(Received for publication, November 14, 1995; and in revised form, February 12, 1996)
From the
In terminally differentiated ileal villus
Na-absorptive cells, epidermal growth factor (EGF)
stimulates NaCl absorption and its component brush border
Na
/H
exchanger, acting via
basolateral membrane receptors, and as we confirm here, a brush border
tyrosine kinase. In the present study we show that brush border
phosphatidylinositol 3-kinase (PI 3-kinase) is involved in EGF
stimulation of NaCl absorption and brush border
Na
/H
exchange. In rabbit ileum
studied with the Ussing chamber-voltage clamp technique, EGF
stimulation of active NaCl absorption is inhibited by the selective PI
3-kinase inhibitor wortmannin. PI 3-kinase, a largely cytosolic enzyme,
translocates specifically to the brush border of ileal absorptive cells
following EGF treatment. This translocation occurs as early as 1 min
after EGF treatment and remains increased at the brush border for at
least 15 min. EGF also causes a rapid (1 min) and large
(4-5-fold) increase in brush border PI 3-kinase activity.
Involvement of PI 3-kinase activity in intestinal Na
absorption is established further by studies done in the human
colon cancer cell line, Caco-2, stably transfected with the intestinal
brush border isoform of the Na
/H
exchanger, NHE3 (Caco-2/NHE3 cells). Brush border
Na
/H
exchange activity was measured
using the pH-sensitive fluorescent dye
2`7`-bis(carboxyethyl)5-(6)-carboxyfluorescein. EGF added to the
basolateral surface but not apical surface of Caco-2/NHE3 cells
increased brush border Na
/H
exchange
activity. The EGF-induced increase in brush border
Na
/H
exchange activity was completely
abolished in cells pretreated with wortmannin. EGF treatment caused
increased tyrosine phosphorylation of PI 3-kinase in both ileal brush
border membranes and Caco-2/NHE3 cells, suggesting that a tyrosine
kinase upstream of the PI 3-kinase is involved in the EGF effects on
Na
absorption. In conclusion, the present study
provides evidence in two separate intestinal models, the ileum and a
human colon cancer cell line, that PI 3-kinase is an intermediate in
EGF stimulation of intestinal Na
absorption.
We reported previously that in terminally differentiated rabbit
ileal villus cells, epidermal growth factor (EGF) ()increases NaCl absorption acting via basolateral membrane
(BLM) receptors(1) . Biochemically very little is known about
the steps involved between binding of EGF to its receptor at the BLM
and stimulation of NaCl absorption, other than the EGF effects involve
tyrosine kinases at both the brush border membranes (BBMs) and
BLMs(1) . In addition, EGF increased tyrosine phosphorylation
of an ileal brush border protein of 85 kDa, (
)and the
tyrosine kinase inhibitor genistein inhibited tyrosine phosphorylation
of several ileal brush border proteins, including one of 85
kDa(1) .
Phosphatidylinositol 3-kinase (PI 3-kinase) consists of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit that is tyrosine phosphorylated in vivo(2, 3, 4, 5) . Since the enzyme forms a complex with growth factor receptors and with oncogene products, it has been implicated in the growth signaling mechanism of receptors and oncogenes that act through tyrosine kinases. In addition, PI 3-kinase is emerging as a critical enzyme in intracellular signaling pathways in nonmitogenic stimuli(6, 7, 8) . However, in nonmitogenic cells the nature and site of interaction of PI 3-kinase with other signaling molecules have not been established.
The majority of PI 3-kinase activity is found in the soluble fraction of resting cells, although a significant fraction is associated with the membrane in growth factor-stimulated and/or oncogene-transformed fibroblasts (9, 10) and following the addition of agonists such as thrombin(7) . Recruitment of PI 3-kinase from the cytosol to the plasma membrane, where its substrate phosphatidylinositides are enriched, is part of PI 3-kinase-mediated signaling in stimulated cells.
Activation of some
plasma membrane transport proteins including GLUT-4, water channels,
and H pump involves exocytic movement of the
transporters(11, 12, 13, 14) . PI
3-kinase activation is involved in insulin-stimulated GLUT-4
translocation from an intracellular compartment to the plasma membrane
in skeletal muscle cells(15, 16) and
insulin-stimulated glucose uptake in 3T3-L1 adipocytes(17) .
Whether PI 3-kinase is similarly involved in regulation of transport
processes in epithelial cells is unknown.
Ileal
Na-absorbing cells are polarized epithelial cells in
the villus and upper crypt. Their cell surface is divided into two
domains, the luminally facing apical domain which is separated by tight
junctions from the basolateral domain. We have shown that the signal
transduction processes stimulated by BLM receptors in these cells are
distinct in these two domains(18, 19) . We now
demonstrate a role for PI 3-kinase in regulation of Na
absorption in a polarized epithelial cell. We show that EGF acts
on ileal BLM receptors to increase selectively the amount and activity
of apical membrane PI 3-kinase and that PI 3-kinase is involved in EGF
stimulation of apical membrane Na
/H
exchange.
Usually six pieces of ileum from a single animal were studied
simultaneously. Unless specified, the bathing solution consisted of
Ringer's HCO composed (in mM) of: 115 NaCl,
25 NaHCO
, 2.4 K
HPO
, 0.4
KH
PO
, 1.2 CaCl
, 1.2
MgCl
; the pH was 7.4 after gassing with 95% O
,
5% CO
. Ten mM glucose was added to the serosal and
10 mM mannitol to the mucosal bathing fluids at the time of
mounting the tissue. Effects of EGF (200 ng/ml) added to the serosal
surface were determined over a 15-min flux period in otherwise
untreated ileum, starting 15 min after EGF addition, and in tissue
preincubated with wortmannin (100 nM) added to the ileal
mucosal plus serosal surfaces.
The
glucose transport buffer contained (in mM): 80 mannitol, 40
MOPSO, 11.4 Tris, pH 6.5, 2 EGTA, 5 Mg(gluconate), 60 NaCl,
0.1 D-glucose ([
H]glucose, 20
µCi/ml, DuPont NEN).
Na
initial rates
of uptake and [
H]glucose uptake were measured by
mixing 15 µl of membranes with 30 µl of transport buffer at 25
°C, as described (23, 24) . For
Na
uptake, the reaction was stopped
electronically after 3, 5, or 8 s by injection of 1 ml of ice-cold stop
solution. [
H]Glucose uptake was stopped after 90
s, a time not during the linear phase of uptake. Glucose equilibrium
values were determined after a 90-min incubation at 25 °C. Reaction
mixture was then vacuum filtered through nitrocellulose filters,
0.45-µM pore size (Millipore, Bedford, MA) and rinsed with
6 ml of stop solution. Stop solutions contained (in mM) either
40 mannitol, 90 potassium gluconate, 15.6 MES, 20 Tris, pH 8.0; or 40
mannitol, 90 potassium gluconate, 20 MOPSO, 5.6 Tris, pH 6.5. The
filters were dissolved in 4 ml of scintillant, and radioactivity was
determined in a liquid scintillation spectrometer (Beckman L5 7500).
Initial uptake rates of Na
/H
exchange
were expressed in pmol/mg of protein/s using linear regression
analyses; glucose uptake was expressed as pmol/mg of protein.
Figure 1:
EGF stimulates ileal villus active NaCl
absorption. Ileal mucosa was exposed under voltage-clamped conditions
to 200 ng/ml EGF on the serosal surface and the effect on
mucosal-to-serosal (J) and serosal-to-mucosal (J
) fluxes of
Na
and
Cl
(net fluxes represent J
- J
) determined.
Data shown represent the effect of EGF (cross-hatched bars) on
ileal Na
and Cl
transport
15-30 min after EGF addition compared with same parameters during
two 20-min control (black bars) flux periods in the same
tissue before the addition of EGF. Results are means ± S.E.; n = 7, n = number of animals studied.
I
and fluxes are expressed in µEq/cm
-h,
and conductance (G) is expressed in
millisiemens/cm
. p values represent a comparison
of EGF and control periods in the same tissue (paired t test). NS, not significant.
The PI 3-kinase inhibitor wortmannin added to the ileal mucosal and
serosal surfaces (100 nM) did not alter basal active
electrolyte transport (Fig. 2). Also, the D-glucose-stimulated increase in Na absorption was not altered by wortmannin (3.0 ± 0.5 versus 2.9 ± 0.5 µEq/cm
-h in control
and wortmannin treated tissue, respectively, n = 7; not
significant).
Figure 2:
Wortmannin does not change basal ileal
active Na and Cl
transport. Ileal
mucosa was exposed under voltage-clamped conditions to 100 nM wortmannin on the mucosal and serosal surfaces and the effect
determined over two 20-min flux periods. Data shown represent
I
, fluxes, and conductance in wortmannin (cross-hatched bars) and solvent control (black bars;
0.02% Me
SO) exposed tissue from the same animals studied
simultaneously. Abbreviations, units, and n are the same as in Fig. 1. p values compare wortmannin effect with solvent
time control, and are all not significant (paired t test).
The effects of wortmannin on the transport effects
caused by serosal EGF were determined (Fig. 3). The addition of
wortmannin prevented the EGF effect to increase the mucosal-to-serosal
and net Na and Cl
fluxes. Wortmannin
did not alter the magnitude of the increase in serosal-to-mucosal
Cl
flux or the short lived increase in I
seen with EGF (0.3 ± 0.1 versus 0.3 ± 0.05
µEq/cm
-h in the absence versus in the presence
of wortmannin, respectively, n = 7, ns), suggesting
that wortmannin only affects the EGF-stimulated NaCl-absorptive
process.
Figure 3:
Increase in ileal NaCl absorption by EGF
is prevented by pretreatment with wortmannin. Ileal mucosa was exposed
under voltage-clamped conditions to 200 ng/ml EGF on the serosal
surface and the effect determined over a 15-min flux period with
determination of mucosal-to-serosal and serosal-to-mucosal fluxes of Na
and
Cl
.
Studies were performed in the absence of wortmannin (0.02%
Me
SO solvent control) and in tissue pretreated for 40 min
with wortmannin (100 nM) on the mucosal and serosal surfaces.
Data shown represent effect of EGF on ileal Na
and
Cl
transport in the absence (black bars) and
presence (cross-hatched bars) of wortmannin and represent
changes in I
, fluxes, and conductance 15-30 min
after EGF addition compared with same parameters during two 20-min flux
periods in same tissue before the addition of EGF. Results are means
± S.E.; n = 7. p values represent
magnitude of EGF effects in presence versus absence of
wortmannin (paired t test). Abbreviations are the same as in Fig. 1.
Brush border vesicles made
from ileum exposed in vitro to EGF exhibited increased
Na/H
exchange compared with control
vesicles (Fig. 4). This was exerted only on Na
uptake in the presence of an acid inside pH gradient and not in
the absence of a pH gradient. Genistein (100 µM) freeze
thawed into control vesicles inhibited basal
Na
/H
exchange, again via an effect
only on Na
uptake in the presence of an acid inside pH
gradient. When genistein was freeze thawed into brush border vesicles
made from ileum exposed in vitro to EGF,
Na
/H
exchange was reduced even more
than the decrease in control vesicles caused by genistein, to a rate
not significantly different from control/genistein vesicles. In
contrast, genistin did not alter basal Na
/H
exchange or the EGF-stimulated brush border
Na
/H
exchange (data not shown).
Figure 4:
EGF and genistein effects on ileal brush
border Na/H
exchange. Sheets of ileal
mucosa were exposed to EGF (200 ng/ml) or control for 15 min. Ileal
villus cells were then prepared by scraping and BBM vesicles prepared
by Mg precipitation under conditions such that intravesicular ATP was
approximately 50 µM(34) . Vesicles were snap
frozen in membrane buffer with and without genistein (100
µM) and studied the next day. Na
uptake
was performed over a period of linear uptake (3, 5, and 8 s) with
Na
/H
exchange determined as
Na
uptake with an intravesicular acid pH gradient
(pH
8.0/pH
6.5) minus Na
uptake with no pH gradient (pH
6.5/pH
6.5). Results from a representative experiment are shown with
similar data having been obtained in five identical experiments. Slopes
of this single experiment are in parentheses. EGF increased
brush border Na
/H
exchange by
approximately 25% (control Na
/H
exchange 18.4 ± 4.3 pmol/mg protein-s, n =
5 versus EGF Na
/H
exchange
22.6 ± 5.1, n = 5, p < 0.02).
Genistein caused an approximately 20% decrease in control
Na
/H
exchange (14.4 ± 3.9
pmol/mg protein-s, n = 5, p < 0.02 compared
with control); with Na
/H
exchange
rate in EGF + genistein (15.9 ± 4.9 pmol/mg protein-s, n = 5) and control + genistein not being
significantly different.
In
contrast to these results, Na-dependent D-glucose uptake was not affected by EGF treatment or by
genistein (Table 1). Thus, brush border vesicles made from
EGF-exposed ileum maintain a memory of in vitro exposure to
EGF with a prolonged stimulation of Na
/H
exchange. Also, a brush border tyrosine kinase(s) is involved in
control of brush border basal Na
/H
exchange and the EGF-induced increase in brush border
Na
/H
exchange.
BBM and BLM (50 µg) protein were separated by 7% SDS-PAGE, and Western analysis done with monoclonal antibodies to the 85-kDa subunit of PI 3-kinase. Under basal conditions both BBM and BLM contain similar amounts of PI 3-kinase (Fig. 5A). One min of EGF treatment caused a 2-fold increase (234 ± 40%, n = 3, p < 0.01) in the amount of BBM PI 3-kinase, whereas a 15-min exposure showed a 40 ± 14% (n = 3, p < 0.05) increase (Fig. 5B). No change was seen in the amounts of PI 3-kinase in the BLM 30 s after EGF treatment (data not shown). Thus EGF specifically causes an increase in amount of PI 3-kinase in the BBM of rabbit ileal villus cells.
Figure 5: Effect of EGF on the amount of ileal BBM and BLM PI 3-kinase. Panel A, PI 3-kinase is a BBM and BLM protein under basal conditions. Fifty µg of BBM and BLM proteins were separated by SDS-PAGE (7%) transferred to nitrocellulose filters, and probed with monoclonal antibodies to PI 3-kinase. The arrow indicates the p85 subunit of PI 3-kinase. This experiment is representative of three with similar results. Panel B, EGF increases the BBM PI 3-kinase amount. Intact rabbit ileum was exposed in vitro to EGF (200 ng/ml) for 1 or 15 min and BBMs prepared from villus cells. Fifty µg of BBM proteins were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with monoclonal antibodies to PI 3-kinase. This experiment is representative of three with similar results.
Studies were done to determine whether the BBM PI 3-kinase associated with the cytoskeleton or plasma membrane under both basal and EGF-treated conditions and whether EGF treatment caused a rearrangement of the BBM PI 3-kinase from one compartment to the other. 150 µg of BBM protein was lysed using a buffer containing 1% Triton X-100. The detergent-soluble and -insoluble fractions were separated on 7% SDS-PAGE, transferred to nitrocellulose, and probed with monoclonal antibody to PI 3-kinase. The total BBM PI 3-kinase activity from control and EGF-exposed tissue was present in the detergent-soluble fraction (Fig. 6), suggesting that PI 3-kinase is not associated with the villus cell cytoskeleton as defined as the Triton X-100-insoluble fraction. No PI 3-kinase was detected in the detergent-insoluble fraction either in BBM prepared from control or EGF-exposed ileum.
Figure 6: PI 3-kinase is present only in the ileal BBM Triton X-100-soluble fraction. BBM from control and EGF-treated tissue were solubilized in a buffer containing Triton X-100 (as described for immunoprecipitation of PI 3-kinase under ``Methods''), and the detergent-soluble and -insoluble fractions were probed by Western analysis for PI 3-kinase. This experiment is representative of three with similar results.
To determine if BBM PI 3-kinase is tyrosine phosphorylated in response to EGF, we separately immunoprecipitated PI 3-kinase and tyrosine-phosphorylated proteins from BBM from control and EGF-treated ileum (1 min). The immunoprecipitated proteins were separated by SDS-PAGE, and Western analysis done with an anti-phosphotyrosine antibody. There was approximately a 5-fold increase in the tyrosine phosphorylation of PI 3-kinase in EGF-treated tissue compared with simultaneously studied control tissue (Fig. 7). When immunoprecipitated with anti-phosphotyrosine antibody, increased tyrosine phosphorylation of an 85-kDa protein was seen. These results show that the p85 subunit of brush border PI 3-kinase has increased tyrosine phosphorylation in response to EGF (Fig. 7).
Figure 7: EGF increases tyrosine phosphorylation of ileal BBM PI 3-kinase. 250 µg of BBM from control and EGF-exposed ileum were solubilized as described under ``Methods,'' and immunoprecipitations (Ipt) were done with polyclonal antibodies to either PI 3-kinase (PI 3-K) or phosphotyrosine (P-Tyr). The immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with anti-phosphotyrosine antibodies. This experiment is representative of three with similar results. First lane from left, BBM from control ileum immunoprecipitated with anti-phosphotyrosine polyclonal antibody; second lane, BBM from ileum exposed to EGF (200 ng/ml, 1 min) immunoprecipitated with anti-phosphotyrosine antibody; third lane, BBM from control ileum immunoprecipitated with anti-PI 3-kinase polyclonal antibody; fourth lane, BBM from ileum exposed to EGF (200 ng/ml, 1 min) immunoprecipitated with anti-PI 3-kinase antibody. The arrow on the right indicates the position of p85 subunit of PI 3-kinase.
We next measured the PI 3-kinase activity in both BBM and BLM to determine whether exposure to EGF activated this enzyme. A 4-5-fold increase was seen in the PI 3-kinase activity as early as 1 min in BBM prepared from EGF-treated tissue (Fig. 8A), and the activity remained elevated (2-fold) 15 min after the addition of EGF. No change was seen in the BLM PI 3-kinase activity 30 s following EGF treatment (Fig. 8B).
Figure 8:
EGF increases the activity of ileal BBM
but not BLM PI 3-kinase. BMB or BLM (250 µg) was lysed as described
under ``Methods,'' and PI 3-kinase was immunoprecipitated
using a polyclonal antibody. The immunoprecipitates were washed and
lipid kinase assay performed on immunoprecipitated PI 3-kinase in
buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM EGTA, 5 mM MgCl, 30
µCi of [
-
P]ATP (20 µM),
and phosphatidylinositol. The lipid products were extracted and
analyzed by thin layer chromatography. Unlabeled phosphatidylinositol
4-phosphate used as a standard was run in parallel and visualized by
exposure to iodine vapors. The arrow in the figure shows the
position of the phosphatidylinositol 4-phosphate, which separates at
the same position as phosphatidylinositol 3-phosphate (PI 3-P)
generated by the kinase reaction. Panel A, EGF increases BBM
PI 3-kinase activity at 1 and 15 min. BBM was prepared from control and
EGF-treated tissue (1 and 15 min). Panel B, EGF does not
increase BLM PI 3-kinase activity (30 s). BLM was prepared from
isolated villus cells exposed to control or EGF (30 s). These
experiments are representative of four with similar
results.
The EGF-stimulated increase in PI 3-kinase activity was inhibited in the presence of 100 nM wortmannin (Fig. 9). These results show that stimulation of intestinal absorbing cells with EGF increases the amount, activity, and tyrosine phosphorylation of BBM PI 3-kinase, whereas the BLM PI 3-kinase amount and activity remain unchanged.
Figure 9: Wortmannin inhibits the EGF-induced increase in ileal BBM PI 3-kinase activity. Intact rabbit ileum was exposed in vitro to EGF (200 ng/ml) for 1 min in the absence or presence of wortmannin (100 nM, 30-min preincubation) and BBMs prepared from villus cells. PI 3-kinase was immunoprecipitated from the BBM, and the lipid kinase assay was performed on the immunoprecipitates as described in the legend of Fig. 8. These experiments are representative of three with similar results.
Figure 10:
EGF stimulates brush border
Na/H
exchange activity in Caco-2/NHE3
cells; an effect prevented by wortmannin. Stably transfected
Caco-2/NHE3 cells were acid selected as described under
``Methods.'' Cells were serum deprived overnight. Cells
loaded with BCECF were allowed to complete a
Na
-dependent recovery of pH
from
an NH
Cl-induced acid load, with Na
added
only to the apical surface. The trace starts when a steady-state
pH
in the presence of apical Na
medium has been achieved. Panel A, effect of basolateral
administration of EGF (100 ng/ml) on Na
/H
exchange activity. EGF in TMA
medium was added
to the basolateral chamber of the cuvette after cells reached
steady-state pH
. The EGF-induced alkalization
represents stimulation of brush border Na
/H
exchange. Panel B, wortmannin inhibits the EGF-induced
increase in Na
/H
exchange activity.
Cells were pretreated with wortmannin (100 nM, 30 min) during
the dye loading period. The cells were perfused with Na
medium from the apical side until a steady-state pH
was reached, and then EGF was added as described above.
These traces are representative of three separate
experiments.
We measured the
Na/H
exchange in transfected
Caco-2/NHE3 cells pretreated with wortmannin for 30 min at room
temperature. Wortmannin (100 nM) was added to both the apical
and basolateral surfaces. Wortmannin by itself did not alter the
Na
/H
exchange activity (data not
shown). Subsequent addition of EGF to these cells demonstrated that the
stimulation of the Na
/H
exchange by
EGF (Fig. 10A) was completely inhibited when the cells
were pretreated with wortmannin (Fig. 10B).
Figure 11: EGF stimulates PI 3-kinase activity in Caco-2/NHE3 cells. Untransfected Caco-2 and Caco-2/NHE3 cells were serum starved overnight. Cells were stimulated with EGF (100 ng/ml, 1 min) and lysed in RIPA buffer as described under ``Methods.'' PI 3-kinase activity was measured as described in Fig. 8, using lysates of Caco-2/NHE3 (panel A) or Caco-2 (panel B) cells immunoprecipitated with anti-phosphotyrosine antibody. This experiment is representative of four with similar results.
In terminally differentiated (nondividing) intestinal
epithelial cells, EGF does not act as a mitogen but, as shown
previously (1) and confirmed here, stimulates active NaCl
absorption. Ileal NaCl absorption is stimulated by serosal but not
mucosal EGF(1) . This supports other evidence that EGF
receptors are present on the basolateral but not apical membranes of
adult intestinal epithelial cells. Thompson and others have shown by
immunofluorescence that EGF binds to the BLM and not BBM of rat small
intestinal epithelial cells (27, 28, 29) .
Similar separation of intestinal cell EGF receptors to one cell pole
and the regulated function involving a brush border
Na/H
exchange to the other, is seen
in Caco-2 cells, which are stably transfected with the brush border
isoform Na
/H
exchanger NHE3
(Caco-2/NHE3 cells). EGF stimulated brush border
Na
/H
exchange in Caco-2/NHE3 cells,
when added to the basolateral but not apical surface. A similar
observation has been reported by Bishop and Wen (30) . They
found that levels of basolateral EGF receptors in polarized Caco-2
cells were 15-fold greater than apical receptors and that no
bioactivity was detected with apical EGF treatment. This suggests that
the polarized Caco-2/NHE3 cells exhibit a distribution of EGF receptors
similar to that of the ileum and link this basolateral EGF receptor to
stimulation of brush border Na
/H
exchange.
We provide several types of evidence linking brush
border PI 3-kinase to EGF stimulation of NaCl absorption and brush
border Na/H
exchange. (i) Studied
with the Ussing chamber-voltage clamp technique, wortmannin, a
selective, potent, and irreversible inhibitor of PI
3-kinase(31) , inhibited the increase in neutral NaCl
absorption induced by EGF (Fig. 3) and the EGF stimulation of
brush border Na
/H
exchanger in
Caco-2/NHE3 cells (Fig. 10). (ii) EGF stimulation of PI 3-kinase
activity and amount occurred only at the BBM. (iii) EGF stimulation of
PI 3-kinase amount and activity in the BBM was a very rapid event
occurring as quickly (1 min) as we could isolate brush borders. The
wortmannin effect was not nonspecific or toxic since it did not alter
ileal basal Na
and Cl
fluxes, D-glucose stimulated increase in ileal Na
absorption, or EGF-stimulated ileal Cl
secretion. It has been demonstrated previously that at
concentrations up to 100 nM wortmannin does not directly
inhibit any other enzyme activity so far investigated including insulin
receptor tyrosine kinase or phosphatidylinositol 4-kinase(32) ,
myosin light chain kinase, protein kinase A, protein kinase C, cyclic
GMP-dependent protein kinase(33) , the components of the
mitogen-activated protein kinase cascade(34, 17) , or
S6 kinase(32) .
Our studies have demonstrated an asymmetric plasma membrane activation of PI 3-kinase as part of EGF-induced signal transduction. Small but equal amounts of PI 3-kinase were present in both BBMs and BLMs under basal conditions. EGF treatment, however, caused a rapid increase in the amount and activity of PI 3-kinase specifically to the BBM and not BLM. The increase in PI 3-kinase activity was rapid, occurring by 1 min; was higher at 1 min than at 15 min; and correlated with a greater amount of BBM PI 3-kinase at 1 min than at 15 min of EGF treatment. PI 3-kinase is a cytosolic enzyme and is known to translocate following its activation by an agonist to the plasma membrane where its substrates are present(7) . There was no change in the BLM PI 3-kinase activity following EGF treatment. This suggests that the transport effects seen with EGF do not involve the association of PI 3-kinase with the basolateral EGF receptor. This is consistent with a previous demonstration that the EGF receptor lacks the PI 3-kinase-binding tyrosine phosphorylated Tyr-X-X-Met motif and binds to p85 very weakly (35) or not at all(36) .
EGF treatment for 1 min also increased the PI
3-kinase activity 2 fold in Caco-2/NHE3 (Fig. 11A).
However, the increase in PI 3-kinase activity in Caco-2/NHE3 cells
following EGF treatment could only be detected in anti-phosphotyrosine
immunoprecipitates, suggesting activation of a subpopulation of PI
3-kinase, which was activated by tyrosine phosphorylation. Of note, EGF
also increased the tyrosine phosphorylation of ileal brush border PI
3-kinase, suggesting that tyrosine phosphorylation activates BBM PI
3-kinase, which is involved in the regulation of Na absorption. In fact, the semiquantitative aspects of PI 3-kinase
activation shown in this study are consistent with tyrosine
phosphorylation being primarily responsible for brush border PI
3-kinase activation. At 1 min, EGF increases the amount of brush border
PI 3-kinase by approximately 2-fold; activity is increased
approximately 4-5 fold; and tyrosine phosphorylation
approximately 5-fold. Thus the similarity in the EGF stimulation of
tyrosine phosphorylation and the activity of brush border PI 3-kinase
is consistent with the increase in activity, likely via tyrosine
phosphorylation, being the major mechanism of PI 3-kinase activation,
although translocation also contributes. Whether tyrosine
phosphorylation of PI 3-kinase occurs at the brush border or in the
cytosol and whether only tyrosine-phosphorylated PI 3-kinase moves to
the brush border are unknown.
Of note is that EGF treatment did not alter the PI 3-kinase activity in untransfected Caco-2 cells (Fig. 11B). The significance of this observation is not known, although the results would seem to indicate a role for NHE3 in the increase in tyrosine-phosphorylated and activated PI 3-kinase present in the apical membrane.
We reported previously suggestive
evidence that a brush border tyrosine kinase was involved in the EGF
effects on Na and Cl
transport
transduced from BLM receptors(1) . Genistein added separately
to the ileal mucosal or serosal surfaces inhibited EGF stimulation of
NaCl absorption. In contrast, carbachol effects on NaCl absorption,
acting by a BLM M
receptor, were inhibited by mucosal but
not serosal genistein. Thus the sidedness of genistein effects on this
preparation can separate apical from BLM tyrosine kinase involvement.
In this study we provide more direct evidence that a brush border
tyrosine kinase is involved in the regulation of basal brush border
Na
/H
exchange and in the increase in
brush border Na
/H
exchange caused by
EGF. The fact that brush borders of ileal villus cells made from
EGF-exposed ileum maintain an increase in
Na
/H
exchange indicates that
basolateral EGF acts via a prolonged biochemical mechanism at the
apical membrane. That genistein but not the negative control, genistin,
inhibits both the basal brush border Na
/H
exchange (1) as well as the increase in
Na
/H
exchange caused by EGF indicates
the involvement of a brush border tyrosine kinase in both. Whether the
same tyrosine kinase is involved in the regulation of basal and the
EGF-induced increase in brush border Na
/H
exchange is not known, although the decrease of both processes to
the same level of Na
/H
exchange
suggests an overlapping mechanism. Since these studies suggest that
EGF-induced tyrosine phosphorylation of PI 3-kinase is what activates
it to regulate brush border Na
absorption it is
likely, but not proven, that the tyrosine kinase that mediates the
EGF-induced increase in brush border Na
/H
exchange is the same as that which activates PI 3-kinase. The
identity of the tyrosine kinase that activates brush border PI 3-kinase
is unknown.
Our data suggest that the ileal BBM PI 3-kinase may be
restricted to the membrane and not associated with the cytoskeleton,
although this interpretation is entirely based on definition of
cytoskeleton as the Triton X-100-insoluble fraction of brush border.
Ileal BBM was solubilized in a buffer containing 1% Triton X-100, and
the Triton-soluble and -insoluble fractions were probed for PI
3-kinase. The BBM PI 3-kinase was found only in the Triton-soluble
fraction under both control and EGF stimulated conditions. No PI
3-kinase was detected in the detergent-insoluble fraction in either
control or EGF-stimulated conditions. This suggests that PI 3-kinase
may not associate with the apical membrane cytoskeleton in ileal
absorbing cells, as defined as the Triton X-100-insoluble fraction. The
lack of any PI 3-kinase activity associated with ileal BBM cytoskeleton
also suggests that cytoskeletal rearrangement may not be the mechanism
by which PI 3-kinase regulates the Na/H
exchanger, which is also present in the detergent-soluble
fraction of the BBM. This is relevant, since activated PI 3-kinase
associates with cytoskeleton (Triton X-100 insoluble fraction) in
multiple other cells. For instance, PI 3-kinase associates with
membrane cytoskeleton in thrombin-exposed platelets(7) . In
stimulated neutrophils, there is a strong correlation between the
production of phosphatidylinositol 3,4-diphosphate and
phosphatidylinositol 3,4,5-trisphosphate and reorganization of actin
filaments, suggesting that the D-3 phosphorylated polyphosphoinositides
are involved in cytoskeletal rearrangement(37) . However, it
may be pointed out that none of these studies has actually demonstrated
a physical association of PI 3-kinase and a cytoskeletal component.
Regulation of a Na/H
exchanger by
PI 3-kinase in nonpolarized Chinese hamster ovary fibroblasts and mouse
mammary gland cells was described in a recent study by Ma et al.(38) . The PI 3-kinase pool involved in regulation was
linked to the platelet-derived growth factor receptor. Mutations in the
platelet-derived growth factor receptor COOH-terminal sequence involved
in PI 3-kinase binding and inhibition of PI 3-kinase (using the
inhibitor LY 294002) prevented platelet-derived growth factor
stimulation of the Na
/H
exchanger.
Not clarified in this study was the number or identity of steps between
PI 3-kinase stimulation and the regulation of the
Na
/H
exchange activity. Furthermore,
the isoform of the Na
/H
exchanger
involved was not specified in this study, although it likely was NHE1,
the housekeeper isoform that is involved in the regulation of
intracellular pH and volume. The involvement of PI 3-kinase in
regulation of NHE3 in the brush border and in the regulation of
Na
absorption shown in the study represents a
different mechanism, not even being in the same plasma membrane domain
as the receptor that initiates signal transduction. Thus PI 3-kinase
regulates multiple NHE isoforms and can do so at steps removed from the
involved plasma membrane receptor.
Our study demonstrates that PI
3-kinase regulates Na absorption in both the ileal
villus absorbing cells and the colon cancer cell line Caco-2/NHE3. It
remains to be determined whether the Na
/H
exchanger is regulated directly by PI 3-kinase or through
intermediate signaling molecules. Na
/H
exchangers have been shown to contain proline-rich
sequences(39) , which could allow a direct association of PI
3-kinase through its SH3 group to the exchanger. It is possible that
the D-3 polyphosphoinositides themselves act as second messengers, such
that these lipids can be the signals rather than precursors of signals.
Since phospholipase C-
and PI 3-kinase both use the same
substrate, it is possible that, like one of the products of PLC-
activation (inositol trisphosphate), the products of PI 3-kinase are
themselves second messengers. However, another possibility is that PI
3-kinase activation may be an intermediate step in a cascade of
signaling events that begins at the BLM with EGF binding to its
receptor and leads to the activation of the brush border
Na
/H
exchanger several steps
downstream from PI 3-kinase activation.