c-Jun enhances intestinal epithelial restitution after wounding by increasing phospholipase C-γ1 transcription

Abstract

c-Jun is an activating protein 1 (AP-1) transcription factor and implicated in many aspects of cellular functions, but its exact role in the regulation of early intestinal epithelial restitution after injury remains largely unknown. Phospholipase C-γ1 (PLCγ1) catalyzes hydrolysis of phosphatidylinositol 4,5 biphosphate into the second messenger diacylglycerol and inositol 1,4,5 triphosphate, coordinates Ca²⁺ store mobilization, and regulates cell migration and proliferation in response to stress. Here we reported that c-Jun upregulates PLCγ1 expression and enhances PLCγ1-induced Ca²⁺ signaling, thus promoting intestinal epithelial restitution after wounding. Ectopically expressed c-Jun increased PLCγ1 expression at the transcription level, and this stimulation is mediated by directly interacting with AP-1 and CCAAT-enhancer-binding protein (C/EBP) binding sites that are located at the proximal region of the rat PLCγ1 promoter. Increased levels of PLCγ1 by c-Jun elevated cytosolic free Ca²⁺ concentration and stimulated intestinal epithelial cell migration over the denuded area after wounding. The c-Jun-mediated PLCγ1/Ca²⁺ signal also plays an important role in polyamine-induced cell migration after wounding because increased c-Jun rescued Ca²⁺ influx and cell migration in polyamine-deficient cells. These findings indicate that c-Jun induces PLCγ1 expression transcriptionally and enhances rapid epithelial restitution after injury by activating Ca²⁺ signal.

the gastrointestinal (GI) epithelium is exposed to a wide variety of luminal noxious substances. Acute mucosal injury occurs commonly during critical pathological conditions, such as trauma, thermal injury, shock, and the period after massive surgical operations (7, 8, 20, 41). Early epithelial restitution is an important repair modality in the GI mucosa and occurs as a consequence of epithelial cell migration over the damaged area after superficial injury, a process that is independent of cell proliferation (4, 20, 36, 37). This rapid reepithelialization is a complex process of coordinated cell migration and highly regulated by multiple extracellular and intracellular factors. Cytosolic free Ca²⁺ ([Ca²⁺]_cyt) plays an important role in the regulation of intestinal epithelial cell (IEC) migration over the wounded area after injury, and increasing [Ca²⁺]_cyt stimulates epithelial restitution (25, 26, 28, 31, 34). Ca²⁺ entry attributable to store depletion is often called store-operated Ca²⁺ entry (SOCE) and is mediated by Ca²⁺-permeable channels termed store-operated Ca²⁺ channels (SOCs) (1, 2, 21). Phospholipase C-γ1 (PLCγ1) catalyzes hydrolysis of phosphatidylinositol 4,5 biphosphate into the second messenger diacylglycerol and inositol 1,4,5 triphosphate, which alters Ca²⁺ store mobilization and increases [Ca²⁺]_cyt through SOCE after wounding. However, the exact mechanism by which PLCγ1 cellular abundance is regulated remains to be fully elucidated.

c-Jun protein is an activating protein 1 (AP-1) transcription factor that is involved in many aspects of cellular processes and functions (40, 43). Disrupted expression of the c-jun gene in murine hepatocytes prevents the emergence of hepatocellular carcinoma (6), and c-Jun is also sufficient for stimulation of anchorage-independent growth of Rat1a cells (15). Fibroblasts lacking the c-jun gene exhibit the defects in cell proliferation and apoptosis in response to genotoxic stress (5, 13). Inhibition of c-Jun expression reduces cell migration and invasion through downregulation of c-Src (22) and ERK (39, 40) and hyperactivation of ROCK-II kinase (12). In GI mucosa, c-Jun expression levels increase significantly after stress-induced mucosal injury, whereas decreasing the levels of c-Jun by polyamine depletion delays the recovery of damaged mucosa (45, 46).

The purpose of this study was to test the hypothesis that c-Jun regulates PLCγ1 expression, thus enhancing SOCE-mediated Ca²⁺ influx and stimulating cell migration after wounding. First, we determined whether c-Jun positively regulates PLCγ1 expression, especially its role at the transcriptional level. Second, we examined whether ectopically expressed c-Jun increases PLCγ1-mediated Ca²⁺ influx through SOCE and promotes IEC migration after wounding, whereas c-Jun silencing decreased PLCγ1, reduced SOCE, and inhibited cell migration. Third, we investigated whether PLCγ1 silencing prevents c-Jun-induced SOCE and cell migration after wounding. Our results show that c-Jun enhances PLCγ1 expression through its transcriptional activation and stimulates IEC migration over the wounded area by increasing PLCγ1/Ca²⁺ signal.

MATERIALS AND METHODS

Chemicals and cell culture.

Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media, Lipofectamine 2000, and dialyzed FBS were obtained from Invitrogen (Carlsbad, CA), and biochemicals were obtained from Sigma (St. Louis, MO). The antibodies recognizing PLCγ1 (cat. no. 610028) and STIM1 (cat. no. 610954) were purchased from BD Biosciences (San Jose, CA), and c-Jun (catalog no. SC-166540) was from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody against actin (cat. no. CP01) was purchased from EMD Millipore (Danvers, MA). L-α-difluoromethylornithine (DFMO) was from Genzyme (Cambridge, MA).

The IEC-6 cell line, derived from normal rat intestinal crypt cells (23), was purchased from the ATCC at passage 13 and used at passages 15–20 as described previously (25, 28, 34). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated FBS. Stable Cdx2-transfected cells (IEC-Cdx2L1) were developed by Suh and Traber (42) and maintained as described in our previous studies (24, 35). The expression vector and the LacSwitch System (Stratagene, La Jolla, CA) was used for directing the conditional expression of the Cdx2 gene, and Isopropyl β-D-1-thiogalactopyranoside (IPTG) served as the inducer for the gene expression. Before experiments, IEC-Cdx2L1 cells were grown in DMEM containing 4 mM IPTG for 16 days to induce cell differentiation as described earlier (24).

Recombinant adenoviral construction and infection.

Adenoviral vectors expressing c-Jun (AdcJun) were constructed using the Adeno-X Expression system (Clontech, Mountain View, CA) according to the protocol recommended by the manufacturer with a slight modification as described previously (17, 18). Briefly, the full-length cDNA of human wild-type c-Jun was cloned into the pShuttle by digesting the BamHI/HindIII and ligating the resultant fragments into the XbaI site of the pShuttle vector. pAdeno-c-Jun (AdcJun) was constructed by digesting the pShuttle construct with PI-SceI/I-CeuI and ligating the resultant fragment into the PI-SceI/I-CeuI sites of the pAdeno-X adenoviral vector. Recombinant adenoviral plasmids were packaged into infectious adenoviral particles by transfecting human embryonic kidney (HEK)-293 cells using Lipofectamine Plus reagent (GIBCO, Gaithersburg, MD). Titers of the adenoviral stock were determined by standard plaque-forming assay. Recombinant adenoviruses were screened for the expression of the introduced gene by Western blot analysis using anti-c-Jun antibody. pAdeno-X, which was the recombinant replication-incompetent adenovirus carrying no exogenous cDNA insert (Adnull), was grown and purified as described above and served as a control adenovirus. Cells were infected by AdcJun or Adnull (100 pfu/cell), and cell samples were collected for various measurements 24 or 48 h after the infection.

RNA interference.

The small interfering RNA (siRNA) specifically targeting the c-Jun mRNA (sic-Jun) or PLCγ1 mRNA (siPLCγ1) was 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 sic-Jun, siPLCγ1, or C-siRNA was transfected into cells as described previously (9, 34). Briefly, for each 60-mm cell culture dish, 10 μl of the 20 μM stock sic-Jun, siPLCγ1, or C-siRNA were 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 incubation.

RT-PCR and qRT-PCR analysis.

Total RNA was isolated by using the RNeasy Mini Kit (Qiagen, Valencia, CA). Equal amounts of total RNA (5 μg) were transcribed to synthesize single-strand cDNA with an RT-PCR kit (Invitrogen). RT-PCR was performed as described in our earlier studies (26, 31, 50). To quantify the PCR products (the amounts of mRNA) of PLCγ1, an invariant mRNA of β-actin was used as an internal control. qRT-PCR was performed using Applied Biosystems Instruments (Foster City, CA) specific primers, probes, and software as described in our previous publications (49, 50). The levels of PLCγ1 mRNA were quantified by qRT-PCR analysis and normalized with GAPDH levels.

Reporter plasmids and luciferase assays.

The PLCγ1 promoter was cloned from rat genomic DNA. The construct of full-length PLCγ1 promoter luciferase (Luc) reporter, F-Luc (1038-bp regulatory region upstream of the PLCγ1 gene fused to the Luc reporter gene) and its four deleted mutants F1-Luc (−761/+92), F2-Luc (−652/+92), F3-Luc (−252/+92), and F4-Luc (−116/+92) were generated using respective primer pairs whose sequences are listed in Table 1. The point mutants of AP-1 and/or CCAAT-enhancer-binding protein (C/EBP) binding sites of PLCγ1 promoter driving Luc reporter were generated using the QuikChange site-directed mutagenesis kit and performed according to the manufacturer’s instructions (Stratagene, La Jolla, CA). By using the F2-Luc construct of the PLCγ1 promoter as a template, two synthetic oligonucleotide primers were designed whose sequences are listed in Table 1, each of which was complementary to the opposite strand of template DNA and contained the desired mutation. The oligonucleotide primers were extended during temperature cycling, and incorporation of the primers generated the mutated plasmid. After digestion with DpnI, 4 µl of products was used to transform XL-1 competent cells provided by the mutagenesis kit. Mutations of various binding sites within the PLCγ1 promoter were verified by DNA sequencing. Transient transfection was performed using the Lipofectamine kit as recommended by the manufacturer (Invitrogen). Cells were collected 48 h after the transfection, and luciferase activity was examined using the Bright-Glo luciferase assay system as recommended by the manufacturer (Promega, Madison, WI). The luciferase activity from individual constructs was normalized by Renilla-driven luciferase activity.

Table 1.

Primers and mutation of PLCγ1 promoter sequences

Primer	Sequence
F-Luc forward	5′-AAGAACCTCAGTGGATTATC-3′
F1-Luc forward	5′-CTAAGATAAAACCAGAGCTT-3′
F2-Luc forward	5′-GTCCGAGGGTCTGAGGTGCC-3′
F3-Luc forward	5′-GGCGGGGTTGCGTGCGTGAC-3′
F4-Luc forward	5′-GGGCGGGGGTCGTCCCTTGG-3′
Reverse	5′-CCATGCTGCAGCGACCTCGG-3′
AP-1 mutation	5′-CCGTGCGCGCAGCCCCCTGACGGGCCGC-3′
	5′-GCGGCCCGTCAGGGGGCTGCGCGCACGG-3′
C/EBP mutation	5′-CGGCAGGACCGAGGTGTGTTCCCTCCGCCTTCTG-3′
	5′-CAGAAGGCGGAGGGAACACACCTCGGTCCTGCCG-3′

Chromatin immunoprecipitation.

Cells were infected with the AdcJun or Adnull for 48 h and then fixed with 1% formaldehyde to cross link chromatin. Chromatin immunoprecipitation (ChIP) analysis was performed using the Active Motif ChIP-IT kit (Carlsbad, CA), following the manufacturer’s recommendations with minor modification as described previously (19, 49). Briefly, cells were suspended in lysis buffer and gently dounced on ice with 10 strokes to aid in nuclei release. After centrifugation, the nuclear pellet was suspended in digestion buffer, and the chromatin was sheared with Enzymatic Shearing Cocktail. The sheared DNA samples were centrifuged, and the supernatants were collected and precleared with protein G beads. The precleared DNA samples were then incubated with the anti-c-Jun antibody or control IgG overnight with constant rotation. The immunocomplexes were captured by addition of protein G beads, and the immunoprecipitated DNA was collected from the beads using ChIP elution buffer. DNA-protein cross links were reversed and deproteinized, and DNA was recovered and amplified by PCR. Primers to amplify the PLCγ1 promoter containing AP-1 and C/EBP sites were 5′-GCAGCCTGCCCAGGTAA-3′ and 5′-GTGTTAAGTCC-GCCTTCTGC-3′. Primers to amplify the proximal region of the GAPDH promoter (a negative control) were 5′-TACTAGCGGTTTTACGGGCG-3′ and 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′. The DNA isolated through IgG ChIP was used as a negative control. The input DNA, obtained from chromatin that was processed (cross linked and reversed) similarly to the samples, served as a positive control for PCR effectiveness.

Western blot analysis.

Cell samples, placed in SDS sample buffer, 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 1× PBS-Tween 20 (PBS-T). Immunological evaluation was then performed for 1 h in 5% milk/PBS-T buffer containing 1 μg/ml of the specific antibody against c-Jun or PLC-γ1 protein. The filters were subsequently washed with 1× PBS-T and incubated for 1 h with the second antibody conjugated to peroxidase by protein cross linking with 0.2% glutaraldehyde. After being extensively washed with 1× PBS-T, the immunocomplexes on the membranes were reacted for 1 min with chemiluminiscence reagent (NEL-100 DuPont NEN). Antibodies used in this study were validated in our previous publications (30, 31).

Measurement of [Ca²⁺]_cyt.

Detailed digital imaging methods employed for measuring [Ca²⁺]_cyt were described in our previous studies (25, 26, 28–30). Briefly, cells were plated on 25-mm coverslips and incubated with the culture medium containing 3.3 µM fura-2 AM for 30 min 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 (Opelco, Washington, DC) coupled by fiberoptics to a Pulnix charge-coupled device video camera (Stanford Photonics, Stanford, CA). Image acquisition and analyses were performed with a Metamorph Imaging System (Universal Imaging, Bedford Hills, NY). The final values of [Ca²⁺]_cyt were obtained from fura-2 fluorescent emission excited at 380 and 340 nm from calibrated ranges as described previously (3, 28–30, 33, 35).

Measurement of cell migration.

Migration assays were carried out as described previously (3, 9, 28–30). 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 7–10 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 the number of migrating cells per millimeter of scratch.

Statistical analysis.

All data for migration experiments are expressed as means ± SE from six dishes in one experiment and repeated three times (n = 3). Measurement of [Ca²⁺]_cyt, qRT-PCR, and immunoblotting analyses 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 (10), and values of P < 0.05 were considered significant.

RESULTS

Increased c-Jun induces PLCγ1 expression levels.

To determine the role of c-Jun in the regulation of PLCγ1 expression, we examined the effect of increasing c-Jun on the levels of PLCγ1 mRNA and protein in differentiated IEC-Cdx2L1 cells. The AdcJun was used in this study because it is shown to infect a variety of cultured rat and human epithelial cells with >90% efficiency (17, 18). As shown in Fig. 1A, the levels of c-Jun protein were increased by ~10-fold after AdcJun infection at 100 pfu/cell for 48 h. Infection with Adnull at the same concentration failed to induce c-Jun level. Transient infection with AdcJun increased the levels of PLCγ1 protein by approximately sevenfold compared with that observed in cells infected with Adnull although it had no effect on STIM1 protein levels. In addition, c-Jun overexpression also induced PLCγ1 mRNA levels as measured by qRT-PCR (Fig. 1B). We also examined the effect of c-Jun on PLCγ1 expression in IEC-6 and Caco-2 cells and demonstrated that ectopically expressed c-Jun by infection with AdcJun had a similar stimulatory action in PLCγ1 expression in these two lines of IECs (data not shown). In addition, neither AdcJun nor Adnull affected cell viability as measured by Trypan blue staining (data not shown). These findings indicate that c-Jun enhances PLCγ1 expression in IECs.

Fig. 1.

Ectopic c-Jun overexpression induces PLCγ1 expression. A: representative immunoblots of c-Jun, PLCγ1, and STIM1 proteins. Differentiated IEC-Cdx2L1 cells were infected with AdcJun or Adnull at a multiplicity of infection of 100 pfu/cell, and whole cell lysates were harvested 48 h after infection for Western blot analysis. Actin immunoblotting was performed as an internal control for equal loading. B: relative levels of PLCγ1 mRNA in the samples as described in A. Values are means ± SE of data from 3 separate experiments (n = 3). *P < 0.05 compared with cells infected with Adnull analyzed by one-way ANOVA followed by Duncan’s test.

c-Jun increases PLCγ1 transcription through AP-1 and C/EBP binding sites within its promoter.

To define the possible mechanism by which c-Jun upregulates PLCγ1 expression, we first examined the effect of increased c-Jun on PLCγ1 gene transcription. To do so, the PLCγ1 promoter was cloned from rat genomic DNA, which contained AP-1 and C/EBP binding sites as illustrated in Fig. 2A, a, top. To map the c-Jun-responsive region of the PLCγ1 promoter, different reporter constructs were also prepared containing deletions of AP-1 or C/EBP site in the PLCγ1 promoter (Fig. 2A, a). As shown in Fig. 2A, b, the elements that contained a 652-bp fraction were required for basal and regulatory PLCγ1 expression in IECs, as the 5′ deletions of the PLCγ1 promoter gradually decreased to basal levels of reporter gene activity. The results presented in Fig. 2B further show that ectopic expression of the c-Jun stimulated PLCγ1 promoter activity and that this induction was mediated through AP-1 and C/EBP sites within its promoter region. The F2-Luc reporter activity (−652-bp promoter fragment containing AP-1 and C/EBP sites) increased significantly after c-Jun overexpression, indicating that c-Jun-responsive elements were included within this segment. However, this increased transcription by c-Jun was abolished when cells were transfected with either F3-Luc (−252-bp promoter fragment containing no AP-1 site) or F4-Luc (−116-bp promoter fragment lacking both AP-1 and C/EBP binding sites). To further determine the exact function of AP-1 and C/EBP sites within the rat PLCγ1 promoter region, different AP-1, C/EBP, and both point mutants of the PLCγ1 promoter were generated as indicated in Fig. 2C using the site-directed mutagenesis kit as described in materials and methods. The point mutation of the AP-1 and/or C/EBP sites within the rat PLCγ1 promoter region partially but significantly prevented the induction in PLCγ1 promoter by c-Jun. The induced activity was decreased by ~30% with AP1Mut-Luc and by ~40% with C/EBPMut-Luc reporter, respectively (Fig. 2D, middle). Moreover, c-Jun-induced PLCγ1 activation was completely prevented when both AP-1 and C/EBP sites were point mutated (AP1+C/EBPMut-Luc) (Fig. 2D, right). Under these conditions, there were no significant differences in the levels of PLCγ1 promoter activity between cells infected with Adnull and cells overexpressing c-Jun.

Fig. 2.

c-Jun regulates PLCγ1 transcription by direct interaction with PLCγ1 promoter. A: defining of active sequence in PLCγ1 promoter by deletion. a: schematic representation of PLCγ1 promoter. Computationally predicted transcription factor binding motifs were marked, and different sizes of PLCγ1 promoter-fraction-driving-luciferase (Luc) reporters were constructed. b: basal activity of various deletion mutants of PLCγ1 promoter. IEC-Cdx2L1 cells were transfected with different deletion mutants of PLCγ1 promoter, and the levels of luciferase reporter activity were measured 24 h after the transfection. Values are means ± SE from three separate assays (n = 3). B: changes in luciferase reporter activity of deletion constructs after c-Jun overexpression. IEC-Cdx2L1 cells were infected with either AdcJun or Adnull at a concentration of 100 pfu/cell for 24 h and then transfected with different PLCγ1 promoter luciferase reporter deletion constructs as shown in A,a. The levels of luciferase activity were assayed 24 h after transfection (n = 3). *P < 0.05 compared with cells infected with Adnull analyzed by one-way ANOVA followed by Duncan’s test. C: schematic representation of point mutation of rat PLCγ1 promoter. F2-Luc (L) reporter was used as the wild-type (WT) template. Mutant luciferase reporter with AP-1 binding site (AP1 Mut-L), C/EBP binding site (C/EBPMut-L), and both sites (AP1+C/EBP Mut-L) alteration were constructed. D: reporter activity from studies used various constructs described in C. *P < 0.05 compared with cells infected with Adnull. +P < 0.05 compared with cells transfected with AdcJun and F2-L.

ChIP analysis was used to further examine the in vivo association of c-Jun with the rat PLCγ1 promoter. Nuclear fractions were immunoprecipitated using a specific anti-c-Jun antibody in cells infected with the AdcJun or Adnull, and the associated DNA was purified. With the use of specific PCR primers, a 244-bp PCR product was obtained, which matched the sequence of the PLCγ1 promoter from −402 to −159 (containing AP-1 and C/EBP sites) relative to the transcriptional start site. c-Jun was found to bind the PLCγ1 promoter in vivo, as shown using an anti-c-Jun antibody in cells overexpressing c-Jun (Fig. 3, top, lane 7). This association was specific for c-Jun because no PCR product was detectable in AdcJun-infected cells when using a nonspecific antibody (IgG; Fig. 3, top, lane 5) or when using primers to an unrelated promoter such as the GAPDH promoter (Fig. 3, bottom, lane 7). These results indicate that c-Jun stimulates PLCγ1 expression at the transcription level possibly via interacting with AP-1 and C/EBP binding sites located within the rat PLCγ1 promoter region.

Fig. 3.

c-Jun binds to the rat PLCγ1 promoter. Association of c-Jun with the PLCγ1 promoter was analyzed by chromatin immunoprecipitation assay by using anti-c-Jun antibody. Nonspecific IgG immunoprecipitation was used as a negative control. The fragment of PLCγ1 promoter in pull-down materials was detected by PCR analysis. The expected size of PCR product was 244 bp. Three experiments were performed that showed similar results (n = 3).

c-Jun promotes cell migration by enhancing PLCγ1-mediated Ca²⁺ signaling.

Our previous studies have shown that PLCγ1 regulates cell migration through SOCE-mediated Ca²⁺ signaling (29, 30). Therefore, we further investigated the biological consequences of c-Jun-induced PLCγ1 expression in intestinal epithelial restitution after wounding in differentiated IEC-Cdx2L1 cells. As shown, ectopically expressed c-Jun increased Ca²⁺ influx following cyclopiazonic acid (CPA)-induced store depletion and also enhanced cell migration after wounding (Fig. 4). Exposure to CPA resulted in an initial transient increase in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺, which was apparently due to Ca²⁺ mobilization from intracellular Ca²⁺ stores. Addition of extracellular Ca²⁺ to the cell superfusate after store depletion by CPA caused a sustained increase in [Ca²⁺]_cyt because of the SOCE. Levels of SOCE following store depletion in cells infected with AdcJun were increased by ~35% compared with those observed in cells infected with Adnull (Fig. 4, A and B, right) although there were no changes in the levels of resting [Ca²⁺]_cyt between two groups (Fig. 4B, left). Moreover, cells migrating over the wounded area after injury were increased by ~20% in AdcJun-infected cells compared with that in cells infected with Adnull (Fig. 4C).

Fig. 4.

c-Jun overexpression increases Ca²⁺ influx and cell migration. A: representative records of Ca²⁺ influx after store depletion by CPA. IEC-Cdx2L1 cells were infected with AdcJun or Adnull, and Ca²⁺ levels were measured as described in materials and methods. a: control cells. b: cells infected with Adnull. c: cells infected with AdcJun. 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 from three separate experiments (n = 3). *P < 0.05 compared with cells infected with Adnull analyzed by one-way ANOVA followed by Duncan’s test. C: summarized data showing cell migration 6 h after wounding in cells described in A. Values are means ± SE from six dishes and repeated three times (n = 3). *P < 0.05 compared with cells infected with Adnull.

To examine the role of loss-of-function of c-Jun in cell migration after wounding, sicJun was used to reduce c-Jun levels in IECs. Transfection with sicJun for 48 h decreased c-Jun levels by ~85%, which was associated with a significant decrease in PLCγ1 content (Fig. 5, A and B). As shown in Fig. 5B, c-Jun silencing specifically decreased PLCγ1 transcription as indicated by a decrease in the levels of PLCγ1 mRNA and its promoter activity (Fig. 5C). c-Jun silencing had no effect on the levels of resting [Ca²⁺]_cyt, but it inhibited CPA-induced Ca²⁺ influx (Fig. 5, D and E, right). The level of CPA-induced Ca²⁺ influx was decreased by ~55% in c-Jun-silenced cells compared with those observed in cells transfected with C-siRNA. Importantly, c-Jun silencing impaired epithelial restitution after wounding, and the number of cells migrating over the denuded area 6 h after wounding was decreased by ~40% in the c-Jun-silenced population of IECs. In addition, neither sicJun nor C-siRNA altered cell viability as measured by Trypan blue staining (data not shown).

Fig. 5.

c-Jun silencing decreases PLCγ1 expression and cell migration. A: representative immunoblots of c-Jun and PLCγ1 proteins. Parental undifferentiated IEC-6 cells were transfected with C-siRNA or sicJun, and whole cell lysates were harvested 48 h thereafter. Actin immunoblotting was performed as an internal control for equal loading. B: relative levels of PLCγ1 mRNA in the samples as described in A. *P < 0.05 compared with cells transfected with C-siRNA analyzed by one-way ANOVA followed by Duncan’s test. C: levels of luciferase reporter activity after c-Jun silencing. D: representative records of Ca²⁺ influx after store depletion by CPA. a: control cells. b: cells transfected with C-siRNA. c: cells transfected with sicJun. E: 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. *P < 0.05 compared with cells transfected with C-siRNA. F: summarized data showing cell migration 6 h after wounding in cells described in A, repeated three times (n = 3). *P < 0.05 compared with cells transfected with C-siRNA.

To determine whether PLCγ1 silencing prevents c-Jun-induced SOCE and cell migration, the siRNA targeting PLCγ1 (siPLCγ1) was used to specifically block endogenous PLCγ1. Cells were transfected with siPLCγ1 for 48 h, followed by infection with either AdcJun or Adnull for an additional 24 h. PLCγ1 protein levels were decreased by ~80% (Fig. 6A) in cells transfected with siPLCγ1 or cotransfected with siPLCγ1 and AdcJun. PLCγ1 silencing prevented the induction in CPA-induced Ca²⁺ influx in cells overexpressing c-Jun (Fig. 6B, right) although it failed to alter the levels of resting [Ca²⁺]_cyt. PLCγ1 silencing also abolished c-Jun-induced stimulation of epithelial restitution after wounding (Fig. 6C). The number of cells migrating over the denuded area 6 h after wounding was decreased by ~70% after PLCγ1 silencing in cell-overexpressing c-Jun. Taken together, our findings indicate that c-Jun promotes IEC migration over the wounded area by activating PLCγ1-mediated Ca²⁺ signaling.

Fig. 6.

Effect of PLCγ1 silencing on Ca²⁺ influx and cell migration in cells overexpressing c-Jun. A: representative immunoblots of c-Jun and PLCγ1 proteins. IEC-Cdx2L1 cells were transfected with either C-siRNA or siPLCγ1 for 48 h, followed by infection with either Adnull or AdcJun for an additional 24 h. 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 = 3). *P < 0.05 compared with cells infected with Adnull. +P < 0.05 compared with cells infected with AdcJun and C-siRNA analyzed by one-way ANOVA followed by Duncan’s test. C: summarized data showing cell migration 6 h after wounding in cells described in A. Values are means ± SE from six dishes and repeated three times (n = 3). *P < 0.05 compared with cells infected with Adnull. +P < 0.05 compared with cells infected with AdcJun and C-siRNA.

Polyamines regulate cell migration by altering c-Jun/PLCγ1/Ca²⁺ signaling.

Our previous studies demonstrate that polyamine depletion by treatment with DFMO decreases cellular PLCγ1 abundance and inhibits cell migration after wounding (29, 30, 32, 33). In this study, we further determined the possibility that polyamine depletion repressed cell migration by reducing PLCγ1-mediated Ca²⁺ signaling through inhibition of c-Jun. After cells were exposed to DFMO for 2 days, they were infected with either Adnull or AdcJun. The levels of c-Jun and PLCγ1 proteins, Ca²⁺ influx, and cell migration were examined 48 h after the infection in the presence of DFMO. As shown in Fig. 7, PLCγ1 expression levels decreased significantly after polyamine depletion but were returned to near normal levels when DFMO-treated cells were infected with AdcJun. Ca²⁺ influx studies show that ectopic overexpression of c-Jun rescued resting [Ca²⁺]_cyt and restored Ca²⁺ influx attributable to SOCE to near normal levels in polyamine-deficient cells (Fig. 7D). Furthermore, c-Jun overexpression also prevented the inhibition of cell migration after wounding in polyamine-deficient cells (Fig. 7E). These results suggest that polyamines enhance cell migration after wounding, at least partially, by activating PLCγ1/Ca²⁺ signaling as a result of the stimulation of c-Jun expression.

Fig. 7.

Effects of ectopic c-Jun overexpression on the PLCγ1 expression and cell migration in polyamine-deficient cells. IEC-Cdx2L1 cells were grown in control cultures and cultures containing 5 mM DFMO for 2 days and then infected with either Adnull or AdcJun for an additional 48 h in the presence of DFMO. A: representative immunoblots for c-Jun and PLCγ1 as measured by Western blot analysis. B: relative levels of PLCγ1 mRNA in the samples as described in A (n = 3). *P < 0.05 compared with control; +P < 0.05 DFMO-treated cells infected by Adnull analyzed by one-way ANOVA followed by Duncan’s test. C: PLCγ1 promoter F2-Luc reporter activity was measured in cells as described in A. After cells were grown as stated above, they were cotransfected with the pPLCγ1-luc; luciferase activities were measured 24 h later. *P < 0.05 compared with control. +P < 0.05 compared with cells treated with DFMO and Adnull. D: summarized data showing resting [Ca²⁺]_cyt (left) and the amplitude of CPA-induced Ca²⁺ influx (right) from cells described in A. *P < 0.05 compared with control. +P < 0.05 compared with cells treated with DFMO and Adnull. E: summarized data showing cell migration 6 h after wounding in cells described in A. Values are means ± SE from six dishes in one experiment and repeated three times (n = 3). *P < 0.05 compared with control. +P < 0.05 compared with cells treated with DFMO and Adnull.

DISCUSSION

Early epithelial restitution is crucial for maintenance of the intestinal mucosal integrity under physiological and various pathological conditions, but the exact mechanism underlying this early rapid mucosal repair remains poorly understood. PLCγ1 is implicated in the control of intracellular Ca²⁺ homeostasis via association with small GTPases such as Rac1 and upregulates epithelial restitution after wounding (30). PLCγ1 inhibition reduces levels of inositol 1,4,5-trisphosphate (IP3), inhibits the formation of Rac1/PLCγ1 complexes, decreases [Ca²⁺]_cyt influx attributable to SOCE, and reduces IEC migration over the wounded area after superficial injury (29, 30). Here, we provide new evidence showing that the AP-1 transcription factor c-Jun stimulates PLCγ1 expression at the transcription level, thereby advancing our understanding of the mechanism by which PLCγ1 expression is regulated during early epithelial restitution after wounding.

The results reported in the present study clearly show that ectopically expressed c-Jun stimulates the transcription of the PLCγ1 gene in IECs. Our results further show that c-Jun induces PLCγ1 gene transcription through direct interaction with AP-1 and C/EBP sites within its proximal promoter. Several pieces of evidence from the present studies demonstrate that AP-1 and C/EBP sequences are necessary for PLCγ1 activation by c-Jun. First, the basal levels of PLCγ₁ transcription reached maximum in the F2-Luc, but its activity progressively decreased with the deletion of 5′ upstream fraction. This observation suggests that there are essential motifs within the sequence, which are responsive to c-Jun. Second, increased PLCγ1 activity by c-Jun overexpression was abolished when AP-1 and C/EBP binding sites were eliminated from the promoter region. Third, ChIP assay revealed that c-Jun bound to the PLCγ1 promoter with 244-bp fraction (−159 ~-402) containing AP-1 and C/EBP sites in vivo. Finally, AP-1 and C/EBP point mutations prevented the induction in PLCγ1 promoter activity in cells overexpressing c-Jun.

The data obtained in the present study suggest that c-Jun-mediated activation of PLCγ1 expression is of physiological significance because increasing the levels of endogenous c-Jun has a similar effect of PLCγ1-induced SOCE on rapid epithelial restitution after wounding as reported previously (29). Induced c-Jun increased [Ca²⁺]_cyt and enhanced epithelial cell migration after wounding, whereas inhibition of c-Jun expression decreased [Ca²⁺]_cyt and repressed cell migration. An increasing body of evidence indicates that SOCE is critical for maintaining sustained increases in [Ca²⁺]_cyt and in refilling Ca²⁺ into the store in nonexcitable cells, including IECs (26, 27, 48). These results are consistent with our previous studies (29, 30) that show that elevated [Ca²⁺]_cyt is a major mediator for the stimulation of IEC migration and that PLCγ1 plays a critical role in regulating Ca²⁺ homeostasis in response to injury. Induction of PLCγ1 by c-Jun increases [Ca²⁺]_cyt at least partially through IP3-sensitive signaling pathway because stimulation of PLCγ1 increases IP3 and promotes Ca²⁺ influx attributable to SOCE (14, 16). Consistent with our present findings, Huang et al. (11) have recently reported that the ectopic overexpression of c-Jun enhances cell proliferation and migration in Schwann cells.

The present study also shows that polyamines regulate IEC migration after wounding by altering c-Jun/PLCγ1/Ca²⁺ signaling. Polyamines, including spermidine, spermine, and their precursor putrescine, are intimately involved in a wide variety of distinct biological functions (32, 38, 44, 45, 47). Increased levels of cellular polyamines after acute mucosal injury enhance early mucosal restitution after injury (24–27). It has been shown that polyamines are absolutely necessary for c-Jun expression and that polyamine depletion represses c-Jun transcription (46, 47). As reported in our previous studies (26, 29, 30, 33, 48), polyamines modulate [Ca²⁺]_cyt and cell migration after wounding partially by altering PLCγ1 expression. The present study provides new evidence that polyamine-regulated expression of c-Jun stimulates PLCγ1 transcription, thus enhancing Ca²⁺ influx and cell migration.

In summary, our results indicate that c-Jun positively regulates PLCγ1 expression at the transcription level and enhances IEC restitution by activating PLCγ1-mediated Ca²⁺ signaling. Ectopically expressed c-Jun increases cellular PLCγ1 abundance, induces Ca²⁺ influx through SOCE, and stimulates epithelial restitution after wounding. Mechanistically, c-Jun enhances PLCγ1 transcription by directly interacting with AP-1 and C/EBP binding sites located at the proximal region of PLCγ1 promoter. These findings suggest that c-Jun functions as an upstream regulator of PLCγ1-induced Ca²⁺ signaling pathway in response to mucosal injury, and its stimulatory effect on early intestinal epithelial restitution results primarily from the PLCγ1-mediated Ca²⁺ signals.

GRANTS

This work was supported by Merit Review Awards from the Department of Veterans Affairs (J. N. Rao and J.-Y. Wang) and by grants from National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (DK-57819, DK-61972, and DK-68491 to J.-Y. Wang).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

P.-Y.W., S.R.W., L.X., and J.C. performed experiments; P.-Y.W., L.X., J.C., J.-Y.W., and J.N.R. analyzed data; P.-Y.W., J.-Y.W., and J.N.R. interpreted results of experiments; P.-Y.W., S.R.W., and J.N.R. prepared figures; P.-Y.W. and J.N.R. drafted manuscript; J.-Y.W. edited and revised manuscript; J.-Y.W. and J.N.R. approved final version of manuscript; J.N.R. conceived and designed the research.