Departments of 1 Surgery, 4 Physiology, and 5 Pathology, University of Maryland School of Medicine and 2 Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201; and 3 Department of Medicine, School of Medicine, University of California, San Diego, California 92103
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ABSTRACT |
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Early mucosal restitution occurs by epithelial cell migration to reseal superficial wounds after injury. Differentiated intestinal epithelial cells induced by forced expression of the Cdx2 gene migrate over the wounded edge much faster than undifferentiated parental cells in an in vitro model. This study determined whether these differentiated intestinal epithelial cells exhibit increased migration by altering voltage-gated K+ (Kv) channel expression and cytosolic free Ca2+ concentration ([Ca2+]cyt). Stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 and Kv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1 cells nor parental IEC-6 cells expressed voltage-dependent Ca2+ channels. The increased expression of Kv channels in differentiated IEC-Cdx2L1 cells was associated with an increase in whole cell K+ currents, membrane hyperpolarization, and a rise in [Ca2+]cyt. The migration rates in differentiated IEC-Cdx2L1 cells were about four times those of parental IEC-6 cells. Inhibition of Kv channel expression by polyamine depletion decreased [Ca2+]cyt, reduced myosin stress fibers, and inhibited cell migration. Elevation of [Ca2+]cyt by ionomycin promoted myosin II stress fiber formation and increased cell migration. These results suggest that increased migration of differentiated intestinal epithelial cells is mediated, at least partially, by increasing Kv channel activity and Ca2+ influx during restitution.
voltage-gated potassium channels; intracellular calcium; membrane potential; restitution; Cdx2 gene; differentiation; polyamines
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INTRODUCTION |
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EPITHELIAL CELLS LINE THE human intestinal mucosa and form an important barrier to a wide array of noxious substances and invasive enteric pathogens in the lumen. Early mucosal restitution refers to resealing of superficial wounds to this barrier as a consequence of epithelial cell migration into the defect, a process independent from epithelial cell proliferation (31, 38, 39). This early rapid mucosal reepithelialization is the function of differentiated intestinal epithelial cells, which are localized in the surface of the mucosa, rather than of undifferentiated epithelial cells, which are within the proliferative zone of the crypts. However, most of the studies (7, 10, 24-27, 33) that used an in vitro system mimicking the early cell division-independent stage of epithelial restitution employed undifferentiated intestinal crypt cells such as the IEC-6 line as a model. We (36) have demonstrated that differentiated intestinal epithelial cells induced by forced expression of the Cdx2 gene, which encodes a transcription factor controlling intestinal epithelial cell differentiation (42, 43), migrate over the wounded edge much faster than undifferentiated parental IEC-6 cells. Although these differentiated intestinal epithelial cells appear to provide an excellent in vitro model for restitution, the exact mechanism through which the rate of differentiated cell migration is increased remains to be elucidated.
The activity of voltage-gated K+ (Kv) channels controls membrane potential (Em), which regulates cytosolic free Ca2+ concentration ([Ca2+]cyt) by governing the driving force for Ca2+ influx (48, 50). [Ca2+]cyt is an important intracellular second messenger that regulates a large number of physiological functions (2, 8, 44). [Ca2+]cyt is controlled by extracellular Ca2+ influx and Ca2+ release from intracellular Ca2+ stores (mainly sarcoplasmic and endoplasmic reticulum) (3, 5, 32). Ca2+ influx partially depends on the Ca2+ driving force, which is determined by the transmembrane Ca2+ gradient and Em (11-13, 18). In eukaryotic cells, Em is primarily determined by K+ permeability (PK), which is directly related to the function and number of membrane K+ channels (11-13, 18). Decreasing the number of Kv channels by inhibiting their gene expression and/or attenuating K+ channel activity results in membrane depolarization. Because Em is a major determinant of the driving force for Ca2+ influx when the transmembrane Ca2+ gradient is constant and Ca2+ entry is a major source for [Ca2+]cyt, membrane depolarization would decrease [Ca2+]cyt in cells lacking L-type voltage-dependent Ca2+ channels (VDCC) (13, 30, 48). In contrast, membrane hyperpolarization would increase the Ca2+ driving force, enhance Ca2+ influx, and increase [Ca2+]cyt in the nonexcitable cells.
Our (37, 48) previous studies have demonstrated that
intestinal epithelial cells do not express VDCC and that induced activation of Kv channels causes membrane hyperpolarization, enhances Ca2+ entry by increasing the driving force for
Ca2+ influx, raises [Ca2+]cyt,
and promotes cell migration after wounding in undifferentiated parental
IEC-6 cells. Expression of the Kv channel genes in IEC-6 cells requires
cellular polyamines, including spermidine, spermine, and their
precursor putrescine. Depletion of cellular polyamines by inhibition of
ornithine decarboxylase (ODC), a key enzyme for polyamine synthesis,
with -difluoromethylornithine (DFMO) decreases Kv channel
expression, causes membrane depolarization, reduces [Ca2+]cyt, and decreases cell migration
(48). We (37) have further demonstrated that
RhoA of small GTPases is a downstream target of elevated
[Ca2+]cyt after activation of K+
channels by increased cellular polyamines and that
Ca2+-activated RhoA activity increases the formation of
actomyosin stress fibers in migrating cells during restitution.
The current study tests the hypothesis that differentiated intestinal epithelial cells exhibit increased migration after wounding by altering Kv channel expression and [Ca2+]cyt. First, we compared the Kv channel expression, Em, and resting [Ca2+]cyt in differentiated intestinal epithelial cells (stable Cdx2-transfected IEC-6 line) with those in undifferentiated parental cells (IEC-6 line). Second, we determined whether inhibition of Kv channel expression by polyamine depletion decreased [Ca2+]cyt in differentiated intestinal epithelial cells and further investigated whether manipulating [Ca2+]cyt, either by increase or decrease, altered cell migration. Third, we determined whether observed changes in [Ca2+]cyt affected the cellular distribution of nonmuscle myosin II.
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MATERIALS AND METHODS |
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Materials. Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed fetal bovine serum (dFBS) were obtained from GIBCO-BRL (Gaithersburg, MD), and biochemicals were from Sigma (St. Louis, MO). The primary antibody, an affinity-purified rabbit polyclonal antibody against Kv1.1 or Kv1.5, was purchased from Alomone Labs. The specific rabbit polyclonal antibody against nonmuscle myosin II was obtained from Biomedical Technologies (Stoughton, MA). Anti-rabbit IgG, FITC isomer conjugate, and ionomycin were purchased from Sigma. DFMO was purchased from Ilex Oncology (San Antonio, TX).
Cell culture and general experimental protocol. The IEC-6 cell line was purchased from American Type Culture Collection at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (35). IEC-6 cells originated from intestinal crypt cells, as judged by morphological and immunologic criteria. They are nontumorigenic and retain the undifferentiated character of intestinal epithelial crypt cells. Stock cells were maintained in T-150 flasks in DMEM supplemented with 5% heat-inactivated FBS, 10 µg insulin, and 50 µg gentamicin sulfate/ml. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2. Stock cells were subcultured once a week at 1:20, and the medium was changed three times per week. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative, and passages 15-20 were used in the experiments. There were no significant changes of biological function and characterization from passages 15 to 20.
The stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1 cells; kind gift of Dr. P. G. Traber, University of Pennsylvania, Philadelphia, PA) were developed and characterized by Suh and Traber (42). The expression vector, the LacSwitch system (Stratagene, La Jolla, CA), was used for directing the conditional expression of Cdx2, and isopropylElectron microscopy. Cells were fixed at room temperature in 2.5% glutaraldehyde-3.2% paraformaldehyde buffered with 0.1 M sodium cacodylate (pH 7.4). Cells were then postfixed in 2% osmium tetroxide in the same buffer, dehydrated, and embedded in Epon as described previously (36). Ultrathin sections were examined in an electron microscope.
RT-PCR.
Total RNA was prepared by the acid guanidinium
thiocyanate-phenol-chloroform extraction method (6).
Specific primers for Kv channel - (pore forming subunit) and
-subunits (cytoplasmic regulatory subunit), L-type VDCC
1- and
1-subunits, and transient receptor
potential channels (TRPC) were designed from the cDNA sequences of the
coding regions corresponding to the channel genes (Table
1). These particular sequences were
chosen on the basis of previously established specificity
(48-50, 53). RT-PCR was performed as we
(47) described previously. To quantify the PCR products
(the amounts of mRNA) of Kv, VDCC, and TRPC, an invariant mRNA of
-actin was used as an internal control. The optical density values
for each band on the gel were measured by a gel documentation system
(UVP, Upland, CA), and the channel signals were normalized to the
optical density values in the
-actin signals (48).
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Western blot analysis. Cell samples, dissolved in SDS sample buffer (250 mM Tris · HCl, pH 6.8, 2% SDS, 20% glycerol, and 5% mercaptoethanol), were sonicated and centrifuged at 2,000 rpm for 15 min. The protein concentration of the supernatant was measured by the Bradford method (4), and each lane was loaded with 25 µg of protein equivalent. The supernatant was boiled for 5 min and then subjected to electrophoresis on 10% acrylamide gels according to the method of Laemmli (23). Briefly, after the transfer of protein onto nitrocellulose filters, the filters were incubated overnight at 4°C in 5% nonfat dry milk in 1× PBS-Tween 20. Immunologic evaluation was then performed for 90 min in 1% BSA-PBS-Tween 20 buffer containing affinity-purified antibody against Kv1.1 or Kv1.5 channel protein. The filters were subsequently washed with 1× PBS-Tween 20 and incubated with an IgG second antibody conjugated to peroxidase by protein cross-linking with 0.2% glutaraldehyde. The immunocomplexes on the filters were reacted for 1 min with chemiluminescence reagent (NEL-100, DuPont NEN).
Electrophysiological measurements.
Whole cell K+ currents (IK) were
recorded with an Axopatch-1D amplifier and a DigiData 1200 interface
(Axon Instruments, Foster City, CA) by the patch-clamp technique
(50). Patch pipettes (2-4 M) were made on a Sutter
electrode puller using borosilicate glass tubes and fire polished on a
Narishige microforge. Step-pulse protocols and data acquisition were
performed with pCLAMP software. Currents were filtered at 1-2 kHz
(
3 dB) and digitized at 2-4 kHz with the Axopatch-1D amplifier.
To record optimal IK(v), we replaced
CaCl2 with equimolar MgCl2 in the bath
solution. Series resistance and capacitance were routinely compensated
(for 60-80%) by adjusting the internal circuitry of the
patch-clamp amplifier. Leakage currents were subtracted with the P/
4
protocol in pCLAMP software. Em in single IEC-6
or IEC-Cdx2L1 cells was measured in current-clamp mode
(I = 0) using whole cell patch-clamp techniques. All
experiments were performed at room temperature (22-24°C).
Measurement of [Ca2+]cyt. The digital imaging methods used for measuring [Ca2+]cyt were as previously described (51). Briefly, IEC-6 or IEC-Cdx2L1 cells were plated on 25-mm coverslips 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-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 (510 nm emission; 380 and 360 nm excitation) 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). Video frames containing images of fura 2 fluorescence from cells and the corresponding background images (fluorescence from fields devoid of cells) were digitized at a resolution of 512 horizontal × 480 vertical pixels and 8 bits using a Matrix LC imaging board operating in an IBM-compatible computer. Images were acquired at a rate of one averaged image every 3 s when [Ca2+]cyt was changing and every 60 s when [Ca2+]cyt was relatively constant. [Ca2+]cyt was calculated from fura 2 fluorescent emission excited at 380 and 360 nm using the ratio method (32). In most experiments, multiple cells (usually 10-15 cells) were imaged in a single field, and one arbitrarily chosen peripheral cytosolic area (4-6 × 4-6 pixels) from each cell was spatially averaged.Measurement of cell migration. The migration assays were carried out as we (47, 48) described previously. 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 incubated as described for stock cultures. The cells were fed on day 2 and migration tested on day 4. To initiate migration, we scratched the cell layer with a single-edge razor blade cut to ~27 mm in length. The scratch began at 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 performed in triplicate, and the results were reported as the number of migrating cells per millimeter of scratch.
Nonmuscle myosin II staining. The immunofluorescence procedure was carried out according to the method of Vielkind and Swierenga (45) with minor changes (37). The primary antibody recognizes the 200-kDa nonmuscle myosin II in immunoblots of IEC-Cdx2L1 cell extracts and does not cross-react with other cytoskeletal proteins (36). Nonspecific slides were incubated without antibody to nonmuscle myosin II. Slides were viewed through a Zeiss confocal microscope (model LSM410).
HPLC analysis of cellular polyamines.
The cellular polyamine content was determined as previously described
(46). Briefly, after the cells were washed three times with ice-cold Dulbecco's PBS, we added 0.5 M perchloric acid. The
cells were then frozen at 80°C until ready for extraction, dansylation, and HPLC. The standard curve encompassed 0.31-10 µM. Values that fell >25% below the curve were considered not detectable. Protein was determined by the Bradford method
(4). The results are expressed as nanomoles of polyamines
per milligram of protein.
Statistical analysis. All data are expressed as means ± SE from six dishes. Autoradiographic and immunofluorescence labeling results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined using Dunnett's multiple-range test (14).
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RESULTS |
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Changes in cell migration in differentiated intestinal epithelial
cells.
Forced expression of the Cdx2 gene in the stable
Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) induced a
significant development of differentiated phenotype as indicated by
electron microscopic features (Fig.
1A) and molecular evidence
(Fig. 1B). Nontransfected parental IEC-6 cells showed a
simple monolayer of flat epithelial cells with no evidence of cellular
differentiation (Fig. 1). However, the IEC-Cdx2L1 cells treated with 4 mM IPTG for 16 days exhibited multiple morphological and molecular
characteristics of intestinal epithelial differentiation (Fig. 1).
These enterocyte-like cells were polarized, showed lateral membrane
interdigitations, a well-demarcated basal lamina, and microvilli at the
apical pole and also expressed brush-border enzymes such as
sucrase-isomaltase.
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Kv channel expression in differentiated IEC-Cdx2L1 cells.
The mRNA expression of Kv1.1 and Kv1.5 channels increased significantly
in differentiated IEC-Cdx2L1 cells that exhibited increased migration
after wounding. As shown in Fig. 2, the
mRNA levels of Kv1.1 and Kv1.5 in differentiated IEC-Cdx2L1 cells were ~2.2- and ~1.9-fold greater, respectively, than those of
undifferentiated parental IEC-6 cells. On the other hand, there
were no significant differences in mRNA expression of Kv2.1, Kv4.3,
Kv9.3, and Kv1.1 between differentiated IEC-Cdx2L1 cells and
parental IEC-6 cells. The mRNA levels of Kv1.1 and Kv1.5 channels in
differentiated IEC-Cdx2L2 cells were similar to those observed in
IEC-Cdx2L1 cells (data not shown). We also examined the effects of
G418, hygromycin B, and IPTG on Kv channel expression and demonstrated that mRNA levels of Kv1.1, Kv1.5, Kv2.1, Kv4.3, Kv9.3, and Kv
1.1 in
the empty vector-transfected IEC-6 cells were indistinguishable from
those in nontransfected parental IEC-6 cells. Exposure of nontransfected parental IEC-6 cells to IPTG or
Cdx2-transfected IEC-6 cells before treatment with IPTG to
induce differentiation was not associated with increased expression of
Kv1.1 and Kv1.5 channels (data not shown).
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Effect of hyperpolarized Em on
[Ca2+]cyt in IEC-Cdx2L1
cells.
In excitable cells, such as smooth muscle cells and neurons, VDCC are
major pathways for Ca2+ influx. Therefore, membrane
depolarization would open VDCC and promote Ca2+ influx
(28, 32, 44). In contrast, nonexcitable cells, including intestinal epithelial cells, do not express VDCC and membrane depolarization would decrease the Ca2+ driving force and
attenuate Ca2+ influx through store-operated
Ca2+ channels that may be formed by TRPC (49, 52,
53). As shown in Fig. 5, both
differentiated IEC-Cdx2L1 cells and parental IEC-6 cells expressed
TRPC1 and TRPC5, which encode the Ca2+-permeable channels
involved in capacitative Ca2+ entry in mammalian cells
(52, 53). The level of TRPC1 mRNA in IEC-Cdx2L1 cells
increased significantly and was approximately twofold greater than in
parental IEC-6 cells (Fig. 5A). Although TRPC4 and
TRPC6 were highly expressed in rat pulmonary artery smooth muscle
cells, they were not detectable in IEC-Cdx2L1 and IEC-6 cells by RT-PCR
analysis (Fig. 5, B and D). Neither
differentiated IEC-Cdx2L1 cells nor parental IEC-6 cells expressed VDCC
(Fig. 5, E and F). The pore-forming
(1-subunit) and regulatory subunits (
1-subunit) of L-type VDCC were not detectable in both
IEC-Cdx2L1 and IEC-6 cells but were highly expressed in rat pulmonary
artery smooth muscle cells. These results indicate that differentiated IEC-Cdx2L1 cells do not express VDCC but express TRPC1 and TRPC5 channels that may be responsible for the capacitative Ca2+
entry in intestinal epithelial cells.
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Effect of inhibition of Kv channel expression by polyamine depletion on [Ca2+]cyt and cell migration. It has been shown (48) that Kv channel expression in intestinal epithelial cells requires polyamines and that depletion of cellular polyamines decreases expression of the Kv channel genes and reduces IK(v). Exposure of IEC-Cdx2L1 cells to 5 mM DFMO (a specific inhibitor for ODC) for 4 days almost completely depleted cellular polyamines. Putrescine and spermidine were undetectable, whereas spermine was decreased by >65% on day 4 in the DFMO-treated IEC-Cdx2L1 cells (data not shown).
Polyamine depletion by DFMO significantly inhibited expression of Kv1.1 and Kv1.5 channels (Fig. 7) but had no effect on expression of Kv2.1, Kv4.3, Kv9.3, and Kv
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Effect of increasing
[Ca2+]cyt on cell
migration.
The relationship between [Ca2+]cyt and cell
migration in differentiated IEC-Cdx2L1 cells was further examined by
using the Ca2+ ionophore ionomycin. Exposure to 1 µM
ionomycin reversibly increased [Ca2+]cyt by
promoting Ca2+ influx regardless of the presence or absence
of polyamines (Fig. 9A). In
normal cells, [Ca2+]cyt was dramatically
increased after the addition of ionomycin for 5 min (from 141 ± 7 to 485 ± 23 nM, n = 10, P < 0.05). When ionomycin was washed out, [Ca2+]cyt rapidly
returned to basal levels (Fig. 9Aa). Exposure of polyamine-deficient cells to ionomycin also remarkably increased [Ca2+]cyt ( from 88 ± 4 to 340 ± 15 nM, n = 10, P < 0.05), but the peak of
ionomycin-induced Ca2+ influx was significantly reduced
compared with that of controls (Fig. 9, Aa vs.
Ab). This reduced response of DFMO-treated cells to
ionomycin was apparently due to a decrease in the Ca2+
driving force as a result of inhibition of Kv channel expression by
polyamine depletion (Fig. 7).
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Effect of [Ca2+]cyt on
distribution of nonmuscle myosin II.
To determine the possible mechanism by which
[Ca2+]cyt mediates cell migration in
differentiated intestinal epithelial cells, the effects of changes in
[Ca2+]cyt, either decreased or increased, on
cellular distribution of nonmuscle myosin II were examined in control
and polyamine-deficient IEC-Cdx2L1 cells. As shown in Fig.
10A, a network of long
stress fibers that traversed the cytoplasm was observed in the control group. This thick network of cortical myosin II fibers was just beneath
the plasma membrane. Exposure of control cells to the Ca2+-free medium during migration significantly decreased
the formation of myosin II stress fibers (Fig. 10, A vs.
B). The distribution of myosin II stress fibers was sparse
and devoid of long stress fiber formation. Polyamine depletion
by DFMO also affected cellular organization of nonmuscle myosin II in
differentiated IEC-Cdx2L1 cells (Fig. 10, A vs.
C). Long stress fibers disappeared in polyamine-deficient cells, and there were no distinct myosin II stress fibers in the cytoplasm. On the other hand, either elevation of
[Ca2+]cyt by treatment with ionomycin in
polyamine-deficient cells (Fig. 10D) or spermidine given
together with DFMO (Fig. 10E) restored the distribution of
nonmuscle myosin II to near normal. The distribution of nonmuscle
myosin II stress fibers in cells treated with DFMO but exposed to
ionomycin after wounding or cells grown in the presence of DFMO plus
spermidine was indistinguishable from that of control cells (Fig. 10,
A vs. D and E). In contrast, removal of extracellular Ca2+ after wounding completely prevented
the restoration of the distribution of nonmuscle myosin II by exogenous
spermidine in polyamine-deficient cells (Fig. 10, E vs.
F). These results indicate that reorganization of nonmuscle
myosin II and the formation of stress fibers in migrating IEC-Cdx2L1
cells are significantly regulated by elevation of
[Ca2+]cyt during restitution after wounding.
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DISCUSSION |
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Early rapid intestinal mucosal reepithelialization following superficial wounding is a complex process that includes the flattening, spreading, migrating, and repolarizing of differentiated columnar epithelial cells, but the exact mechanisms involved in this primary repair modality are still unclear. We (36) have recently demonstrated that differentiated intestinal epithelial cells induced by forced expression of the Cdx2 gene (IEC-Cdx2L1 cells) migrate over the wounded edge much faster than undifferentiated parental crypt cells (IEC-6 line) in an in vitro model mimicking the early cell division-independent stage of epithelial restitution. These findings (36) are of important biological significance because the rapid mucosal restitution of superficial wounds in vivo is the function of differentiated intestinal epithelial cells from the surface of the mucosa rather than from the undifferentiated epithelial cells within the crypts. In this study, we provide direct evidence to support the contention that activation of Kv channel expression and the resultant elevation of [Ca2+]cyt due to enhanced Ca2+ driving force play a critical role in the process by which the rate of differentiated intestinal epithelial cell migration is increased after wounding.
Differentiated IEC-Cdx2L1 cells highly expressed Kv1.1 and Kv1.5
channels, associated with an increase in IK(v)
and membrane hyperpolarization. At the molecular level, the Kv channels
in mammalian cells are composed of the pore-forming -subunits and the regulatory
-subunits (16). It has been shown
(11-13) that the function and number of Kv channels
are major determinants of Em in many types of
cells. In resting cells, Em is a function of the
Na+, K+, and Cl
concentration
gradients across the plasma membrane and the relative ion permeability.
Because transmembrane PK > PNa > PCl
(PK/PNa/PCl = 1:0.04:0.45) is predominant under physiological conditions, Em is controlled primarily by
PK and K+ concentration gradients.
PK (and thus Em) is
directly related to IK(v), which is dependent on
the total number of functional K+ channels and
single-channel (unitary) current (18, 29). When K+ channel opens or K+ channel expression
rises, PK is increased, leading to membrane hyperpolarization (13, 30). As shown in Figs. 2 and 3, the levels of Kv1.1 and Kv1.5 channel mRNAs and proteins in differentiated IEC-Cdx2L1 cells are approximately twofold higher than those of undifferentiated parental IEC-6 cells, indicating that membrane hyperpolarization in differentiated epithelial cells results, at least
partially, from the increased expression of Kv channels.
Em regulates [Ca2+]cyt through controlling the Ca2+ driving force for Ca2+ influx in nonexcitable cells that do not express VDCC (13, 48). [Ca2+]cyt, which regulates a large number of biological functions, is controlled by Ca2+ influx through Ca2+-permeable channels in the plasma membrane and Ca2+ release from intracellular Ca2+ stores (32, 44). Under physiological conditions, extracellular Ca2+ concentration is 1.6~1.8 mM, ~10,000-20,000-fold higher than the resting [Ca2+]cyt (50-150 nM), which provides a seemingly inexhaustible supply of Ca2+ for its diverse intracellular function. The transmembrane Ca2+ influx depends on the Ca2+ driving force (i.e., the electrochemical gradient across the plasma membrane), which is predominantly regulated by Em while the Ca2+ concentration gradient is constant (11-13, 18). In nonexcitable cells, including epithelial cells and lymphocytes, membrane hyperpolarization raises [Ca2+]cyt by increasing the Ca2+ driving force, whereas membrane depolarization reduces [Ca2+]cyt by decreasing the Ca2+ driving force (13, 30, 48). However, in excitable cells such as neurons and muscle cells that highly express VDCC, membrane depolarization increases [Ca2+]cyt by opening VDCC (28, 44). Although differentiated IEC-Cdx2L1 cells did not express VDCC, they highly expressed TRPCs (Fig. 5), which are Ca2+-permeable channels responsible for capacitative Ca2+ entry (49, 52, 53). It is possible that the elevation of [Ca2+]cyt in differentiated IEC-Cdx2L1 cells (Fig. 6) is partially due to the increase in capacitative Ca2+ entry via TRPCs following membrane hyperpolarization induced by activation of Kv channel expression. Because passive Ca2+ leakage, receptor-operated Ca2+ channels, and nonselective cation channels all contribute to Ca2+ influx (15, 30, 34, 44, 54), other Ca2+ channels also may be involved in the process leading to the induction of [Ca2+]cyt in differentiated IEC-Cdx2L1 cells.
Elevation of [Ca2+]cyt in differentiated
IEC-Cdx2L1 cells is a major mediator for the increased migration after
wounding. Our (37, 48) previous studies have shown that Kv
channels play a critical role in the regulation of cell migration by
controlling Em and
[Ca2+]cyt in undifferentiated parental IEC-6
cells. Expression of Kv channels requires cellular polyamines, and
depletion of cellular polyamines by treatment with DFMO decreases Kv
channel gene expression, results in membrane depolarization, reduces
[Ca2+]cyt, and inhibits cell migration. To
determine the role of increased expression of Kv channels and the
resultant elevation of [Ca2+]cyt in the
process of epithelial migration in differentiated IEC-Cdx2L1 cells
after wounding, we examined the effects of inhibition of Kv channel
expression by polyamine depletion on
[Ca2+]cyt and cell motility in the IEC-Cdx2L1
cells. As shown in Fig. 7, depletion of cellular polyamines by
treatment with DFMO resulted in a remarkable decrease in levels of
Kv1.1 and Kv1.5 channel mRNAs and proteins, although it negligibly
affected expression of Kv2.1, Kv4.3, Kv9.3, and Kv1.1 channels (data
not shown). Reduced expression of Kv1.1 and Kv1.5 channels was
associated with significant decreases in both
[Ca2+]cyt and cell migration in
differentiated IEC-Cdx2L1 cells (Fig. 8). Exogenous spermidine given
together with DFMO not only completely prevented the inhibition of Kv
channel expression but also restored cell migration to normal levels.
These findings are consistent with those observed (37, 48)
in undifferentiated parental IEC-6 cells and strengthen the evidence
that the activation of Kv channels plays an important role in the
regulation of cell migration during early restitution by controlling
Em and [Ca2+]cyt,
which is regulated by cellular polyamines.
The decrease in the migration rate in the polyamine-deficient cells is primarily due to the decrease in [Ca2+]cyt rather than to the alteration of differentiation, because polyamine depletion by DFMO fails to affect characteristics of differentiated phenotype in IEC-Cdx2L1 cells (36). In support of this possibility, removal of extracellular Ca2+ from the culture medium immediately after wounding almost completely prevented the restoration of cell migration by exogenous spermidine in polyamine-deficient IEC-Cdx2L1 cells (Fig. 8, B and C). This contention is further supported by the results presented in Fig. 9 showing that increasing [Ca2+]cyt by treatment with the Ca2+ ionophore ionomycin significantly increased cell migration in polyamine-deficient IEC-Cdx2L1 cells.
To investigate how elevated [Ca2+]cyt modulates cell migration during restitution in differentiated intestinal epithelial cells, we examined the distribution of cytoskeletal protein nonmuscle myosin II in IEC-Cdx2L1 cells in the presence or absence of Ca2+. Nonmuscle myosin II is a major cellular motor molecule of the intestinal epithelial cells (9, 17) and is implicated in the formation of stress fibers that regulate cell adhesion, spreading, and motility (1, 17, 20, 31, 41). Dysfunction of myosin II, by either microinjection of myosin II antibody, antisense RNA, or recombination dominant negative mutants, significantly decreases cell migration in nonmuscle cells (22, 40). Figure 10 shows that [Ca2+]cyt regulates the migration of differentiated IEC-Cdx2L1 cells at least partially through alteration of the formation of actomyosin stress fibers. When [Ca2+]cyt was decreased by exposure to the Ca2+-free medium or inhibition of Kv channel expression via polyamine depletion, the number of long stress fibers of myosin decreased significantly, and in some cells they disappeared completely from the cytoplasm. In contrast, increased [Ca2+]cyt via treatment with the Ca2+ ionophore ionomycin in polyamine-deficient cells promoted the formation of myosin II stress fibers. These results are consistent with data from other studies (19-21) finding that the formation and function of stress fibers are regulated by Ca2+-dependent signaling pathways. Although the exact mechanisms involved in downstream targeting in this process are still unknown, we (37) have recently demonstrated that the stimulation of myosin II stress fiber formation by polyamines is a result of Ca2+-induced activation of RhoA protein in undifferentiated parental IEC-6 cells.
In summary, these results indicate that differentiated IEC-Cdx2L1 cells highly express Kv1.1 and Kv1.5 channel mRNAs and proteins, which are associated with a significant increase in IK(v) and membrane hyperpolarization. Because IEC-Cdx2L1 cells do not express VDCC, the membrane hyperpolarization in differentiated intestinal epithelial cells increases the Ca2+ driving force for Ca2+ influx and raises [Ca2+]cyt. Inhibition of Kv channel expression by depletion of cellular polyamines with DFMO decreases [Ca2+]cyt and inhibits cell migration during restitution. Increasing [Ca2+]cyt by exposure to the Ca2+ ionophore ionomycin after wounding promotes cell migration in normal and polyamine-deficient IEC-Cdx2L1 cells. Elevation of [Ca2+]cyt also increases the formation of myosin II stress fibers in differentiated IEC-Cdx2L1 cells, while decreased [Ca2+]cyt after inhibition of Kv channel expression or removal of extracellular Ca2+ results in the reorganization of myosin II, along with a marked reduction of stress fibers. These findings suggest that differentiated intestinal epithelial cells exhibit increased migration after wounding, at least partially, by the activation of Kv channel expression, leading to the increase in the Ca2+ driving force for Ca2+ influx during restitution.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants DK-57819 (to J.-Y. Wang), HL-54043, and HL-64945 (both to J. X.-J. Yuan) and a Merit Review Grant from the Department of Veterans Affairs (to J.-Y. Wang). J. X.-J. Yuan is an Established Investigator of the American Heart Association and J.-Y. Wang is a Research Career Scientist for the Medical Research Service of the Department of Veterans Affairs.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene St., Baltimore, MD 21201 (E-mail: jwang{at}smail.umaryland.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00361.2001
Received 30 July 2001; accepted in final form 7 November 2001.
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