Involvement of Integrins in Osmosensing and Signaling toward Autophagic Proteolysis in Rat Liver*
Stephan vom Dahl
,
Freimut Schliess,
Regina Reissmann,
Boris Görg,
Oliver Weiergräber,
Mariana Kocalkova
,
Frank Dombrowski
and
Dieter Häussinger
From the
Division of Gastroenterology, Hepatology and Infectious Diseases,
Heinrich-Heine-University, Moorenstrasse 5, D-40225-Düsseldorf, Germany
and the
Institute of Pathology,
Otto-von-Guericke-University, D-40225 Magdeburg, Germany
Received for publication, October 18, 2002
, and in revised form, March 26, 2003.
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ABSTRACT
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Inhibition of autophagic proteolysis by hypoosmotic or amino acid-induced
hepatocyte swelling requires osmosignaling toward p38MAPK; however,
the upstream osmosensing and signaling events are unknown. These were studied
in the intact perfused rat liver with a preserved in situ environment
of hepatocytes. It was found that hypoosmotic hepatocyte swelling led to an
activation of Src (but not FAK), Erks, and p38MAPK, which was
prevented by the integrin inhibitory hexapeptide GRGDSP, but not
its inactive analogue GRGESP. Src inhibition by PP-2 prevented
hypoosmotic MAP kinase activation, indicating that the integrin/Src system is
located upstream in the osmosignaling toward p38MAPK and Erks.
Inhibition of the integrin/Src system by the RGD motif-containing peptide or
PP-2 also prevented the inhibition of proteolysis and the decrease in
autophagic vacuole volume, which is otherwise observed in response to
hypoosmotic or glutamine/glycine-induced hepatocyte swelling. These
inhibitors, however, did not affect swelling-independent proteolysis
inhibition by phenylalanine. In line with a role of p38MAPK in
triggering the volume regulatory decrease (RVD), PP-2 and the RGD peptide
blunted RVD in response to hypoosmotic cell swelling. The data identify
integrins and Src as upstream events in the osmosignaling toward MAP kinases,
proteolysis, and RVD. They further point to a role of integrins as osmo- and
mechanosensors in the intact liver, which may provide a link between cell
volume and cell function.
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INTRODUCTION
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Changes in cell hydration within a narrow, physiological range markedly
affect carbohydrate and protein metabolism, hepatic bile flow, gene
expression, and cellular susceptibility to environmental stress
(16).
The regulation of cell function by cell hydration requires structures, which
sense hydration changes (osmosensing) and intracellular signaling pathways
toward effector sites (osmosignaling).
Progress has been made in identifying signal transduction pathways linking
cell volume changes to alterations in cell function
(7). For example, the
MAPK,1
p38MAPK, activation is critical for creating volume-regulatory ion
fluxes in response to hypoosmotic swelling in perfused rat liver
(8) and HTC liver cells
(9). Likewise, proteolysis
inhibition by cell swelling strongly depends on activation of the
p38MAPK in perfused rat liver
(10). Specific inhibition of
the p38MAPK abolishes the antiproteolytic effects exerted by
hypoosmolarity and glutamine, but is without effect on cell swelling under
these conditions (10). Another
component involved in swelling-dependent proteolysis inhibition is the
microtubular system. Colchicine blocks hypoosmotic proteolysis inhibition, but
not p38MAPK activation upon hypoosmolarity
(11). These data show that the
microtubule-dependent element in hydration-dependent proteolysis signaling is
obviously localized downstream of p38MAPK activation. In contrast
to agonists that cause changes of cell hydration, the antiproteolytic action
of non-swelling amino acids, e.g. phenylalanine, resides on the
activation of other signaling events, such as activation of mammalian target
of rapamycin (mTOR) and p70 ribosomal S6 protein kinase (p70S6K
kinase)
(1214),
which can clearly be differentiated from the swelling-related antiproteolytic
signaling cascade (15).
Whereas in bacteria, plants, and fungi two-component histidine kinases were
identified to be involved in sensing of and subsequent adaptation to adverse
osmotic conditions (16), the
mechanisms of osmosensing in mammalian cells are far from being understood.
Integrins are candidates to be involved in mechanotransduction, i.e.
the conversion of a mechanical stimulus into covalent modifications of
signaling components. Integrins are heterodimers with each subunit having a
single transmembrane domain. They establish cell adhesion to the extracellular
matrix and bind inside the cell to cytoplasmic proteins, which in turn
interact with different signal transduction components and the cytoskeleton
(for reviews see Refs. 17 and
18). In normal liver, the most
important integrins are
1
1,
5
1, and
9
1
(1921).
The present study investigates the role of integrins and Src in hypoosmotic
signaling toward proteolysis inhibition and cell volume regulation in the
isolated perfused rat liver, which most authentically represents the
three-dimensional hepatocyte anchoring to the extracellular matrix, preserved
cell polarity, intact cytoskeleton, and structural/functional cell-cell
interactions. Using the integrin antagonistic peptide GRGDSP and the Src
kinase inhibitor PP-2, an integrin-dependent activation of Src kinases was
localized upstream of swelling-induced p38MAPK signaling toward
inhibition of autophagy. In contrast, the antiproteolytic effect of
phenylalanine, which does not involve cell swelling and p38MAPK
does not depend on GRGDSP-sensitive integrin action and Src activation.
Consistent with inhibition of osmosignaling toward p38MAPK, GRGDSP
and PP2 effectively antagonize the volume regulatory response triggered by
hypoosmotic swelling. The findings suggest a role of integrins in hepatic
osmosensing and transforming hepatocyte swelling into a physiological
response.
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EXPERIMENTAL PROCEDURES
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Liver PerfusionLivers from male Wistar rats (160230
g), fed a standard diet, were perfused in an open non-recirculating manner at
a flow rate of 3.54.5 ml/min/g. The perfusion medium was
bicarbonate-buffered Krebs-Henseleit saline plus lactate (2.1 mmol/liter) and
pyruvate (0.3 mmol/liter), incubated with O2/CO2 (19:1,
v/v), at a temperature of 37 °C. For changing the osmolarity, the NaCl
concentration (115 mmol/liter) was varied, resulting in corresponding changes
of osmolarity. Additions were made either by the use of micropumps (20
µl/min) or by dissolution in Krebs-Henseleit buffer. Inhibitors were
dissolved in Me2SO. Viability of the livers was assessed by
monitoring effluent oxygen concentration and measurement of lactate
dehydrogenase leakage from livers, which did not exceed 1520
milliunits/min/g of liver.
Monitoring and Assays in Liver PerfusionEffluent perfusate
pH was monitored continuously with a pH-sensitive electrode, and the perfusion
pressure was detected by a pressure transducer (Hugo Sachs Electronics,
Hugstetten, Germany). Basal portal pressure was 35 cm H2O
and was not affected by the compounds used in this study. The intracellular
water space was calculated from the difference of washout profiles of
simultaneously infused [14C]urea and [3H]inulin as
described previously (22). In
fed animals, the cell water under control conditions was 551 ± 10
µl/g (n = 28). Proteolysis was determined in separate perfusion
experiments as 3H label release from rats that had been injected
intraperitoneally with 50 µCi of L-4,5-[3H]leucine 16
h prior to the perfusion experiment as described previously
(23). The rate of proteolysis
was set to 100% under normotonic control conditions, due to different labeling
of hepatic proteins after intraperitoneal injection, and the extent of
inhibition of proteolysis was determined 30 min after institution of the
respective condition, a time point, when a new steady state had been
reached.
In bile experiments, livers were perfused with 100 µmol/liter
[3H]taurocholate (1 µCi/liter). Bile was collected at 2-min
intervals. Bile flow was assessed by gravimetry, assuming a specific mass of 1
g/ml. Taurocholate excretion into bile was determined by liquid scintillation
counting of the radioactivity present in bile, based on the specific
radioactivity of [3H]taurocholate in influent perfusate.
Preparation of Cultured HepatocytesLiver parenchymal cells
were isolated from the livers of male Wistar rats (200 g body wt.) by
collagenase perfusion as described previously by Meijer et al.
(24). The cells were plated on
fibronectin-coated culture dishes (17 µg/dish, diameter 60 mm,
1
x 106 cells) and maintained in Krebs-Henseleit buffer (KHB)
with 6 mmol/liter glucose, equilibrated in a humidified atmosphere
(air/CO2, 19:1, v/v) at 37 °C. After 4 h in KHB, the cells were
cultured for another 48 h in Dulbecco's modified Eagle's medium (DMEM)
containing 5% fetal calf serum and 1% penicillin/streptomycin, insulin (100
nmol/liter), 1% glutamine, dexamethasone (100 nmol/liter), sodium selenite (30
nmol/liter), and aprotinin (1 µg/ml). Fresh DMEM was added after 24 h.
After a total cultivation time of 48 h, cells were cultured in normoosmotic
DMEM without additions containing 1000 mg/liter glucose for 4 h. After
starvation for 4 h in normoosmotic medium, cells were either exposed to
hypoosmolar (205 mosmol/liter) or normoosmotic control medium (305
mosmol/liter) for 2 min. If indicated, cells were incubated with PP-2 (20
µmol/liter) or GRGDSP (250 µmol/liter) 20 min prior to installing
hypoosmolarity or the normoosmotic control condition, respectively. At the end
of experimental treatment, medium was removed from the culture, and cells were
immediately lysed at 4 °C using lysis buffer containing 20 mmol/liter
Tris-HCl (pH 7.4), 140 mmol/liter NaCl, 10 mmol/liter NaF, 10 mmol/liter
sodium pyrophosphate, 1% Triton X-100, 1 mmol/liter EDTA, 1 mmol/liter EGTA, 1
mmol/liter sodium vanadate, 20 mmol/liter
-glycerophosphate, and
protease inhibitor mixture (Roche Applied Science). The homogenized lysates
were centrifuged at 20,000 x g at 4 °C, and protein
analyses were performed as described below. Protein concentrations were
determined according to Bradford
(25).
Tissue Processing for Immune Complex Kinase Assays and Western Blot
AnalysisRat livers were perfused for 130 min with isoosmotic
perfusion medium, thereafter with hypoosmotic perfusion medium (185
mosmol/liter). The desired osmolarity was achieved by omission of 60
mM NaCl. When indicated, inhibitors were present for 30 min prior
to institution of hypoosmotic perfusion conditions or addition of amino acids.
For immune complex assay and Western blot determinations, liver lobes from
perfused liver were excised at the respective time points (0, 2, 5, 10, 20,
and 30 min after installation of hypoosmotic perfusion conditions), dounced
with an Ultraturrax (Janke & Kunkel, Staufen, Germany) at 0 °C in
lysis buffer containing 20 mmol/liter Tris-HCl (pH 7.4), 140 mmol/liter NaCl,
10 mmol/liter NaF, 10 mmol/liter sodium pyrophosphate, 1% Triton X-100, 1
mmol/liter EDTA, 1 mmol/liter EGTA, 1 mmol/liter sodium vanadate, 20
mmol/liter
-glycerophosphate, and protease inhibitor mixture.
Immune Complex Kinase Assays and Western Blot AnalysisThe
lysed samples from the perfused liver or hepatocytes were centrifuged at 4
°C, and aliquots of the supernatant were incubated with 1.5 µg of an
antibody recognizing Erk-1 and Erk-2 for 2 h at 4 °C. Immune complexes
were collected by using protein A-Sepharose 4B (Sigma), washed three times
with lysis buffer and four times with kinase buffer (10 mmol/liter Tris-HCl
(pH 7.4), 150 mmol/liter NaCl, 10 mmol/liter MgCl2, and 0.5
mmol/liter dithiothreitol), and incubated with 1 mg/ml MBP in the presence of
10 µCi [
-32P]ATP for 30 min at 37 °C. The reactions
were stopped by adding 2x gel loading buffer, and activity of Erk-2 was
monitored via autoradiography after sodium dodecyl sulfatepolyacrylamide gel
electrophoresis (12.5% gel). To perform SDS-PAGE and Western blot analysis an
identical volume of 2x gel loading buffer containing 200 mmol/liter
dithiothreitol (pH 6.8) was added to the lysates. After heating to 95 °C
for 5 min, the proteins were subjected to SDS-PAGE (50 µg protein/lane,
7.5% gels). Following electrophoresis, gels were equilibrated with transfer
buffer (39 mmol/liter glycine, 48 mmol/liter Tris-HCl, 0.03% SDS, 20%
methanol). Proteins were transferred to nitrocellulose membranes using a
semi-dry transfer apparatus (Amersham Biosciences). Blots were blocked
overnight in 1% bovine serum albumin solubilized in 20 mmol/liter Tris-HCl, pH
7.5, containing 150 mmol/liter NaCl and 0.1% Tween 20 and then incubated for
34 h with antibodies raised against [Tyr(P)418]Src,
[Tyr(P)529]Src, Src, [Tyr(P)397]FAK, FAK,
[Thr(P)180/Tyr(P)182]p38MAPK and p38 at a
dilution of 1:5,000. Following washing and incubation for 2 h with horseradish
peroxidase-coupled anti-rabbit-IgG antibody (1:10,000), the blots were washed
again and developed using enhanced chemiluminescent detection (Amersham GmbH,
Freiburg, Germany). Densitometric analysis was performed with the E. A. S. Y.
RH system (Herolab, Wiesloch, Germany).
Electron MicroscopyFor electron microscopic morphometry,
fixation of the liver lobes was performed as described previously
(11) by perfusion of
glutaraldehyde (3%) in Krebs-Henseleit medium for 30 s. From the fixed livers
small cubes of
1 mm3 were cut, postfixed for 2 h with 2%
osmium tetroxide, 2% uranylacetate, and 1.5% lead citrate in PBS buffer,
dehydrated in a graded series of ethanol and embedded in Epon 812. Thin
sections for electron microscopy were placed on copper grids, stained with
uranyl acetate and lead citrate, and were examined with a EM 900 electron
microscope (Zeiss, Oberkochem, Germany).
Quantitative Evaluation of Intracellular OrganellesThe
autophagic vacuoles were defined as bits of cytoplasm sequestered from the
remaining cytoplasm by one or two membranes. The morphology of autophagic
vacuoles has been described in detail elsewhere
(26). The square fields, which
were defined by the copper grids (127 µm x 127 µm) were used as
test fields and systematically searched for autophagic vacuoles at a
magnification of x10,500. The area of cytoplasm that was examined ranged
between 7,000 and 12,000 µm2 (n = 6). The area of the
AV was measured at magnification x21,000. Low power electron micrographs
of the test fields were mounted, and the area of the hepatocytic cytoplasm was
calculated by count pointing method (144 test points). The fractional volume
of autophagic vacuoles, which is defined as the volume of autophagic vacuoles
per volume of liver cell cytoplasm (Vav/Vc) was
calculated as described previously
(10,
27).
Immunocytochemistry and Confocal Laser MicroscopyFor
indirect immunofluorescence microscopy, rat livers were perfused for 120 min
under isoosmotic conditions, and liver lobes were instantly fixed for
cryosectioning in liquid nitrogen. When present, latrunculin B (2
µmol/liter) had been added 30 min prior to fixation of liver lobes. Liver
sections were obtained using a cryotom CM 350 S (Leica, Bensheim, Germany) at
a thickness of 5 µm. Air-dried samples were fixed using for 10 min at 4
°C and washed five times with ice-cold PBS. Immediately after washing
samples were incubated with Phalloidin-FITC (Sigma) at a dilution of 1:500 in
PBS containing 5% bovine serum albumine for 2 h at room temperature.
Subsequently samples were washed again three times with ice-cold PBS.
Immunostained liver perfusion samples were analyzed with a Leica TCS NT
confocal laser scanning system (Leica, Bensheim, Germany) DM IRB inverted
microscope. Images were acquired from a channel at a wavelength of 488 nm.
MaterialsThe integrin antagonistic GRGDSP and the inactive
control peptide GRGESP were from Bachem (Heidelberg, Germany). The antibody
raised against Erk-1/Erk-2 was from Upstate (Charlottesville, VA). Antibodies
recognizing [Tyr(P)397]FAK, [Tyr(P)418]Src,
[Tyr(P)529]-Src and total Src were from BIOSOURCE (Camarillo, CA).
Anti-[Thr(P)180/Tyr(P)182]p38MAPK antibody
was from Promega (Madison, WI). The antibodies raised against total FAK and
total p38 were from Santa Cruz Biotechnology. [
-32P]ATP,
L-[4,5-3H]leucine, [3H]inulin, and
[14C]urea were from Amersham Biosciences. L-lactic acid
was from Roth (Karlsruhe, Germany). Glutaraldehyde was purchased from Serva
(Heidelberg, Germany). PP-2 was from Biomol (Plymouth, PA), and PP-3 and
latrunculin B were from Calbiochem (Bad Soden, Germany). Dulbecco's modified
Eagle's medium, fetal bovine serum, and gentamicin were purchased from
Biochrom (Berlin, Germany). Fibronectin was purchased from Invitrogen
(Karlsruhe, Germany). Enzymes were from Roche Applied Science. Insulin,
dexamethasone, and glutamine were from Sigma. Penicillin/streptomycin was from
Invitrogen. All other chemicals were from Merck (Darmstadt, Germany).
StatisticsData from different perfusion experiments are
given as means ± S.E. (number of independent experiments). Conditions
were compared by Student's t test. Differences were considered
significant at p < 0.05.
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RESULTS
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Involvement of Integrins and Src in Hypoosmotic Signal
TransductionHypoosmotic (185 mosmol/liter) liver perfusion induced
a transient Src phosphorylation on Tyr418, which was maximal (2.8
± 0.6-fold, n = 4) at about 2 min. The increase in
Src-Tyr418 phosphorylation was accompanied by a transient
dephosphorylation of Src on Tyr529. According to earlier findings
(10,
28), hypoosmolarity induced a
transient activation of the MAP kinases Erk-1/Erk-2 and p38, which was maximal
between 5 and 10 min (Fig. 1).
Normoosmotic control perfusions were without effect on Erk-1/Erk-2 and
p38MAPK (10) and
neither altered Src phosphorylation on Tyr418 and
Tyr529, nor FAK phosphorylation on Tyr397
(Fig. 1).

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FIG. 1. Activation of Src and MAP kinases by hypoosmotic hepatocyte swelling in
perfused rat liver and its inhibition by Src inhibitor PP-2 and the integrin
receptor antagonistic peptide GRGDSP, but not its inactive analogue
GRGESP. Livers from fed rats were perfused for 130 min with isoosmotic
perfusion medium. Then, osmolarity was lowered from 305 to 185 mosmol/liter
(A). Liver samples were taken immediately before (zero time point)
and during perfusion with hypoosmotic medium (2, 5, 10, 20, and 30 min after
onset of hypoosmotic perfusion conditions). Infusion of inhibitors was started
30 min prior to installation of hypoosmolarity (B).
Src-Tyr418 and Src-Tyr529 phosphorylation, dual p38
phosphorylation and FAK-Tyr397 phosphorylation were addressed by
Western blot analyses using the respective phosphospecific antibodies. In
addition, expression of Src, FAK, and p38 was monitored by probing Western
blots with antibodies recognizing the respective total protein. Erk-1/Erk-2
activity was measured with an immune complex assay using MBP as a substrate.
Representative results of 35 independent experiments are shown.
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Infusion of the integrin antagonistic peptide GRGDSP (10 µmol/liter),
but not the inactive analogue GRGESP (10 µmol/liter) prevented the
hypoosmotic stimulation of Src-Tyr418 phosphorylation and
activation of the MAP kinases Erk-1/Erk-2 and p38
(Fig. 1B and
Table 1). Likewise, PP-2 (250
nmol/liter), an inhibitor of Src kinases
(29), abolished the
hypoosmotic increase of Src-Tyr418 phosphorylation and activation
of the MAP kinases (Fig.
1B and Table
1). In the presence of the inhibitors no significant effect of
hypoosmotic perfusion on Src-Tyr529 phosphorylation could be
observed (Table 1).
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TABLE I Effects of the integrin antagonistic peptide GRGDSP, its inactive
analogue GRGESP and the Src inhibitor PP-2 on hypoosmotic
Src-Tyr418 and dual phosphorylation of p38 and Erk-1/Erk-2
activation in perfused rat liver
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Further experiments were performed in isolated liver cells plated on
fibronectin. 48-h cultured cells were exposed to hypoosmotic (205
mosmol/liter) or normoosmotic (305 mosmol/liter) medium for 2 min.
Hypoosmolarity induced an 2.7 ± 0.6-fold increase of
Src-Tyr418 phosphorylation, which was blunted to 0.8 ± 0.1-
and 1.1 ± 0.1-fold in the presence of GRGDSP (250 µmol/liter) or
PP-2 (20 µmol/liter), respectively (n = 5). In a similar way,
GRGDSP and PP-2 reduced the hypoosmotic p38 activation from 2.3 ±
0.4-fold under hypoosmotic control conditions to 0.9 ± 0.1- and 1.3
± 0.2-fold (n = 5) in the presence of the respective
inhibitors (Fig. 2). The
findings support the suggestion that direct rather than indirect effects of
GRGDSP and PP-2 account for the inhibitory effects found in the intact
liver.

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FIG. 2. Sensitivity of hypoosmotic activation of Src and p38MAPK
toward inhibition by GRGDSP and PP-2 in cultured rat hepatocytes. Liver
parenchymal cells were isolated from livers of male Wistar rats by collagenase
perfusion. After a total cultivation time of 48 h, cells were starved for 4 h.
After this time, cells were either exposed to hypoosmolar (205 mosmol/liter)
or normoosmotic control medium (305 mosmol/liter) for 2 min. If present,
inhibitors (GRGDSP (RGD) or PP-2) had been added 4 h prior to installation of
hypoosmotic conditions. At the end of this treatment, medium was removed from
the culture, cells were immediately lysed and phosphorylation of
Src-Tyr418, and dual phosphorylation of p38 was addressed by
Western blot analysis as described in the legend to
Fig. 1. Results were normalized
to total blotted protein, as measured by determination of total p38 and Src.
Representative results of 45 independent experiments are shown.
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The Integrin/Src System Is Involved in Regulatory Volume
DecreaseAs shown recently, p38MAPK activation in
response to hypoosmotic cell swelling is also involved in regulatory volume
decrease (RVD) (8), which
manifests within about 10 min of hypoosmotic exposure as a net K+
and Cl- release from the hepatocytes through Ba2+-,
DIDS-, and quinidine-sensitive ion channels
(30). This RVD response only
partially restores cell volume, and after its completion the cells are left in
a slightly swollen state (30).
When perfused livers are suddenly exposed to hypoosmotic fluid (225
mosmol/liter), a net K+ release of 12.2 + 0.5 µmol/g of liver is
observed, which is completed within 415 ± 11 s
(Table 3), and the residual
cell volume increase after completion of RVD is 13.4 + 0.8%
(Table 2). As shown recently
(8), inhibition of
p38MAPK blunts and delays this volume regulatory net K+
release and renders the cells in a more swollen state. As shown in
Table 2, prevention of
swelling-induced p38MAPK activation by GRGDSP or PP-2 rendered the
cells in a significantly more swollen state following hypoosmotic exposure.
Likewise, volume regulatory net K+ efflux was significantly
decreased and delayed in the presence of GRGDSP and PP-2, compared with the
presence of their inactive analogues GRGESP and PP-3, respectively
(Table 3). These data suggest
that the integrin/Src system is also involved in triggering RVD via
p38MAPK activation.
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TABLE III Influence of the integrin antagonistic peptide GRGDSP, the Src
inhibitor PP-2, and p38MAPK inhibitor SB 203 580 on the extent and
time course of cell volume regulatory net K+ release in response to
hypoosmotic exposure (225 mosmol/liter) of isolated perfused rat
liver
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TABLE II Cell hydration changes in perfused rat liver under the influence of
anisoosmotic conditions and amino acids in presence or absence of the integrin
inhibitory peptide GRGDSP, its inactive analogue GRGESP, and the Src inhibitor
PP-2
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Role of the Integrin/Src System in Proteolysis Control by Cell
VolumeAs shown previously
(10,
11,
31,
32), hypoosmotic cell swelling
led to a rapid and transient inhibition of proteolysis in perfused rat liver
(Fig. 3) due to an inhibition
of autophagic vacuole formation. Upon normoosmotic reexposure, proteolysis
rate returned to baseline levels, indicating the reversibility of the process.
GRGDSP, a hexapeptide with integrin receptor antagonistic activity
(33), but not its inactive
analogue GRGESP, prevented the swelling-dependent decrease of autophagic
proteolysis in perfused rat liver (Fig.
3A). In the presence of GRGESP, the decrease of
proteolysis upon hyposmolar exposure (225 mosmol/liter) was 23.1 ± 3.4%
(n = 6), whereas GRGDSP reduced this effect by about 80% to 5.6
± 2.1% (n = 4). Also Src inhibition by PP-2 abolished the
antiproteolytic effect of hypoosmotic cell swelling. In the presence of PP-2,
hypoosmotic proteolysis inhibition was only 3.1 ± 1.3% (n = 4)
compared with an inhibition by 21.9 ± 2.9% (n = 3) in the
presence of PP-3, a biologically inactive analogue of PP-2
(Fig. 3B). Likewise,
in the presence of a higher degree of hypoosmolarity (185 mosmol/liter),
proteolysis inhibition was 33.2 ± 1.4% under control conditions
(n = 6) and was blunted to 9.6 ± 2.9% (n = 4) in the
presence of GRGDSP (10 µmol/liter), an inhibitory effect, which was not
observed with GRGESP (hypoosmotic proteolysis inhibition 31.0 ± 3.4%
(n = 3)). Neither PP-3 nor GRGESP had any significant influence on
swelling-induced proteolysis inhibition.

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FIG. 3. Antiproteolytic effect of hypoosmolarity and its sensitivity to the
integrin receptor antagonist GRGDSP (A) and Src inhibitor PP-2
(B) in perfused rat liver. Livers from fed rats were prelabeled
in vivo by intraperitoneal injection of 150 µCi of
[3H]leucine, and 3H label release into effluent was
monitored as a measure of hepatic proteolysis in liver perfusion experiments.
Due to different labeling of the animals in vivo, the release of
radioactivity was set to 100% during control conditions. Infusion of integrin
antagonistic peptide GRGDSP (Ref.
33, ) or its inactive
analogue GRGESP () was started at 100 min of perfusion time
(A). In B, either PP-2 (250 nmol/liter, ), an
inhibitor of Src activation
(29) or its inactive analogue
PP-3 (250 nmol/liter, ( )
(50)) was infused 30 min prior
to installation of hypoosmotic perfusion conditions. Neither GRGDSP, GRGESP,
PP-2, nor PP-3 had a significant effect on basal proteolysis activity. Data
are given as means ± S.E. and are from 45 separate perfusion
experiments, respectively.
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Also under conditions of isoosmotic hepatocyte swelling, i.e. by
addition of amino acids, an involvement of integrin-dependent Src activation
in triggering the inhibition of autophagic proteolysis could be demonstrated.
Glutamine is known to exert its antiproteolytic action mainly via an increase
in cell hydration ((23,
31,
34), compare also
Table 2). Whereas glutamine
inhibited proteolysis by 14.0 ± 1.2% (n = 6) under control
conditions (Fig. 4A),
this effect was decreased to 3.9 ± 1.0% (n = 4) in the
presence of the integrin antagonistic peptide GRGDSP (10 µmol/liter).
Likewise, in livers from 24-h starved animals, in which the swelling potency
of amino acids is increased due to an up-regulation of concentrative amino
acid transport systems in the plasma membrane
(35,
36), the strong inhibition of
proteolysis by glutamine+ glycine (2 mmol/liter, each) by 34.6 ± 0.8%
(n = 4) was diminished to 9.8 ± 1.0% (n = 3) in the
presence of PP-2 (250 nmol/liter, Fig.
4B). This residual antiproteolytic activity of these
amino acids in the presence of PP-2 resembles that obtained after
p38MAPK inhibition
(10) and is ascribed to
ammonia formation during breakdown of these amino acids with consecutive
alkalinization of the degradative compartments
(34). In contrast to glutamine
and glycine, phenylalanine does not induce hepatocyte swelling and its
antiproteolytic action is p38MAPK-independent and involves
mechanisms distinct from hepatocyte swelling
(10,
11,
32). As shown in
Fig. 5, there was no effect of
GRGDSP on the antiproteolytic effect of phenylalanine. These data suggest that
dependence on the integrin/Src system is a feature of proteolysis control by
cell volume, but not of proteolysis control in general, i.e. also by
cell volume-independent mechanisms.
Integrins are linked to the actin cytoskeleton
(17). Thus, the role of
microfilaments in hypoosmotic signaling toward proteolysis inhibition was
assessed after destruction of microfilaments by latrunculin B. Latrunculin B
(2 µmol/liter) induced sustained cholestasis in perfused rat liver
(Fig. 6A), and as
shown in Fig. 7,
microscopically polymerized actin was no longer visible in
immunofluorescence-labeled thin sections of perfused rat liver. However,
despite destruction of actin filaments, in the presence of latrunculin the
antiproteolytic effect of hypoosmotic cell swelling was fully preserved
(Fig. 6B). These
findings indicate that integrin-dependent proteolysis regulation by cell
swelling does not require the integrity of microfilaments.

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FIG. 7. The effect of latrunculin B (2 µmol/liter) on microfilaments in
perfused rat liver. Morphological analysis of the distribution of actin G
was obtained in slices from perfused rat liver by freeze sectioning under
control conditions after a 90-min isoosmotic perfusion period without
inhibitor (A, C, E) or after prior infusion of latrunculin B (2
µmol/liter) from 60 to 90 min perfusion time (B, D, F).
Microfilaments were visualized by means of immunofluorescence labeling with
phalloidin-targeted mouse anti-actin-G antibodies. Representative results are
shown from three independent perfusion experiments for each condition and show
native transmission microscopic views (A and B,
magnification x43), projection scans (C and D,
magnification x43), and enlarged pericellular areas (E and
F, magnification x128). For further details, see
"Experimental Procedures."
|
|
Sequestration of Autophagic VacuolesMorphometric analysis
of electron micrographs (Table
4) from perfused rat liver was performed under hypoosmotic
conditions and in the presence of phenylalanine, a non-swelling amino acid
with strong antiproteolytic activity. Shifting ambient osmolarity from 305
(control) to 185 mosmol/liter (hypoosmotic) significantly decreased the
fractional volume occupied by autophagic vacuoles
(Vav/Vc) within 30 min from 51.8 ± 1.9 x
10-4 (n = 7) by about 47% to 27.3 + 3.0 x
10-4 (n = 6), indicating that hypoosmotic proteolysis
inhibition is due to an inhibition of autophagic vacuole formation
(10). The hypoosmolarity
induced decrease of Vav/Vc was fully suppressed by PP-2
(250 nmol/liter) and significantly inhibited by GRGDSP (10 µmol/liter), but
not GRGESP (10 µmol/liter, Table
4). In line with the known strong antiproteolytic effect of
phenylalanine (37), which
neither causes significant ammonia production nor changes of liver cell
hydration (10,
11,
32), phenylalanine also caused
a marked decrease of Vav/Vc
(Table 4). The
phenylalanine-dependent decrease of autophagic vacuole formation, however, was
insensitive to inhibition by PP-2 (Table
4). In view of the fact that the swelling-independent mechanisms
of regulation of protein degradation do not require intact microtubules
(32) and activation of
p38MAPK (11) the
data suggest that the swelling-dependent (hypoosmolarity, glutamine, glycine)
and swelling-independent proteolysis regulation mechanisms (phenylalanine)
converge at the level of formation of autophagic vacuoles (sequestration
step), with the former being mediated by integrin-triggered Src-dependent
p38MAPK activation, and the latter being integrin/Src-and
p38MAPK-independent.
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|
TABLE IV Effects of hypoosmotic exposure and phenylalanine on fractional volume
(Vav/Vc) of autophagic vacuoles (AVs) in the perfused
rat liver in presence of the integrin antagonistic peptide GRGDSP, its
inactive analogue GRGESP, the Src inhibitor PP-2, or the p38MAPK
inhibitor SB 203580
|
|
 |
DISCUSSION
|
---|
Cell hydration changes critically determine cell function by initiating
signal transduction (osmosignaling). Here evidence is presented that integrins
play a role in sensing hepatocyte swelling induced by hypoosmolarity or amino
acid accumulation in perfused rat liver, i.e. an intact organ model
authentically preserving hepatocyte polarity and three-dimensional anchoring
to the extracellular matrix. Based on experiments with the fibronectin-derived
hexapeptide GRGDSP (33) and
the Src inhibitor PP-2 (29),
this study shows a role of integrin-mediated activation of Src-type kinases as
a trigger of p38MAPK and Erk-1/Erk-2 activation by hepatocyte
swelling (Figs. 1 and
2). In line with this, the
swelling-related reduction of autophagosome volume
(Table 4), proteolysis
inhibition (Figs. 3 and
4) and the RVD and cell
swelling induced by hypoosmolarity (Tables
2 and
3), which represent functional
consequences of p38MAPK activation, are blunted by the RGD peptide
and PP-2, respectively. Proteolysis inhibition by phenylalanine, which does
not involve cell swelling and p38MAPK
(10) is insensitive to the RGD
peptide and PP-2 (Fig. 5),
indicating that the inhibitors do not generally interfere with the regulation
of autophagic proteolysis and specifically impair the swelling-related signal
transduction toward proteolysis.
In contrast to results from the isolated perfused rat liver
(23) and 24-h cultured rat
hepatocytes (32), no
antiproteolytic response to hypoosmolarity was found in freshly isolated
suspended rat hepatocytes
(38). Likewise, hypoosmolarity
activates Erk-1/Erk-2 in perfused rat liver (Refs.
8 and
10 and this study) and
cultured hepatocytes (39), but
not in freshly isolated cells
(40). In view of the present
study, the absence of MAP kinase activation and proteolysis regulation by
hypoosmolarity in suspended, but not in 48-h cultured hepatocytes
(Fig. 2) may reflect a deficit
in sensing hypoosmotic swelling due to the lack of integrin-mediated adherence
to the extracellular matrix. On the other hand, in freshly isolated suspended
hepatocytes hypoosmolarity activates PI 3-kinase leading to increased glycogen
and fatty acid synthesis (40,
41) and taurocholate uptake
(42). Further, hypoosmolarity
sensitizes these cells to proteolyis inhibition by amino acids
(38), which depends on
ribosomal S6 phosphorylation in a rapamycin-sensitive manner
(13). This suggests that
multiple osmosensing mechanism exist in hepatocytes, which could be
differentially linked to intracellular signaling pathways.
Although hypoosmotic swelling was shown to induce reorganization of the
actin cytoskeleton in isolated hepatocytes
(43) and FAK tyrosine
phosphorylation in HepG2 hepatoma
(44) and intestine 407 cells
(45) and FAK is involved in
mechanosensing in fibroblasts
(46), it seems questionable
that actin filaments and FAK play a role in integrin-mediated signaling toward
swelling-induced proteolyis inhibition in perfused rat liver. Disruption of
the actin cytoskeleton by cytochalasins prevents integrin signaling toward FAK
and Erk-1/Erk-2 in NIH-3T3 cells
(47), abolishes hypoosmotic
FAK Tyr phosphorylation in HepG2 cells
(44) and produces a pronounced
cholestasis in perfused rat liver
(32). However, cytochalasin
treatment does not interfere with the antiproteolytic response to
hypoosmolarity in the latter system
(32). Likewise, latrunculin B,
which disturbs actin organization by a mechanism distinct from that of
cytochalasins, induces cholestasis and disrupts the actin cytoskeleton in
perfused rat liver but does not impair hypoosmotic proteolysis inhibition
(Figs. 6 and
7). Secondly,
[Tyr(P)397]FAK phosphorylation, which is present already under
normoosmotic conditions in perfused rat liver, does not increase in response
to hypoosmolarity (Fig. 1).
Finally, livers are perfused in absence of serum, a condition known to prevent
Src recruitment by FAK (17).
FAK-dependent and independent pathways of Src activation are triggered by
integrins (17) and such
independent pathways are apparently involved in swelling-induced MAP kinase
signaling toward proteolysis inhibition under the conditions employed in the
present study. Potential integrin partners in osmosensing and signaling
include tetraspan proteins such as the osmotically regulated CD9
(48) and caveolin, which
mediates the hypoosmotic activation of volume regulatory anion channels in
endothelial cells (49). A link
of integrins to the microtubular system may also play a role in generating
antiproteolytic signals apart from/downstream of hypoosmotic
p38MAPK activation.
Our current working hypothesis is outlined in
Fig. 8. Integrins sense
hepatocyte swelling, leading to activation of Src-type kinases, which in turn
mediate activation of Erk-1/Erk-2 and p38MAPK. Impairment of
integrin-matrix interaction and inhibition of Src-type kinases, but not
disruption of the actin cytoskeleton prevents the p38MAPK-dependent
inhibition of autophagy due to cell swelling and the regulatory volume
decrease triggered by hypoosmolarity. Thus, integrins may act as cell volume
sensors at least in response to hepatocyte swelling. As in bacteria, plants,
and fungi (16), multiple
osmosensing mechanisms probably also exist in mammalian cells, and future work
will reveal their relative contributions.
 |
FOOTNOTES
|
---|
* This work was supported by Grant SFB 575 from the Deutsche
Forschungsgemeinschaft (DFG). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18
U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed: Division of Gastroenterology,
Hepatology, and Infectious Diseases, Heinrich-Heine-University, Moorenstr. 5,
D-40225 Düsseldorf, Germany. Tel.: 0049-211-811-8764; Fax:
0049-211-811-8752; E-mail:
dahlv{at}uni-duesseldorf.de.
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; AV,
autophagic vacuole; DMEM, Dulbecco's modified Eagle's medium; Erk,
extracellular signal-regulated kinase; FAK, focal adhesion kinase; MBP, myelin
basic protein; PBS, phosphate-buffered saline; PI, phosphoinositide; RVD,
regulatory volume decrease. 
 |
ACKNOWLEDGMENTS
|
---|
We thank M. Mroz and N. Eichhorst for diligent technical assistance in the
perfusion experiments.
 |
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