Early epithelial restitution occurs as a consequence of intestinal epithelial cell (IEC) migration after wounding, and its defective regulation is implicated in various critical pathological conditions. Polyamines stimulate intestinal epithelial restitution, but their exact mechanism remains unclear. Canonical transient receptor potential-1 (TRPC1)-mediated Ca2+ signaling is crucial for stimulation of IEC migration after wounding, and induced translocation of stromal interaction molecule 1 (STIM1) to the plasma membrane activates TRPC1-mediated Ca2+ influx and thus enhanced restitution. Here, we show that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca2+ signaling by altering the ratio of STIM1 to STIM2. Increasing cellular polyamines by ectopic overexpression of the ornithine decarboxylase (ODC) gene stimulated STIM1 but inhibited STIM2 expression, whereas depletion of cellular polyamines by inhibiting ODC activity decreased STIM1 but increased STIM2 levels. Induced STIM1/TRPC1 association by increasing polyamines enhanced Ca2+ influx and stimulated epithelial restitution, while decreased formation of the STIM1/TRPC1 complex by polyamine depletion decreased Ca2+ influx and repressed cell migration. Induced STIM1/STIM2 heteromers by polyamine depletion or STIM2 overexpression suppressed STIM1 membrane translocation and inhibited Ca2+ influx and epithelial restitution. These results indicate that polyamines differentially modulate cellular STIM1 and STIM2 levels in IECs, in turn controlling TRPC1-mediated Ca2+ signaling and influencing cell migration after wounding.
- mucosal injury
- early rapid repair
- cell migration
- capacitative calcium entry
- intestinal epithelial cells
- surface biotinylation
- stromal interaction molecule 1 and 2
- canonical transient receptor potential-1
the successful repair of damaged mucosa and ulcers in the gut requires epithelial cell decisions that regulate signaling networks controlling gene expression, survival, migration, and proliferation. Early restitution is an important repair modality in the intestine after injury, and it rapidly reseals superficial wounds by migrating visible remaining intestinal epithelial cells (IECs) from areas adjacent to, or just beneath, the injured surface to cover the defect, a process independent of cell proliferation (12, 24, 41, 50). This rapid epithelial restitution is a complex process that is highly regulated by numerous extracellular and intracellular factors including cellular polyamines (22, 30, 51). The natural polyamines (spermidine, spermine, and their precursor putrescine) are organic cations found in all eukaryotic cells and are implicated in many aspects of distinct cellular functions (9, 14, 43). The control of cellular polyamine levels has been thought for many years to be critical point of central convergence for the multiple signaling pathways driving epithelial cell motility and proliferation (38, 49). Polyamines stimulate early mucosal restitution of gastric and duodenal mucosal erosions in stressed rats (50, 51) and are essential for the stimulation of cell migration in an in vitro model mimicking the early cell division-independent stage of intestinal epithelial restitution (22, 30, 35). However, the precise mechanism by which polyamines enhance epithelial restitution at the molecular level remains to be fully elucidated.
Cytosolic free Ca2+ ([Ca2+]cyt) plays an important role in the regulation of cell motility and migration (31, 52), and increasing [Ca2+]cyt promotes epithelial restitution after wounding in both in vivo (50) as well as in vitro models (31–37). Ca2+ entry due to store depletion via store-operated channels (SOCs) is referred to as store-operated Ca2+ entry (SOCE) and greatly contributes to maintaining a sustained increase in intracellular [Ca2+]cyt and the refilling of Ca2+ into the stores (6, 23, 27). In nonexcitable cells including IECs, the canonical transient receptor potential-1 (TRPC1) protein functions as a Ca2+-permeable channel and is activated by Ca2+ store depletion (1, 34, 42). TRPC1-mediated Ca2+ signaling is crucial for epithelial restitution, because inhibition of TRPC1-mediated Ca2+ influx by TRPC1 silencing represses cell migration after wounding (34). Recently, stromal interaction molecule 1 (STIM1) has been shown to act as a sensor of Ca2+ within the store and play an essential role in the activation of TRPC1- and Orail-mediated Ca2+ influx after store depletion (5, 13, 54, 59). Although STIM1 is predominantly located in the endoplasmic reticulum (ER) in unstimulated cells, it rapidly translocates to the plasma membrane after Ca2+ store depletion or in response to various biological stimuli (2, 3, 21, 40). In the plasma membrane, STIM1 interacts with and activates SOCs such as TRPC1 and Orai1, thereby increasing SOCE. Inhibition of the expression of STIM1 reduces SOC activation, whereas overexpression of a constitutively active EF-hand motif mutant STIM1 increases its levels at the plasma membrane region and enhances SOCE (10, 59). Our previous studies (37) show that STIM1 translocation to the plasma membrane increases after wounding and that induced membrane STIM1 stimulates epithelial restitution by enhancing TRPC1-mediated SOCE.
STIM2 is the second member of STIM family, and it is similar to STIM1 in domain architecture, but the mature protein is longer by 69 amino acids than STIM1 (61). STIM2 has almost identical EF-hand-containing N-terminal domains and transmembrane sequences of STIM1, but STIM2 does not traffic to the plasma membrane after store depletion and its role in SOCE is controversial and depends on cell type (16). Initially, STIM2 was identified together with STIM1 as a positive regulator of SOCE in HeLa cells (7), but the reduction in STIM2 expression in other cell types has no effect on SOCE (8, 16). Cells overexpressing STIM2 are associated with an increase in resting [Ca2+]cyt levels (4), and STIM2 knockdown selectively lowers basal [Ca2+]cyt and ER Ca2+ concentrations (7, 44, 45). On the other hand, several studies (7, 26, 44) show that STIM2 is a powerful inhibitor of SOCE. Ectopic STIM2 overexpression inhibits SOCE and reduces a calcium release-activated calcium current in HEK293, PC12, A7r5, and Jurkat-T cells. STIM2 forms heteromers with STIM1 and blocks STIM1-activated SOCE (16), suggesting that STIM1 and STIM2 play a coordinated role in the control of SOCE.
This study was to test the hypothesis that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca2+ signaling by differentially modulating STIM1 and STIM2. First, we determined the influence of manipulating cellular polyamines on the levels of STIM1 and STIM2 expression and their interactions with TRPC1 in IECs. Second, we examined the role and mechanism of STIM2 in the regulation of STIM1-activated SOCE and epithelial restitution. Finally, we determined the implication of STIM1 and STIM2 in polyamine-dependent cell migration after wounding. The results presented herein indicate that increasing the levels of cellular polyamines by ectopic ODC overexpression increases STIM1 but decreases STIM2 levels, whereas polyamine depletion by inhibiting ODC has the opposing effects on expression of STIM1 and STIM2. STIM2 physically interacts with STIM1 but not with TRPC1, and increased STIM2 in polyamine-deficient cells inhibits TRPC1-mediated Ca2+ influx by repressing STIM1 translocation to the plasma membrane after wounding. These results indicate that polyamines stimulate epithelial restitution by enhancing TRPC1-mediated Ca2+ signaling as a result in alteration in the ratio of STIM1 and STIM2.
MATERIALS AND METHODS
Chemicals and cell culture.
Tissue culture medium and dialyzed FBS were from Invitrogen (Carlsbad, CA), and biochemicals were from Sigma (St. Louis, MO). The affinity-purified rabbit polyclonal antibody against TRPC1 was purchased from Alomone Laboratories (Jerusalem, Israel), and the antibodies recognizing STIM1, STIM2, and β-actin were obtained from BD Biosciences (San Diego, CA) and Cell Signaling Technologies (Danvers, MA). The secondary antibody conjugated to horseradish peroxidase was purchased from Sigma. α-difluoromethylornithine (DFMO) was from Genzyme (Cambridge, MA).
The IEC-6 cell line was purchased from the American Type Culture Collection (ATCC) at passage 13. IEC-6 cells were derived from normal rat intestinal crypt cells and were developed and characterized by Quaroni et al. (29). 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. ODC-overexpressing IEC (ODC-IEC) cells were developed from IEC-6 cells as previously described (11, 57) and expressed a more stable ODC variant with full enzyme activity. Stable Cdx2-transfected IEC cells (IEC-Cdx2L1) were developed from IEC-6 cells and maintained as described previously (30, 47, 48). Before experiments, IEC-Cdx2L1 cells were grown in DMEM containing 4 mM isopropyl-β-d-thiogalactopyranoside for 16 days to induce cell differentiation as described earlier (30, 35, 36, 39). The stable STIM1-transfected IEC cells (STIM1-IEC) were developed and characterized as described in our recent publications (37) and expressed a constitutively active STIM1 mutant protein (STIM1 EF-hand motif mutant EF1A3A). Caco-2 cells were purchased from ATCC and maintained in standard culture conditions as previously described (36).
RNA interference and plasmid construction.
The small interfering (si)RNA that was designed to specifically target the coding region of STIM2 (siSTIM2) mRNA was synthesized and purchased from Dharmacon (Lafayette, CO). Scrambled control siRNA (C-siRNA), which had no sequence homology to any known genes, was used as the control. The siSTIM2 and C-siRNA were transfected into cells as previously described (34–37). Briefly, for each 60-mm cell culture dish, 20 μl of the 5 μM stock siSTIM2 or C-siRNA were mixed with 500 μl of Opti-MEM medium (Invitrogen). This mixture was added to a solution containing LipofectAMINE 2000 in 500 μl of 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. The STIM2 expression vector that contains the full-length wild-type STIM2 cDNA was purchased from Origene Technologies (Rockville, MD), in which STIM2 expression was directed by the pCMV promoter. Cells were transfected with the STIM2 expression vectors by using the LipofectAMINE 2000 and performed as recommended by the manufacturer (Invitrogen).
Immunoprecipitation and immunoblotting analysis.
Cell samples, dissolved in ice-cold RIPA-buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 1.0% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 2 μg/ml leupeptin, and 2 mM sodium orthovanadate), were sonicated and centrifuged at 4°C, and the supernatants were collected for immunoprecipitation. Equal amounts of proteins (500 μg) for each sample were incubated with the specific antibody against STIM1 or TRPC1 (4 μg) at 4°C for 3 h, and protein A/G-PLUS-agarose was added and incubated overnight at 4°C. The precipitates were washed five times with ice-cold D-PBS, and the beads were resuspended in SDS sample buffer. For immunoblotting, samples were subjected to electrophoresis on PAGE gels as described previously (36, 37). Briefly, after the transfer of protein onto nitrocellulose membranes, the membranes were incubated for 1 h in 5% nonfat dry milk in 1× TBS-T buffer (Tris-buffered saline, pH 7.4, with 0.1% Tween-20). Immunologic evaluation was then performed overnight at 4°C in 5% nonfat dry milk/TBS-T buffer containing a specific antibody against STIM1, STIM2, or TRPC1. The membranes were subsequently washed with 1× TBS-T and incubated with the secondary antibodies 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).
Surface biotinylation assay.
Cell surface STIM1 protein was detected by cell surface biotinylation (37, 59) using a purification kit (Pierce, Rockford, IL) and performed according to manufacturer instructions. Briefly, after cells were washed twice with ice-cold PBS, they were biotinylated with 0.5 mg/ml sulfo-NHS-SS-biotin/PBS at 4°C for 45 min. Following quench of unreacted biotinylation reagent, the cells were washed and then lysed. After insoluble materials were removed by centrifugation, biotinylated proteins were collected by incubation with immobilized Neutravidin Gel overnight at 4°C. Proteins were eluted by boiling in 5× SDS-PAGE denaturing sample buffer (Pierce) for electrophoresis and Western blotting with the antibody against STIM1. β-Catenin immunoblotting was also examined for verifying equal loading of each lanes.
Measurement of [Ca2+]cyt.
Details of the digital imaging methods employed for measuring [Ca2+]cyt were described in our previous publications (31, 32, 34–37, 52). Briefly, cells were plated on 25-mm coverslips and incubated in culture medium containing 3.3 μM fura-2 AM for 30 min at room temperature (22–24°C) under an atmosphere of 10% CO2 in air. The 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). The final values of [Ca2+]cyt were obtained from fura-2 fluorescent emission excited at 380 and 340 nm from calibrated ranges as described in our previous publications (34, 52).
Measurement of cell migration.
Migration assays were carried out as described in our earlier publications (30–37). Cells were plated at 6.25 × 104/cm2 in DMEM containing FBS on 60-mm dishes thinly coated with Matrigel according to the manufacturer's instructions (BD Biosciences) and were incubated as described for stock cultures. Cells were fed on day 2, and cell migration was assayed 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 7- to 10-mm wide. The migrating cells in six contiguous 0.1-mm squares were counted at ×100 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 were reported as number of migrating cells per millimeter of scratch.
All data are expressed as means ± SE from six dishes. Immunoprecipitation and immunoblotting 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 (15).
Polyamines differentially regulate expression of STIM1 and STIM2.
The focus of the current study was to determine whether polyamines regulate TRPC1-mediated Ca2+ signaling by altering STIM1 and STIM2 levels. To determine the effect of increasing cellular polyamines on STIM1 and STIM2 expression, two clonal populations of IEC cells stably overexpressing ODC (ODC-IEC; Refs. 11, 20, 57) were used in this study. These stable ODC-IEC cells exhibited very high levels of ODC protein and >50-fold increase in ODC enzyme activity. Consistently, the levels of putrescine, spermidine, and spermine in ODC-IEC cells were increased by ∼12-fold, ∼2-fold, and ∼25% respectively, when compared with cells transfected with the control vector lacking ODC cDNA (data not shown), as previously reported (19, 53). As shown in Fig. 1A, ODC-IEC cells displayed a substantial increase in the levels of STIM1 mRNA and protein, but they were associated with a significant inhibition of STIM2 expression. The levels of STIM1 mRNA and protein were increased by more than threefold in stable ODC-IEC cells compared with those observed in cells transfected with the control vector, whereas the levels of STIM2 mRNA and protein were decreased by >55%. These distinct effects of increasing polyamine content on expression of STIM1 and STIM2 were not simply due to clonal variation since two stable clones, ODC-IEC-C1 and ODC-IEC-C2, showed similar responses. These results indicate that increasing the levels of cellular polyamines stimulates STIM1 but it represses STIM2 expression in IECs.
To further determine if decreasing the levels of cellular polyamines has opposite effect on expression of STIM1 and STIM2, cellular polyamines were depleted by inhibiting ODC activity with DFMO treatment. Consistent with our previous studies (30, 31, 52), exposure of IEC-6 cells to 5 mM DFMO for 4 and 6 days completely inhibited ODC enzyme activity and almost totally depleted cellular polyamines. The levels of putrescine and spermidine were undetectable on days 4 and 6 after treatment with DFMO, and spermine had decreased by ∼60% (data not shown). As shown in Fig. 1B, inhibition of polyamine biosynthesis by DFMO decreased levels of STIM1 mRNA and protein, but it increased STIM2 expression. The steady-state levels of STIM1 mRNA and protein were decreased by ∼50% in cells treated with DFMO for 4 and 6 days, whereas the levels of STIM2 mRNA and protein were more than twofold of the control values after polyamine depletion. Addition of exogenous putrescine (10 μM) given together with DFMO not only restored STIM1 expression to normal level but also prevented the increase in the levels of STIM2 mRNA and protein. Spermidine (5 μM) had an effect equal to that of putrescine on expression of STIM1 and STIM2 when it was added to cultures that contained DFMO (data not shown). These results indicate that polyamine depletion decreases STIM1 abundance but stimulates STIM2 expression.
Polyamines regulate TRPC1-mediated Ca2+ influx by altering the ratio of STIM1 to STIM2.
Our previous studies (37) demonstrate that induced STIM1/TRPC1 association at the plasma membrane increases SOCE in IECs and enhances epithelial restitution after wounding. To determine if polyamines regulate TRPC1-mediated SOCE and epithelial restitution by altering the ratio of STIM1 to STIM2, the following two sets of experiments were carried out. First, we examined changes in the physical interaction of TRPC1 with STIM1 and STIM2 after increasing or decreasing the levels of cellular polyamines. As shown in Fig. 2A, immunoprecipitation of STIM1 resulted in coimmunoprecipitation of both TRPC1 and STIM2 in parental IEC-6 cells, differentiated IEC-Cdx2L1 cells, and Caco-2 cells, whereas immunoprecipitation of TRPC1 only caused coimmunoprecipitation of STIM1 but not of STIM2 (Fig. 2B). Increasing the levels of cellular polyamines by ectopic ODC overexpression in stable ODC-IEC cells decreased STIM1/STIM2 complex, but it increased STIM1/TRPC1 association (Fig. 2C). In contrast, depletion of cellular polyamines by DFMO enhanced the STIM1/STIM2 association and reduced STIM1/TRPC1 complex, which was completely prevented by exogenous putrescine given together with DFMO (Fig. 2D). These results indicate that TRPC1 physically interacts with STIM1 but not with STIM2 and that polyamines stimulate STIM1/TRPC1 association by increasing the ratio of STIM1 to STIM2.
Second, we determined the role of polyamine-induced STIM1/TRPC1 complex in the regulation of SOCE and epithelial restitution after wounding. As shown in Fig. 3A, induced STIM1/TRPC1 complex by increasing cellular polyamines in stable ODC-IEC cells enhanced Ca2+ influx after store depletion with CPA, although there were no significant changes in the levels of resting [Ca2+]cyt and transient Ca2+ release from the store depletion. The level of store depletion-induced Ca2+ influx was increased by ∼80% in stable ODC-IEC cells compared with those observed in cells transfected with control vector. On the other hand, polyamine depletion-induced inhibition of STIM1/TRPC1 complex was associated with decreases in resting [Ca2+]cyt and store depletion-induced Ca2+ influx by CPA (Fig. 3B). The levels of resting [Ca2+]cyt and the amplitude of store depletion-induced Ca2+ influx in DFMO-treated cells were decreased by ∼25 and ∼60%, respectively. Furthermore, increased Ca2+ influx in stable ODC-IEC cells was accompanied by stimulation of epithelial restitution as indicated by an increase in cell migration after wounding (Fig. 3C). The number of cells migrating over the denuded area was increased by ∼45% in stable ODC-IEC cells. Consistently, the decreased resting [Ca2+]cyt and inhibited Ca2+ influx in DFMO-treated cells were associated with repression of epithelial restitution after wounding (by ∼70%, Fig. 3D). Restoration of the levels of STIM1/TRPC1 complex by exogenous putrescine given together with DFMO not only returned resting [Ca2+]cyt and store depletion-induced Ca2+ influx to near normal levels but also restored cell migration after wounding in polyamine-deficient cells. These findings indicate that polyamine-induced STIM1/TRPC1 association enhances Ca2+ influx and stimulates epithelial restitution.
STIM2 inhibits TRPC1-mediated Ca2+ influx and epithelial restitution by repressing STIM1 translocation to the plasma membrane.
To further define the exact role of STIM2 in the regulation of Ca2+ influx and epithelial restitution, three sets of experiments were performed. First, we determined the effect of overexpression of the wild-type STIM2 gene on levels of [Ca2+]cyt and cell migration in Caco-2 cells. This cell line was chosen for this study because it represents human intestinal epithelial cells and provides an excellent model for transient transfection. When Caco-2 cells were transfected with the expression vector containing the corresponding STIM2 cDNA under the control of pCMV promoter, levels of STIM2 protein were increased by approximately sixfold at 48 and 72 h after the transfection (Fig. 4A). The vector that lacked exogenous STIM2 cDNA was used as a negative control in this experiment and did not alter STIM2 level. In addition, transfection with the STIM2 expression vector had no effect on levels of STIM1 protein. As shown in Fig. 4, B and C, forced expression of the STIM2 gene decreased store depletion-induced Ca2+ influx by CPA, although it had no effect on the resting [Ca2+]cyt level. Levels of store depletion-induced Ca2+ influx were decreased by ∼50% at 48 and by ∼70% at 72 h after STIM2 transfection. Furthermore, ectopic expression of the STIM2 gene also repressed cell migration after wounding (Fig. 4D). The numbers of cell migration were decreased by ∼25% at 48 h and ∼55% at 72 h after the transfection. These results clearly show that ectopic STIM2 overexpression reduces Ca2+ influx due to SOCE and inhibits epithelial cell migration after wounding.
Second, we examined changes in the levels of STIM1 at the plasma membrane in cells overexpressing STIM2 after wounding. Consistent with our previous studies (37), levels of STIM1 at the plasma membrane increased dramatically after wounding as measured by surface biotinylation assays (Fig. 5A, left), although there were no significant changes in levels of total STIM1. The increase in STIM1 translocation to the plasma membrane occurred within 15 min after wounding and remained additional elevated between 30 and 60 min. The levels of plasma membrane STIM1 at 30 and 60 min after wounding were approximately sixfold the prewounding control level. The levels of STIM1 at the plasma membrane were returned to near normal level at 360 min after wounding (data not shown). β-Catenin, a membrane-associated protein, served as loading control in this study and exhibited no changes in its levels at the plasma membrane region after wounding. However, this rapid induction in STIM1 translocation to the plasma membrane after wounding was suppressed by STIM2 overexpression, because there was only a marginal increase in the levels of STIM1 at the plasma membrane in STIM2-transfected cells after wounding (Fig. 5A, right). Interestingly, increased levels of endogenous STIM2 by polyamine depletion also prevented the increase in STIM1 translocation to the plasma membrane after wounding (Fig. 5B). DFMO-treated cells exhibited a decreased levels of STIM1 at the plasma membrane compared with those observed in control cells after wounding. These findings indicate that induced STIM2 represses STIM1 translocation to the plasma membrane after wounding, thus reducing STIM1/TRPC1 interaction.
Third, we determined the influence of ectopic STIM2 overexpression on Ca2+ influx and cell migration in stable STIM1-IEC cells, which were recently developed and characterized in our laboratory (37). The stable STIM1-expressing cells highly expressed constitutively active STIM1 as reported previously (37) and exhibited a significant increase in both resting [Ca2+]cyt and Ca2+ influx through SOCE after depletion of store Ca2+ by CPA (Fig. 6, A and B). Stable STIM1-IEC cells also displayed an increase in the levels of cell migration after wounding, whereas ectopic STIM2 overexpression not only completely prevented increases in resting [Ca2+]cyt and CPA-induced Ca2+ influx but also totally abolished the stimulation of epithelial restitution (Fig. 6C). There were no significant differences in levels of [Ca2+]cyt, Ca2+ influx induced by CPA, and cell migration after wounding between control cells and stable STIM1-IEC cells transfected with STIM2. These results show that STIM2 is a negative regulator of STIM1/TRPC1-mediated Ca2+ signaling and epithelial restitution after injury.
Polyamines stimulate epithelial restitution by both decreasing STIM2 and increasing STIM1 levels.
To determine the implication of STIM1 and STIM2 in polyamine-induced epithelial restitution, we first examined changes in epithelial restitution in polyamine-deficient cells after STIM2 silencing by transfection with siRNA targeting STIM2 mRNA (siSTIM2). As shown in Fig. 7A, transfection of DFMO-treated cells with siSTIM2 prevented the increased levels of STIM2 protein, although it had no additional effect on decreased STIM1 content. STIM2 silencing in polyamine-deficient cells enhanced epithelial restitution as indicated by an increase in cell migration after wounding; this stimulatory effect on cells migration was further increased by overexpressing STIM1 (Fig. 7B). Second, we determined the effect of increasing the levels of STIM2 by ectopic overexpression of the STIM2 gene on epithelial restitution in stable ODC-IEC cells with high content of cellular polyamines (Fig. 7, C and D). Stable ODC-IEC cells were associated with both decreased STIM2 and increased STIM1 levels and also exhibited a stimulation of epithelial restitution. STIM2 overexpression in stable ODC-IEC cells inhibited polyamine-induced epithelial restitution as shown in a decrease in cell migration after wounding. In addition, transfection with the STIM2 expression vector failed to alter the levels of STIM1 in stable ODC-IEC cells. These findings indicate that polyamines promote epithelial restitution through TRPC1-mediated Ca2+ signaling by altering the ratio of STIM1 and STIM2.
Cellular polyamines are absolutely required for mucosal repair in the intestine (49–52), but few specific functions of polyamines at cellular and molecular levels are defined. Our previous studies have shown that activation of TRPC1-mediated Ca2+ influx is crucial for normal early epithelial restitution (34) and that induced STIM1 translocation to the plasma membrane after wounding activates this signaling pathway and promotes cell migration (37). The present studies further demonstrate that STIM2 functions as an inhibitor of STIM1/TRPC1-mediated Ca2+ influx through competitive inhibition of STIM1 binding to TRPC1 and represses epithelial restitution, thus advancing our understanding of the regulation of STIM1/TRPC1-mediated Ca2+ signaling after injury. The most significant of the new findings reported in this study, however, is that polyamines differentially modulate expression of STIM1 and STIM2 in IECs and that polyamines stimulate epithelial restitution by enhancing STIM1/TRPC1-mediated Ca2+ signaling through alterations in the ratio of STIM1 and STIM2.
The findings reported here clearly show that increasing cellular polyamines stimulated STIM1 but inhibited STIM2 expression, whereas depletion of cellular polyamines reduced STIM1 but induced STIM2 levels (Fig. 1). The opposing roles of cellular polyamines in STIM1 and STIM2 expression are not surprising, because polyamines regulate expression of various target genes positively or negatively through different mechanisms (9, 17, 28). For example, polyamines upregulate expression of c-Myc (19), c-Jun (28), CDK4 (55, 56), occludin (58), and E-cadherin genes (19), but these compounds repress expression of p53 (62), TGFβ/Smads (18), JunD (17, 55), ATF2 (56), MEK-1 (53), and CUG-binding protein-1 genes (11). Several studies (25, 43) aimed at characterizing the molecular aspects of polyamines further show that polyamines stimulate gene expression primarily by increasing the mRNA synthesis, but they repress gene expression predominantly by destabilizing mRNAs and/or inhibiting translation (19, 28, 58). How polyamines differentially regulate expression of STIM1 and STIM2 remains an open question, but we have preliminary evidence that supports the implication of RNA-binding protein HuR in this process. HuR functions as a powerful enhancer of mRNA stability and translation (62, 63), and its subcellular trafficking and activity are tightly regulated by polyamines (58, 64). A wide range of computer search for target mRNAs identified the STIM1 as a putative HuR target and that there are several computationally predicted hits of the HuR motif in 3′-untranslated region of the STIM1 mRNA. Interventions to decrease cellular polyamines decrease Chk2-dependent HuR phosphorylation (20) and also inhibit HuR/STIM1 mRNA association (unpublished data). The exact role and mechanism of HuR in the control of STIM1 mRNA stability and translation in the presence or absence of cellular polyamines are the focus of our ongoing studies.
Our results also indicate that STIM1 physically interacted with both STIM2 and TRPC1 in IECs, whereas STIM2 only directly bound to STIM1 but not to TRPC1. Considering the distinct roles of STIM1 and STIM2 in the regulation of SOCs in certain cell types (26, 45), it is likely that STIM2 represses STIM1/TRPC1-mediated SOCE by competitively inhibiting STIM1 binding to TRPC1. In support of this possibility, decreased levels of STIM2 by increasing cellular polyamines in stable ODC-IEC cells were associated with an induction in STIM1/TRPC1 association, whereas increased STIM2 levels following polyamine depletion were accompanied by a reduction in STIM1/TRPC1 interaction (Fig. 2). Consistent with our previous studies (31, 33, 52), decreased levels of Ca2+ influx in polyamine-deficient cells also exhibited delayed epithelial restitution after wounding, which was prevented by adding exogenous polyamine putrescine (Fig. 3) or STIM2 silencing (Fig. 7). In stable ODC-IEC cells with a reduction of endogenous STIM2, the increases in the levels of Ca2+ influx due to SOCE and cell migration after wounding were prevented by ectopic STIM2 overexpression. These findings provide new evidence showing that decreased TRPC1-mediated Ca2+ signaling and subsequent inhibition of epithelial restitution in polyamine-deficient cells results, at least partially, from decreased STIM1/TRPC1 association as a result of an increase in STIM2 levels.
STIM2 inhibits STIM1/TRPC1-mediated Ca2+ influx and epithelial restitution by repressing subcellular redistribution of STIM1 after wounding. Unlike STIM1, biotinylation and FACs analysis indicate that STIM2 does not traffic to the plasma membrane after store depletion (8, 44), although STIM2 expression is dynamically regulated according to cell status (4). Generally, the expression pattern of STIM2 often opposes that of STIM1, whose expression levels do not change upon T-lymphocyte differentiation (7) or increase during the differentiation of adipocytes and myoblasts (26, 45, 46). As reported previously (37), relocalization of STIM1 to the plasma membrane increased after wounding in normal IEC monolayer, and this wounding-induced STIM1 redistribution increases Ca2+ influx through SOCs and it occurs before an induction of cell migration after injury. Reduction in the levels of STIM1 at the plasma membrane by STIM1 silencing represses SOCE and inhibits epithelial restitution (37). The data presented in Fig. 5 further show that both ectopic STIM2 overexpression and increasing endogenous levels of STIM2 by polyamine depletion abolished an increase in STIM1 translocation to the plasma membrane after wounding. Moreover, increased the levels of STIM2 also prevented STIM1-induced stimulation of Ca2+ influx and cell migration after wounding in cells stably overexpressing constitutively active STIM1 mutant (37) (Fig. 6). These results suggest that formation of STIM2/STIM1 heteromers suppress STIM1 translocation to the plasma membrane after wounding, thus inhibiting SOCE and epithelial restitution.
In summary, these results indicate that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca2+ signaling by differentially modulating expression of STIM1 and STIM2. Increasing the levels of cellular polyamines stimulates STIM1 but inhibits STIM2 expression, whereas polyamine depletion decreases STIM1 but increases STIM2 levels. Experiments investigating the biological functions of polyamine-regulated STIMs further show that induced STIM1/TRPC1 complex through an induction in the ratio of STIM1/STIM2 by increasing cellular polyamines enhanced TRPC1-mediated Ca2+ influx and stimulated epithelial restitution after injury, while decreased STIM1/TRPC1 association via a reduction in the ratio of STIM1/STIM2 following polyamine depletion decreased Ca2+ influx and repressed cell migration. The present study also shows that formation of STIM1/STIM2 heteromers in cells overexpressing STIM2 or after polyamine depletion suppressed STIM1 translocation to the plasma membrane after wounding, thus inhibiting TRPC1-mediated Ca2+ signaling. Because polyamines are required for early mucosal restitution (50, 51) and their cellular levels increase dramatically after injury (22, 30, 51), these findings suggest that polyamine-regulated Ca2+ signaling through alteration in STIM1/TRPC1 association plays an important role in the maintenance of intestinal epithelial integrity under physiological and pathological conditions.
This work was supported by Merit Review Grants from the Department of Veterans Affairs (to J. N. Rao and J. Y. Wang) and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-57819, DK-61972, and DK-68491 (to J. Y. Wang). J.-Y. Wang is a Senior Research Career Scientist, Medical Research Service, US Department of Veterans Affairs.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: J.N.R. and J.-Y.W. conception and design of research; J.N.R., N.R., R.Z., T.Z., L.L., and L.X. performed experiments; J.N.R., N.R., R.Z., T.Z., L.L., L.X., D.J.T., and J.-Y.W. analyzed data; J.N.R. and D.J.T. interpreted results of experiments; J.N.R. and J.-Y.W. prepared figures; J.N.R. and J.-Y.W. drafted manuscript.