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POLYAMINES ARE REQUIRED FOR PHOSPHOLIPASE-Cγ1 EXPRESSION

PROMOTING INTESTINAL EPITHELIAL RESTITUTION AFTER WOUNDING

Jaladanki N. Rao,1,3 Lan Liu,1,3 Tongtong Zou,1,3 Bernard S. Marasa,1,2 Dessy Boneva,1 Shelley

R. Wang,3 Debra L. Malone,1,4 Douglas J. Turner,1,3 and Jian-Ying Wang1,2,3

1Cell Biology Group, Department of Surgery, 2Department of Pathology, University of Maryland

School of Medicine, and 3Baltimore Veterans Affairs Medical Center, Baltimore, MD; and

4Medical Service, US Air Force, Bethesda, MD

Running Head: PLC-γ1 and intestinal epithelial restitution

Submitted to: Am J Physiol-GI and Liver Physiology

Address Correspondence to: Dr. Jian-Ying Wang

Department of Surgery

Baltimore Veterans Affairs Medical Center

10 North Greene Street,

Baltimore, MD 21201

Phone: 410-605-7000 Ext. 5678

Fax: 410-605-7949

E-mail: jwang@smail.umaryland.edu

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Page 1 of 32Articles in PresS. Am J Physiol Gastrointest Liver Physiol (September 14, 2006). doi:10.1152/ajpgi.00282.2006

Copyright © 2006 by the American Physiological Society.

Revised 9-3-2006 Ms. G-00282-2006R1

ABSTRACT

Intestinal mucosal restitution occurs by epithelial cell migration, rather than proliferation,

to reseal superficial wounds after injury. Polyamines are essential for stimulation of intestinal

epithelial cell (IEC) migration during restitution in association with their ability to regulate Ca2+

homeostasis, but the exact mechanism by which polyamines induce cytosolic free Ca2+ ([Ca2+]cyt)

concentration remains unclear. Phospholipase-Cγ1 (PLC-γ1) catalyzes the formation of inositol-

1,4,5-trisphosphate (IP3) that is implicated in the regulation of [Ca2+]cyt by modulating Ca2+ store

mobilization and Ca2+ influx. The current study tested the hypothesis that polyamines are

involved in PLC-γ1 activity regulating [Ca2+]cyt and cell migration after wounding. Depletion of

cellular polyamines by α-difluoromethylornithine (DFMO) inhibited PLC-γ1 expression in

differentiated IECs (stable Cdx2-transfected IEC-6 cells) as indicated by substantial decreases in

levels of PLC-γ1 mRNA and protein, and its enzyme product IP3. Polyamine-deficient cells also

displayed decreased [Ca2+]cyt and inhibited cell migration. Decreased levels of PLC-γ1 by

treatment with U-73122 or transfection with siRNA specifically targeting PLC-γ1 (siPLC-γ1)

also decreased IP3, reduced resting [Ca2+]cyt and Ca2+ influx after store depletion, and suppressed

cell migration in control cells. In contrast, stimulation of PLC-γ1 by 2,4,6-trimethyl-N-(meta-3-

trifluoromethylphenyl)-benzenesulfonamide (m-3M3FBS) induced IP3, increased [Ca2+]cyt, and

promoted cell migration in polyamine-deficient cells. These results indicate that polyamines are

absolutely required for PLC-γ1 expression in IECs and that polyamine-mediated PLC-γ1

signaling stimulates cell migration during restitution as a result of increased [Ca2+]cyt.

Key Words: mucosal injury; early mucosal repair; cell migration; capacitative Ca2+ entry; Ca2+

influx; Cdx2 gene, intestinal epithelium

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INTRODUCTION

Early epithelial restitution is an important repair modality in the gastrointestinal 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 (6,8,20,39,48). Defective regulation of

this process underlies various critical pathological states such as mucosal bleeding and ulcers,

disruption of epithelial integrity, and gut barrier dysfunction. This rapid reepithelialization is a

complex process that is highly regulated by numerous extracellular and intracellular factors, but

its exact mechanism is still unclear. Polyamines, including spermidine, spermine and their

precursor putrescine, are organic cations found in all eukaryotic cells and have been intimately

implicated in a wide variety of distinct biological functions (16,42). Polyamines are shown to

stimulate early mucosal repair of gastric and duodenal injury in vivo (50,51) and enhance

epithelial cell migration in an in vitro model (18,27,52) that mimics the early cell division-

independent stage of epithelial restitution. Studies from our laboratory (29,30,53) have further

demonstrated that cellular polyamines stimulate epithelial cell migration during restitution

primarily by controlling intracellular free Ca2+ ([Ca2+]cyt) concentration. However, little is

known about the exact process by which polyamines modulate [Ca2+]cyt in intestinal epithelial

cells (IECs) except these compounds are involved in the activation of voltage-gated K+ (Kv)

channels (29).

Phospholipase C (PLC) is an important regulatory enzyme that catalyzes hydrolysis of

the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) to generate diacylglycerol (DAG)

and inositol-1,4,5-trisphosphate (IP3), both of which are implicated in the regulation of a variety

of cellular processes (3,11,17,54,56). It is well known that DAG functions as the protein kinase

C (PKC) activator (15) and that IP3 acts as the Ca2+-mobilizing messenger resulting in the release

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of Ca2+ from the IP3-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma

membrane Ca2+ permeable channels (21,47). To date, three isoforms of PLC have been

identified in mammalian cells: β, γ, and δ; but their expression is cell type-dependent in various

tissues. Activity of these PLC isoforms is regulated through different signaling pathways and

has distinct biological roles in the signal transduction cascade. Although the activation

mechanism of PLC-δ is unknown, PLC-β isoenzymes are activated by agonists whose receptors

are coupled to heterotrimeric G proteins, while regulation of PLC-γ activity is implicated in its

activation with, and phosphorylation by, receptor tyrosine kinases (12,19). Among the PLC-γ

isoenzymes, the PLC-γ1 is expressed ubiquitously, whereas PLC-γ2 is expressed commonly in

cells of hematopoietic origin (36,38). Several studies have shown that treatment with growth

factor induces PLC-γ1 activation, resulting in the enhancement of cell motility (1,55). In

contrast, pharmacological inhibition of PLC-γ1 activity represses cell migration and reduces cell

invasiveness in breast, prostate and glioblastoma multiform cancer cell lines (25,45). Piccolo et

al (24) have reported that EGF induces a phosphoinositide-3 kinase dependent translocation of

PLC-γ1 at the leading edge of migrating cells in a wound healing assay, suggesting that induced

PLC-γ1 is relevant in cell migration during epithelial repair.

Receptor-operated (ROC) and store-operated (SOC) Ca2+ influx pathways have been

described in non-excitable cells including IECs for many years, but functional properties and

molecular identity of channels supporting ROC and SOC Ca2+ influx remain elusive and are the

focus of intensive investigation. Our previous studies (29,33) have demonstrated that canonical

transient receptor potential (TRPC)1 protein is highly expressed in IECs and functions as SOC

channels mediating capacitative Ca2+ entry (CCE) after store depletion. Recently, it has also

been found that PLC-γ1 is necessary for activation of TRPC channels in human keratinocytes

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and is implicated in the regulation of SOC-mediated Ca2+ influx (46). The current study

determined whether polyamines regulate [Ca2+]cyt by altering PLC-γ1 activity and if polyamine-

induced PLC-γ1 plays a role in intestinal epithelial restitution after wounding. The data

presented herein demonstrate that polyamines are absolutely required for PLC-γ1 expression in

IECs and that induced PLC-γ1 signaling stimulates cell migration during epithelial restitution as

a result of increased [Ca2+]cyt through CCE. Some of these data have been published previously

in abstract form (32).

MATERIALS AND METHODS

Chemicals and supplies. Disposable culture ware was purchased from Corning Glass

Works (Corning, NY). Tissue culture media, isopropyl-β-D-thiogalactopyranoside (IPTG),

LipofectAMINE, and dialyzed fetal bovine serum (dFBS) were obtained from Invitrogen

(Carlsbad, CA), and biochemicals were from Sigma (St. Louis, MO). The primary antibody, an

affinity-purified mouse monoclonal antibody against PLC-γ1, PLC-γ2, or PLC-β1 was purchased

from Upstate Biotechnology (Lake Placid, NY). U-73122 was purchased from Biomol Research

Laboratories (Plymouth Meeting, PA), while m-3M3FBS was obtained from Calbiochem (San

Diego, CA). D-myo-Inositol-1,4,5-triphosphote (IP3) [3H] Biotrak assay kit was purchased from

Amersham Biosciences (Arlington Heights, IL). D,L-α-difluoromethylornithine (DFMO) was

purchased from Ilex Oncology Inc. (San Antonio, TX).

Cell culture. Stable Cdx2-transfected IEC-6 cells were developed and characterized by

Suh and Traber (41) and were a kind gift from Dr. Peter G. Traber (Baylor College of Medicine,

Houston, TX). The expression vector, the LacSwitch System (Stratagene, La Jolla, CA), was

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used for directing the conditional expression of the Cdx2 gene, and isopropyl-β-D-

thiogalactopyranoside (IPTG) served as the inducer for the gene expression (43). IEC-6 cells,

derived from normal rat intestinal crypts, were transfected with pOPRSVCdx2 by electroporation

technique, and clones resistant to selection medium containing 0.6 mg G418/ml and 0.3 mg

hygromycin B/ml were isolated and screened for Cdx2 expression by Northern blot, RNase

protection assays, and electrophoretic mobility shift assay. Stock-stable Cdx2-transfected IEC-6

(IEC-Cdx2L1) cells were grown in DMEM supplemented with 5% heat-inactivated FBS, 10

μg/ml insulin, and 50 μg/ml gentamicin sulfate. Before experiments, IEC-Cdx2L1 cells were

grown in DMEM containing 4 mM IPTG for 16 days to induce cell differentiation as described

in our earlier publications (27-29, 33).

RNA Interference. The siRNA that was designed to specifically cleave PLC-γ1 mRNA

(siPLC-γ1) was synthesized and purchased from Dharmacon Inc (Lafayette, CO). Scrambled

control siRNA (C-siRNA), which had no sequence homology to any known genes, was used as

the control. For each 60-mm cell culture dish, 20 µl of the 5 µM stock siPLC-γ1 or C-siRNA

was mixed with 500 µl of Opti-MEM medium (Invitrogen). This mixture was gently added to a

solution containing 6 µl of LipofectAMINE 2000 in 500 µl of Opti-MEM. The solution was

incubated for 15 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 24- or 48-h incubation.

Reverse transcription and PCR. Total RNA was isolated by using 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, Carlsbad, CA). The specific sense and

antisense primers for PLC-γ1 included 5’-ACACGCTGTCTTTTTGGC-3’ and 5’-

CCTTGTAGTCGAAGAGAG-3’, and the expected size of PLC-γ1 fragments was 627 bp.

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Reverse transcription and PCR was performed as described in our earlier publications (29,33).

To quantify the PCR products (the amounts of mRNA) of PLC-γ1, an invariant mRNA of β-actin

was used as an internal control. The optical density (OD) values for each band on the gel were

measured by a gel documentation system (UVP Inc, Upland, CA), and their signals were

normalized to the OD values in the β-actin signals.

Western blot analysis. Cell samples, placed in SDS sample 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 phenylmethyl-sulfonyl fluoride, 20 μg/ml aprotinin, 2 μg/ml leupeptin, and 2 mM

sodium orthovanadate), were sonicated and centrifuged (12,000 rpm) at 4oC for 15 min. The

supernatant from cell samples was boiled for 5 min and then subjected to electrophoresis on

7.5% SDS-PAGE gels according to Laemmli (13). After the transfer of protein onto

nitrocellulose filters, the filters were incubated for 1 h in 5% non-fat dry milk in 1× 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 specific antibody against PLC-γ1 protein. The filters

were subsequently washed with 1× PBS-T and incubated for 1 h with the second antibody

conjugated with horseradish peroxidase for 1 h at room temperature. The immunocomplexes on

the membranes were reacted for 1 min with enhanced chemiluminiscence reagent (NEL-100;

DuPont NEN).

Measurement of cellular IP3. Cellular IP3 levels were measured by using Biotrak assay

system that was purchased from Amersham Biosciences. After different treatments, cells were

rapidly mixed with ice-cold 20% perchloric acid and kept on ice for 20 min. The preparations

were centrifuged (2,000 rpm) at 4ºC for 15 min, and the supernatants were removed and

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neutralized with KOH (10 M) to pH 7.5. The preparations were re-centrifuged, and the

supernatants were collected and utilized for IP3 assay. Levels of IP3 were measured by the

competitive binding assay system with highly isomeric specificity. Assays were assessed for

their linearity with respect to various incubation conditions and the results were expressed as

pmol/mg protein.

Measurement of [Ca2+]cyt. Details of the digital imaging methods employed for

measuring [Ca2+]cyt were described in our previous publications (28,29,33,53). Briefly, cells

were plated on 25-mm cover slips and incubated in culture medium containing 3.3 μM fura-2

AM for 30-40 min at room temperature (22-24°C) under an atmosphere of 10% CO2 in air. 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 ratio imaging of [Ca2+]cyt was obtained

from fura-2 fluorescent emission excited at 380 and 340 nm (17,46).

Measurement of cell migration. The migration assays were carried out as described in

our earlier publications (27-29,33,53). Cells were plated at 6.25 × 104/cm2 in DMEM plus dFBS

on 60-mm dishes thinly coated with Matrigel according to the manufacturer’s instructions and

were incubated as described for stock cultures. The cells were fed on day 2 and migration was

tested on day 4. To initiate migration, the cell layer was scratched with a single edge razor blade

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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 number

of migrating cells per millimeter of scratch.

Polyamine analysis. The cellular polyamines content was analyzed by high-performance

liquid chromatographic (HPLC-) analysis as previously described (18,49). Briefly, after the cells

were washed three times with ice-cold D-PBS, 0.5 M perchloric acid was added, and the cells

were frozen at -80°C until ready for extraction, dansylation, and HPLC- analysis. The standard

curve encompassed 0.31-10 µM. Values that fell >25% below the curve were considered

undetectable. The results are expressed as nanomoles of polyamines per milligram of protein.

Statistical analysis. All data are expressed as means ± SE from six dishes. PCR and

immunoblotting results were repeated three times. The significance of the difference between

means was determined by analysis of variance. The level of significance was determined using

the Duncan’s multiple-range test (10).

RESULTS

Changes in PLC-γ1 expression and its enzyme product IP3 following polyamine

depletion. Induced expression of the Cdx2 gene by treatment of stable IEC-Cdx2L1 cells with 4

mM IPTG for 16 days resulted in a significant development of differentiated phenotype. These

differentiated IEC-Cdx2L1 cells exhibited multiple morphological and molecular characteristics

of intestinal epithelial differentiation as indicated by polarization, development of lateral

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membrane interdigitations and microvilli at the apical pole, and expression of brush-border

enzymes such as sucrase-isomaltase (data not shown). Because these differentiated IEC-Cdx2L1

cells migrate over the wounded edge much faster than undifferentiated parental IEC-6 cells after

injury (27,29,31), they provide an excellent model for the current study.

To determine the role of cellular polyamines in the regulation of PLC-γ1 expression,

differentiated IEC-Cdx2L1 cells were cultured in the DMEM containing 5 mM DFMO, a

specific inhibitor of polyamine synthesis, for 4 and 6 days. Exposure to DFMO completely

depleted putrescine within 48 h, but it took 4 days to totally deplete spermidine and substantially

decreased spermine (by ~60%) (data not shown). Similar results have been published in our

previous studies (27,29,48,49). Results presented in Fig. 1 show that depletion of cellular

polyamines by DFMO significantly inhibited PLC-γ1 expression in differentiated IEC-Cdx2L1

cells. The levels of PLC-γ1 mRNA in the cells treated with DFMO for 4 and 6 days were

decreased by ~80% (Fig. 1Aa), which were paralleled by decreases in PLC-γ1 protein (Fig.

1Ab). The levels of PLC-γ1 protein in the cells exposed to DFMO for 4 and 6 days were

decreased by ~75%. Consistently, the decreased levels of PLC-γ1 protein in polyamine-deficient

cells were associated with a reduction of its enzyme product IP3 (Fig. 1B). The levels of IP3

were decreased by ~60% in cells exposed to DFMO for 4 and 6 days. In the presence of DFMO,

addition of exogenous putrescine (10 µM) to the cultures not only prevented the decreased levels

of PLC-γ1 mRNA and protein but also restored the IP3 levels to near normal. Spermidine (5

µM) had an effect equal to putrescine on levels of PLC-γ1 when it was added to cultures that

contained DFMO (data not shown). We also examined changes in other mammalian PLC-

isozymes, including PLC-γ2 and PLC-β1, in the presence or absence of cellular polyamines and

demonstrated that there were no significant differences in levels of these PLC proteins between

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control cells and cells exposed to DFMO alone or DFMO plus putrescine for 4 and 6 days (Fig.

1C). These results clearly indicate that polyamines are required for PLC-γ1 expression and that

decreasing cellular polyamines inhibits PLC-γ1 formation, thus leading to the reduction of IP3 in

intestinal epithelial cells.

Polyamine depletion-mediated reduction of IP3 was associated with decreases in

[Ca2+]cyt and cell migration. As shown in Fig. 2, reduced levels of IP3 following polyamine

depletion decreased the resting [Ca2+]cyt and inhibited Ca2+ influx after Ca2+ store depletion

induced by cyclopiazonic acid (CPA). 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, when the

CPA-induced transient rise in [Ca2+]cyt returned to basal level, caused a sustained increase in

[Ca2+]cyt because of the capacitative Ca2+ entry. In polyamine-deficient cells, levels of resting

[Ca2+]cyt and store depletion-induced Ca2+ influx were decreased by ~50%. These decreased

levels of [Ca2+]cyt were also accompanied by a significant inhibition of cell migration after

wounding (Fig. 2C). The numbers of cells migrating over the wounded edge were decreased by

~70% in DFMO-treated cells. Restoration of IP3 by exogenous putrescine given together with

DFMO not only returned the resting [Ca2+]cyt and store depletion-induced Ca2+ influx to near

normal levels but also abolished the inhibition of cell migration in polyamine-deficient cells.

Effect of PLC-γ1 inhibition on [Ca2+]cyt and cell migration. To elucidate the exact

relationship between PLC-γ1 and intestinal epithelial restitution, the following two studies were

carried out. First, we examined the effects of decreased levels of PLC-γ1 by treatment with its

specific chemical inhibitor U-73122 on [Ca2+]cyt and cell migration. Results presented in Fig. 3A

show that exposure of control differentiated IEC-Cdx2L1 cells (without DFMO) to U-73122

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dose-dependently decreased the levels of PLC-γ1 protein, which were also associated with a

significant decrease in IP3 (Fig. 3B). When U-73122 at different concentrations was added to the

medium, levels of IP3 were decreased by ~17% at 1 µM, ~68% at 2 µM, and ~75% at 5 µM,

respectively. The reduced levels of IP3 by U-73122 decreased resting [Ca2+]cyt and inhibited the

store depletion-induced Ca2+ influx (Fig. 3C). Levels of resting [Ca2+]cyt in cells exposed to U-

73122 was decreased by ~25%, while Ca2+ influx after Ca2+ store depletion was decreased by

~50%. Treatment with U-73122 also inhibited cell migration after wounding (Fig. 3D). In U-

73122-treated cells, the numbers of cells migrating over the wounded edge were decreased by

~21% at 1 µM, ~66% at 2 µM, and ~80% at 5 µM, respectively.

Second, we examined changes in levels of [Ca2+]cyt and cell migration after inhibition of

PLC-γ1 expression by siRNA specifically targeting PLC-γ1 mRNA (siPLC-γ1). These specific

siPLC-γ1 nucleotides were designed to cleave rat PLC-γ1 mRNA by activating endogenous

RNase H and to have a unique combination of specificity, efficacy, and reduced toxicity (33).

Initially, we determined the transfection efficiency of the siRNA nucleotides in differentiated

IEC-Cdx2L1 cells and demonstrated that >95% of cells were positive when they were

transfected with a fluorescent FITC-conjugated siPLC-γ1 for 24 h (data not shown). As shown

in Fig. 4A, transfection with the siPLC-γ1 inhibited expression of PLC-γ1 in differentiated IEC-

Cdx2L1 cells. Levels of PLC-γ1 protein were decreased by ~70% at 24 h and ~85% at 48 h after

the transfection. To determine the specificity of siPLC-γ1 used in this study, we reprobed the

membrane with anti-PLC-β1 antibody and showed that levels of PLC-β1 protein were not

affected when cells were transfected with siPLC-γ1 (Fig. 4Aa). Inhibition of PLC-γ1 expression

by siPLC-γ1 also decreased IP3, and its levels were decreased by ~70% compared with those

observed in control cells and cells transfected with control siRNA (C-siRNA). Decreased levels

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of IP3 by siPLC-γ1 reduced resting [Ca2+]cyt and inhibited Ca2+ influx after Ca2+ store depletion

(Fig. 5A and B). The levels of resting [Ca2+]cyt were decreased by ~40%, while store depletion-

induced Ca2+ influx was decreased by ~50% in differentiated IEC-Cdx2L1 cells transfected with

siPLC-γ1 for 48 h. Furthermore, inhibition of PLC-γ1 expression and the subsequent decrease in

[Ca2+]cyt by siPLC-γ1 suppressed cell migration after wounding (Fig. 5C). The rate of cell

migration was decreased by ~36% in cells transfected with the siPLC-γ1 for 48 h (Fig. 5D).

Transfection with C-siRNA at the same concentrations showed no inhibitory effects on PLC-γ1

expression, [Ca2+]cyt, and cell migration. In addition, neither siPLC-γ1 nor C-siRNA affected

cell viability as measured by Trypan blue staining (data not shown). These findings indicate that

inhibition of PLC-γ1 expression decreases [Ca2+]cyt and represses cell migration during

restitution after wounding.

Effect of increased PLC-γ1 on [Ca2+]cyt and cell migration in polyamine-deficient cells.

In this study, the synthetic compound m-3M3FBS, which is shown to specifically increase PLC-

γ (2), was used to stimulate PLC-γ1 expression in polyamine-deficient cells. Results presented

in Fig. 6 show that stimulation of PLC-γ1 by treatment with m-3M3FBS not only prevented the

decrease in store depletion-induced Ca2+ influx but also stimulated cell migration in polyamine-

deficient cells. When different concentrations of m-3M3FBS were added to the culture medium

containing DFMO, they dose-dependently increased levels of PLC-γ1 protein and IP3. Levels of

IP3 in polyamine-deficient cells were increased by ~40% at 5 µM m-3M3FBS, ~50% at 10 µM,

and ~110% at 25 µM. Induced IP3 by m-3M3FBS also consistently increased resting [Ca2+]cyt

and promoted Ca2+ influx after store depletion (Fig. 6C). When polyamine-deficient cells were

exposed to 25 µM m-3M3FBS for 6 h, the resting [Ca2+]cyt was increased by ~25%, while the

store depletion-induced Ca2+ influx was increased by ~65%. Furthermore, treatment with m-

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3M3FBS increased cell migration after wounding in polyamine-deficient cells (Fig. 6D). The

numbers of cells migrating over the wounded edge were increased by ~30% at 5 µM, ~45% at 10

µM, and ~68% at 25 µM. These results indicate that polyamine-induced PLC-γ1 expression

increases [Ca2+]cyt and promotes intestinal epithelial cell migration after wounding.

DISCUSSION

Epithelial cell migration is a primary process during early rapid mucosal repair after

superficial wounds in the gastrointestinal tract, which absolutely requires cellular polyamines.

Our previous studies have demonstrated that polyamines enhance epithelial cell migration, at

least partially, by regulating [Ca2+]cyt concentration, because decreased levels of cellular

polyamines reduced [Ca2+]cyt and inhibited cell migration after wounding (29,30,33,53). The

present study supports and extends our previous observations by demonstrating that polyamines

are necessary for PLC-γ1 expression and that induced PLC-γ1 plays a critical role in the

regulation of Ca2+ homeostasis during intestinal epithelial restitution. Decreased expression of

PLC-γ1 by polyamine depletion with DFMO decreased the formation of IP3 (Fig. 1), which was

associated with significant decreases in resting [Ca2+]cyt and Ca2+ influx through CCE (Fig. 2).

Furthermore, inhibition of PLC-γ1 signaling in normal IEC-Cdx2L1 cells (without DFMO) by

either treatment with its chemical inhibitor, U-73122, (Fig. 3) or transfection with siRNA

targeting the specific coding region of PLC-γ1 mRNA also decreased [Ca2+]cyt and repressed cell

migration (Fig. 5). In contrast, increased levels of PLC-γ1 protein in DFMO-treated cells by its

specific chemical activator, m-3M3FBS, increased [Ca2+]cyt and promoted cell migration in the

absence of cellular polyamines (Fig. 6).

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The findings reported herein clearly show that depletion of cellular polyamines inhibits

expression of PLC-γ1 in differentiated IECs. To provide insight into the molecular basis for

PLC-γ1 inhibition after polyamine depletion, the results presented in Fig. 1A indicate that levels

of PLC-γ1 mRNA decreased significantly in cells treated with DFMO for 4 and 6 days, which

was paralleled by a reduction of PLC-γ1 protein. This inhibition of PLC-γ1 expression in

DFMO-treated cells were completely prevented by addition of exogenous putrescine, indicating

that the observed changes in PLC-γ1 expression must be related to polyamine depletion rather

than to the nonspecific effect of DFMO. This inhibitory effect of polyamine depletion on PLC-

γ1 expression is specific, because there were no significant differences in levels of other

mammalian PLC-isoforms such as PLC-γ2 and PLC-β1 between control cells and cells exposed

to DFMO alone or DFMO plus putrescine for 4 and 6 days (Fig. 1C). Although the exact

mechanism by which polyamine depletion decreases PLC-γ1 mRNA remains unknown, the

current study suggests that regulation of PLC-γ1 expression by polyamines appears to occur at

the transcriptional level. In support of this possibility, our previous studies (14,23,57) and others

(4,5) have shown that polyamines are implicated in both transcription and posttranscription of

various genes encoding different cellular signaling proteins and that decreases in mRNAs

following polyamine depletion result predominantly from the inhibition of their gene

transcription. On the other hand, decreasing polyamines increases cellular signaling factors

primarily by stabilizing their mRNAs and proteins (57,58). For example, polyamine depletion

decreases c-myc and c-jun mRNAs in IECs by repressing their gene transcription but failing to

affect their mRNA stability (23), while decreasing cellular polyamines increases levels of p53

and JunD by stabilizing their mRNAs without effect on gene transcription (14,57). Clearly,

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further studies are needed to define the molecular process by which polyamines regulate

transcription of the PLC-γ1 gene in IECs.

The data from the current studies also show that polyamine-modulated PLC-γ1 plays a

critical role in the regulation of [Ca2+]cyt concentration, at least in part, through IP3-sensitive

signaling pathway in IECs. Inhibition of PLC-γ1 expression by polyamine depletion decreased

the level of IP3, which was associated with a decrease in [Ca2+]cyt due to the reduction of CCE

(Fig. 2). Consistently, inhibition of PLC-γ1 expression in normal IEC-Cdx2L1 cells by

treatment with U-73122 (Fig. 3) or transfection with siRNA targeting PLC-γ1 mRNA also

decreases IP3 and reduced [Ca2+]cyt (Fig. 5), while stimulation of PLC-γ1 by m-3M3FBS in

polyamine-deficient cells increased IP3 and promoted Ca2+ influx through CCE (Fig. 6). These

findings are consistent with results from others who have demonstrated that IP3 triggers the

release of Ca2+ from intracellular Ca2+ store through binding to IP3 receptors and results in

activation of Ca2+ influx via SOC channels (21,22,26). However, it also has been reported that

PLC-γ1 augments Ca2+ entry induced by either a G protein-coupled receptor agonist or Ca2+

store depletion through its direct interaction with other signaling molecules such as TRPC3 and

TRPC4, but independent of its lipase activity (17,44). Several studies further show that the

interaction of PLC-γ1 with TRPC3 requires the partial pleckstrin homology (PH) domain and

that the partial PH domain of PLC-γ1 interacts with a complementary partial PH-like domain in

TRPC3 to elicit lipid binding and cell surface expression of TRPC3 (9,35,46). Our previous

studies have demonstrated that IECs do not express TRPC3, but highly express TRPC1 that

functions as SOC channels mediating Ca2+ influx after store depletion. Interestingly, Tu et al

(44) have recently found that PLC-γ1 activates SOC channels in human keratinocytes by

16

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Revised 9-3-2006 Ms. G-00282-2006R1

interacting with TRPC1, but not with TRPC4. It is unclear at present whether polyamine-

modulated PLC-γ1 directly binds to and regulates TRPC1 channels in IECs.

It is of physiological significance that polyamines regulate expression of PLC-γ1 in IECs,

because inhibition of PLC-γ1 signaling by polyamine depletion (Fig. 2) or specific siRNA

targeting PLC-γ1 (Fig. 5) decreased [Ca2+]cyt and inhibits intestinal epithelial cell migration after

wounding. Under biological conditions, the pool of intracellular polyamines is dynamically

regulated by polyamine biosynthesis, uptake, and degradation (40). Cellular levels of

polyamines are changed rapidly, either increased or decreased, in response to various

physiological and pathological stimuli, leading to the activation or inactivation of different

cellular signaling pathways. It has been shown that levels of tissue polyamines in the damaged

gastrointestinal mucosa are dramatically increased, which stimulates early rapid mucosal

restitution in rats (50,51). As reported in our previous studies (28-30,53), elevated [Ca2+]cyt is a

major mediator for the stimulation of intestinal epithelial cell migration following an increase in

cellular polyamines, but the exact mechanisms by which polyamines regulate Ca2+ influx and

store Ca2+ release remain largely unknown. A series of studies from our laboratory has

demonstrated that polyamines regulate [Ca2+]cyt partially by governing membrane potential (Em)

through control of Kv channel expression in IECs (29,34,53). Depletion of cellular polyamines

inhibits Kv channel activity as indicated by a decrease in voltage-gated K+ currents and

membrane depolarization, contributing to the reduction of [Ca2+]cyt through shrinkage of the

driving force for Ca2+ influx. The current study provides a strong evidence for the role of

polyamine-modulated PLC-γ1 signaling in the control of [Ca2+]cyt concentration during epithelial

restitution after injury.

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Revised 9-3-2006 Ms. G-00282-2006R1

In summary, these results indicate that polyamines are necessary for PLC-γ1 expression

and that induced PLC-γ1 is implicated in the signaling pathway of control of intracellular Ca2+

homeostasis during epithelial restitution after wounding. Depletion of cellular polyamines

inhibits PLC-γ1 expression, reduces levels of IP3, and decreases [Ca2+]cyt, thereby repressing

intestinal epithelial cell migration. In addition, inhibition of PLC-γ1 expression by treatment

with its chemical inhibitor or transfection with the specific siRNA targeting PLC-γ1 mRNA in

normal IECs also decreases [Ca2+]cyt and causes the inhibition of cell migration. In contrast,

increased PLC-γ1 by m-3M3FBS increases [Ca2+]cyt and promotes cell migration in polyamine-

deficient cells. These findings suggest that PLC-γ1 is a biological regulator for control of

[Ca2+]cyt in IECs under physiological and pathological conditions and plays a major role in

polyamine-dependent intestinal epithelial cell migration during restitution.

ACKNOWLEDGEMENTS

This work was supported by Merit Review Grant from the Department of Veterans

Affairs and by National Institutes of Health Grants DK-57819, DK-61972, and DK-68491. J-Y.

Wang is a Research Career Scientist, Medical Research Service, US Department of Veterans

Affairs.

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FIGURE LEGENDS

Fig. 1. Changes in phospholipase-Cγ1 (PLC-γ1) expression and inositol-1,4,5-trisphosphate (IP3)

levels in control differentiated IEC-Cdx2L1 cells and cells treated with either α-

difluoromethylornithine (DFMO) alone or DFMO plus putrescine (PUT). Before

experiments, stable IEC-Cdx2L1 cells were grown in DMEM containing 4 mM isopropyl

β-D-thiogalactopyranoside (IPTG) for 16 days to induce cell differentiation. These

differentiated IEC-Cdx2L1 cells were grown in DMEM containing either DFMO (5 mM)

alone or DFMO plus PUT (10 µM) for 4 and 6 days, and then total RNA and whole cell

lysates were harvested for various measurements. A: changes in PLC-γ1 expression. a,

mRNA levels as measured by RT-PCR analysis. The first-strand cDNAs, synthesized

from total cellular RNA, were amplified with the specific sense and antisense primers,

and PCR-amplified products displayed in agarose gel for PLC-γ1 (~627 bp) and β-actin

(~244 bp). b: representative immunoblots of Western analysis in cells from described in

Aa. Twenty micrograms of total protein were applied to each lane, and immunoblots

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were hybridized with the antibody specific for PLC-γ1 (~135 kDa). After the blot was

stripped, actin (~42 kDa) immunoblotting was performed as an internal control for equal

loading. c, quantitative analysis derived from densitometric scans of immunoblots of

PLC-γ1 as described in Ab. Values are means ± SE of data from 3 separate experiments;

relative levels of PLC-γ1 protein were corrected for loading as measured by densitometry

of actin. B. levels of IP3 in cells described in A. Values are means ± SE of data from 6

dishes. * P < 0.05 compared with control and DFMO + PUT. C. representative

immunoblots of Western analysis for PCL-γ2 and PCL-β1 proteins in cells described in

A. Three experiments were performed that showed similar results.

Fig. 2. Changes in resting [Ca2+]cyt, Ca2+ influx after cyclopiazonic acid (CPA)-induced Ca2+

store depletion, and cell migration in differentiated IEC-Cdx2L1 cells described in Fig. 1.

A: representative records of [Ca2+]cyt changes measured in peripheral areas of control

cells and cells exposed to DFMO or DFMO plus PUT for 4 days. The Ca2+ stores were

depleted by treatment with CPA in the absence of extracellular Ca2+ (0Ca2+). B:

summarized data showing resting [Ca2+]cyt concentrations (left) and the amplitude of

CPA-induced Ca2+ influx (right) from cells described in A. Values were means ± SE (n =

20). 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, as described in MATERIALS

AND METHODS. Values were means ± SE of data from 6 dishes. * P < 0.05 compared

with control cells and cells exposed to DFMO plus PUT.

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Fig. 3. Effect of inhibition of PLC-γ1 by treatment with U-73122 on levels of IP3, [Ca2+]cyt, and

cell migration in differentiated IEC-Cdx2L1 cells. After cells were grown in standard

DMEM for 4 days, they were exposed to U-73122 at different concentrations for 6 h. A:

changes in PLC-γ1 protein. a, representative immunoblots of Western blot analysis.

Levels of PLC-γ1 were identified by using a specific antibody; and actin immunoblotting

was performed as an internal control for equal loading. b, quantitative analysis derived

from densitometric scans of immunoblots of PLC-γ1 as described in Ab. Values are

means ± SE of data from 3 separate experiments. * P < 0.05 compared with cells treated

without U-73122 (Control). B: levels of IP3 in cells described in A. Values are means ±

SE of data from 6 dishes. * P < 0.05 compared with control cells. C: representative

records showing time course of [Ca2+]cyt changes after exposure to CPA in the absence

(0Ca2+) or presence of extracellular Ca2+: a) control; and b) cells exposed to U-73122 (5

µM). D: changes in cell migration in cells described in A. Cells migration was assayed 6

h after wounding, and values are means ± SE from 6 dishes. * P < 0.05 compared with

control cells.

Fig. 4. Effect of treatment with small interfering (si)RNA targeting the PLC-γ1 mRNA coding

region (siPLC-γ1) on levels of PLC-γ1 protein and IP3 in differentiated IEC-Cdx2L1

cells. A: changes in PLC-γ1 and PLC-β1 expression. a, representative immunoblots of

Western blot analysis for PLC-γ1 and PLC-β1 proteins; and b, quantitative analysis of

immunoblots of PLC-γ1 by densitometry from Aa. Cells were transfected with either

control siRNA (C-siRNA) or siPLC-γ1 by LipofectAMINE technique. Whole cell

lysates were harvested 24 and 48 h after transfection, and levels of PLC-γ1 and PLC-β1

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proteins were measured by Western immunoblot analysis. Actin immunoblotting was

performed as an internal control for equal loading. Values are means ± SE of data from 3

separate experiments. B: levels of IP3 in cells described in A. Values are means ± SE of

data from 6 dishes. * P < 0.05 compared with cells transfected with C-siRNA.

Fig. 5. Changes in [Ca2+]cyt and cell migration in differentiated IEC-Cdx2L1 cells described in

Fig. 4. A: representative records showing the time course of [Ca2+]cyt changes induced by

exposure to CPA in the absence (0Ca2+) or presence of extracellular Ca2+: a) control

cells; b), cells transfected with C-siRNA for 48 h; and c) cells transfected with siPLC-γ1

for 48 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 = 25).

*P < 0.05 compared with control cells and cells transfected with C-siRNA. C: images of

cell migration 6 h after wounding by removal of part of the monolayer: a) control cells;

b) cells transfected with C-siRNA; and c) cells transfected with siPLC-γ1. After cells

were transfected for 48 h, cell migration was assayed 6 h after wounding. D:

summarized data showing rates of cell migration after wounding in cells described in C.

Values are means ± SE of data from 6 dishes. * P < 0.05 compared with controls and

cells transfected with C-siRNA.

Fig. 6. Effect of induction of PLC-γ1 by treatment with m-3M3FBS on levels of IP3, [Ca2+]cyt,

and cell migration in polyamine-deficient IEC-Cdx2L1 cells. A: representative

immunoblots of Western blot analysis for PLC-γ1 protein. Differentiated IEC-Cdx2L1

cells were grown in culture containing 5 mM DFMO for 4 days and then exposed to

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Revised 9-3-2006 Ms. G-00282-2006R1

different concentrations of m-3M3FBS for 6 h. Levels of PLC-γ1 protein were measured

by Western immunoblot analysis, while actin immunoblotting was performed as an

internal control for equal loading. B: levels of IP3 in cells described in A. Values are

means ± SE of data from 6 dishes. * P < 0.05 compared with cells treated without m-

3M3FBS (control). C: representative records showing the time course of [Ca2+]cyt

changes after exposure to CPA in the absence (0Ca2+) or presence of extracellular Ca2+:

a) cells treated with DFMO alone; and b) cells treated with DFMO and then exposed to

m-3M3FBS (25 μM). Three separate experiments were performed that showed similar

results. D: summarized data showing rates of 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.

26

Page 26 of 32

Fig. 1

A

627-

244-

4 Days 6 Days

bp DF

MO

+P

UT

Con

trol

DF

MO

DF

MO

+P

UT

Con

trol

DF

MO

-PLC-γγγγγ1

-βββββ-actin

B

C

a. PLC-γγγγγ1 mRNA

140- -PLC-γγγγγ2

kDa

-PLC-βββββ1150-

42- -Actin

1 2 3 4 5 6

0

4

8

12

16

IP

3 le

vels

(pm

ol/m

g pr

otei

n)

* *

1 2 3 4 5 6

b. PLC-γγγγγ1 ProteinkDa

135-

42-

-PLC-γγγγγ1

-Actin

1 2 3 4 5 6c. Relative Protein Levels

PL

C- γγγγ γ

1 pr

otei

n

* *

0.0

0.5

1.0

1.5

2.0

1 2 3 4 5 6

Page 27 of 32

BC

ell m

igra

tion

(cel

ls/m

m)

0

200

400

A

Fig. 2

Rat

io (F

340/F

380)

0

1

2

3

CPA0Ca2+

CPA0Ca2+

a. Control b. DFMO c. DFMO+PUT

0

1

2

3

CPA0Ca2+ 2 min

Rat

io (F

340/F

380)

Con

trol

DFM

O

DFM

O+P

UT

**

*

Resting [Ca2+]cyt

Ca2+ influxControl DFMO DFMO

+PUT

C

Page 28 of 32

Fig. 3

C

B R

atio

(F34

0/F38

0)

A

Cel

l mig

rati

on (c

ells

/mm

)

0 1 2 5

*

U-73122 (μμμμμM)

0

150

300

450

600

*

IP3 l

evel

s (pm

ol/m

g pr

otei

n)

0 1 2 5

0

1

2

3

CPA0Ca2+

CPA0Ca2+

a. Control b. U-73122

2 min

D

0

6

12

18

* *

U-73122 (μμμμμM)

kDa 0 1 2 5

-PLC-γγγγγ1

-Actin

135-

42-

U-73122 (μμμμμM)

0.0

0.6

1.2

1.8

PL

C- γγγγ γ

1 pr

otei

n

1 2 3 4 1 2 3 4

a. PLC-γγγγγ1 protein b. Relative levels

*

*

Page 29 of 32

Fig. 4

B

**

a. PLC proteins

Rel

ativ

e P

LC

- γγγγ γ1

leve

ls

-PLC-γγγγγ1

A

1 2 3 4-Actin

kDa

135-

42-

Control

C-siRNA

siPLC-γγγγγ1

(24 h

)

siPLC-γγγγγ1

(48 h

)

0

1

2

1 2 3 4

b. Relative levels

0

5

10

15

20

IP

3 le

vels

(pm

ol/m

g pr

otei

n)

**

1 2 3 4

-PLC-βββββ1150-

Page 30 of 32

Fig. 5

B

0

1

2

3

0Ca2+CPA

Rat

io (F

340/F

380)

a. Control b. C-siRNA c. siPLC-γγγγγ1A

0Ca2+CPA

0

1

2

3

Rat

io (F

340/F

380)

Con

trol

C-s

iRN

A

siP

LC

- γγγγ γ1

*

*

Resting [Ca2+]cyt Ca2+ influx

a. Control b. C-siRNA c. siPLC-γγγγγ1C

0

150

300

450

Control C-siRNA siPLC-γγγγγ1

Cel

l mig

rati

oin

(cel

ls/m

m)

D

*

0Ca2+CPA

2 min

Page 31 of 32

Fig. 6

C

A

B-Actin

-PLC-γγγγγ1

42-

0 5 10 25

m-3M3FBS (μμμμμM)

Cel

l mig

rati

on (c

ells

/mm

)

0

100

200

300

135-

kDa

0

1

2

3

Rat

io (F

340/F

380)

2 min0Ca2+

CPA

0Ca2+

CPA

a. DFMO b. DFMO+m-3M3FBS

D

**

*

0

5

10

15

20

0 5 10 25

m-3M3FBS (μμμμμM)

0 5 10 25

m-3M3FBS (μμμμμM)

IP

3 lev

els

(pm

ol/m

g pr

otei

n)

*

**

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