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Am J Physiol Gastrointest Liver Physiol 290: G782-G792, 2006. First published November 10, 2005; doi:10.1152/ajpgi.00441.2005
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MUCOSAL BIOLOGY

TRPC1 functions as a store-operated Ca2+ channel in intestinal epithelial cells and regulates early mucosal restitution after wounding

Jaladanki N. Rao,1,4 Oleksandr Platoshyn,5 Vera A. Golovina,3 Lan Liu,1,4 Tongtong Zou,1,4 Bernard S. Marasa,1,2,4 Douglas J. Turner,1,3 Jason X.-J. Yuan,5 and Jian-Ying Wang1,2,4

Departments of 1Surgery, 2Pathology, and 3Physiology, University of Maryland School of Medicine, and 4Baltimore Veterans Affairs Medical Center, Baltimore, Maryland; and 5Department of Medicine, University of California-San Diego, La Jolla, California

Submitted 19 September 2005 ; accepted in final form 9 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) results from Ca2+ release from intracellular stores and extracellular Ca2+ influx through Ca2+-permeable ion channels and is crucial for initiating intestinal epithelial restitution to reseal superficial wounds after mucosal injury. Capacitative Ca2+ entry (CCE) induced by Ca2+ store depletion represents a major Ca2+ influx mechanism, but the exact molecular components constituting this process remain elusive. This study determined whether canonical transient receptor potential (TRPC)1 served as a candidate protein for Ca2+-permeable channels mediating CCE in intestinal epithelial cells and played an important role in early epithelial restitution. Normal intestinal epithelial cells (the IEC-6 cell line) expressed TRPC1 and TPRC5 and displayed typical records of whole cell store-operated Ca2+ currents and CCE generated by Ca2+ influx after depletion of intracellular stores. Induced TRPC1 expression by stable transfection with the TRPC1 gene increased CCE and enhanced cell migration during restitution. Differentiated IEC-Cdx2L1 cells induced by forced expression of the Cdx2 gene highly expressed endogenous TRPC1 and TRPC5 and exhibited increased CCE and cell migration. Inhibition of TRPC1 expression by small interfering RNA specially targeting TRPC1 not only reduced CCE but also inhibited cell migration after wounding. These findings strongly suggest that TRPC1 functions as store-operated Ca2+ channels and plays a critical role in intestinal epithelial restitution by regulating CCE and intracellular [Ca2+]cyt.

Ca2+ influx; intracellular Ca2+; voltage-gated K+ channels; polyamines; mucosal injury; rapid mucosal repair; canonical transient receptor potential 1

EARLY MUCOSAL RESTITUTION is an important primary repair modality in the gastrointestinal tract. Defective regulation of this process underlies diseases including stress and peptic ulcers, Crohn's disease and ulcerative colitis, mucosal injury or erosions induced by Helicobacter pylori infection, or nonsteroidal anti-inflammatory drug-induced mucosal bleeding. Mucosal restitution occurs as a consequence of intestinal epithelial cell migration to reseal superficial wounds, a process independent of cell proliferation (27, 40, 43). This rapid mucosal reepithelialization after superficial wounds is a complex process that is highly regulated by numerous extracellular and intracellular factors, but its exact mechanism is still unclear. Our previous studies (11, 34–37, 50) and those of others (39, 43) have established the importance of cytosolic free Ca2+ concentration ([Ca2+]cyt) in the control of intestinal epithelial cell migration during restitution and have identified a novel mechanism by which voltage-gated K+ (Kv) channels regulate Ca2+ influx after wounding. In intestinal epithelial cells and other nonexcitable cells, [Ca2+]cyt is increased mainly by Ca2+ influx through Ca2+-permeable channels in the plasma membrane and Ca2+ release from intracellular Ca2+ stores such as the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) (14, 25, 26). Although mobilization from intracellular stores results in a transient increase in [Ca2+]cyt and modulates cell motility, sustained intestinal epithelial cell migration during restitution depends on extracellular Ca2+ influx (11, 34–37, 43, 50).

Ca2+ entry due to store depletion is referred to as capacitative Ca2+ entry (CCE) and is mediated by Ca2+-permeable channels termed store-operated Ca2+ channels (SOCs). CCE through SOCs contributes to maintaining a sustained increase in [Ca2+]cyt and the refilling of Ca2+ into stores (5, 6, 25, 29, 39). Although the participation of Kv channels is critical for the control of Ca2+ influx through regulation of the membrane potential (Em) that governs the driving force for Ca2+ influx during restitution, the exact channel proteins that participate in forming SOCs and mediate CCE in intestinal epithelial cells have not yet been defined. The early leads in the hunt to characterize the molecular basis of the store refilling pathway are provided by results obtained from phototransduction in Drosophila melanogaster. There are three functionally related genes initially cloned and isolated in photoreceptor cells, including the transient receptor potential (TRP) gene (24), the TRP-like gene (31), and the TRP-{gamma} gene (52). To date, seven short mammalian cDNA homologs of Drosophila TRP channels have been identified, termed canonical TRP (TRPC)1–TRPC7 (3, 7, 19, 28, 30, 53, 54, 56). Expression of TRPC genes in oocytes or mammalian cells results in the formation of Ca2+-permeable cation channels that are activated by release/depletion of Ca2+ from internal stores (3, 19, 28, 30, 56), whereas inhibition of TRPC gene expression attenuates SOC currents (Isoc) and prevents the increase in [Ca2+]cyt due to CCE (23, 55). TRPCs have been proposed as molecular candidates for SOCs in nonexcitable cells for many years, but studies examining the functional effects induced by ectopic expression of TRPC genes have shown that characteristics of the expressed activity are distinct from those of endogenous SOC activity (23, 55). In addition, cells transfected with TRPC cDNAs have also shown increased receptor-operated, or basal, Ca2+ influx in some cases (8, 46). These observations suggest that only a few TRPC gene products meet the functional criteria for SOCs. TRPC1, a member of the TRPC subfamily, is highly expressed in a variety of cell types including epithelial cells (18, 35, 47). Recently, TRPC1 has been shown to function as a protein forming native SOCs and is implicated in the store-operated Ca2+ influx mechanism in salivary gland cells (18).

On the basis of the fact that normal intestinal epithelial cells lack voltage-dependent Ca2+ channels (VDCC) but express TRPCs (35, 50), the present study tested the hypotheses that TRPC1 is a candidate protein for Ca2+-permeable channels mediating CCE in normal intestinal epithelial cells and plays an important role in early epithelial restitution after injury. First, we characterized the functional expression of TRPCs and SOCs in IEC-6 cells derived from normal rat small intestinal crypts. Second, we determined the changes in CCE and cell migration during restitution in IEC-6 cells stably transfected with TRPC1 cDNA. Finally, we investigated whether decreased expression of TRPC1 by small interfering (si)RNA targeting a specific site of the TRPC1 mRNA coding region decreased CCE and inhibited cell migration in differentiated IEC-Cdx2L1 cells that highly express endogenous TRPCs. Some of these data have been published previously in abstract form (38).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media, isopropyl-beta-D-thiogalactopyranoside (IPTG), LipofectAMINE, and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA), and other biochemicals were obtained from Sigma (St. Louis, MO). The primary antibody, an affinity-purified rabbit polyclonal antibody against TRPC1 or TRPC5, was purchased from Alomone Laboratories (Jerusalem, Israel). Flourescein-conjugated goat anti-rabbit antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Plasmid construction and transfection. The full-length cDNA of human TRPC1 (~3.8 kb) was inserted into the NotI and ApaI sites of expression vector pcDNA3.0(+) (Invitrogen) with the cytomegalovirus promoter (pcDNA-TRPC1) (51). IEC-6 cells were transfected with pcDNA-TRPC1 or pcDNA3.0(+) vectors containing no TRPC1 cDNA using a LipofectAMINE kit and performed as recommended by the manufacturer (Invitrogen). After a 3-h period of incubation, the transfection medium was replaced by standard growth medium containing 5% FBS for 2 days before exposure to the selection medium. These transfected cells were selected for TRPC1 integration by incubation with selection medium containing 0.6 mg/ml G418, and clones resistant to the selection medium were isolated, cultured, and screened for TRPC1 expression by RT-PCR using specific TRPC1 primers (Table 1) and Western blot analysis with specific anti-TRPC1 antibody.


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  		Table 1. Oligonucleotide sequences of primers used for RT-PCR

 
RNA interference. The siRNA that was designed to specifically cleave TRPC1 mRNA was synthesized (42, 45) and purchased from Sequitur (Natick, MA). siRNA nucleotides were screened against the GenBank database (GenBank Accession No. U31110), and no matches were found to other targeted genes. Scrambled control siRNA (C-siRNA), which had no sequence homology to any known genes, was used as the control. For each 60-mm cell culture dish, 15 µl of the 20 µM stock siRNA or C-siRNA were mixed with 300 µl of Opti-MEM (Invitrogen). This mixture was gently added to a solution containing 15 µl LipofectAMINE 2000 in 300 µl Opti-MEM. The solution was incubated for 20 min at room temperature and gently overlaid onto monolayers of cells in 3 ml of medium, and cells were harvested for various assays after a 48-h incubation.

Cell culture and general experimental protocol. IEC-6 and IEC-18 cell lines were purchased from the American Type Culture Collection (ATCC) at passage 13. These two cell lines were derived from normal rat intestinal crypt cells and were developed and characterized by Quaroni et al. (32). Stock cells were maintained in T-150 flasks in DMEM supplemented with 5% heat-inactivated FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2, and passages 15–20 were used in the experiments. There were no significant changes of biological function and characterization of IEC-6 cells at passages 15–20 (20, 49). The stable IEC-Cdx2L1 cells were developed and characterized by Suh and Traber (35) and were kind gifts from Dr. Peter G. Traber (Baylor College of Medicine, Houston, TX). The expression vector, the LacSwitch System (Stratagene, La Jolla, CA), was used for directing the conditional expression of the Cdx2 gene, and IPTG served as an inducer for the gene expression. Stock stable IEC-Cdx2L1 cells were grown in DMEM as described above. Before experiments, IEC-Cdx2L1 cells were grown in DMEM containing 4 mM IPTG for 16 days to induce cell differentiation as described in our previous studies (33, 35, 44). The Caco-2 cell line (a human colon carcinoma cell line) was also obtained from ATCC at passage 16 and was maintained similarly to the IEC-6 cell line except that it was maintained in an atmosphere of 95% air-5% CO2. The medium used was Eagle's MEM with 10% heat-inactivated FBS, and passages 18–23 were used for the experiments.

In the first series of experiments, we characterized the functional expression of TRPCs and SOCs in normal IEC-6 cells. Cells were grown in control cultures for 4 days, and the monolayers were then washed three times with ice-cold Dulbecco's PBS. Different solutions were added according to the assays to be conducted. Expression of TRPC mRNAs was examined by RT-PCR analysis using various specific primers (Table 1), whereas levels of TRPC proteins were measured by Western immunoblot analysis using specific anti-TRPC antibodies. Whole cell Isoc was recorded by the patch-clamp technique (10), and resting [Ca2+]cyt and CCE were measured by digital cellular imaging methods (50).

In the second series of experiments, we determined whether increased TRPC1 expression by stable transfection of the TRPC1 cDNA altered CCE and cell migration during restitution. IEC-6 cells were transfected with pcDNA-TRPC1 expression vectors, and stable clones that highly expressed TRPC1 were selected and used in this study. Cells transfected with pcDNA(+) vector lacking TRPC1 cDNA served as a control. CCE generated by Ca2+ influx after depletion of the Ca2+ store was measured as described above, and cell migration was assayed 6 h after removal of part of the cell layers (33, 50).

In the third series of experiments, we investigated whether inhibition of endogenous TRPC1 expression by transfection with TRPC1 siRNA decreased CCE and inhibited cell migration after wounding in differentiated IEC-Cdx2L1 cells. After cells were grown in control DMEM for 2 days, they were transfected with either TRPC1 siRNA or C-siRNA. Changes in CCE generated by Ca2+ influx and cell migration were measured 48 h after the transfection.

RT-PCR. Total RNA was isolated using the RNeasy Mini Kit (Qiagen; Valencia, CA). Equal amounts of total RNA (5 µg) were transcribed to synthesize single-strand cDNA with a RT-PCR kit (Invitrogen). The specific sense and antisense primers for TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, L-type VDCC-{alpha}1, and VDCC-beta1 subunits were designed from the cDNA sequences of the coding regions corresponding to the genes (Table 1). These particular sequences were chosen on the basis of previously established specificity (10, 22). RT-PCR was performed as described in our earlier studies (35, 50). To quantify the PCR products (the amounts of mRNA) of TRPCs, an invariant mRNA of beta-actin was used as an internal control. The optical densiometry (OD) values for each band on the gel were measured by a Gel Documentation System (UVP; Upland, CA), and their signals were normalized to the OD values in the beta-actin signals.

Western blot analysis. Cell samples, placed in SDS sample buffer, were sonicated and centrifuged (12,000 rpm) at 4°C for 15 min. The supernatant from cell samples was boiled for 5 min and then subjected to electrophoresis on 7.5% SDS-PAGE gels according to Laemmli (17). After the transfer of protein onto nitrocellulose filters, filters were incubated for 1 h in 5% nonfat dry milk in 1x PBS-Tween 20 [PBS-T; 15 mM NaH2PO4, 80 mM Na2HPO4, 1.5 M NaCl (pH 7.5), and 0.5% (vol/vol) Tween 20]. Immunological evaluation was then performed for 1 h in 1% BSA-PBS-T buffer containing 1 µg/ml of specific antibody against TRPC1 or TRPC5 protein. The filters were subsequently washed with 1x PBS-T and incubated for 1 h with the second antibody conjugated with horseradish peroxidase for 1 h at room temperature. The immunocomplexes on the membranes were reacted for 1 min with Chemiluminiscence Reagent (NEL-100 DuPont NEN).

Electrophysiological measurements. Whole cell Isoc was recorded with an Axiopatch-1D amplifier using patch-clamp techniques (10). Patch pipettes (2–4 M{Omega}) were made on a Sutter electrode puller using borosilicate glass tubes and fire polished on a Narishige microforge. Voltage stimuli were delivered from a holding potential of 0 mV using voltage steps from –120 to 0 or +20 mV. Current traces recorded before the activation of SOCs was activated by passive depletion of store Ca2+ using 10 mM cyclopiazonic acid (CPA). The bath (extracellular) solution for recording optimal Isoc contained (in mM) 120 Na methane sulfonate, 20 Ca2+ aspartate, 0.5 3,4-diaminopyridine, 10 glucose, and 10 HEPES (pH 7.4 with methane sulfonic acid). The (intracellular) pipette solution contained (in mM) 138 Cs aspartate, 1.15 EGTA, 1 Ca(OH)2, 2 Na2-ATP, and 10 HEPES (pH 7.2). These ionic conditions eliminated the currents through K+ and Cl– channels. In Ca2+-free bath solution, Ca2+ aspartate was replaced by equimolar Na aspartate to maintain osmolarity.

CPA was dissolved in DMSO to make a stock solution of 30 mM. Aliquots of the stock solution were then diluted 1:3,000 into the bath solution or culture medium to make a final concentration of 10 µM CPA (pH 7.4). pH values of all solutions were checked after the addition of the chemicals and readjusted to 7.4.

Measurement of [Ca2+]cyt. Details of the digital imaging methods employed for measuring [Ca2+]cyt have been described in our previous studies (34–36, 50). Briefly, either parental IEC-6 cells or IEC-Cdx2L1 cells were plated on 25-mm coverslips and incubated in culture medium containing 3.3 µM fura-2 AM for 30–40 min at room temperature (22–24°C) under an atmosphere of 10% CO2 in air. Fura-2 AM-loaded cells were then superfused with standard bath solution for 20–30 min at 22–24°C to wash away extracellular dye and permit intracellular esterases to cleave cytosolic fura-2 AM into active fura-2. Fura-2 fluorescence from the cells and background fluorescence were imaged using a Nikon Diaphot microscope equipped for epifluorescence. Fluorescent images were obtained using a microchannel plate image intensifier (Amperex XX1381, Opelco; Washington, DC) coupled by fiber optics to a Pulnix charge-coupled device video camera (Stanford Photonics; Stanford, CA). Image acquisition and analysis were performed with a Metamorph Imaging System (Universal Imaging). [Ca2+]cyt was calculated from fura-2 fluorescent emission excited at 380 and 340 nm using the ratio method (29).

Measurement of cell migration. Migration assays were carried out as described in our earlier studies (11, 34–37, 50). Cells were plated at 6.25 x 104 cells/cm2 in DMEM plus FBS on 60-mm dishes thinly coated with Matrigel according to the manufacturer's instructions and incubated as described for stock cultures. The cells were fed on day 2, and migration was tested on day 4. To initiate migration, the cell layer was scratched with a single-edge razor blade cut to ~27 mm in length. The scratch was made over the diameter of the dish and extended over an area of 7–10 mm wide. The migrating cells in six contiguous 0.1-mm squares were counted at x100 magnification beginning at the scratch line and extending as far out as the cells had migrated. All experiments were carried out in triplicate, and the results are reported as numbers of migrating cells per millimeter of scratch.

Statistical analysis. All data are expressed as means ± SE from six dishes. PCR and autoradiographic results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined using the Duncan's multiple-range test (13).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of TRPC mRNAs and proteins in normal intestinal epithelial cells. To determine the potential involvement of TRPCs in CCE in normal intestinal epithelial cells, basal expression of TRPC genes was examined in IEC-6 cells. This line of cells was used as a model, because IEC-6 cells represent normal intestinal epithelial cells (32) and are extensively used and widely accepted as an in vitro system for cell division-independent stage of epithelial restitution (21, 34, 35, 41, 50). Total cellular RNAs isolated from rat brain tissue served as a positive control in this study. As shown in Fig. 1A, IEC-6 cells expressed mRNAs of TRPC1 and TRPC5, which encode Ca2+-permeable channels involved in CCE in mammalian cells (23, 29). Although TRPC2, TRPC3, TRPC4, TRPC6, and TRPC7 were highly expressed in brain tissue, they were not detectable in IEC-6 cells by RT-PCR analysis. Consistent with our previous studies (35, 50), IEC-6 cells did not express the pore-forming ({alpha}1) and regulatory (beta1) subunits of VDCC, although both VDCC-{alpha}1 and VDCC-beta1 subunits were highly expressed in rat brain tissue. Furthermore, expressed mRNAs of TRPC1 and TRPC5 in IEC-6 cells were paralleled by expression of their proteins as measured by Western blot analysis (Fig. 1B). To extend these findings observed in IEC-6 cells, we further examined the expression of TRPC1 and TRPC5 in IEC-18 cells (another line of normal rat crypt cells) and Caco-2 cells (a line of human colon epithelial cells). As shown in Fig. 1C, both IEC-18 and Caco-2 cells also expressed TRPC1 and TRPC5 proteins. These results clearly show that TRPC1 and TRPC5 are present in intestinal epithelial cells.


Figure 1
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  		Fig. 1. Expression of canonical transient receptor potential (TRPC) channels and voltage-dependent Ca2+ channels (VDCC) in intestinal epithelial cells. Cells were cultured in standard DMEM containing 5% dialyzed fetal bovine serum (FBS) for 4 days, and total cellular RNA and protein were harvested for RT-PCR and Western immunoblot analysis. A: mRNA levels in IEC-6 cells. First-strand cDNAs, synthesized from total cellular RNA, were amplified with specific sense and antisense primers (see Table 1), and PCR-amplified products are displayed in agarose gel for TRPC1 (~372 bp), TRPC2 (~560 bp), TRPC3 (~850 bp), TRPC4 (~415 bp), TRPC5 (~340 bp), TRPC6(~327 bp), TRPC7 (~563 bp), VDCC-{alpha}1 (~372 bp), VDCC-beta1 (~549 bp), and beta-actin (~244 bp). RNA isolated from rat brain tissue served as a positive control. M, molecular mass marker. B: protein levels in cells described in A. Whole cell lysates were applied to each lane (20 µg) and subjected to electrophoresis on a 10% acrylamide gel. Levels of TRPC1 (~220 kDa) and TRPC5 (~95 kDa) proteins were identified by probing nitrocellulose membranes with the specific antibodies as described in MATERIALS AND METHODS. After the blot was stripped, actin (~45 kDa) immunoblotting was performed as an internal control for equal loading. C: representative immunoblots of Western immunoblot analysis for TRPC1 and TRPC5 proteins in IEC-18 and Caco-2 cells. Three separate experiments were performed that showed similar results.

 
SOC activity and CCE in IEC-6 cells. To determine the possibility that TRPCs are implicated in CCE generated by Ca2+ influx, we examined whether the expression of TRPC1 and TRPC5 was associated with SOCs in IEC-6 cells. Activity of SOC, or Isoc, is critically determined by the total number of functional SOC proteins expressed in the plasma membrane. If TRPC1 and TRPC5 encode the proteins involved in forming native SOCs, expressed proteins of TRPC1 and TRPC5, as shown in Fig. 1, would increase SOC activity. To determine this possibility, we measured whole cell Isoc in IEC-6 cells. Whole cell Isoc was elicited in IEC-6 cells held at 0 mV to inactivate voltage-dependent Na2+ channels and VDCCs by a series of test potentials ranging between –120 and 20 mV from a holding potential of 0 mV (which inactivates voltage-dependent Na2+ channels and VDCCs). The inward currents at negative test potentials were mainly generated by Ca2+ influx, and the outward currents at positive potentials were putatively generated by Cs+ efflux because 1) the permeability ratios for Ca2+ and Na+ are usually on the order of 10:1 under physiological conditions with extracellular Ca2+ in millimolar ranges (1, 4, 30); and 2) the permeability of SOCs to Na+, K+, and Cs+ is equal (15). The results presented in Fig. 2 clearly show that the amplitude of Isoc in IEC-6 cells was activated by passive depletion of store Ca2+ through treatment with 10 µM CPA, an inhibitor of Ca2+-Mg2+-ATPase in the ER. CPA-induced Isoc in IEC-6 cells was significantly inhibited by lanthanum, a chemical inhibitor of SOCs (15, 16, 22). Because IEC-6 cells do not express VDCCs and other TRPCs, these results suggest that expressed proteins of TRPC1 and TRPC5 subunits contribute to forming different kinds of SOCs and play a critical role in CCE in intestinal epithelial cells.


Figure 2
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  		Fig. 2. Whole cell store-operated Ca2+ channel (SOC) current (Isoc) activated by cyclopiazonic acid (CPA)-induced passive depletion of intracellular Ca2+ stores in the IEC-6 cells described in Fig. 1. A: images of IEC-6 cells. The arrow indicates the pipette. B: representative families of Isoc elicited by 300-ms voltage steps from –120 to +20 mV in 20-mV increments before (control) and after 10-min application of CPA (10 µM) in the absence or presence of 50 µM lanthanum (Lan). C: summarized data showing the current-voltage (I-V) relationship of Isoc in the IEC-6 cells described in B. CPA-induced Isoc was significantly inhibited by exposure to Lan. Data are expressed as means ± SE; n = 16.

 
To determine if the observed SOC activity was associated with CCE, levels of [Ca2+]cyt were measured after depletion of store Ca2+ by CPA. As shown in Fig. 3, exposure of IEC-6 cells to CPA, by blocking Ca2+ sequestration into the ER/SR, induced a transient increase in [Ca2+]cyt because of leakage of Ca2+ from the ER to the cytosol in the absence of extracellular Ca2+ concentration ([Ca2+]o). The CPA-induced [Ca2+]cyt transient in cells bathed in Ca2+-free solution declined back to the baseline level with the depletion of store Ca2+. Under these conditions, restoration of [Ca2+]o induced a rise in [Ca2+]cyt, which was apparently due to CCE. Blockade of SOCs by lanthanum prevented the transient increase in [Ca2+]cyt induced by CPA. On the other hand, the membrane depolarization induced by raising the extracellular K+ concentration from 4.7 to 40 mM did not affect [Ca2+]cyt (data not shown) because IEC-6 cells did not express VDCCs (35, 50). Taken together, these findings indicate that normal intestinal epithelial cells express TRPC1 and TRPC5, which are paralleled by CCE generated by Ca2+ influx through SOCs.


Figure 3
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  		Fig. 3. Capacitative Ca2+ entry (CCE) in the IEC-6 cells described in Fig. 1. A: representative records showing the time course of cytosolic Ca2+ concentration ([Ca2+]cyt) measured at the indicated times [basal (a), cells treated with CPA in the absence of extracellular Ca2+ (CPA-0Ca2+; b), cells exposed to CPA in the presence of extracellular Ca2+ (CPA-Ca2+; c), cells exposed CPA plus Lan in the presence of extracellular Ca2+ (CPA-Ca2+-Lan; d), and cells washed with control solution (washout; e)]. B: pseudocolor images showing [Ca2+]cyt measured at times (a, b, c, d, and e) corresponding to those shown in A. The 380-nm excitation image (black and white image) of the cells used for measuring [Ca2+]cyt is shown in the first column to clarify the size of cells (scale bars = 10 µm). Three separate experiments were performed that showed similar results.

 
Ectopic expression of the TRPC1 gene increases CCE. To further define the role of TRPCs in CCE in intestinal epithelial cells, stable TRPC1-transfected IEC-6 cells (TRPC1-transfected IEC cells) were developed and characterized. The expression vector encoding the full-length cDNA of human TRPC1 under the control of the cytomegalovirus promoter was constructed (Fig. 4A). The results presented in Fig. 4B show that four clones that were resistant to the selection medium containing 0.6 mg/ml G418 highly expressed TRPC1 mRNA and protein. Levels of TRPC1 mRNA and protein were approximately fivefold the value of parental IEC-6 cells. There were no significant differences in levels of TRPC1 mRNA and protein between parental IEC-6 cells and the clone that was transfected with the control vector containing no TRPC1 cDNA (null) but also exposed to the same dose of G418.


Figure 4
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  		Fig. 4. Characterization of stable TRPC-transfected IEC cells. A: structure of expression vector [null (a) and TRPC1 expression vector (b)]. The complete open reading frame of human TRPC1 cDNA was cloned to the expression pcDNA3.1(+) vector. B: expression of TRPC mRNA (a) and protein (b). IEC-6 cells were transfected with the TRPC1 expression vector by the LipofectAMINE technique, and clones resistant to the selection medium containing 0.6 mg/ml G418 were isolated and screened for TRPC1 expression by RT-PCR and Western blot analysis. Total RNA and whole cell lysates from each stable TRPC1-transfected clone (C1 and C2) and cells transfected with the null vector (control) were harvested, and levels of TRPC1 mRNA and protein were measured by RT-PCR and Western immunoblot analysis. Actin mRNA and protein were examined as internal controls for equal loading. Three separate experiments were performed that showed similar results.

 
Consistently, these stable TRPC1-transfected IEC cells were associated with a significant increase in levels of CCE (Fig. 5). Although resting [Ca2+]cyt and transient Ca2+ release from the store depletion were unchanged, sustained Ca2+ influx through CCE after Ca2+ store depletion was increased by approximately twofold in TRPC1-transfected IEC cells (TRPC1-C1 and TRPC1-C2). Importantly, the patterns of increased Ca2+ influx activity associated with TRPC1 expression displayed characteristics identical to endogenous SOCs in parental IEC-6 cells. We also examined changes in CCE in other TRPC1 independently transfected clones such as TRPC1-C3 and TRPC1-C4 and demonstrated that levels of Ca2+ influx through SOCs were similar to those observed in TRPC1-C1 and TRPC1-C2. These findings indicate that TRPC1 is implicated in CCE in intestinal epithelial cells and provide a fundamental basis for studies to define the role of TRPC1 in intestinal epithelial restitution after wounding.


Figure 5
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  		Fig. 5. Resting [Ca2+]cyt and CCE induced by CPA in the stable TRPC1-transfected IEC (IEC-TRPC1) cells described in Fig. 4. A: representative records of [Ca2+]cyt changes measured in peripheral areas of parental IEC-6 cells (null) and stable IEC-TRPC1 cells. The Ca2+ store was depleted by treatment with CPA in the absence of extracellular Ca2+ (0Ca2+). B: summarized data showing [Ca2+]cyt (a) and the amplitude of CPA-induced Ca2+ influx (b) in control and IEC-TRPC1 cells. Data are expressed as means ± SE; n = 30. *P < 0.05 compared with null vector-transfected IEC-6 cells.

 
Stable TRPC1-transfected IEC cells exhibit increased migration after wounding. To determine the effect of forced expression of the TRPC1 gene on early rapid mucosal repair after injury, an in vitro model that mimics the early cell division-independent stages of epithelial restitution was used, as described in our previous studies (34, 35, 50) and those of others (21, 41). As shown in Fig. 6, the migration rate of stable TRPC1-transfected IEC cells increased significantly during restitution after wounding. The numbers of cell migrating over the wounded edge in TRPC1-transfected IEC cells were increased by ~50% compared with parental IEC-6 cells that were transfected with the null vector after wounding. Increased migration in TRPC1-transfected IEC cells is not simply due to clonal variation, because identical results were observed when four independently transfected clones (TRPC1-C1, TRPC1-C2, TRPC1-C3, and TRPC1-C4) were analyzed. These findings demonstrate that increased TRPC1 and the resultant induction of CCE in stable TRPC1-transfected IEC cells increase intestinal epithelial cell migration during restitution.


Figure 6
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  		Fig. 6. Changes in cell migration after wounding in the IEC-TRPC1 cells described in Fig. 4. A: images of cell migration after wounding by removal of part of the monolayer [0 h after wounding (a), 6 h after wounding in parental IEC-6 cells (control; b), 6 h after wounding in IEC-TRPC1-C1 cells (c), 6 h after wounding in IEC-TRPC1-C2 cells (d), 6 h after wounding in IEC-TRPC1-C3 cells (e), and 6 h after wounding in IEC-TRPC1-C4 cells (f)]. Cells were grown in standard DMEM for 4 days, and cell migration was assayed 6 h after wounding. B: summarized data showing rates of cell migration after wounding in control and TRPC1-IEC cells. Data are expressed as means ± SE from 6 dishes. *P < 0.05 compared with IEC-6 cells transfected with the vector containing no TRPC1 cDNA.

 
Changes in TRPC expression and CCE in differentiated IEC-Cdx2L1 cells. It has been shown that forced expression of the cdx2 gene in IEC-6 cells induces the development of a differentiated phenotype (33, 44). These differentiated IEC-Cdx2L1 cells were polarized, exhibited multiple morphological characteristics of villus-type enterocytes, and also expressed brush-border enzymes such as sucrase-isomaltase (data not shown). As shown in Fig. 7A, a, differentiated IEC-Cdx2L1 cells expressed higher basal levels of TRPC1 and TRPC5 than parental IEC-6 cells. mRNA levels of TRPC1 and TRPC5 in differentiated IEC-Cdx2L1 cells were ~4.6- and ~1.6-fold greater, respectively, than those of parental IEC-6 cells. Increased levels of TRPC1 and TRPC5 mRNAs were paralleled by increases in their proteins (Fig. 7B,b). Levels of TRPC1 and TRPC5 proteins in differentiated IEC-Cdx2L1 cells were ~5.2 and ~1.5 times greater than those in parental IEC-6 cells. In addition, increased expression of TRPCs in IEC-Cdx2L1 cells did not result from clonal variation, because similar results were observed when another Cdx2-transfected clone, the IEC-Cdx2L2 line, was analyzed (data not shown).


Figure 7
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  		Fig. 7. Changes in TRPC expression and CCE in parental IEC-6 and stable cdx2-transfected IEC-6 (IEC-Cdx2L1) cells. A: expression of TRPC1 and TRPC5 mRNA (a) and protein (b). Before the experiments, IEC-Cdx2L1 cells were grown in DMEM containing 5% FBS in the presence of 4 mM isopropyl beta-D-thiogalactopyranoside (IPTG; the inducer for gene expression) for 16 days to induce cell differentiation. Both parental IEC-6 cells and differentiated IEC-Cdx2L1 cells were then cultured in DMEM for 4 days, and total RNA and whole cell lysates were harvested for measurements of TRPC expression. Levels of TRPC mRNA and protein were measured by RT-PCR and Western immunoblot analysis. Actin mRNA and immunoblotting were performed as internal controls for equal loading. Three separate experiments were performed that showed similar results. B: representative records of [Ca2+]cyt measured in peripheral areas of parental IEC-6 cells (a) and differentiated IEC-Cdx2L1 cells (b) as described in A. C: summarized data showing resting [Ca2+]cyt (a) and the amplitude of CPA-induced Ca2+ influx (b) in the parental IEC-6 cells and differentiated IEC-Cdx2L1 cells described in B. Data are expressed as means ± SE; n = 25. *P < 0.05 compared with parental IEC-6 cells.

 
The high levels of TRPC expression in differentiated IEC-Cdx2L1 cells were associated with significant increases in both resting [Ca2+]cyt and the amplitude of CCE (Fig. 7, B and C). Although there were no statistically significant differences in the CPA-induced initial transient increase in [Ca2+]cyt (resulting from Ca2+ store depletion) between differentiated IEC-Cdx2L1 cells and parental IEC-6 cells, there was a sustained increase in [Ca2+]cyt because of CCE in IEC-Cdx2L1 cells. Maximal levels of [Ca2+]cyt induced by CCE were ~1,255 ± 61 nM in differentiated IEC-Cdx2L1 cells and ~690 ± 33 nM in parental IEC-6 cells. Furthermore, increased CCE in differentiated IEC-Cdx2L1 cells was associated with a significant increase in cell migration after wounding. The numbers of cells migrating over the wounded edge in differentiated IEC-Cdx2L1 cells were almost four times that of parental IEC-6 cells at 6 h after wounding (from 105 ± 5 cells/mm in IEC-6 cells to 412 ± 12 cells/mm in IEC-Cdx2L1 cells). These results strongly suggest that increased expression of TRPC1 and TRPC5 in differentiated IEC-Cdx2L1 cells are implicated in the increase in CCE and play an important role in the stimulation of cell migration after wounding.

Effect of inhibition of endogenous TRPC1 expression on CCE and cell migration. Because differentiated IEC-Cdx2L1 cells are associated with a high expression of endogenous TRPCs and increased CCE, they provide an excellent model for the present study. To determine the roles of TRPCs in CCE and epithelial restitution, siRNA nucleotides complementary to TRPC1 mRNA were used to specially block endogenous TRPC1 in differentiated IEC-Cdx2L1 cells. These specific siRNA nucleotides were designed to cleave rat TRPC1 mRNA by activating endogenous RNase H and have a unique combination of specificity, efficacy, and reduced toxicity (45). Initially, we determined the transfection efficiency of siRNA nucleotides in differentiated IEC-Cdx2L1 cells and demonstrated that >95% of cells were positive when they were transfected with fluorescent FITC-conjugated C-siRNA for 24 h (data not shown). As shown in Fig. 8, exposure to TRPC1 siRNA dramatically decreased the levels of TRPC1 mRNA and protein in differentiated IEC-Cdx2L1 cells. Levels of TRPC1 protein were decreased by ~80% when cells were exposed to siRNA nucleotides for 24 or 48 h. Transfection with C-siRNA at the same concentrations showed no inhibitory effects. In addition, neither TRPC1 siRNA nor C-siRNA affected cell viability as measured by trypan blue staining (data not shown).


Figure 8
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  		Fig. 8. Effect of treatment with small interfering (si)RNA targeting the TRPC1 mRNA coding region (siTRPC1) on levels of TRPC1 mRNA (A) and protein (B) in differentiated IEC-Cdx2L1 cells as described in Fig. 7. Differentiated IEC-Cdx2L1 cells were transfected with either control siRNA (C-siRNA) or siTRPC1 at the concentration of 0.5 µg/ml by the LipofectAMINE technique. Total RNA and whole cell lysates were harvested 24 and 48 h after transfection, and levels of TRPC1 mRNA and protein were measured by RT-PCR and Western immunoblot analysis, respectively. Actin mRNA and immunoblotting assays were performed as internal controls for equal loading. Three separate experiments were performed that showed similar results.

 
Decreased levels of TRPC1 by the transfection with TRPC1 siRNA inhibited CCE due to store depletion and decreased cell migration after wounding in differentiated IEC-Cdx2L1 cells. The increase in [Ca2+]cyt due to CPA-induced CCE in cells treated with TRPC1 siRNA was decreased by ~60% (Fig. 9). Furthermore, inhibition of TRPC1 expression and the subsequent decrease in CCE by TRPC1 siRNA also impaired intestinal epithelial restitution after wounding (Fig. 10). The number of cell migrating over the denuded area in cells transfected with TRPC1 siRNA was decreased by ~27% 6 h after wounding compared with that observed in IEC-Cdx2L1 cells transfected with C-siRNA. C-siRNA had no effects on both CCE (Fig. 9) and cell migration (Fig. 10). These findings strongly suggest that TRPCs play a critical role in the stimulation of cell migration after wounding.


Figure 9
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  		Fig. 9. Effect of inhibition of TRPC1 expression by treatment with siTRPC1 on the resting [Ca2+]cyt and CPA-induced Ca2+ influx in differentiated IEC-Cdx2L1 cells as described in Fig. 8. A: representative records of [Ca2+]cyt changes measured in peripheral areas of control differentiated IEC-Cdx2L1 cells (a) and differentiated IEC-Cdx2L1 cells transfected with either siTRPC1 (b) or C-siRNA (c) for 48 h. B: summarized data showing the resting [Ca2+]cyt (a) and the amplitude of CPA-induced Ca2+ influx (b) in the differentiated IEC-Cdx2L1 cells described in A. Data are expressed as means ± SE; n = 25. *P < 0.05 compared with IEC-Cdx2L1 cells transfected with C-siRNA.

 

Figure 10
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  		Fig. 10. Effect of inhibition of TRPC1 expression by treatment with siTRPC1 on cell migration after wounding in differentiated IEC-Cdx2L1 cells. A: summarized data showing cell migration after wounding in parental IEC-6 cells and differentiated IEC-Cdx2L1 cells. Cells were grown in standard DMEM for 2 days and transfected with either TRPC1 siRNA or C-siRNA for 48 h, and part of the monolayer was then removed. Cell migration was measured 6 h after wounding. Data are means ± SE from 6 dishes. *P < 0.05 compared with IEC-Cdx2L1 cells transfected with C-siRNA. B: representative images of cell migration in the differentiated IEC-Cdx2L1 cells described in A [6 h after wounding in cells transfected with C-siRNA (a) and 6 h after wounding in cells transfected with siTRPC1 (b)].

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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[Ca2+]cyt regulates distinct biological functions in a variety of excitable and nonexcitable cells. Ca2+ homeostasis is a dynamic process involving Ca2+ entry, release from internal Ca2+ stores, and store refilling (5, 6, 29). This complex process is cell type dependent and highly controlled by numerous factors. There are at least three classes of Ca2+-permeable channels functionally expressed in the plasma membrane in mammalian cells: 1) VDCCs, which are regulated by Em; 2) receptor-operated Ca2+ channels, which are regulated by binding ligands with their respective receptors; and 3) SOCs, which are regulated by the capacity of Ca2+ in the ER (2, 29, 46). Excitable cells, such as neurons and muscle cells, highly express VDCCs that are activated by membrane depolarization, leading to an increase in [Ca2+]cyt. In contrast, nonexcitable cells, including intestinal epithelial cells, do not express VDCC (46). Ca2+ influx in nonexcitable cells depends on both the Ca2+ driving force (the electrochemical gradient across the plasma membrane) and the activity of specific Ca2+-permeable channels that are activated by receptor activation and store depletion (2, 46). Because [Ca2+]o is maintained stably at 1.6–1.8 mM, which is ~10,000- to 20,000-fold higher than the resting [Ca2+]cyt (50–150 nM) under physiological conditions, Ca2+ influx is critical for the maintenance of [Ca2+]cyt in a variety of nonexcitable cell types (2, 8, 9, 29, 46).

An increasing body of evidence indicates that CCE generated by Ca2+ influx through SOCs is a critical mechanism involved in maintaining sustained increases in [Ca2+]cyt and in refilling Ca2+ into the ER in nonexcitable cells (2, 8, 46). However, the data presently available do not provide any convincing evidence linking the localization and expression of TRPC proteins with the SOC function in intestinal epithelial cells. The present study clearly shows that intestinal epithelial cells expressed TRPC1 and TPRC5 (Fig. 1) and exhibited the typical Isoc and CCE resulting from the Ca2+ influx after depletion of the Ca2+ store by CPA (Figs. 2 and 3). Inhibition of Isoc by lanthanum was associated with an apparent decrease in CCE, suggesting that CCE by Ca2+ influx is mediated through SOCs in IEC-6 cells. Furthermore, IEC-6 cells stably transfected with TRPC1 cDNA expressed higher levels of TRPC1 (Fig. 4) and displayed a significant increase in CCE generated by Ca2+ influx (Fig. 5). This induction of CCE did not result from an increase in the basal Ca2+ permeability of the cells, because there were no differences in basal levels of [Ca2+]cyt between stable TRPC1-transfected IEC cells and parental IEC-6 cells (Fig. 5). Because the characteristics of Ca2+ influx in stable TRPC1-transfected IEC cells were similar to those of SOC activity observed in parental IEC-6 cells, these results show, for the first time, an association between the levels of TRPC1 protein and CCE and strongly suggest that TRPC1 is a candidate protein for the SOC mechanism in normal intestinal epithelial cells.

The association between TRPC1 expression and SOCs in IEC-6 cells is further supported by our demonstration that transfection of differentiated IEC-Cdx2L1 cells with TRPC1 siRNA resulted in a decrease in endogenous TRPC1 and a significant attenuation of CCE. IEC-Cdx2L1 cells were chosen in this study based on the following reasons: 1) this line of cells represents another normal intestinal epithelial cell line that is associated with a differentiated phonotype (44); 2) differentiated IEC-Cdx2L1 cells highly express endogenous TRPCs and display higher CCE compared with parental IEC-6 cells (Fig. 7); and 3) they exhibit increased cell migration after wounding (33, 35). As shown in Figs. 8 and 9, specific inhibition of TRPC1 alone by transfection with TRPC1 siRNA dramatically decreased levels of [Ca2+]cyt due to the reduction of CPA-induced CCE, suggesting that TRPC1 is a predominant heterotetrameric subunit for the composition of the native SOC in intestinal epithelial cells. On the basis of our interesting findings showing that forced expression of the TRPC1 gene increased CCE in IEC-6 cells and that decreased levels of endogenous TRPC1 inhibited CCE in differentiated IEC-Cdx2L1 cells, it is strongly suggested that TRPC1 functions as the SOC and is implicated in the SOC mechanism of intestinal epithelial cells. Consistent with our current findings, Liu et al. (18) have recently reported that TRPC1 is involved in store-operated Ca2+ influx in salivary gland cells. Although the exact mechanism by which TRPC1 is activated after depletion of the Ca2+ store remains unknown, it has been shown that TRPC1-associated SOC activity in salivary gland cells is independent of the formation of phosphatidylinositol (4,5)-bisphosphate and inositol (1,4,5)-trisphosphate (18, 57).

The most significant of the new findings reported in this study, however, is that TRPC1 plays an important role in the stimulation of intestinal epithelial cell migration during restitution by regulating [Ca2+]cyt through CCE. As shown in Figs. 5 and 6, forced expression of the TRPC1 gene by stable transfection with TRPC1 cDNA not only increased levels of CCE but also enhanced cell migration over the denuded area after wounding. Because increased cell migration in stable TRPC1-transfected IEC cells was observed in all four independent transfected clones, it is suggested that the increase in cell migration must be related to the induction of TRPC1-associated CCE rather than to clonal variation. The results presented in Figs. 9 and 10 further indicate that decreased levels of endogenous TRPC1 by transfection with TRPC1 siRNA reduced CCE in differentiated IEC-Cdx2L1 cells, which was associated a significant inhibition of cell migration after wounding. These findings are consistent with our previous studies (11, 12, 34–37, 50) and others (40, 43), which have demonstrated that a constant influx of Ca2+ from extracellular fluid to the cytosol is absolutely required for the stimulation of intestinal epithelial cell migration during restitution. However, the present study provides novel information showing that TRPC1 activity plays a critical role in the elevation of [Ca2+]cyt by increasing CCE through SOCs after wounding in intestinal epithelial cells.

The exact cellular signal leading to the activation of SOCs during intestinal epithelial restitution remains to be elucidated. The present study suggests that the product of the TRPC1 gene is the molecule mediating Ca2+ influx into the cytosol in normal intestinal epithelial cells. However, the signal that regulates expression of the TRPC1 gene or is transmitted from the internal Ca2+ store to the plasma membrane to trigger activation of SOCs after wounding is still unknown. Our previous studies (11, 33–37, 48, 50) and those of others (21, 41) have demonstrated that cellular polyamines regulate intestinal epithelial restitution in vivo as well as in vitro and that polyamines are required for the stimulation of cell migration during restitution by modulating [Ca2+]cyt homeostasis. Levels of cellular polyamines are rapidly increased after wounding, and depletion of cellular polyamines decreases [Ca2+]cyt due to the reduction of CCE (33–35, 50). Polyamine depletion inhibits expression of Kv channel proteins and decreased activity of Kv channels, thus resulting in membrane depolarization in intestinal epithelial cells (35, 50). This depolarized Em in polyamine-deficient cells significantly contributes to the decrease in [Ca2+]cyt because it greatly reduces the driving force for Ca2+ influx. It is not clear at present that cellular polyamines are implicated in the expression and activation of TRPC1 in intestinal epithelial cells after wounding.

In summary, the present study demonstrates that TRPC proteins, especially TRPC1 and TRPC5, are endogenously present in intestinal epithelial cells, which are associated with typical Isoc and CCE. Stable expression of TRPC1 cDNA in IEC-6 cells increases levels of TRPC1 protein and induces an increase in SOCs. Increased Ca2+ influx activity in stable TRPC1-transfected IEC cells displays characteristics similar to the endogenous SOC in parental IEC-6 cells. Specific inhibition of TRPC1 expression by transfection with TRPC1 siRNA prevents the increase in SOCs in differentiated IEC-Cdx2L1 cells. Importantly, this study provides evidence indicating that TRPC1 plays a critical role in intestinal epithelial restitution after injury by regulating CCE and [Ca2+]cyt. Stable TRPC1-transfected IEC cells exhibit increased cell migration during restitution, whereas decreased levels of TRPC1 inhibited cell migration after wounding. These findings strongly suggest that TRPC1 functions as a SOC in intestinal epithelial cells and is implicated in the regulation of mucosal restitution under various biological and pathological conditions.


   GRANTS
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 ABSTRACT
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REFERENCES
 
This work was supported by a Merit Review Grant from the Department of Veterans Affairs (to J.-Y. Wang) and by National Institutes of Health Grants DK-57819, DK-61972, and DK-68491 (to J.-Y. Wang) and HL-64945, HL-54043, and HL-66012 (to J. X.-J. Yuan). J.-Y. Wang is a Research Career Scientist of the Medical Research Service, United States Department of Veterans Affairs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 N. Greene St., Baltimore, MD 21201 (e-mail: jwang{at}smail.umaryland.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Arnon A, Hamlyn JM, and Blaustine MP. Na+ entry via store-operated channels modulates Ca2+ signaling in arterial myocytes. Am J Physiol Cell Physiol 278: C163–C173, 2000.[Abstract/Free Full Text]
  2. Berridge MJ, Lipp P, and Bootman MD. The calcium entry pas de deux. Science 287: 1604–1605, 2000.[Free Full Text]
  3. Birnbaumer L, Zhu X, Jiang M, Boulay G, Peyton M, Vannier B, Brown D, Platano D, Sadeghi H, Stefani E, and Birnbaumer M. On the molecular basis and regulation of cellular capacitative calcium entry: roles for Trp proteins. Proc Natl Acad Sci USA 93: 15195–15202, 1996.[Abstract/Free Full Text]
  4. Blaustein MP. Physiological effects of endogenous ouabain: control of intracellular Ca2+ store and cell responsiveness. Am J Physiol Cell Physiol 264: C1367–C1387, 1993.[Abstract/Free Full Text]
  5. Bootman MD. The elemental principles of calcium signaling. Cell 83: 675–678, 1995.[CrossRef][Web of Science][Medline]
  6. Bootman MD, Lipp P, and Berridge MJ. The organization and functions of local Ca2+-signals. J Cell Sci 114: 213–222, 2001.
  7. Clapham DE, Runnels LW, and Strubing C. The TRP ion channel family. Nature Rev Neurosci 2: 387–396, 2001.[Web of Science][Medline]
  8. Clapham DE. TRP channels as cellular sensors. Nature 426: 517–524, 2003.[CrossRef][Medline]
  9. Fleischman BK, Washabau RJ, and Kotlikoff MI. Control of resting membrane potential by delayed rectifier potassium currents in ferret airway smooth muscle cells. J Physiol 469: 625–638, 1993.[Abstract/Free Full Text]
  10. Golovina VA, Platoshyn O, Bailey CL, Wang J, Limsuwan A, Sweeney M, Rubin LJ, and Yuan JXJ. Upregulated TRP and enhanced capacitative Ca2+ entry in human pulmonary artery myocytes during proliferation. Am J Physiol Heart Circ Physiol 280: H746–H755, 2001.[Abstract/Free Full Text]
  11. Guo X, Rao JN, Liu L, Rizvi M, Turner DJ, and Wang JY. Polyamines regulate beta-catenin tyrosine phosphorylation via Ca2+ during intestinal epithelial cell migration. Am J Physiol Cell Physiol 283: C722–C735, 2002.[Abstract/Free Full Text]
  12. Guo X, Rao JN, Liu L, Zuo TT, Turner DJ, and Wang JY. Regulation of adherens junctions and epithelial paracellular permeability: novel function for polyamines. Am J Physiol Cell Physiol 285: C1174–C1187, 2003.[Abstract/Free Full Text]
  13. Harter JL. Critical values for Duncan's new multiple range test. Biometrics 16: 671–685, 1960.[CrossRef][Web of Science]
  14. Himmel HA, Whorton AR, and Strauss HC. Intracellular calcium, currents, and stimulus-response coupling in endothelial cells. Hypertension 21: 112–117, 1993.[Abstract/Free Full Text]
  15. Kerschbaum HH and Cahalan MD. Monovalent permeability, rectification, and ionic block of store-operated calcium channels in Jurkat T lymphocytes. J Gen Physiol 111: 521–537, 1998.[Abstract/Free Full Text]
  16. Kerschbaum HH and Cahalan MD. Single-channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes. Science 283: 836–839, 1999.[Abstract/Free Full Text]
  17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1990.[CrossRef]
  18. Liu X, Wang W, Singh BB, Lockwich T, Jadlowiec J, Connell BO, Wellner R, Zhu MX, and Ambudkar IS. Trp1, a candidate protein for the store-operated Ca2+ influx mechanism in salivary gland cells. J Biol Chem 275: 3403–3411, 2000.[Abstract/Free Full Text]
  19. Liu X and Ambudkar IS. TRPC1 is required for functional store-operated Ca2+ channels: role of acidic amino acid residues in the S5–S6 region. J Biol Chem 278: 11337–11343, 2003.[Abstract/Free Full Text]
  20. Liu L, Li L, Rao JN, Zou T, Zhang HM, Boneva D, Bernard MS, and Wang JY. Polyamine-modulated expression of c-myc plays a critical role in stimulation of normal intestinal epithelial cell proliferation. Am J Physiol Cell Physiol 288: C89–C99, 2005.[Abstract/Free Full Text]
  21. McCormack SA, Viar MJ, and Johnson LR. Polyamines are necessary for cell migration by a small intestinal crypt cell line. Am J Physiol Gastrointest Liver Physiol 264: G367–G374, 1993.[Abstract/Free Full Text]
  22. McDaniel SS, Platoshyn O, Wang J, Yu J, Sweeney M, Krick S, Rubin LJ, and Yuan JXJ. Capacitative Ca2+ entry in agonist-induced pulmonary vasoconstriction. Am J Physiol Lung Cell Mol Physiol 280: L870–L880, 2001.[Abstract/Free Full Text]
  23. Minke B and Cook B. TRP channels and signal transduction. Physiol Rev 82: 429–472, 2002.[Abstract/Free Full Text]
  24. Montell C and Rubin GM. Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron 2: 1313–1323, 1989.[CrossRef][Web of Science][Medline]
  25. Moore TM, Brough GH, Babal P, and Stevens T. Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1. Am J Physiol Lung Cell Mol Physiol 275: L574–L582, 1998.[Abstract/Free Full Text]
  26. Nilius B, Viana F, and Droogmans G. Ion channels in vascular endothelium. Annu Rev Physiol 59: 145–170, 1997.[CrossRef][Web of Science][Medline]
  27. Nusrat A, Delp C, and Madara JL. Intestinal epithelial restitution: characterization of cell culture model and mapping of cytoskeletal elements in migrating cells. J Clin Invest 89: 1501–1511, 1992.[Web of Science][Medline]
  28. Okada T, Inoue R, Yamazaki K, Maeda A, Kurosaki T, Yamakuni T, Tanaka I, Shimizu S, Ikenaka K, Imoto K, and Mori Y. Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. J Biol Chem 274: 27359–27370, 1999.[Abstract/Free Full Text]
  29. Parekh AB and Penner R. Store depletion and calcium influx. Physiol Rev 77: 901–930, 1997.[Abstract/Free Full Text]
  30. Phillip S, Cavalie A, Freichel M, and Flockerzi. A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL. EMBO J 15: 6166–6171, 1996.[Web of Science][Medline]
  31. Phillips AM, Bull A, and Kelly LE. Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene. Neuron 8: 631–642, 1992.[CrossRef][Web of Science][Medline]
  32. Quaroni A, Wands J, Trelstat RL, and Isselbacher KJ. Epithelial cell cultures from rat small intestine. J Cell Biol 80: 248–265, 1979.[Abstract/Free Full Text]
  33. Rao JN, Li L, Li J, Bass BL, and Wang JY. Differentiated intestinal epithelial cells exhibit increased migration through polyamines and myosin II. Am J Physiol Gastrointest Liver Physiol 277: G1149–G1158, 1999.[Abstract/Free Full Text]
  34. Rao JN, Li L, Golovina VA, Platoshyn O, Strauch ED, Yuan JXJ, and Wang JY. Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration. Am J Physiol Cell Physiol 280: C993–C1007, 2001.[Abstract/Free Full Text]
  35. Rao JN, Platoshyn O, Li L, Guo X, Golovina VA, , Yuan JXJ, and Wang JY. Activation of K+ channels and increased migration of differentiated intestinal epithelial cells after wounding. Am J Physiol Cell Physiol 282: C885–C898, 2002.[Abstract/Free Full Text]
  36. Rao JN and Wang JY. Ca2+ signaling in epithelial restitution. In: Gastrointestinal Mucosal Repair and Experimental Therapeutics, edited by Cho CH and Wang JY. Basel, Switzerland: Karger, 2002, vol. 25, p. 29–42.[Medline]
  37. Rao JN, Guo X, Liu L, Zou T, Murthy KS, Yuan JXY, and Wang JY. Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution. Am J Physiol Cell Physiol 284: C848–C859, 2003.[Abstract/Free Full Text]
  38. Rao JN, Boneva D, Guo X, Liu L, Zou T, Zhang HM, and Wang JY. Transient receptor potential channel-1 (TRPC1) regulates capacitative Ca2+ entry and intestinal epithelial cell migration during restitution (Abstract). Gastroenterology 125: A537, 2004.
  39. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Borisy G, Parson JT, and Horwitz AR. Cell migration: integrating signals from front to back. Science 302: 1704–1709, 2003.[Abstract/Free Full Text]
  40. Rutten MJ and Ito S. Morphology and electrophysiology of guinea pig gastric mucosal repair in vitro. Am J Physiol Gastrointest Liver Physiol 244: G171–G182, 1983.[Abstract/Free Full Text]
  41. Santos MF, McCormack SA, Guo Z, Okolicany J, and Johnson LR. Rho proteins play a critical role in cell migration during the early phases of mucosal restitution. J Clin Invest 100: 216–225, 1997.[Web of Science][Medline]
  42. Sharp PA. RNA interference-2001. Genes Dev 15: 485–490, 2001.[Free Full Text]
  43. Silen W and Ito S. Mechanism for rapid-epithelialization of the gastric mucosal surface. Annu Rev Physiol 47: 217–229, 1985.[CrossRef][Web of Science][Medline]
  44. Suh E and Traber PG. An intestine-specific homeobox gene regulates proliferation and differentiation. Mol Cell Biol 16: 619–625, 1996.[Abstract]
  45. Sweeney M, Yu Y, Platoshyn O, Zhang S, McDaniel SS, and Yuan JXJ. Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation. Am J Physiol Lung Cell Mol Physiol 283: L144–L155, 2002.[Abstract/Free Full Text]
  46. Tsien RW and Tsien RY. Calcium channels, stores, and oscillations. Annu Rev Cell Biol 6: 715–760, 1990.[CrossRef][Web of Science][Medline]
  47. Wang J, Laurier LG, Sims SM, and Preiksaitis HG. Enhanced capacitative calcium entry and TRPC channel gene expression in human LES smooth muscle. Am J Physiol Gastrointest Liver Physiol 284: G1074–G1083, 2003.[Abstract/Free Full Text]
  48. Wang JY and Johnson LR. Luminal polyamines stimulate repair of gastric mucosal stress ulcers. Am J Physiol Gastrointest Liver Physiol 259: G584–G592, 1990.[Abstract/Free Full Text]
  49. Wang JY, McCormack SA, Viar MJ, Wang HL, Tzen CY, Scott RE, and Johnson LR. Decreased expression of protooncogenes c-fos, c-myc, and c-jun following polyamine depletion in IEC-6 cells. Am J Physiol Gastrointest Liver Physiol 265: G331–G338, 1993.[Abstract/Free Full Text]
  50. Wang JY, Wang J, Golovina VA, Li L, Platoshyn O, and Yuan JXJ. Role of K+ channel expression in polyamine-dependent intestinal epithelial cell migration. Am J Physiol Cell Physiol 278: C303–C314, 2000.[Abstract/Free Full Text]
  51. Wes PD, Chevesich J, Jeromin A, Rosenberg C, and Stetten G. TRPC1, a human homolog of a Drosophila store-operated channel. Proc Natl Acad Sci USA 92: 9652–9656, 1995.[Abstract/Free Full Text]
  52. Xu XZ, Chien F, Butler A, Salkoff L, and Montel C. TRP{gamma}, a Drosophila TRP-related subunit, forms a regulated cation channel with TRPL. Neuron 26: 647–657, 2000.[CrossRef][Web of Science][Medline]
  53. Xu SZ and Beech DJ. TrpC1 is a membrane-spanning subunit of store-operated Ca2+ channels in native vascular smooth muscle cells. Circ Res 88: 84–87, 2001.[Abstract/Free Full Text]
  54. Zhu X, Jiang M, Peyton M, Boulay G, Hurst R, and Birnbaumer L. Trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry. Cell 85: 661–671, 1996.[CrossRef][Web of Science][Medline]
  55. Zhu X and Birnbaumer L. Calcium channels formed by mammalian trp homologues. News Physiol Sci 13: 211–217, 1998.[Abstract/Free Full Text]
  56. Zitt C and Schultz G. Expression of TRPC3 in Chinese hamster ovary cells results in calcium activated cation currents not related to store depletion. J Cell Biol 138: 1333–1341, 1997.[Abstract/Free Full Text]
  57. Zitt C, Halaszovich CR, and Luckhoff A. The TRP family of cation channels: probing and advancing the concepts on receptor-activated calcium entry. Prog Neurobiol 66:243–264, 2003.



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