Department of Surgery, Yale University School of Medicine, New Haven 06520-8062; and Connecticut Veterans Affairs Health Care System, West Haven, Connecticut 06516
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the intestinal epithelium undergoes
complex deformations during normal function, nutrient absorption,
fasting, lactation, and disease, the effects of deformation on
intestinal mucosal biology are poorly understood. We previously
demonstrated that 24 h of cyclic deformation at an average 10%
deformation every 6 s stimulates proliferation and modulates
brush-border enzyme activity in human intestinal Caco-2 cell
monolayers. In the present study we sought potential mechanisms for
these effects. Protein kinase C (PKC) activity increased within 1 min
after initiation of cyclic deformation, and the PKC- and -
isoforms translocated from the soluble to the particulate fraction.
Cyclic deformation also rapidly increased tyrosine kinase activity.
Tyrosine phosphorylation of several proteins was increased in the
soluble fraction but decreased in the particulate fraction by cyclic
deformation for 30 min. Inhibition of PKC and tyrosine kinase signals
by calphostin C, G-06967, and erbstatin attenuated or blocked cyclic
deformation-mediated modulation of Caco-2 DNA synthesis and
differentiation. These results suggest that cyclic deformation may
modulate intestinal epithelial proliferation and brush-border enzyme
activity by regulating PKC and tyrosine kinase signals.
alkaline phosphatase; dipeptidyl dipeptidase; protein kinase C-
isoform; protein kinase C-
isoform; tyrosine phosphorylation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ALTHOUGH THE INTESTINAL mucosa is exposed to complex patterns of physical deformation during peristalsis, villous motility, and diverse disease states (8, 11, 21, 23, 25-28, 33, 35-37), the effects of deformation on intestinal epithelial biology are poorly understood. Because it is difficult to regulate physical deformation in the intestinal mucosa in vivo without altering the entire regulatory milieu, we used a subclone of the human intestinal Caco-2 cell line (29) as a model to study the effects of deformation on the intestinal epithelial cell. These cells spontaneously differentiate at confluence into cells exhibiting many of the morphological and functional characteristics of mature enterocytes and represent a useful in vitro model to study normal human intestinal epithelium (18). To simulate physical deformation, we utilized a computer-regulated deformation apparatus that modulates the physical deformation and relaxation of cultured intestinal epithelial cell monolayers.
We have previously reported that 24-h exposure of human intestinal Caco-2 cells to an average 10% deformation at 10 cycles/min stimulates cell proliferation and modulates brush-border enzyme activity, dipeptidyl dipeptidase (DPDD), and alkaline phosphatase (AKP) (4). The frequency and amplitude of deformation are within the range of deformation on mucosa during peristalsis (28) or villous motility (36). Protein kinase C (PKC) and tyrosine kinase signals are known to regulate important cellular events, including gene expression, proliferation, and differentiation of enterocytes in vivo (10, 19) and in vitro (2, 3, 7). Furthermore, rhythmic deformation of endothelial and smooth muscle cell monolayers has been observed to modulate PKC and tyrosine kinase activity (30, 38).
In this study we therefore sought to determine whether rhythmic deformation modulates PKC and tyrosine kinase activity in Caco-2 cells and whether cyclic deformation-mediated alterations of proliferation and brush-border enzyme activity of Caco-2 cells are regulated by modulating PKC and tyrosine kinase-mediated signals.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
Tissue culture medium, FCS, phenylmethylsulfonyl fluoride (PMSF),
leupeptin,
1-chloro-3-tosylamido-7-amino-L-2-hepatone
(TLCK), N-tosyl-L-phenylalanine
chloromethyl ketone (TPCK), and sodium orthovanadate were obtained from
Sigma Chemical (St. Louis, MO). Transferrin, anti-phosphotyrosine
peroxidase, biotin-labeled synthetic tyrosine kinase substrates,
phosphopeptide standard, piceatannol, 2,2'-azino-bis-(3-ethylbenz-thiazolin-6-sulfonic acid) diammonium salt (ABTS), and streptavidin-coated microtiter plates were purchased from Boehringer Mannheim (Indianapolis, IN). PepTag C1 peptide, phosphatidyl serine, and gel solubilization solution were purchased from Promega (Madison, WI). Polyclonal antibody to PKC-, G-06967, genistein, and erbstatin were obtained from GIBCO (Gaithersburg, MD).
Monoclonal antibody to PKC-
was obtained from Upstate Biotechnology (Lake Placid, NY). Calphostin C was purchased from Calbiochem (La
Jolla, CA). Nitrocellulose membranes, enhanced chemiluminescence (ECL)
kits for Western blotting protein detection, and the peroxidase-coupled sheep anti-mouse and donkey anti-rabbit antibodies were purchased from
Amersham (Arlington Heights, IL).
[3H]thymidine was
obtained from Dupont-NEN (Boston, MA). Unless otherwise noted all other
reagents were purchased from Sigma Chemical or Fisher Scientific
(Springfield, NJ).
Cells. The Caco-2 cells used for these studies were a clonal subpopulation selected for enterocytic differentiation (29). Stock cultures were maintained in DMEM containing 10% FCS, 10 µg/ml transferrin, 25 mmol/l glucose, 2 mmol/l glutamine, 1 mmol/l pyruvate, 15 mmol/l HEPES, 100 U/ml penicillin G, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 95% air-5% CO2.
Application of rhythmic deformation. Caco-2 cells were seeded at a density of 30,000 cells/cm2 on collagen I-coated flexible-bottomed six-well plates (Flexercell, McKeesport, PA). Either at confluence (for enzyme activity and detection of PKC isoforms and phosphorylated tyrosine) or 30-40% confluence (for cell number and [3H]thymidine uptake), plates were placed in a deformation unit (Flexercell) consisting of a vacuum manifold in a tissue culture incubator (5% CO2, 37°C) which was regulated by a solenoid valve controlled by a computer with a timer program. The membrane bottoms were deformed by a known percent elongation by application of a precise vacuum. On release of the vacuum, the membrane bottoms returned to their original conformation. Because cells adhere to the flexible surface of the culture well, the cell experiences the same deformation that is applied to the culture well (4). The deformation on the flexible well during stretch at various vacuum levels (i.e., increasing levels of deformation) has been calculated mathematically by finite element analysis and empirically by measuring with a micrometer the distance between concentric circles (radial deformation) or diametric axes (axial deformation) marked on the membrane. Very little change is observed in the latter; hence, the force on the attached cells is uniaxial. Indeed, we have previously demonstrated that Caco-2 cells subjected to such deformation regimens on these membranes remain adherent and experience uniaxial deformation consistent with deformation of the membrane (4).
In the present study Caco-2 cells were subjected to an average deformation of 10% at 10 cycles/min (3 s of deformation alternating with 3 s in neutral conformation) for the indicated times. Unstretched Caco-2 cell monolayers served as controls. In some experiments, blockers of PKC and tyrosine kinase or the appropriate vehicle (DMSO) were added to the Caco-2 cells before the initiation of cyclic deformation. When calphostin C was added to cells, it was preactivated by exposure to light for 2 h.Cell number. Subconfluent (30-40%) Caco-2 cells grown on a collagen-coated flexible membrane were subjected to an average 10% deformation with 10 cycles/min for 24 h in the presence and absence of either staurosporine (20 ng/ml) or genistein (200 µmol/l). Caco-2 cell monolayers were washed with Ca2+- and Mg2+-free Hanks' balanced salt solution and trypsinized. Cell number was then determined by Coulter counter analysis (Coulter Instruments, Hialeah, FL). The cell viability was higher than 98%.
[3H]thymidine incorporation. After cells were attached to the membrane, cells were exposed to cyclic deformation for 24 h with 1 µCi [3H]thymidine/well, and [3H]thymidine incorporation was assessed by scintillation counting of the TCA- soluble, sodium hydroxide-precipitable fraction of the cell lysate as previously described (4). Calphostin C (10 nmol/l), G-06967 (100 nmol/l), and erbstatin (3 µmol/l) were added simultaneously with [3H]thymidine.
Digestive enzyme activity. AKP and DPDD were assayed as previously described by measuring hydrolysis of the synthetic substrates p-nitrophenyl phosphate and L-alanine-p-nitroaniline, respectively (4).
PKC assay. Caco-2 cell monolayers were washed twice with ice-cold 10 mmol/l phosphate buffer and homogenized by 10 strokes with a type B pestle in 20 mmol/l Tris · HCl (pH 7.4), 1 mmol/l EGTA, 1 mmol/l dithiothreitol (DTT), 1 mmol/l PMSF, 0.2 mmol/l leupeptin, and 5 U/ml aprotinin. The homogenates were centrifuged for 5 min at 600 g, and supernatants were used for PKC assay. PKC activity was assessed by measuring phosphorylation of a synthetic colored substrate, PepTag C1 peptide (P-L-S-R-T-L-S-V-A-A-K; Promega), using highly purified PKC enzyme from rat brain (Promega) as a standard.
Tyrosine kinase assay.
Caco-2 cells were washed twice with ice-cold phosphate buffer and
harvested in lysis buffer containing 50 mmol/l
Tris · HCl (pH 8.0), 150 mmol/l NaCl, 1 mmol/l DTT,
0.5 mmol/l EDTA, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS, 0.1 mmol/l sodium orthovanadate, 100 µg/ml PMSF, 1 µg/ml
aprotinin, and 2 µg/ml leupeptin and homogenized with 10 strokes
using a type B pestle. The homogenates were centrifuged at 10,000 g for 10 min at 4°C, and the
supernatants were used for tyrosine kinase assay. Tyrosine kinase
activity was determined by measuring the transfer of the -phosphate
group from ATP to the biotin-labeled tyrosine kinase substrate
(K-V-E-K-I-G-E-G-T-Y-V-V-Y-K-amide), which corresponds to a partial
amino acid sequence of cell division kinase p34cdc2, by ELISA as
recommended by the manufacturer's instructions (Boehringer Mannheim).
Detection of PKC- and -
isoforms
and phosphorylated tyrosine by Western blotting.
All samples for Western blotting were preliminarily assayed for protein
(bicinchoninic acid; Pierce, Rockford, IL) and diluted with buffer to
equal protein concentrations before loading. Equivalency of loading
across the lanes of the gel was also routinely verified by Coomassie
blue staining.
Analysis of data. Data are presented as means ± SE. Data were analyzed using a paired t-test with a Bonferroni correction. P < 0.05 was considered a significant difference.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyclic deformation rapidly induced PKC activity. We initially examined whether cyclic deformation regulates PKC activity in Caco-2 cells. Confluent Caco-2 cells grown on collagen-coated flexible membranes were exposed to an average 10% deformation at 10 cycles/min for 0-24 h. Basal PKC activity in Caco-2 cells at confluence was 2.6 ± 0.1 U/mg protein. Cyclic deformation rapidly increased cellular PKC activity to a maximum of 80 ± 31% (Fig. 1, P < 0.05, n = 12) by 1 min after initiation of deformation. After 24 h of exposure to cyclic deformation, PKC activity was still slightly but statistically significantly elevated (17 ± 13%, P < 0.05, n = 12).
|
Cyclic deformation rapidly induced and redistributed
PKC- and -
isoforms in subcellular
fractions.
Because activated PKC translocates from the cytosol to the membrane
fraction, we next examined the effects of cyclic deformation on the
subcellular distribution of PKC-
and -
, the predominant PKC
isoforms in Caco-2 cells (7) by Western blotting. Our preliminary studies also confirmed the lack of detection of PKC-
and -
isoforms (not shown). Because PKC activity was rapidly increased by
deformation with the peak at 1 min, we examined the distribution of PKC
isoforms from 6 s to 5 min after initiation of deformation. Cyclic
deformation rapidly translocated PKC-
from the soluble to the
particulate fraction. The peak of translocation of PKC-
occurred at
30 s (Fig. 2A).
Densitometric analysis demonstrated that 28 ± 9% of PKC-
was
associated with the particulate fraction and 72 ± 9% was within
the soluble fraction in static cells
(n = 4). However, cyclic deformation
for 30 s increased the level of the PKC-
isoform in the particulate
fraction up to 47 ± 9% (P < 0.05, n = 4) with a corresponding
decrease in the soluble fraction to 53 ± 9%
(P < 0.05, n = 4). Similarly, cyclic deformation
also rapidly translocated the PKC-
isoform from the soluble to the
particulate fraction (Fig. 2B). In
static cells 56 ± 7% of the PKC-
isoform was located in the
particulate fraction and 44 ± 7% was in the soluble fraction (n = 4). Cyclic deformation for 30 s
rapidly increased the PKC-
isoform from 56 ± 7 to 76 ± 7% (P < 0.05, n = 4) in the particulate fraction
with a decrease from 44 ± 7 to 24 ± 7%
(P < 0.05, n = 4) in the soluble fraction. As a
positive control for these studies, we also replicated the finding of
Bissonnette and colleagues (7) that the exposure of Caco-2 cells to 1 µmol/l of PMA for 20 min completely
(P < 0.05, n = 4) translocated PKC-
but not
PKC-
from the soluble to the particulate fraction (data not shown).
|
Rhythmic deformation of Caco-2 monolayer also rapidly stimulated tyrosine kinase activity. We next examined the effects of repetitive deformation on tyrosine kinase activity. As in the PKC study, cyclic deformation was applied to confluent Caco-2 cells for 0-24 h before tyrosine kinase activity assay. At 100% confluence, cellular tyrosine kinase activity in Caco-2 cells was 2.1 ± 0.3 mU/mg protein. Cyclic deformation rapidly increased cellular tyrosine kinase activity. The peak occurred at 5 min after initiation of deformation with a 41.0 ± 13.5% (P < 0.05, n = 15) increase compared with that in control (Fig. 3). Tyrosine kinase activity then rapidly decreased toward control levels after 10-30 min of deformation. However, chronic exposure of Caco-2 cells to cyclic deformation for 24 h demonstrated a sustained increase (49.2 ± 18.0%, P < 0.05, n = 15) in tyrosine kinase activity compared with that in static control cells.
|
Repetitive deformation rapidly increased phosphorylation of tyrosine residues in several proteins in soluble fraction but decreased phosphotyrosine in particulate fraction. We next directly examined the effect of deformation on the tyrosine phosphorylation of cellular proteins. Western blotting revealed that exposure to cyclic deformation rapidly increased tyrosine phosphorylation in several cellular proteins in the soluble fraction with peaks at 30 min (Fig. 4). Densitometric analysis demonstrated that cyclic deformation for 30 min increased tyrosine phosphorylation by 1,397 ± 290, 389 ± 98, 176 ± 37, and 92 ± 18% in 50-, 60-, 70-, and 125-kDa molecular mass proteins, respectively (P < 0.05 for each, n = 4), above that in control cells. In contrast, tyrosine phosphorylation of cellular proteins in the particulate fraction was decreased by 73 ± 21, 92 ± 8, 78 ± 17, and 89 ± 10% in 50-, 60-, 70-, and 125- kDa proteins, respectively, by cyclic deformation for 30 min (Fig. 4; P < 0.05 for each, n = 4). In two additional independent experiments, we compared phosphorylation of these tyrosine phosphoproteins at the 0- and 30-min time points in multiple lanes on the same gels and observed similar effects, demonstrating that the observed differences in band intensity were not artifacts of lane position on the gels (data not shown).
|
The deformation-induced increase in cell number was blocked by PKC and tyrosine kinase inhibitors. We next directly examined the effects of inhibitors of PKC and tyrosine kinase activity on the mitogenic effect of deformation. Subconfluent Caco-2 cells were exposed to cyclic deformation (average 10% deformation at 10 cycles/min) for 24 h, and cell numbers were counted by a Coulter counter after trypsinization. Cyclic deformation increased cell number by 44.5 ± 7.8 to 54 ± 7% (P < 0.05, n = 6) above that (890,000 ± 73,800 cells) in static controls (Fig. 5). This deformation-mediated stimulation of cell number was completely blocked by the PKC inhibitor staurosporine (Fig. 5A) and by the tyrosine kinase inhibitor genistein (Fig. 5B). Because of the less than optimal specificity of staurosporine and genistein, we used more specific inhibitors of PKC and tyrosine kinase activity for the following studies.
|
Blockade of PKC and tyrosine kinase inhibited deformation-mediated
stimulation of [3H]thymidine
incorporation in Caco-2 cells.
Caco-2 cells at 30-40% confluence were used for
[3H]thymidine
incorporation studies. In static control monolayers Caco-2 cells accumulated 168,400 ± 7,238 counts/min
[3H]thymidine/well
during 24-h incubation at 37°C (Table
1). Exposure of Caco-2 cells to an average
10% deformation at 10 cycles/min for 24 h increased
[3H]thymidine
incorporation by 26.4 ± 5.3% (P < 0.05, n = 12). This change is
similar to what we have previously observed (4). Deformation-mediated
stimulation of Caco-2
[3H]thymidine
incorporation was completely inhibited by the PKC inhibitor calphostin
C (10 nmol/l) and by the tyrosine kinase inhibitor erbstatin (3 µmol/l) but was potentiated by the PKC- inhibitor G-06967 (100 nmol/l). Although neither calphostin C nor erbstatin affected
[3H]thymidine uptake
in static control cells, G-06967 slightly but significantly
(P < 0.05) increased
[3H]thymidine uptake
in static control cells.
|
PKC and tyrosine kinase inhibition completely prevented cyclic
deformation-mediated modulation of DPDD brush-border enzyme activity
and attenuated AKP activity.
To examine the effects of cyclic deformation on differentiation, we
exposed confluent Caco-2 cells to cyclic deformation for 24 h. Cyclic
deformation increased DPDD activity by 38 ± 9%
(P < 0.001, n = 12) compared with that
in static cells (Table 2). However, the
stretch-associated increase in DPDD activity was completely blocked by
PKC inhibition with either calphostin C or G-06967, as well as by the
tyrosine kinase inhibitor erbstatin. Neither PKC nor tyrosine kinase
inhibition influenced DPDD activity in static control cells. In
contrast to DPDD, AKP activity in stretched cells was decreased by 33.5 ± 2.6% (P < 0.0005, n = 12) compared with their static
controls (Table 3). The addition of PKC, PKC-, or
tyrosine kinase inhibitors decreased AKP activity in static controls by
13.0 ± 6.1 to 21.9 ± 1.6% (n = 12, P < 0.05). However, treatment
with each inhibitor prevented any further decrease in AKP activity by
cyclic deformation (Table 3).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intestinal epithelial cells are exposed to various conditions, including fasting, intestinal motility, feeding, altered luminal nutrient concentrations, ionic composition, pressure, and villous contraction or motility, which cause diverse deformation patterns of the intestinal mucosa (11, 21, 23, 28, 33, 35, 36). Modulation of intestinal epithelial proliferation and differentiation in vivo under such conditions are well described (12, 13, 15, 24), but it remains unknown whether the modulation of enterocyte biology by these factors is deformation mediated. Because it is difficult to regulate deformation frequency and amplitude in the intestinal mucosa in vivo and to isolate the effects of deformation from other physiological parameters, we have previously used a computer-regulated deformation apparatus to repetitively deform cultured human intestinal epithelial Caco-2 cell monolayers (4). Caco-2 cells are derived from a colon cancer but spontaneously differentiate into polarized cells with many morphological and functional properties of mature enterocytes and are a common in vitro model for intestinal epithelial biology (9, 18, 29).
Intestinal epithelial deformation is likely to represent a complex summation of peristaltic contraction, physical interaction of the mucosa with luminal contents, alterations in villus shape, and alterations in cell shape. Intestinal circular smooth muscle contraction transmits contractile forces across the mucosa to luminal contents. Because luminal contents are largely noncompressible, this would increase pressure on the mucosa and change intestinal epithelial shape (6, 26). Longitudinal smooth muscle contraction may also induce mucosal deformation as small bowel segments themselves move and bend within the abdomen.
Villus contraction may also induce intestinal epithelial deformation. Spontaneous repetitive intestinal villus contraction is mediated by muscularis mucosa smooth muscle fibers oriented along the villus. Individual villi contract 0-15 times/min (36). Videomicroscopy (37) suggests a dramatic decrease in villus length. Water absorption, vagal stimulation, amino acids, and fatty acids increase contractile frequency; vagotomy and sympathetic stimulation inhibit it (8, 36, 37). Villus and epithelial deformations also occur during lactation (22) and ingestion of fiber (33) and after small bowel resection (35).
Enterocytes also change shape during normal gut function and during contact with luminal contents or opposing mucosal surfaces in vitro (25), presumably because of compression from within the lumen by the passage of a bolus of noncompressible luminal contents as well as friction or drag forces from luminal contents which move individual villi and further induce strain on the intestinal epithelial cells (23). Finally, intestinal epithelial cells actively deform during lipid absorption (11) or during restitution (5).
Although intestinal epithelial deformation in vivo is complex, we chose
to study the effects on cultured Caco-2 cells of a regular and rhythmic
deformation pattern of physiologically relevant amplitude and frequency
(28, 36) to facilitate reproducible analysis of intracellular signaling
at acute time points. We found that cyclic deformation rapidly
stimulated PKC activity and translocated PKC- and -
from a
soluble to a particulate subcellular fraction. Deformation also rapidly
stimulated intracellular tyrosine kinase activity. The tyrosine
phosphorylation of several cellular proteins rapidly increased in the
soluble fraction and tyrosine phosphoprotein content in the particulate
fraction decreased in parallel. PKC and tyrosine kinase inhibitors
blocked the effects of deformation on Caco-2 proliferation and
differentiation.
The finding that deformation initiated rapid PKC and tyrosine kinase
signals in Caco-2 cells is consistent with observations in vascular
endothelial cells (30) and myocytes (31), albeit at very different
deformation parameters. Interestingly, colonic mucosal PKC activity is
altered by dietary fat and fiber (10). Although these agents could act
directly on the colonocyte, alterations in bowel contractility and
mucosal deformation could also contribute to such PKC modulation.
Because PKC activation is often accompanied by PKC isozyme
translocation (20), we next examined the subcellular distribution of
the PKC- and -
isoforms. In static Caco-2 cells, one-third and
two-thirds of total PKC-
, a
Ca2+-dependent PKC isoform, were
located in the particulate and soluble fraction, respectively, whereas
two-thirds and one-third of total PKC-
, a
Ca2+-independent PKC isoform, were
found in the particulate and soluble fraction, respectively, consistent
with previous observations (7). However, deformation rapidly
translocated both the PKC-
and -
isoforms from the soluble to the
particulate fraction. Thus cyclic deformation may stimulate Caco-2 PKC
via both Ca2+-dependent and
-independent PKC isozymes.
Altered PKC and tyrosine kinase activity is associated with altered intestinal mucosal proliferation and differentiation in vivo (10, 19) and in cultured Caco-2 cells (2, 3). Cyclic deformation for 24 h increased both cell number and thymidine uptake in proliferating Caco-2 cells and modulated Caco-2 brush-border enzyme specific activity in confluent cells. Although intestinal brush-border enzyme activities may increase together in differentiation (18), intestinal marker enzyme activities may also be selectively increased or decreased by hormones and peptides (12, 17), PKC inhibition (3), and refeeding (15). We therefore next examined whether deformation-mediated alterations of Caco-2 proliferation and differentiation were sensitive to modulation of PKC and tyrosine kinase activity.
Indeed, deformation-stimulated increases in cell number were significantly inhibited by staurosporine and genistein, which inhibit PKC and tyrosine kinase activity, respectively. Although thymidine uptake does not necessarily mirror proliferation in all settings and must therefore be interpreted cautiously, we have previously reported that deformation increases cell number and thymidine uptake equivalently (4). The hypothesis that the PKC and tyrosine kinase signals induced by deformation might mediate the mitogenic effects of deformation may thus also be supported by observations that calphostin C (a more specific PKC inhibitor) and erbstatin (a tyrosine kinase inhibitor with different specificity) blocked deformation-mediated increases in thymidine uptake without altering control monolayer thymidine uptake.
Intestinal brush-border enzyme specific activity is a common intestinal epithelial differentiation marker (18). Deformation substantially increased Caco-2 DPDD activity and decreased AKP activity. PKC and tyrosine kinase signaling may also mediate deformation effects on Caco-2 differentiation, because PKC or tyrosine kinase blockade prevented these effects. Although 1 µmol/l calphostin C stimulated DPDD and inhibited AKP in static cells in another study (3), the 10 nmol/l calphostin C used here did not significantly affect DPDD.
The mechanism of the deformation-induced decrease in AKP activity awaits elucidation. Because enterocytes secrete intestinal AKP (1), the apparent downregulation of cellular AKP by deformation could reflect increased secretion of this protein. However, we could not demonstrate such a phenomenon by Western blot (M. D. Basson and D. H. Alpers, unpublished data).
G-06967, which inhibits PKC-, increased thymidine uptake in static
cells and potentiated deformation-induced thymidine uptake. G-06967
augmentation of static Caco-2 proliferation is consistent with
suggestions that PKC-
induction inhibits vascular smooth muscle cell
proliferation (32). PKC-
, -
, and -
are downregulated in
proliferative colonic adenomas and carcinomas compared with colonocytes
(34). Although the role of PKC-
in modulating static and
deformation-induced Caco-2 proliferation awaits further study, these
data suggest that PKC-
is unlikely to be a critical mediator of the
mitogenic effects of deformation in Caco-2 cells. G-06967 also
decreased AKP in static cells and slightly but not significantly decreased DPDD. However, the effects of deformation on brush-border enzyme activity were substantially attenuated by G-06967 treatment and
did not achieve statistical significance. Thus a PKC-
signal may be
involved in the effects of deformation on Caco-2 differentiation.
In summary, cyclic deformation rapidly stimulates PKC activity in human
intestinal Caco-2 cells, translocating both
Ca2+-dependent (PKC-) and
Ca2+-independent (PKC-
)
isoforms from the soluble to the particulate fraction. Cyclic
deformation also rapidly modulates tyrosine kinase activity and
tyrosine phosphorylation of proteins in the soluble and particulate
fractions. Certainly, chronic cyclic deformation could conceivably
modulate the expression of other PKC isoforms in a signal cascade by
regulating a specific PKC isoform, stimulate interaction between one or
more PKC isozymes and tyrosine kinases, or regulate other intracellular
signals. Deformation-associated PKC and tyrosine kinase signals may
also modulate other intestinal epithelial characteristics since, for
instance, PKC modulates transepithelial chloride and glucose transport
(14, 16). However, these results suggest that
deformation-mediated PKC and tyrosine kinase signals may
influence proliferation and differentiation of human intestinal Caco-2
cells. PKC-
, in particular, may be involved in deformation
effects on differentiation but not proliferation.
![]() |
ACKNOWLEDGEMENTS |
---|
The technical assistance of Mark Brown and Dr. Hiroyuki Kito is gratefully acknowledged.
![]() |
FOOTNOTES |
---|
This work was supported in part by funding from the Veterans Administration and by the March of Dimes (6-FY-96-0624) (both to M. D. Basson).
Address for reprint requests: M. D. Basson, Dept. of Surgery, Yale Univ. School of Medicine, 333 Cedar St., PO Box 208062, New Haven, CT 06520-8062.
Received 29 April 1997; accepted in final form 24 April 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alpers, D. H.,
A. Mahmood,
M. Engle,
F. Yamagishi,
and
K. DeSchryver-Kecskemeti.
The secretion of intestinal alkaline phosphatase (IAP) from the enterocyte.
J. Gastroenterol.
7:
63-67,
1994.
2.
Basson, M. D.,
N. J. Emenaker,
and
Z. Rashid.
Regulation of human Caco-2 intestinal epithelial cell brush border enzyme activity by modulation of tyrosine phosphorylation.
Cell Tissue Res.
292:
553-562,
1998[Medline].
3.
Basson, M. D.,
and
F. Hong.
Modulation of human Caco-2 intestinal epithelial cell phenotype by protein kinase C inhibitors.
Cell Biol. Int.
19:
1025-1032,
1995[Medline].
4.
Basson, M. D.,
G. D. Li,
F. Hong,
O. Han,
and
B. E. Sumpio.
Amplitude-dependent modulation of brush border enzymes and proliferation by cyclic deformation in human intestinal Caco-2 monolayers.
J. Cell. Physiol.
168:
476-488,
1996[Medline].
5.
Basson, M. D., I. M. Modlin, G. Turowski, and
J. A. Madri. Enterocyte-matrix interactions in the
healing of mucosal injury. Eur. J. Gastroenterol. Hepatol. 5, Suppl. 3: S21-S28,
1993.
6.
Bertuzzi, A.,
S. Salinari,
R. Mancinelli,
and
M. Pescatori.
Peristaltic transport of a solid bolus.
J. Biomech.
16:
459-464,
1983[Medline].
7.
Bissonnette, M.,
X. Y. Tien,
S. M. Niedziela,
S. C. Hartmann,
B. P. Frawley, Jr.,
H. K. Roy,
M. D. Sitrin,
R. L. Perlman,
and
T. A. Brasitus.
1,25 (OH)2 vitamin D3 activates PKC- in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i:
Am. J. Physiol.
267 (Gastrointest. Liver Physiol. 30):
G465-G475,
1994
8.
Carr, K. E.,
C. Bullock,
S. S. Ryan,
M. G. McAlinder,
and
F. C. Boyle.
Radioprotectant effects of atropine on small intestinal villous shape.
J. Submicrosc. Cytol. Pathol.
23:
569-577,
1991[Medline].
9.
Chantret, I.,
A. Barbat,
E. Dussaulx,
M. G. Brattain,
and
A. Zweibaum.
Epithelium polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines.
Cancer Res.
48:
1936-1942,
1988[Abstract].
10.
Davidson, L. A.,
J. R. Lupton,
Y. H. Jiang,
W. C. Chang,
H. M. Aukema,
and
R. S. Chapkin.
Dietary fat and fiber alter rat colonic protein kinase C isozyme expression.
J. Nutr.
125:
49-56,
1995[Medline].
11.
Friedman, H. I.,
and
R. R. Cardell, Jr.
Alterations in the endoplasmic reticulum and Golgi complex of intestinal epithelial cells during fat absorption and after termination of this process.
Anat. Rec.
188:
77-101,
1977[Medline].
12.
Galluser, M.,
R. Belkhou,
J. N. Freund,
I. Duluc,
N. Torp,
M. Danielsen,
and
F. Raul.
Adaptation of intestinal hydrolases to starvation in rats: effect of thyroid function.
J. Comp. Physiol. [B]
161:
357-361,
1991[Medline].
13.
Goodlad, R. A.,
J. A. Plumb,
and
N. A. Wright.
Epithelial cell proliferation and intestinal absorption function during starvation and refeeding in rat.
Clin. Sci. (Colch.)
74:
301-306,
1988[Medline].
14.
Hirsch, J. R.,
D. D. F. Loo,
and
E. M. Wright.
Regulation of Na+/glucose cotransporter expression by protein kinases in Xenopus laevis oocytes.
J. Biol. Chem.
271:
14740-14746,
1996
15.
Hodin, R. A.,
J. R. Graham,
S. Meng,
and
M. P. Upton.
Temporal pattern of rat small intestinal gene expression with refeeding.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G83-G89,
1994
16.
Huang, X. P.,
X. T. Fan,
J. F. Desjeux,
and
M. Castagna.
Bile acids, non-phorbol-ester-type tumor promoters, stimulate the phosphorylation of protein kinase C substrates in human platelets and colon cell line HT-29.
Int. J. Cancer
52:
444-450,
1992[Medline].
17.
Inoue, Y.,
J. P. Grant,
and
P. J. Snyder.
Effects of glutamine-supplemented total parenteral nutrition on the recovery of the small intestine after starvation atrophy.
JPEN J. Parenter. Enteral Nutr.
17:
165-170,
1993[Abstract].
18.
Jumarie, C.,
and
C. Malo.
Caco-2 cells cultured in a serum free medium as a model for the study of enterocytic differentiation in vitro.
J. Cell. Physiol.
149:
24-33,
1992.
19.
Kahl-Rainer, P.,
J. Karner-Hanusch,
W. Weiss,
and
B. Marian.
Five of six protein kinase C isozymes present in normal mucosa show reduced protein levels during tumor development in the human colon.
Carcinogenesis
15:
779-782,
1994[Abstract].
20.
Kraft, A. S.,
and
W. B. Anderson.
Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane.
Nature
301:
621-623,
1983[Medline].
21.
Lee, J. S.
Contraction of villi and fluid transport in dog jejunal mucosa in vitro.
Am. J. Physiol.
221:
488-495,
1971[Medline].
22.
Lichtenberger, L. M.,
and
J. S. Trier.
Changes in gastrin levels, food intake and duodenal mucosal growth during lactation.
Am. J. Physiol.
237 (Endocrinol. Metab. Gastrointest. Physiol. 6):
E98-E105,
1979[Medline].
23.
Madara, J. L.
Epithelial cells develop membrane wounds-and recover.
Gastroenterology
96:
1360-1361,
1989[Medline].
24.
Mayhew, T. M.
Striated brush border of intestinal absorptive epithelial cells: stereological studies on microvillous morphology in different adaptive states.
J. Electron Microsc. (Tokyo)
16:
45-55,
1990.
25.
McNeil, P. L.,
and
S. Ito.
Gastrointestinal cell plasma membrane wounding and resealing in vivo.
Gastroenterology
96:
1238-1248,
1989[Medline].
26.
Miftakhov, R.,
and
D. Wingate.
Biomechanics of small bowel motility.
Med. Eng. Phys.
16:
406-415,
1994[Medline].
27.
Moore, R.,
S. Carlson,
and
J. L. Madara.
Villus contraction aids repair of intestinal epithelium after injury.
Am. J. Physiol.
257 (Gastrointest. Liver Physiol. 20):
G274-G283,
1989
28.
Otterson, M. F.,
and
M. G. Sarr.
Normal physiology of small intestinal motility.
Surg. Clin. North Am.
73:
1173-1192,
1993[Medline].
29.
Peterson, M. D.,
and
M. S. Mooseker.
Characterization of the enterocyte-like brush border cytoskeleton of the Caco-2BBe clones of the human intestinal cell line, Caco-2.
J. Cell Sci.
102:
581-600,
1992[Abstract].
30.
Rosales, O. R.,
and
B. E. Sumpio.
Changes in cyclic deformation increase inositol triphosphate and diacylglycerol in endothelial cells.
Am. J. Physiol.
262 (Cell Physiol. 31):
C956-C962,
1992
31.
Sadoshima, J. I.,
and
S. Izumo.
Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism.
EMBO J.
12:
1681-1692,
1993[Abstract].
32.
Sasagyru, T.,
C. Kosaka,
M. Hirata,
J. Masuda,
K. Shimokado,
M. Fujishima,
and
J. Ogata.
Protein kinase C-mediated inhibition of vascular smooth muscle cell proliferation: the isoforms that may mediate G1/S inhibition.
Exp. Cell Res.
208:
311-320,
1993[Medline].
33.
Tasman-Jones, C.,
A. L. Jones,
and
R. L. Owen.
Jejunal morphological consequences of dietary fiber in rats (Abstract).
Gastroenterology
74:
1102,
1978.
34.
Wali, R. K.,
M. Bissonnnette,
S. Khare,
B. Aquino,
S Niedziela,
M. Sitrin,
and
T. A. Brasitus.
Protein kinase C isoforms in the chemopreventive effects of a novel vitamin D3 analogue in rat colonic tumorigenesis.
Gastroenterology
111:
118-126,
1996[Medline].
35.
Williamson, R. C. N.
Intestinal adaptation. Structural, functional and cytokinetic changes.
N. Engl. J. Med.
298:
1393-1402,
1978[Medline].
36.
Womack, W. A.,
J. A Barrowman,
W. H. Graham,
J. N. Benoit,
P. R. Kvietys,
and
D. N. Granger.
Quantitative assessment of villous motility.
Am. J. Physiol.
252 (Gastrointest. Liver Physiol. 15):
G250-G256,
1987
37.
Womack, W. A.,
P. K. Tygart,
D. Mailman,
P. R. Kvietys,
and
D. N. Granger.
Villous motility: relationship to lymph flow and blood flow in the dog jejunum.
Gastroenterology
94:
977-983,
1988[Medline].
38.
Yano, Y.,
J. Geibel,
and
B. E. Sumpio.
Tyrosine phosphorylation of pp125FAK and paxillin in aortic endothelial cells induced by mechanical deformation.
Am. J. Physiol.
271 (Cell Physiol. 40):
C635-C649,
1996