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Am J Physiol Cell Physiol 294: C1277-C1287, 2008. First published March 5, 2008; doi:10.1152/ajpcell.90635.2007
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Induced TRPC1 expression increases protein phosphatase 2A sensitizing intestinal epithelial cells to apoptosis through inhibition of NF-{kappa}B activation

Bernard S. Marasa,1,2,3 Lan Xiao,1,3 Jaladanki N. Rao,1,3 Tongtong Zou,1,3 Lan Liu,1,3 Jian Wang,4 Emily Bellavance,1,3 Douglas J. Turner,1,3 and Jian-Ying Wang1,2,3

1Cell Biology Group, Department of Surgery and 2Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland; 3Baltimore Veterans Affairs Medical Center, Baltimore, Maryland; and 4Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 18 December 2007 ; accepted in final form 4 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transient receptor potential canonical-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs), and induced TRPC1 expression sensitizes IECs to apoptosis by inhibiting NF-{kappa}B activation. However, the exact mechanism by which increased TRPC1 results in NF-{kappa}B inactivation remains elusive. Protein phosphatase 2A (PP2A) is a widely conserved protein serine/threonine phosphatase that is implicated in the regulation of a wide array of cellular functions including apoptosis. The present study tests the hypothesis that induced TRPC1 expression inhibits NF-{kappa}B activation by increasing PP2A activity through Ca2+ influx in IECs. The expression of TRPC1 induced by stable transfection with the wild-type TRPC1 gene increased PP2A activity as indicated by increases in levels of PP2A proteins and their phosphatase activity. Increased levels of PP2A activity in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1) were associated with decreased nuclear levels of NF-{kappa}B proteins and a reduction in NF-{kappa}B-dependent transcriptional activity, although there were no changes in total NF-{kappa}B protein levels. Inhibition of PP2A activity by treatment with okadaic acid or PP2A silencing with small interfering RNA not only enhanced NF-{kappa}B transactivation but also prevented the increased susceptibility of IEC-TRPC1 cells to apoptosis induced by treatment with tumor necrosis factor-{alpha} (TNF-{alpha})/cycloheximide (CHX). Decreasing Ca2+ influx by exposure to the Ca2+-free medium reduced PP2A mRNA levels, destabilized PP2A proteins, and induced NF-{kappa}B activation, thus blocking the increased sensitivity of IEC-TRPC1 cells to TNF-{alpha}/CHX-induced apoptosis. These results indicate that induced TRPC1 expression increases PP2A activity through Ca2+ influx and that increased PP2A sensitizes IECs to apoptosis as a result of NF-{kappa}B inactivation.

store-operated Ca2+ channels; capacitative Ca2+ entry mechanism; programmed cell death; I{kappa}B; small interfering ribonucleic acid; intestinal epithelium; mucosal homeostasis; transient receptor potential canonical-1

REVERSIBLE PROTEIN PHOSPHORYLATION in eukaryotic cells depends on the activities of protein kinases and protein phosphatases and plays a critical role in the regulation of a variety of cellular processes including apoptosis (27). Protein phosphatase 2A (PP2A) is a ubiquitously expressed protein serine/threonine phosphatase and accounts for a large fraction of phosphatase activity in these cells (15, 27). PP2A functions as a trimeric protein complex consisting of a catalytic subunit (PP2A/C), a scaffold subunit (PP2A/A), and one of the alternative regulatory B subunits (PP2A/B) (15, 18). In a common PP2A functional complex, the PP2A/A subunit mediates interaction of the core dimer (PP2A/C-PP2A/A) with a wide variety of PP2A/B subunits that dictate the functions of individual forms. Since there are multiple families, isoforms, and splice variants of the PP2A/B, they allow generation of amazingly diverse heterotrimeric PP2A holoenzymes with a vast array of substrate specificities (6, 15). It has been shown that PP2A has distinct biological actions and is implicated in numerous cellular functions including metabolism, transcription, translation, ion transport, development, proliferation, differentiation, and apoptosis in different cell types (14, 15, 19, 27, 41). A recent study indicates that PP2A plays an important role in the control of intestinal mucosal homeostasis by regulating intestinal epithelial cell (IEC) apoptosis and that PP2A activity is highly regulated by numerous factors including cellular polyamines (39).

The epithelium of mammalian intestinal mucosa is a rapidly self-renewing tissue in the body and serves a number of important physiological functions, including digestion and absorption, secretion, barrier, and immune functions (35). Maintenance of intestinal epithelial integrity is prerequisite to these functions and depends on a complex interplay between processes involved in cell proliferation, differentiation, migration, and apoptosis (9, 21, 44). Undifferentiated IECs continuously replicate in the proliferating zone within the crypts and differentiate as they migrate up the luminal surface of the colon and the villous tips in the small intestine (21, 32). To maintain stable numbers of enterocytes, cell proliferation must be dynamically counterbalanced by apoptosis, a fundamental biological process involving selective cell deletion to regulate tissue homeostasis (7, 32). Under physiological conditions, apoptosis occurs in the crypt area, where it maintains the critical balance in cell number between newly divided and surviving cells, and at the luminal surface of the colon and villous tips in the small intestine, where differentiated cells are lost (3, 7, 21). Although imbalance between cell proliferation and apoptosis dramatically alters intestinal mucosal tissue homeostasis and has significant pathological consequences such as mucosal neoplasia and atrophy, the exact mechanism underlying this process remains poorly understood.

A significant body of evidence indicates that cytosolic free Ca2+ ([Ca2+]cyt) regulates apoptosis and that increasing [Ca2+]cyt can be pro- or anti-apoptotic, depending on cell type and other factors (1, 28, 29). To carry out different regulatory functions, [Ca2+]cyt has to be flexible yet precisely regulated, and this tight control involves intracellular Ca2+ store depletion and extracellular Ca2+ influx. Mobilization of Ca2+ from intracellular stores causes a transient increase in [Ca2+]cyt, but a sustained increase in [Ca2+]cyt and the refilling of Ca2+ into intracellular stores absolutely requires extracellular Ca2+ influx (30, 45). 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) (1, 30, 33). Although the exact SOCs mediating CCE in IECs remain unclear, our recent study (37) shows that normal IECs express transient receptor potential canonical-1 (TRPC1) and TRPC5 channels and display typical store-operated Ca2+ currents and CCE after Ca2+ store depletion. Induced TRPC1 expression by stable transfection with the wild-type TRPC1 gene increases Ca2+ influx after Ca2+ store depletion, indicating that TRPC1 is an excellent candidate protein for the SOCs and regulates [Ca2+]cyt homeostasis in IECs, but the exact role of TRPC5 in this process is still unknown. We have also demonstrated that TRPC1 channels are implicated in the regulation of apoptosis in IECs and that ectopic expression of the TRPC1 gene sensitizes IECs to apoptosis by inhibiting NF-{kappa}B activation (25). Here, we sought to further investigate whether PP2A plays a role in TRPC1-induced repression of NF-{kappa}B activation. Our results indicate that induced TRPC1 stimulated PP2A expression and increased PP2A phosphatase activity, whereas PP2A inhibition enhanced NF-{kappa}B activation and prevented the TRPC1-induced susceptibility to apoptosis. The data presented herein also demonstrate that increased PP2A expression resulted primarily from an increase in Ca2+ influx through CCE.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and supplies. Disposable culture-ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed fetal bovine serum were purchased from Invitrogen (Carlsbad, CA), and biochemicals were obtained from Sigma Chemical (St. Louis, MO). The affinity-purified rabbit polyclonal antibody against TRPC1 was purchased from Alomone Laboratories (Jerusalem, Israel), and antibodies against NF-{kappa}B subunits p50, p65, and I{kappa}B{alpha} were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against PP2A/C, PP2A/A, and PP2A/B were purchased from Cell Signaling Technology (Boston, MA).

Cell culture and stable TRPC1 gene transfection. The IEC-6 cell line was purchased from the American Type Culture Collection at passage 13. The cell line was derived from normal rat intestinal crypt cells and was developed and characterized by Quaroni et al. (34). Stock cells were maintained in T-150 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2, and passages 15–20 were used in experiments. There were no significant changes of biological function and characterization of IEC-6 cells at passages 15–20 (38, 45).

The stable TRPC1-transfected IEC-6 cells (IEC-TRPC1) were developed and characterized as described in our recent publications (25, 37). The IEC-6 cells were transfected with the pcDNA3.0(+) expression vector containing the full-length cDNA of human TRPC1 under the cytomegalovirus (CMV) promoter (pcDNA-TRPC1) or pcDNA3.0(+) vector containing no TRPC1 cDNA by using the LipofectAMINE kit. The transfected cells were selected for TRPC1 integration by incubation with the selection medium containing 0.6 mg/ml of G418, and clones resistant to the selection medium were isolated, cultured, and screened for TRPC1 expression by RT-PCR using specific TRPC1 primers and Western immunoblotting analysis using specific anti-TRPC1 antibody.

RNA interference. The small interfering RNA (siRNA) specifically targeting the coding region of PP2A/C (siPP2A/C) was purchased from Invitrogen. Scrambled control siRNA (C-siRNA), which had no sequence homology to any known genes, was used as the control. The siPP2A/C and C-siRNA were transfected into cells as described previously (37, 46). Briefly, for each 60-mm cell culture dish, 15 µl of the 20 µM stock siPP2A/C or C-siRNA was mixed with 300 µl of Opti-MEM medium (Invitrogen). This mixture was gently added to a solution containing 10 µl of LipofectAMINE 2000 in 300 µl of Opti-MEM medium. The solution was incubated for 20 min at room temperature and gently overlaid onto the monolayer of cells in 3 ml of medium, and cells were harvested for various assays after 48 h of incubation.

Measurement of NF-{kappa}B-dependent transcriptional activity. The NF-{kappa}B-dependent luciferase reporter gene construct containing the synthetic sequence with four tandem copies of NF-{kappa}B-binding elements was purchased from Clontech. Transient transfection was performed by using the LipofectAMINE kit as recommended by the manufacturer (Invitrogen). Cells were collected 48 h after the transfection, and luciferase activity from individual transfection was normalized by the β-galactosidase activity from cotransfected pCMV β-galactosidase plasmid. The experiments were done in triplicate and are reported as the means of relative light unit/β-galactosidase.

Real-time quantitative PCR analysis. Total RNA was isolated by using the RNeasy Mini kit (Qiagen, Valencia, CA). Equal amounts of total RNA (2 µg) were transcribed to synthesize single-stranded cDNA with an RT-PCR kit (Invitrogen). Real-time quantitative PCR (Q-PCR) was performed using an Applied Biosystems instrument (Foster City, CA) using specific primers, probes, and software (Applied Biosystems) as described in our previous publications (46, 50). The levels of PP2A/C and PP2A/A mRNA were quantified by Q-PCR analysis and normalized with GAPDH levels.

Preparation of cytoplasmic and nuclear proteins and Western blot analysis. Cytoplasmic and nuclear proteins were prepared via the procedure described previously (46, 50), and the protein contents in different preparations were measured using Bradford method (4). Cell samples, placed in SDS sample buffer (250 mM Tris·HCL, pH 6.8, 2% SDS, 20% glycerol, and 5% mercaptoethanol), were sonicated and then centrifuged (10,000 g) at 4°C for 15 min. The supernatant from cell samples was boiled for 5 min and then subjected to electrophoresis on 7.5% acrylamide gels. After the transfer of protein onto nitrocellulose filters, the filters were incubated for 1 h in 5% nonfat dry milk in 1x phosphate-buffered saline/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 the specific antibody against PP2A, p65, p50, caspase-3, or I{kappa}B{alpha} proteins. The filters were subsequently washed with 1x PBS-T and incubated for 1 h with the second antibody conjugated to peroxidase by protein cross-linking with 0.2% glutaraldehyde. After extensive washing with 1x PBS-T, the immunocomplexes on the filters were developed by the enhanced chemiluminescence method according to the manufacturer's instruction (Amersham Pharmacia Biotech, Arlington Height, IL).

Determination of apoptosis. After various experimental treatments, cells were photographed with a Nikon inverted microscope before fixation as described previously (48). Annexin-V staining of apoptosis was carried out by using a commercial apoptosis kit (Clontech Laboratories, Palo Alto, CA) and performed according to the protocol recommended by the manufacturer. Briefly, cells were rinsed with 1x binding buffer and resuspended in 200 µl of 1x binding buffer. Annexin-V (5 µl) was added on slide and incubated at room temperature for 10 min in the dark. Annexin-stained cells were visualized and photographed under fluorescence microscope using a dual filter set for FITC and rhodamine, and the percentage of "apoptotic" cells was determined.

Measurement of the caspase-3 activity. The caspase-3 activity was measured by using the Caspase-3 Colorimetric Assay kit (R&D Systems, Minneapolis, MN) and performed according to the protocol recommended by the manufacturer. Briefly, cells were treated with TNF-{alpha} and cycloheximide (CHX) for different times, washed with ice-cold Dulbecco's-PBS, and scraped from the dishes. The collected cells were washed with Dulbecco's-PBS and then lysed in ice-cold cell lysis buffer {50 mM HEPES, pH 7.4, 0.1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS), 1 mM DTT, 0.1 mM EDTA, and 0.1% Nonidet P-40}. The assay for caspase-3 activity was carried out in a 96-well plate. In each well, there were 50 µl cell lysate (~150 µg of total proteins), 50 µl of reaction buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS, 100 mM NaCl, 10 mM DTT, and 1 mM EDTA), 5 µl of caspase-3 colorimetric substrate, and a caspase-specific peptide that is conjugated to a chromogen, p-nitroanilide. The 96-well plate was incubated at 37°C for 90 min, during which the caspase-3 in the sample presumably cleaved the chromophore p-nitroanilide from the substrate molecule. Absorbency readings at 405 nm were made after the incubation, with the caspase-3 activity being directly proportional to the color reaction. Protein levels of each sample were determined by the method described by Bradford (4).

Measurement of PP2A activity. PP2A activity was determined using the nonradioactive and malachite green-based serine/threonine phosphatase assay kit and performed as recommended by the manufacturer (Upstate Biotechnology, Lake Placid, NY). Briefly, 5 µg of cellular proteins extracted in lysis buffer were incubated with 175 µM of the phosphopeptide (K-R-pT-I-R-R) and the PP2A buffer in a total volume of 25 µl. Reactions were initiated by addition of the phosphopeptide substrate and conducted for 10 min at room temperature and then stopped by addition of malachite green solution. After the solution was left for 15 min to allow for color development, the plates were read at 650 nm with a microplate reader, and the amount of phosphate released was expressed as nmol phosphate/min per unit.

Statistical analysis. All data are expressed as means ± SE from six dishes or three separate experiments. Immunoblotting and immunohistochemical results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined using Duncan's multiple-range test (12).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ectopic expression of the TRPC1 gene increased PP2A levels and induced its phosphatase activity. To determine whether PP2A plays a role in TRPC1-induced susceptibility of IECs to apoptosis, the levels of the PP2A catalytic subunit (PP2A/C), its associated scaffolding regulatory subunit partner (PP2A/A), and secondary regulatory subunit (PP2A/B) were examined in stable TRPC1-expressing IEC-6 cells (IEC-TRPC1), which were recently developed and characterized in our laboratory (37). The stable IEC-TRPC1 cells highly expressed TRPC1 (by ~5-fold) and exhibited a significant increase in Ca2+ influx (by ~2-fold) through CCE after depletion of store Ca2+ by cyclopiazonic acid (data not shown). Results presented in Fig. 1 show that PP2A expression and its phosphatase activity in stable IEC-TRPC1 cells increased significantly when compared with control IEC-6 cells that were transfected with the empty vector containing no TRPC1 cDNA. Levels of PP2A/C mRNA in stable IEC-TRPC1 cells were increased by ~60%, and its protein levels were increased more than threefold the control value. Levels of PP2A/A mRNA and protein in stable IEC-TRPC1 cells were only marginally increased. On the other hand, there were no differences in levels of PP2A/B mRNA (data not shown) and its protein between control IEC-6 cells and stable IEC-TRPC1 cells. Stable IEC-TRPC1 cells also exhibited a significant increase in PP2A phosphatase activity (Fig. 1C). The effect of TRPC1 overexpression on PP2A activity was not simply due to clonal variation since two stable clones, IEC-TRPC1-C1 and IEC-TRPC1-C2, showed identical responses. These results suggest that increasing TRPC1 stimulates PP2A expression and induces its phosphatase activity.


Figure 1
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  		Fig. 1. Changes in levels of protein phosphatase 2A (PP2A) expression and its phosphatase activity in control IEC-6 and stable IEC-TRPC1 cells (IEC, intestinal epithelial cells; TRPC1, transient receptor potential canonical-1). The complete open reading frame of human TRPC1 cDNA was cloned to the expression pcDNA3.1(+) vector, and 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. A: levels of PP2A mRNAs. Total RNA from each of the stable TRPC1-transfected clones (C) and control cells transfected with control vector lacking TRPC1 cDNA were harvested, and levels of TRPC1 mRNA were measured by real-time quantitative PCR analysis. Data were normalized to amount of GAPDH, and values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with control cells. B: representative immunoblots for PP2A proteins. Whole cell lysates were harvested, applied to each lane (20 µg) equally, and subjected to electrophoresis on 10% acrylamide gel. Levels of PP2A proteins were identified by probing nitrocellulose with the specific antibody against either PP2A/C (~36/38 kDa), PP2A/A (~65 kDa), or PP2A/B (~61 kDa). After the blot was stripped, actin (~42 kDa) immunoblotting was performed as an internal control for equal loading. Three experiments were performed that showed similar results. C: changes in levels of PP2A phosphatase activity as measured by the assays described in MATERIALS AND METHODS. Values are means ± SE of data from 6 dishes. *P < 0.05 compared with control cells.

 
Increased PP2A activity was associated with increased I{kappa}B{alpha} and inactivation of NF-{kappa}B. To determine the effect of increased PP2A by TRPC1 overexpression on NF-{kappa}B activation, the levels of cytoplasmic and nuclear NF-{kappa}B (subunits p50 and p65) were examined in control IEC-6 and stable IEC-TRPC1 cells. I{kappa}B{alpha} has been shown to bind to NF-{kappa}B and keep it in the cytoplasm (17, 51); therefore, its total levels and subcellular distribution also were examined in the present study. To monitor the quality and abundance of the cytoplasmic and nuclear fractions, we examined the levels of lamin B (a nuclear protein; Fig. 2A, right) and β-tubulin (a cytoplasmic protein; middle), respectively. Assessment of these markers revealed that there was no contamination between cytoplasmic and nuclear fractions. As shown in Fig. 2, the levels of cytoplasmic I{kappa}B{alpha} and NF-{kappa}B proteins increased markedly in stable IEC-TRPC1 cells, whereas their nuclear abundances decreased compared with those observed in control IEC-6 cells. There were no significant changes in whole cell levels of I{kappa}B{alpha} and NF-{kappa}B proteins between controls and IEC-TRPC1 cells (Fig. 2A). Consistently, stable IEC-TRPC1 cells also displayed decreased NF-{kappa}B transcriptional activity as indicated by reduced levels of NF-{kappa}B-dependent promoter luciferase reporter gene activity (Fig. 2B). These results suggest that increased PP2A activity by TRPC1 overexpression inactivates NF-{kappa}B through a process involving I{kappa}B{alpha} signaling in IECs.


Figure 2
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  		Fig. 2. Changes in subcellular distribution of I{kappa}B{alpha}, NF-{kappa}B (subunits p50 and p65), and levels of NF-{kappa}B-dependent transcriptional activity in control IEC-6 cells and stable IEC-TRPC1 cells. A: representative immunoblots for I{kappa}B{alpha} and NF-{kappa}B proteins in total (left), cytoplasmic (middle), and nuclear fractions (right). Whole cell lysates were harvested from control and stable TRPC1-transfected cells, and cytoplasmic and nuclear proteins were extracted for Western blotting. Levels of I{kappa}B{alpha} and NF-{kappa}B proteins were measured by Western immunoblotting analysis using specific antibodies against I{kappa}B{alpha} or different NF-{kappa}B subunits. After detecting I{kappa}B{alpha} and NF-{kappa}B, blots were reprobed to detect β-actin in total proteins, β-tubulin in cytoplasmic proteins, or lamin B in nuclear proteins to control for equal loading of samples. Three experiments were performed that showed similar results. B: levels of NF-{kappa}B transcriptional activity as measured by NF-{kappa}B-dependent promoter luciferase reporter gene assays. Cells were transfected with the NF-{kappa}B-dependent promoter construct (pNF-{kappa}B-Luc) or control vector by LipofectAMINE technique. Transfected cells were harvested and assayed for luciferase activity after 48 h of incubation. Data of NF-{kappa}B dependent promoter activity were normalized by β-galactosidase activity from cotransfection of pRSV β-galactosidase. Values are means ± SE of data from 6 dishes. *P < 0.05 compared with control cells.

 
Inhibition of PP2A activity decreased I{kappa}B{alpha} and activated NF-{kappa}B. To determine the exact relationship between increased PP2A and NF-{kappa}B inactivation in stable IEC-TRPC1 cells, the following two experiments were carried out. First, we examined the effect of PP2A inhibition on I{kappa}B{alpha} levels and NF-{kappa}B activity by treatment with its chemical inhibitor okadaic acid. Exposure of stable IEC-TRPC1 cells to okadaic acid not only inhibited PP2A phosphatase activity (Fig. 3A) but also decreased the levels of I{kappa}B{alpha} protein in a dose-dependent manner (Fig. 3B). Treatment with okadaic acid also increased the nuclear levels of NF-{kappa}B proteins and correspondingly reduced their cytoplasmic abundances (Fig. 3C), but it did not alter the levels of total NF-{kappa}B proteins (data not shown). Consistently, inhibition of PP2A activity by okadaic acid significantly increased NF-{kappa}B-dependent transcriptional activity (Fig. 3D). In okadaic acid-treated IEC-TRPC1 cells, the levels of NF-{kappa}B-dependent promoter luciferase reporter gene activity were increased by ~1.6-fold at 50 nM and by 2.3-fold at 100 nM, respectively.


Figure 3
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  		Fig. 3. Effect of inhibition of PP2A phosphatase activity by treatment with okadaic acid on I{kappa}B{alpha} levels, subcellular distribution of NF-{kappa}B proteins, and NF-{kappa}B-dependent transcriptional activity in stable IEC-TRPC1 cells. A: levels of PP2A phosphatase activity. Cells were exposed to different concentrations of okadaic acid for 2 h, and levels of PP2A phosphatase activity were measured. Values are means ± SE of data from 6 dishes. *P < 0.05 compared with controls. B: representative immunoblots for I{kappa}B{alpha} protein in cells described in A. Levels of I{kappa}B{alpha} protein were measured by Western immunoblotting analysis, and three experiments were performed that showed similar results. C: levels of cytoplasmic (a) and nuclear (b) NF-{kappa}B subunits p50 and p65 in cells described in A. Cytoplasmic and nuclear proteins were prepared after treatment with okadaic acid, and levels of p50 and p65 were measured using the specific antibody against p50 or p65 protein. After the blots were stripped, β-actin in cytoplasmic proteins and lamin B in nuclear proteins were detected for equal loading. D: changes in levels of NF-{kappa}B-dependent transcriptional activity in cells described in A. After cells were transfected with the NF-{kappa}B-dependent promoter construct or control vector for 46 h, they were exposed to okadaic acid. The luciferase activity was measured 2 h after administration of okadaic acid, and data were normalized by β-galactosidase activity from cotransfection of pRSV β-galactosidase. Values are means ± SE of data from 6 dishes. *P < 0.05 compared with control cells.

 
Second, we examined the changes in levels of I{kappa}B{alpha} and NF-{kappa}B transcriptional activity after inhibition of PP2A expression by transfection with siPP2A/C. These specific siPP2A/C nucleotides were designed to cleave rat PP2A/C mRNA and had a unique combination of specificity, efficacy, and reduced toxicity. Initially, we determined the transfection efficiency of the siRNA nucleotides in stable IEC-TRPC1 cells and demonstrated that >95% of cells were positive when they were transfected with a fluorescent FITC-conjugated siPP2A/C for 24 h (data not shown). As shown in Fig. 4A, transfection with the siPP2A/C inhibited expression of PP2A/C in IEC-TRPC1 cells, and the levels of PP2A/C protein were decreased by ~85% at 48 h thereafter. To determine the specificity of siPP2A/C used in the present study, we reprobed the membrane with anti-PP2A/B antibody and showed that levels of PP2A/B protein were not affected when cells were transfected with siPP2A/C (data not shown). Transfection with siPP2A/C also decreased the levels of PP2A phosphatase activity (Fig. 4B) and reduced I{kappa}B{alpha} protein (Fig. 4A, middle) in IEC-TRPC1 cells, which was associated with a significant increase in NF-{kappa}B nuclear translocation. The levels of nuclear NF-{kappa}B proteins were increased (Fig. 4C, right), whereas cytoplasmic NF-{kappa}B abundances decreased (Fig. 4C, left). Furthermore, the increased nuclear NF-{kappa}B proteins exhibited elevated transcriptional activity, since the levels of NF-{kappa}B-dependent promoter luciferase reporter gene activity were twice the value in control IEC-TRPC1 cells and cells transfected with control-siRNA (Fig. 4D). In addition, neither siPP2A/C nor control-siRNA affected cell viability as measured by Trypan blue staining (data not shown). These findings indicate that inhibition of PP2A by okadaic acid or PP2A/C silencing decreases I{kappa}B{alpha} levels, enhances NF-{kappa}B nuclear translocation, and induces NF-{kappa}B activation.


Figure 4
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  		Fig. 4. Effect of specific inhibition of PP2A expression by small interfering RNA (siRNA) targeting PP2A/C mRNA coding region (siPP2A/C) on phosphatase activity, subcellular distribution of NF-{kappa}B proteins, and NF-{kappa}B-dependent transcriptional activity in stable IEC-TRPC1 cells. A: representative immunoblots for PP2A/C and I{kappa}B{alpha} proteins. Stable IEC-TRPC1 cells were transfected with either control siRNA (C-siRNA) or siPP2A/C, and levels of PP2A/C and I{kappa}B{alpha} proteins were measured 48 h after the transfection by Western immunoblotting analysis. Actin immunoblotting on the stripped blots was performed as an internal control for equal loading. Three separate experiments were performed that showed similar results. B: levels of PP2A phosphatase activity in cells described in A. Values are means ± SE of data from 6 dishes. *P < 0.05 compared with controls. C: levels of cytoplasmic (left) and nuclear (right) NF-{kappa}B subunits p50 and p65 in cells described in A. Cytoplasmic and nuclear proteins were prepared, and levels of p50 and p65 proteins were measured by Western immunoblotting analysis. Levels of β-actin in cytoplasmic proteins and lamin B in nuclear proteins also were detected for equal loading controls. D: changes in NF-{kappa}B transcriptional activity in cells described in A. Values are means ± SE of data from 6 dishes. *P < 0.05 compared with cells transfected with C-siRNA.

 
PP2A/C silencing decreased the susceptibility of IEC-TRPC1 cells to apoptosis. To investigate the biological consequences of inducing PP2A activity following TRPC1 overexpression, we examined its possible involvement in regulating IEC apoptosis. We first examined spontaneous apoptotic cell death without any challenge of apoptotic stimulators after inhibition of PP2A/C expression by using siPP2A/C and demonstrated that decreased PP2A/C failed to directly induce apoptosis. There were no apparent differences in cell viability in IEC-TRPC1 cells transfected with siPP2A/C compared with the cells transfected with C-siRNA as measured by Trypan blue staining assay. Neither morphological features of apoptosis (Fig. 5, A and B, top) nor detectable levels of caspase-3 activity (Fig. 5C) were obtained in IEC-TRPC1 cells after PP2A/C silencing. Second, we determined whether PP2A/C silencing altered the susceptibility of IEC-TRPC1 cells to apoptosis after exposure to TNF-{alpha}/CHX. This apoptotic model was chosen because TNF-{alpha}/CHX-induced apoptosis is widely accepted as a form of programmed cell death induced by a biological apoptotic inducer (5, 48). PP2A/C silencing significantly decreased the susceptibility of IEC-TRPC1 cells to TNF-{alpha}/CHX-induced apoptosis. As shown in Fig. 5, A and B, morphological features characteristic of programmed cell death decreased markedly in IEC-TRPC1 cells transfected with siPP2A/C compared with those observed in the cells transfected with C-siRNA after exposure to TNF-{alpha}/CHX. Morphological assessments of apoptosis were confirmed by changes in caspase-3 activity (Fig. 5C). The levels of caspase-3 activity and percentages of apoptotic cells (Fig. 5D) in siPP2A/C-treated IEC-TRPC1 cells were decreased by ~45% after treatment with TNF-{alpha}/CHX. We also examined the effect of inhibition of PP2A activity by okadaic acid on the susceptibility of stable IEC-TRPC1 cells to apoptosis and demonstrated that treatment with okadaic acid also dose-dependently decreased the levels of apoptotic cell death after exposure to TNF-{alpha}/CHX (data not shown). In addition, PP2A/C silencing or pretreatment with okadaic acid also reduced the sensitivity of IEC-TRPC1 cells to staurosporine-induced apoptosis (data not shown). These results indicate that inhibition of PP2A prevents the increased susceptibility to apoptosis in IEC-TRPC1 cells.


Figure 5
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  		Fig. 5. Changes in apoptotic response to TNF-{alpha}/ cycloheximide (CHX) treatment after specific inhibition of PP2A expression by PP2A siRNA (siPP2A/C) in stable IEC-TRPC1 cells. Cells were transfected with either control siRNA (C-siRNA) or siPP2A/C for 48 h and then exposed to TNF-{alpha}/CHX. Apoptosis was examined 2 and 4 h after exposure to TNF-{alpha}/CHX. A: images of TNF-{alpha}/CHX-induced apoptosis in cells transfected with C-siRNA (left) and siPP2A/C (right): a, cells treated without TNF-{alpha}/CHX (control); b, cells exposed to TNF-{alpha}/CHX for 2 h; and c, cells exposed to TNF-{alpha}/CHX for 4 h. Original magnification, x150. B: images of ApoAlert Annexin-V staining in cells described in A. C: changes in caspase-3 activity cells described in A. Values are means ± SE of data from 6 samples. *P < 0.05 compared with cells transfected with C-siRNA. D: percentage of apoptotic cells in cells described in A. Values are means ± SE of data from 3 experiments. *P < 0.05 compared with cells transfected with C-siRNA.

 
Induced TRPC1 increased the stability of PP2A/c protein through Ca2+ influx. To determine the mechanism by which induced TRPC1 increases PP2A expression, we examined changes in the stability of PP2A proteins in control IEC-6 and IEC-TRPC1 cells. As shown in Fig. 6A, a, the levels of PP2A/C protein in control IEC-6 cells declined rapidly after protein synthesis was inhibited by CHX, with a half-life of ~45 min as calculated by linear regression analysis (Fig. 6B,a). In IEC-TRPC1 cells, however, the stability of PP2A/C protein increased significantly (Fig. 6A,b), and the half-life of PP2A/C protein in IEC-TRPC1 cells was ~220 min (Fig. 6B,a). On the other hand, PP2A/A protein in IEC-6 cells was stable (Fig. 6A, middle), and its half-life was much longer than 240 min. After exposure to CHX, the levels of PP2A/A protein in IEC-TRPC1 cells declined slightly slower than levels observed in control IEC-6 cells, but the difference was not statistically significant (Fig. 6B,b), indicating that induced TRPC1 expression only has a marginal effect on PP2A/A stability. Because TRPC1 acts as a store-operated Ca2+ channel and stable IEC-TRPC1 cells exhibit the increased Ca2+ influx after store depletion, we further examined the role of [Ca2+]cyt in the increased stability of PP2A/C protein in IEC-TRPC1 cells. As shown in Fig. 6, C and D, decreased [Ca2+]cyt by removal of extracellular Ca2+ from the culture medium dramatically decreased the PP2A/C stability when protein synthesis was repressed by treatment with CHX. The half-life of PP2A/C protein in IEC-TRPC1 cells exposed to the Ca2+-free medium was ~47 min. Together, these results clearly indicate that induction of PP2A/C by TRPC1 overexpression results, at least partially, from an increase in PP2A/C protein stability through an elevation of [Ca2+]cyt due to CCE.


Figure 6
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  		Fig. 6. Studies of PP2A protein stability in control IEC-6 and stable IEC-TRPC1 cells with or without extracellular Ca2+.A: representative immunoblots of Western blots for PP2A/C and PP2A/A in cells cultured in the standard DMEM medium (with Ca2+): a, control IEC-6 cells (control); b, stable TRPC1-transfected IEC cells (TRPC1-C1). After cells were cultured in standard DMEM medium for 4 days, CHX at the concentration of 50 µg/ml was added to cultures, and whole cell lysates were harvested at indicated times. Levels of PP2A/C and PP2A/A proteins were assayed by Western blot analysis, and loading of proteins was monitored by actin. B: quantitative analysis of Western immunoblots by densitometry from results described in A: a, PP2A/C protein; b, PP2A/A protein. Values are means ± SE of data from three separate experiments, and relative levels of PP2A were corrected for protein loading as measured by densitometry of actin. *P < 0.05 compared with control IEC-6 cells. C: effect of exposure to Ca2+-free medium on PP2A/C protein stability in stable IEC-TRPC1 cells. The Ca2+-free medium (1.8 mM CaCl2 was replaced by 1.8 mM MgCl2 and an additional 0.1 mM EGTA was added to chelate the residual Ca2+) and CHX (50 µg/ml) were added at the same time, and whole cell lysates were harvested at indicated periods for Western blot analysis. a: cells exposed to the control medium (with Ca2+) and CHX (normal). b: cells exposed to the Ca2+-free medium and CHX (Ca2+-free). D: quantitative analysis of Western immunoblots by densitometry from results described in C. Values are means ± SE of data from three separate experiments. *P < 0.05 compared with cells exposed to the normal medium.

 
Decreased [Ca2+]cyt not only induced NF-{kappa}B activation by decreasing PP2A but also inhibited apoptosis. Removal of extracellular Ca2+ by exposure to the Ca2+-free medium decreased the levels of PP2A/C and PP2A/A mRNAs in IEC-6 cells and IEC-TRPC1 cells (Fig. 7), which were associated with significant reductions of their protein abundances (Fig. 8A). Interestingly, exposure to the Ca2+-free medium also reduced the levels of I{kappa}B{alpha} and induced NF-{kappa}B activation (Fig. 8B). A significant increase in NF-{kappa}B activation occurred at 3 h, and activation peaked at 4 h after exposure to the Ca2+-free medium. We also examined whether decreased PP2A activity by exposure to Ca2+-free medium affects the susceptibility of IEC-TRPC1 cells to TNF-{alpha}/CHX-induced apoptosis. Results presented in Fig. 8C show that PP2A inhibition by removal of extracellular Ca2+ protected IEC-TRPC1 cells against TNF-{alpha}/CHX-induced apoptosis. The percentages of apoptotic cell death declined from ~62% at 3 h and ~87% at 4 h in cells cultured in normal DMEM medium after exposure to TNF-{alpha}/CHX to ~35% and ~56% in cells cultured in the Ca2+-free medium, respectively. These results indicate that decreased [Ca2+]cyt by blocking Ca2+ influx activates NF-{kappa}B by repressing PP2A activity, thus inhibiting apoptosis in IECs.


Figure 7
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  		Fig. 7. Effect of removal of extracellular Ca2+ by exposure to Ca2+-free medium on levels of PP2A mRNA in stable IEC-TRPC1 cells. A: levels of PP2A/C mRNA. B: levels of PP2A/A mRNA. Stable IEC-TRPC1 cells were cultured in standard DMEM medium for 4 days, and total RNA was harvested at different time points after exposure to Ca2+-free medium. Levels of PP2A/C and PP2A/A mRNAs were measured by real-time quantitative PCR analysis. Data were normalized to amount of GAPDH, and values are means ± SE of data from three separate experiments. *P < 0.05 compared with control IEC-6 cells.

 

Figure 8
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  		Fig. 8. Effect of removal of extracellular Ca2+ on levels of PP2A and I{kappa}B{alpha} protein, NF-{kappa}B-dependent transcriptional activity, and apoptotic response to TNF-{alpha}/CHX in IEC-TRPC1 cells. A: representative immunoblots for PP2A/C, PP2A/A, and I{kappa}B{alpha} proteins. Whole cell lysates were harvested at different times after exposure to Ca2+-free medium, and levels of PP2A/C, PP2A/A, and I{kappa}B{alpha} proteins were measured by Western immunoblotting analysis. Three experiments were performed that showed similar results. B: NF-{kappa}B transcriptional activity as measured by NF-{kappa}B-dependent promoter luciferase reporter gene assays in cells described in A. Values are means ± SE of data from 6 dishes. *P < 0.05 compared with cells exposed to Ca2+-free medium for 0 h. C: changes in percentage of apoptotic cell death in cells described in A. Apoptosis was measured 3 and 4 h after exposure to TNF-{alpha}/CHX plus Ca2+-free medium. Data are expressed as means ± SE from 6 samples. *P < 0.05 compared with cells exposed to TNF-{alpha}/CHX alone.

 

   DISCUSSION
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Several lines of evidence indicate that TRPC proteins provide the structural basis for SOCs in mammalian cells (3, 8, 23, 24, 26) and that the pattern of expression of different TRPC isoforms differs among various tissues and is cell type dependent (2, 49). In normal IECs, TRPC1 is expressed as the predominant isoform (37, 38, 45), but its exact functions have not been fully elucidated. Our previous study shows that ectopic expression of the TRPC1 gene in IECs increases store depletion-mediated CCE, whereas TRPC1 silencing by transfection with its specific siRNA decreases Ca2+ influx through reduction of CCE, suggesting that TRPC1 is an excellent candidate protein for the SOC mechanism in the intestinal epithelium (37). To define the biological functions of TRPC1, it has been found that induced TRPC channel activity increases IEC migration during early epithelial restitution after superficial wounding by enhancing TRPC-mediated Ca2+ influx (37, 45). We have recently demonstrated that TRPC1 also plays an important role in regulating cell survival and apoptosis and that induced TRPC1 expression sensitizes IECs to apoptosis by inhibiting NF-{kappa}B activation (25). The present studies further implicate the increased TRPC1 channel activity in the regulation of PP2A expression, thereby advancing our understanding of TRPC functions in the regulation of intestinal mucosal homeostasis.

Data from the current study clearly indicate that induced TRPC1 expression increased levels of PP2A/C and PP2A/A mRNAs and proteins, which was associated with a significant increase in PP2A phosphatase activity (Fig. 1). To our knowledge, this is the first report showing the involvement of TRPC1 in the regulation of PP2A expression. As reported in our previous studies (25, 37), stable IEC-TRPC1 cells exhibit increased Ca2+ influx through CCE after Ca2+ store depletion. It is possible that induced TRPC1 channel activity enhances expression of PP2A/C and PP2A/A by increasing [Ca2+]cyt, although the exact mechanism underlying this process remains unclear. In support of this notion, it has been shown that PP2A gene transcription is altered by manipulation of [Ca2+]cyt (16, 47) and that increasing [Ca2+]cyt stimulates the PP2A/C{alpha}-promoter activity through the cAMP response element located in its proximal region (47). Because the inhibitor of the endoplasmic reticulum Ca2+-ATPase (thapsigargin) and cell-permeable Ca2+ chelator [1,2-bis(O-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetra (acetoxymethyl)ester] were used to increase or decrease [Ca2+]cyt in those studies (16, 17), the involvement of Ca2+-permeable channels in the regulation of PP2A expression was not elucidated. The results from the current study provide direct evidence indicating that TRPC1 functions as SOC channels in normal IECs and plays an important role in the regulation of PP2A expression.

The present study further demonstrates that PP2A modulates the subcellular localization of NF-{kappa}B through a process involving I{kappa}B signaling and that the inactivation of NF-{kappa}B in stable IEC-TRPC1 cells results, at least partially, from the increased PP2A activity. NF-{kappa}B is an inducible transcription factor, and many of the genes that are activated in the initiation of apoptosis are targets of NF-{kappa}B (31, 51). The kinetics of the NF-{kappa}B pathway, from its activation to its return to the resting state, are highly regulated by the status of I{kappa}B phosphorylation. Under nonstress conditions, NF-{kappa}B is sequestered in the cytoplasm by binding to I{kappa}B protein (17). In response to a host of stimuli, I{kappa}B proteins are phosphorylated by I{kappa}B kinase (IKK) and then targeted for polyubiquitination and degradation by proteasome, allowing free NF-{kappa}B to translocate to the nucleus where it activates transcription of its target genes (5, 13, 31). As shown in Fig. 2, increased PP2A activity in IECs overexpressing TRPC1 was associated with the increased levels of cytoplasmic I{kappa}B{alpha} and NF-{kappa}B, whereas inhibition of PP2A activity by treatment with okadaic acid or reduced PP2A expression by transfection with its specific siRNA decreased I{kappa}B{alpha} levels, enhanced NF-{kappa}B nuclear translocation, and increased NF-{kappa}B transcriptional activity (Figs. 3 and 4). Although the mechanism by which PP2A regulates I{kappa}B{alpha} degradation and subcellular distribution of NF-{kappa}B/I{kappa}B{alpha} complex remains unknown, PP2A is shown to directly interact with and regulate IKK activity (14, 22, 42). Inhibition of PP2A activity by treatment with okadaic acid potently activates IKK, induces I{kappa}B phosphorylation and resultant degradation, and enhances NF-{kappa}B nuclear translocation (40, 43).

On the other hand, contrary evidence also has been reported, indicating that PP2A plays a positive role in IKK activation and that inhibition of PP2A activity by okadaic acid attenuates TNF-{alpha}-induced degradation of I{kappa}B (15, 20). It is suggested by numerous studies that once IKK is activated by T-loop phosphorylation, it undergoes further hyperphosphorylation and becomes attenuated in its kinase activity. PP2A may remove all phosphate moieties from IKK, thereby returning IKK to its fully resting state, or perhaps under certain conditions, may remove only the inhibitory phosphates while leaving the activating phosphate intact. To date, whether PP2A plays a negative, positive, or dual role in the regulation of IKK activity remains to be fully resolved. Clearly, further studies are needed to define the mechanism by which PP2A regulates IKK activity and alters the subcellular distribution of NF-{kappa}B in cells overexpressing TRPC1 or after exposure to different stimuli.

The data produced in the present study also suggest that suppression of NF-{kappa}B activation, caused by PP2A activity induced by TRPC1 overexpression, plays an important role in regulating intestinal epithelial homeostasis. As shown in Figs. 5 and 8, decreased PP2A activity by PP2A/C silencing or decreasing [Ca2+]cyt via removal of extracellular Ca2+ prevented the increased susceptibility of IEC-TRPC1 cells to TNF-{alpha}/CHX-induced apoptosis. These results are consistent with the findings from others (39, 42) who have demonstrated that PP2A regulates apoptosis in IECs and that inhibition of PP2A by okadaic acid or depletion of cellular polyamines decreases apoptosis. To determine the mechanism underlying the pro-apoptotic role of PP2A in IECs, inhibition of PP2A activity is shown to increase steady-state levels of Bad and Bcl-2 and their phosphorylation, thereby abolishing cytochrome c release and caspase-9 and caspase-3 activation (39). The current study shows that PP2A is also implicated in the regulation of NF-{kappa}B subcellular distribution and that increased PP2A activity by ectopic expression of the TRPC1 gene inhibits NF-{kappa}B activation by increasing I{kappa}B{alpha} signaling, thus sensitizing IECs to TNF-{alpha}/CHX-induced apoptosis.

The results presented in Fig. 6 show that induced TRPC1 increased the half-life of PP2A/C protein, whereas decreased [Ca2+]cyt by exposure to the Ca2+-free medium prevented the increased stability of PP2A/C protein. This indicates that increased levels of PP2A/C protein in stable IEC-TRPC1 cells are partially due to a decrease in the rate of its protein degradation. These findings are consistent with our previous observations that show that stabilization of E-cadherin, occludin, and RhoA proteins in IECs are mediated by increasing Ca2+ influx after increased cellular polyamines (10, 11, 36). On the other hand, there were no significant changes in the rate of PP2A/A protein degradation between controls and IEC-TRPC1 cells, suggesting that induced PP2A/A by TRPC1 overexpression is mediated through a process independent of its protein stability. Data shown in Fig. 7 further indicate that decreased [Ca2+]cyt by removal of extracellular Ca2+ also decreased the levels of PP2A/C and PP2A/A mRNAs. These results indicate that Ca2+ also is necessary for expression of PP2A/C and PP2A/A mRNAs. It is unclear at present whether decreasing [Ca2+]cyt reduces the levels of PP2A/C and PP2A/A mRNAs by repressing their synthesis or increasing degradation.

In summary, these results indicate that forced expression of the TRPC1 gene in IECs increases PP2A protein levels and induces its phosphatase activity, which are associated with the NF-{kappa}B inactivation. Inhibition of PP2A by treatment with okadaic acid or PP2A/C silencing stimulates NF-{kappa}B nuclear translocation by decreasing I{kappa}B{alpha}, thus increasing NF-{kappa}B-dependent transcriptional activity. Our studies further demonstrate that inhibition of PP2A promotes cell survival when IEC-TRPC1 cells are exposed to TNF-{alpha}/CHX. The present study also helps to further define the mechanism underlying PP2A induction by providing new evidence showing that induced TRPC1 channel activity stimulates PP2A expression by increasing Ca2+ influx due to CCE. Stable IEC-TRPC1 cells display increased half-life of PP2A/C protein, whereas decreased Ca2+ influx by exposure to the Ca2+-free medium prevents the increased stability of PP2A/C. Increasing [Ca2+]cyt through CCE also is necessary for PP2A mRNA expression, because removal of extracellular Ca2+ decreases levels of PP2A/C and PP2A/A mRNAs. These findings suggest that PP2A plays a crucial role in regulation of apoptosis through NF-{kappa}B signaling after changes in TRPC1 channel activity under physiological conditions and thus contributes to the maintenance of intestinal mucosal homoeostasis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was supported by a Merit Review Grant (to J.-Y. Wang) from the Department of Veterans Affairs and by National Institutes of Health Grants DK-57819, DK-61972, and DK-68491 (to J.-Y. Wang). J.-Y. Wang is a Research Career Scientist, Medical Research Service, Department of Veterans Affairs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-Y. Wang, Baltimore Veterans Affairs Medical Center (112), 10 North 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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J. Abramowitz and L. Birnbaumer
Physiology and pathophysiology of canonical transient receptor potential channels
FASEB J, February 1, 2009; 23(2): 297 - 328.
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ajpcell.90635.2007v1
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