Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca²+ signaling by differentially modulating STIM1 and STIM2

Abstract

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 Ca(2+) 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 Ca(2+) influx and thus enhanced restitution. Here, we show that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca(2+) 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 Ca(2+) influx and stimulated epithelial restitution, while decreased formation of the STIM1/TRPC1 complex by polyamine depletion decreased Ca(2+) influx and repressed cell migration. Induced STIM1/STIM2 heteromers by polyamine depletion or STIM2 overexpression suppressed STIM1 membrane translocation and inhibited Ca(2+) influx and epithelial restitution. These results indicate that polyamines differentially modulate cellular STIM1 and STIM2 levels in IECs, in turn controlling TRPC1-mediated Ca(2+) signaling and influencing cell migration after wounding.

Fig. 1.

Changes in stromal interaction molecule 1 and 2 (STIM1 and STIM2) expression after altering the levels of cellular polyamines. A: levels of STIM1 (a) and STIM2 (b) mRNAs and proteins (c) after increasing polyamine levels by ectopic ornithine decarboxylase (ODC) overexpression in stable ODC-intestinal epithelial cell (IEC) cells. IEC-6 cells were infected with either the retroviral vector containing the sequence encoding mouse ODC cDNA or control retroviral vector lacking ODC cDNA (vector). Clones resistant to the selection medium containing 0.6 mg/ml G418 were isolated and screened for ODC expression. Levels of STIM1 and STIM2 mRNAs were examined by quantitative PCR analysis; values are the means ± SE from 3 separate experiments. *P < 0.05, compared with cells infected with control vector. Protein levels were assessed by Western blot analysis, and 3 separate experiments were performed that showed similar results. B: levels of STIM1 (a) and STIM2 (b) mRNAs and proteins (c) after polyamine depletion by treatment with α-difluoromethylornithine (DFMO). IEC-6 cells were grown in the control culture and culture containing DFMO (5 mM) alone or DFMO plus putrescine (Put; 10 μM) for 4 and 6 days. Values are the means ± SE from 3 separate experiments. *P < 0.05, compared with controls and cells treated with DFMO plus Put.

Fig. 2.

Association of STIM1 with STIM2 and TRPC1 after increasing or decreasing the levels of cellular polyamines. A: levels of STIM2 and TRPC1 proteins in the complex immunoprecipitated (IP) by anti-STIM1 antibody (Ab) in different lines of IECs. After whole cells lysates of IEC-6, differentiated IEC-Cdx2L1, and Caco-2 cells were immunoprecipitated by the specific antibody against STIM1, precipitates were separated by performing SDS-PAGE gels. Levels of STIM2 and TRPC1 proteins were measured using Western blot analysis with the antibody against STIM2 or TRPC1. Three separate experiments were performed that showed similar results. B: levels of STIM1 and STIM2 proteins in the complex immunoprecipitated by anti-TRPC1 antibody in different lines of IECs. C: changes in STIM1 association with STIM2 and TRPC1 after increasing the levels of cellular polyamines in stable ODC-IEC cells as described in Fig. 1A. D: changes in STIM1 association with STIM2 and TRPC1 after polyamine depletion by treatment with DFMO. IEC-6 cells were exposed to DFMO alone or DFMO plus Put for 4 days, and whole cell lysates were prepared for IP using anti-STIM1 antibody.

Fig. 3.

Changes in the levels of store-operated Ca²⁺ influx and cell migration after altering the levels of cellular polyamines. A: effect of increasing the levels of cellular polyamines on Ca²⁺ influx after store depletion: left: representative records showing the time course of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) changes after exposure to 10 μM cyclopiazonic acid (CPA) in the absence (0 Ca²⁺) or presence of extracellular Ca²⁺ in stable ODC-IEC cells; right: summarized data showing resting [Ca²⁺]_cyt and amplitude of CPA-induced Ca²⁺ influx. Values are the means ± SE; n = 20. *P < 0.05, compared with cells infected with control vector. B: effect of decreasing the levels of cellular polyamines on Ca²⁺ influx after store depletion. After IEC-6 cells were grown in control culture and cultures containing DFMO alone or DFMO plus Put for 4 days, levels of [Ca²⁺]_cyt were measured. Values are the means ± SE; n = 20. *P < 0.05, compared with controls or cells treated with DFMO plus Put. C: summarized data showing cell migration 6 h after wounding by removal of part of the monolayers in cells described in A. Values are the means ± SE of data from 6 dishes. *P < 0.05, compared with cells infected with control vector. D: summarized data showing cell migration after wounding in cells described in B. Values are the means ± SE of data from 6 dishes. *P < 0.05, compared with controls or cells treated with DFMO plus Put.

Fig. 4.

Effect of ectopic overexpression of STIM2 on store-operated Ca²⁺ influx and cell migration after wounding. A: representative immunoblots of STIM2 and STIM1. After Caco-2 cells were transfected with the STIM2 expression vector or vector lacking STIM2 cDNA (vector) for 48 and 72 h, whole cell lysates were harvested for Western blot analysis to monitor the expression of STIM2, STIM1, and loading control actin. Three separate experiments were performed that showed similar results. B: representative records showing the time course of [Ca²⁺]_cyt changes after exposure to CPA in the absence (0 Ca²⁺) or presence of extracellular Ca²⁺ in cells described in A. C: summarized data showing resting [Ca²⁺]_cyt (left) and the amplitude of CPA-induced Ca²⁺ influx (right) from cells described in B. Values are means ± SE; n = 20. *P < 0.05, compared with controls and cells transfected with vector alone. D: changes in cell migration after wounding in cells described in A. Cell migration was assayed 6 h after part of the monolayer was removed. Values are means ± SE of data from 6 dishes. *P < 0.05, compared with controls and cells transfected with vector alone.

Fig. 5.

Effect of ectopic STIM2 overexpression or increased endogenous STIM2 by polyamine depletion on redistribution of STIM1 to the plasma membrane (PM) during epithelial restitution after wounding. A: levels of STIM1 protein at the PM as measured by surface biotinylation assays in STIM2-transfected cells. After cells were transfected with control vector (left) and STIM2 expression vector (right) for 48 h, the PM fractions were isolated at various times after wounding and biotinylated proteins were detected by Western blot analysis using the antibody against STIM1. β-Catenin immunoblotting was performed as an internal control for equal loading. For measurement of total STIM1 protein, actin immunoblotting was performed as an internal control for equal loading. Three experiments were performed that showed similar results. (B) Levels of STIM1 protein at the PM in polyamine-deficient cells. Cells were grown in control culture medium (left) and medium containing 5 mM DFMO (right) for days; the PM fractions were isolated at various times after wounding for measurement of STIM1 at the PM.

Fig. 6.

Changes in the levels of store-operated Ca²⁺ influx and cell migration after STIM2 overexpression in stable STIM1-transfected IEC cells. A: representative records showing the time course of [Ca²⁺]_cyt changes after exposure to CPA in the absence (0 Ca²⁺) or presence of extracellular Ca²⁺ in IEC cells overexpressing constitutively active STIM1 (STIM1 EF-hand motif mutant EF1A3A; STIM1-IEC) or STIM1-IEC cells transfected with the STIM2 expression vector. After STIM1-IEC cells were transfected with the STIM2 expression vector or control vector for 48 h, the levels of store-operated Ca²⁺ influx was examined. B: summarized data showing resting [Ca²⁺]_cyt (left) and the amplitude of CPA-induced Ca²⁺ influx (right) from cells described in A. Values are means ± SE; n = 20. *,+P < 0.05, compared with controls and STIM1-IEC cells transfected with control vector. C: changes in cell migration after wounding in cells described in A. Cell migration was assayed 6 h after part of the monolayer was removed. Values are means ± SE of data from 6 dishes. *P < 0.05, compared with controls and STIM1-IEC cells transfected with control vector.

Fig. 7.

Effects of decreasing or increasing the levels of STIM2 on cell migration after wounding in polyamine-deficient cells and stable ODC-expressing cells. A: representative blots of STIM2 and STIM1 after STIM2 silencing or STIM2 silencing plus STIM1 overexpression in polyamine-deficient cells. Cell were cultured with DFMO for 2 days and then transfected with siRNA targeting the STIM2 mRNA (siSTIM2) or cotransfected with siSTIM2 and the STIM1 expression vector. After cells were grown for additional 48 h in the presence DFMO, they were harvested for Western blotting analysis. B: changes in cell migration 6 h after wounding in cells described in A. Values are means ± SE of data from 6 dishes. *P < 0.05, compared with controls; +P < 0.05, compared with DFMO; and #P < 0.05, compared with DFMO-cells transfected with siSTIM2 alone. C: representative blots of STIM2 and STIM1 in stable ODC-IEC cells after STIM2 overexpression. After cells were transfected with the STIM2 expression vector or control vector for 48 and 72 h, levels of STIM2 and STIM1 proteins were measured. D: changes in cell migration 6 h after wounding in cells described in C. *,+P < 0.05, compared with controls and ODC-IEC cells transfected with empty vector, respectively.