cells transfected with negative control non-coding siRNA (control siRNA) and with 3 different TRPV6-siRNAs (siRNA#1, 2 and 3). Single bands for TRPV6 and ␤-actin are indicated by arrows and the corresponding molecular weight (kDa), respectively. (B) The graphs represent densitometric analysis of the blots plotted as percentage of control. Values are displayed as mean ± S.E.M. (n = 3) (** Significance (P < 0.01) between control siRNA and siRNATRPV6 transfected T84 cells.

level leading to the inhibition of TRPV6 channel activity in response to E2 treatment.

Discussion We previously reported that 17␤-estradiol induced [Ca2+ ]i rise in colonic epithelial cells [21]. To our knowledge, the endogenous expression and the acute effects of E2 on [Ca2+ ]i involving TRPV6 have not previously been investigated in human colonic epithelial T84 cells. In the present study, we have investigated the response of [Ca2+ ]i to 17␤-estradiol stimulation in T84 cells and the contribution of TRPV6 channels in this response. We provide the first evidence for (i) a direct, rapid and specific action of 17␤-estradiol on Ca2+ entry through activation of TRPV6 channels and (ii) the expression of TRPV6 protein in T84 cells is associated with a response of cells to changes in [Ca2+ ]i evoked by E2 . We found that TRPV6 is endogenously expressed and functionally active in T84 cells. Previous studies have reported that TRPV6 is expressed at the mRNA level in other cell types [9,27]. At the protein levels, quantitative mRNA expression reports indicate a predominant role of TRPV6 in the

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intestine. TRPV6 appears to be the major apical Ca2+ entry channel for intestine [9]. Several other studies indicate that the highly Ca2+ -selective channel TRPV6 is responsible for apical entry of Ca2+ into cells such as enterocytes [11]. In the intestine, TRPV6 expression was found in the human epithelial cells of esophagus, stomach, small intestine and large intestine [12]. In functional terms, using the patch-clamp technique, we characterized TRPV6 channels in T84 cells by demonstrating the main known characteristics of TRPV6 current profiles. Whole-cell TRPV6 currents display an inwardly rectifying Ca2+ current and are functionally Ca2+ and voltage dependent in T84 cells. We found that extracellular Ca2+ is an important determinant of the activity of TRPV6 channel, since this channel shows no activity in low (0.5 mM) extracellular Ca2+ conditions. At 0.5 mM [Ca2+ ]e , current amplitudes were nearly linear, membrane potentials reverse close to zero and show no inward rectification at negative potentials as shown in Fig. 1. However, increasing the [Ca2+ ]e induces a current with amplitude that depends on the [Ca2+ ]e indicating that the currents are carried by Ca2+ ions. We also found that TRPV6 is voltage-dependent channel, since changing the holding potentials to more negative voltages activated TRPV6 current. Estrogen modulation of TRPV6 channel activity to transport Ca2+ in T84 cells was examined using spectrofluorescence measurements and patch-clamp recordings. Estrogen rapidly (3—5 min) and markedly enhanced [Ca2+ ]i which was partially but significantly prevented by ruthenium red, a reportedly effective inhibitor of TRPV6 channel activity [28]. Ruthenium red also reduced the number of cells responding to E2 with a change in intracellular Ca2+ . The kinetic profile of Ca2+ responses to E2 stimulation was found to be rapid transient peak rise followed by a rapid return to resting levels. Both, spectrofluorescence measurements and patch-clamp whole-cell recordings revealed that E2 rapidly elevates intracellular Ca2+ involving Ca2+ entry channel TRPV6. Exposure of cells to ruthenium red prevented the E2 activation of Ca2+ -dependent TRPV6 currents resulting in a reduction of whole-cell conductance close to the conductance recorded under control conditions, indicating that E2 induces Ca2+ entry primarily via TRPV6 and the ruthenium red insensitive rise in Ca2+ may be due to release from intracellular stores or influx through other Ca2+ entry channels. Considering the potentially important functional impact of TRPV6 channels, the pharmacology of this channel is an important topic. So far no specific inhibitor for this channel has been reported as neither ruthenium red, ecanozole, miconazole or La3+ represent specific blockers of TRPV6 channels [28]. Moreover, the need for a specific blocker of TRPV6 stems not only from a possible therapeutic use, but also from the difficulties in clarifying its physiological role in calcium entry/transport and signalling in epithelia. siRNA represents a suitable alternative to functional blockers of channel activity. This method has proven to be a useful and specific tool to inhibit the function of proteins (including ion channels) and represents a promising tool for therapeutic approaches. In this study, we have tested three independent TRPV6 sequences and observed varying levels of successful TRPV6 knockdown. Among the three-siRNA sequences tested, siRNA#3 is the most effec-

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Figure 9 TRPV6-siRNA blocks the effect of E2 on [Ca2+ ]i rise and TRPV6 currents in T84 cells. Calcium-imaging experiments performed in Fura 2 loaded T84 cells. (A) Cells were either treated with functional non-coding control siRNA or with siRNA#3 prior treatment with E2 . Treatment with E2 induced a rapid (<5 min) increase in [Ca2+ ]i in control non-coding siRNA treated T84 cells but not in siRNA#3 treated T84 cells. The results shown are traces of a representative cell with similar results observed in separate experiments. (B) Averaged data normalized and presented as % of [Ca2+ ]i changes form control cells, as indicated in the graph. Data are expressed as mean ± S.E.M. (* Significant differences between control siRNA and siRNA#3 treated cells (*** P < 0.001). (C) Membrane currents recorded from T84 cells under whole-cell mode. Current/voltage relationships of TRPV6 current density stimulated with E2 in: () Control (non-coding siRNA) treated cells (n = 12) and () cells transfected with siRNA#3 (n = 12). Data represent the mean ± S.E.M (* P < 0.02). * Significant differences between non-coding-siRNA transfected (control) and siRNA#3 transfected cells stimulated with E2 .

tive for knockdown TRPV6. Cells transfected with siRNA#3 showed a significant reduction of TRPV6 protein expression and this reduction is associated with a substantial reduction of the activity of TRPV6 in T84 cells. We found a substantial inhibition of the rise of [Ca2+ ]i evoked by E2 treatment in TRPV6-siRNA transfected T84 cells compared to control (untreated) cells. These findings highlight the potential involvement of TRPV6 channels in calcium entry induced by E2 and support the conclusion that E2 induces Ca2+ entry in T84 cells via TRPV6 channels and consequently TRPV6 might be a suitable target for regulating intracellular calcium homeostasis in response to estrogen. The classical cellular mechanism of E2 action is assumed to involve diffusion of the hormone through the plasma membrane, binding to the nuclear receptor and subsequent transcription and protein synthesis [29]. In addition to this genomic mechanism, there is growing evidence for a rapid non-genomic E2 effects in diverse cell types acting via Ca2+ as a second messenger [30—32] and target-

ing protein kinases and ion transport pathways [33]. The most established signal transduction pathways regulating intracellular calcium flux are generally activated by signaling cascades involving activation of a G-protein coupled receptor or a receptor tyrosine kinase of phospholipase C (PLC). PLC hydrolyzes phosphatidyl-inositol-4,5-bisphospate (PIP2) to form inositol [1,3,4] trisphosphate (IP3) that opens the IP3 receptor (IP3R), and liberates Ca2+ from the endoplasmic reticulum and diacylglycerol (DAG) from phosphatidylinositol-4,5-bisphosphate. Accompanying these chains of events, and not necessarily linked to Ca2+ store depletion, is activation of the TRPV channels. Further reports have indicated that TRPV6 share most of the biophysical properties of the calcium-release-activated calcium (CRAC) channel and its activity is regulated by intracellular Ca2+ store depletion [33], supporting the idea that TRPV6 might, in addition to its crucial role in Ca2+ entry into cell, contribute to pathways of Ca2+ store refilling. The details of these mechanisms are incompletely understood at present.

Rapid effects of 17␤-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells In our laboratory, the rapid, non-genomic, responses to steroid hormones have been most extensively studied in absorptive and secretory epithelia. Our group has demonstrated rapid E2 -induced increases in [Ca2+ ]i via PKC and PKA sensitive pathways. These rapid E2 signaling responses are sex steroid selective and female gender-specific in the intestine [34]. Other studies have shown E2 induces Ca2+ entry in various cell types including rat osteoblasts [29], colonic epithelial cells [21], or pancreatic ␤-cells [35]. However, the nature of the Ca2+ entry pathway is still controversial with evidence for involvement of voltage-dependent Ca2+ channels, voltage-independent Ca2+ channels and/or non-selective cation channels. Although the effects of E2 on intracellular Ca2+ have been reported in a wide variety of tissues, the E2 dependent regulation of Ca2+ transport pathways has been limited to investigating E2 effects at the mRNA and protein expression levels of TRPV6 in over-expressing heterologus systems (usually HEK and CHO cells) and thus lacking a normal physiological hormone signal transduction regulation of TRPV6 channels. Much less is known about the rapid regulation of [Ca2+ ]i by 17␤-estradiol involving the highly Ca2+ -selective TRPV6 channel. The present paper is the first report of a rapid effect of 17␤-estradiol on native TRPV6 channel activity. The results reported here indicate that the expression of TRPV6 protein in T84 colonic cells is associated with a response of the cells to changes in [Ca2+ ]i induced by E2 . We have demonstrated that E2 induces an increase in [Ca2+ ]i which was lacking in cells in which TRPV6 expression was knocked-down with siRNA. We conclude the action of E2 on Ca2+ influx is a result of TRPV6 channel modulation possibly mediated via a nonclassical E2 -receptor since its effect occurs within minutes. Further studies are aimed at uncovering the cellular signals involved in transducing E2 activation of TRPV6 and E2 effects on transepithelial Ca2+ absorption.

Acknowledgments This work was supported by Wellcome Trust program grant 040067/Z/93 and by the Higher Education Authority of Ireland PRTLI Cycle 3 Award.

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