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J Biol Chem, Vol. 273, Issue 48, 32304-32311, November 27, 1998
From the Departments of Shear stress, the dragging force generated
by fluid flow, differentially activates extracellular signal-regulated
kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in
bovine aortic endothelial cells (BAEC) (Jo, H., Sipos, K., Go, Y. M., Law, R., Rong, J., and McDonald, J. M. (1997) J. Biol. Chem. 272, 1395-1401). Here, we examine whether
cholesterol-enriched compartments in the plasma membrane are
responsible for such differential regulation. Pretreatment of BAEC with
a cholesterol-binding antibiotic, filipin, did not inhibit
shear-dependent activation of JNK. In contrast, filipin and
other membrane-permeable cholesterol-binding agents (digitonin and
nystatin), but not the lipid-binding agent xylazine, inhibited shear-dependent activation of ERK. The effect of
cholesterol-binding drugs did not appear to be due to membrane
permeabilization, since treatment of BAEC with a detergent, Triton
X-100 which also permeabilizes membranes, did not inhibit
shear-dependent activation of ERK. Furthermore,
shear-dependent activation of ERK, but not JNK, was inhibited by cyclodextrin, a membrane-impermeable cholesterol-binding agent, which removes cell-surface cholesterol. Moreover, the effects of
cyclodextrin were prevented by adding cholesterol during the incubation. These results indicate that cholesterol or
cholesterol-sensitive compartments in the plasma membrane play a
selective and essential role in activation of ERK, but not JNK, by
shear stress. Although exposure to shear stress (1 h) increased the
number of caveolae by 3-fold, treatment with filipin had no effect in
either control or shear-exposed cells suggesting that caveolae density
per se is not a crucial determinant in
shear-dependent ERK activation. In summary, the current
study suggests that cholesterol-sensitive microdomains in the plasma
membrane, such as caveolae-like domains, play a critical role in
differential activation of ERK and JNK by shear stress.
Vascular endothelial cells recognize shear stress by unknown
mechanosensing system(s) that respond both acutely and chronically to
flow by producing autocrine and paracrine factors (1). Through these
endothelial responses, shear stress controls vascular tone, vessel wall
remodeling, binding of blood cells to endothelium, and hemostasis (1).
Shear stress selectively and differentially regulates expression of
many genes that are important in the pathophysiology of vessel wall
function (2-10). Furthermore, a conserved cis-acting shear stress
response element has been identified in many shear-sensitive genes
including platelet-derived growth factor-B, intercellular adhesion
molecule-1, tissue plasminogen activator, and transforming growth
factor How does shear stress selectively and differentially regulate such a
diverse range of nuclear responses? At least some of these responses
appeared to be mediated through regulation of MAP1 kinases (12-16).
Members of the MAP kinase family, ERK, JNK (also known as
stress-activated protein kinase), and p38 kinase, have been proposed as
important signaling components linking extracellular stimuli to
cellular responses including cellular growth, death, differentiation,
and metabolic regulation (17, 18). Recently, shear stress has been
shown to differentially regulate activation of ERK and JNK, and at
least some of shear-stimulated gene expressions are regulated by MAP
kinases (15, 16).
Shear stress activates ERK in a rapid and transient manner (maximum by
5 min which returns to basal by 30 min shear exposure), whereas JNK
activation occurs over a much slower and prolonged time course
(requiring at least 30 min and returning to basal levels after 1 day
shear exposure) than that of ERK (15). In addition, ERK activation is
shear force-dependent (minimum and maximum at 1 and 10 dyn/cm2 shear stress, respectively) whereas that of JNK
activity is either "turned off" under no shear condition or
"turned on" maximally by even a relatively low shear force (0.5 dyn/cm2) (15). Further studies demonstrated that
G Caveolae are non-coated, flask-shaped invaginations of the plasma
membrane (50-100 nm in diameter) and are found in most cell types
including endothelial cells, fibroblasts, smooth muscle cells and
adipocytes (19, 20, 23). However, "flat" caveolae (formed by
dynamic clustering of sphingolipids and cholesterol in the cell
membrane and caveolae-specific proteins such as caveolin) do exist,
although their physiologic significance has not been clearly defined
(19-22). Caveolae are found in both the apical and basal plasma
membranes and carry out at least two different functions as follows: 1)
transport large and small molecules (transcytosis of macromolecules and
potocytosis of ions and the vitamin folate), and 2) act as
transmembrane signaling microdomains (20-24). Caveolae appear to be
formed by self-packaging of "caveolin" along with highly
concentrated cholesterol and glycolipids (25-27). In addition, caveolae contain signaling molecules such as Gi2 The present study was designed to examine whether cholesterol-sensitive
membrane microdomains are involved in the signaling pathways
responsible for the differential activation of ERK and JNK in response
to shear stress. This study shows evidence that cholesterol or
cholesterol-sensitive microdomains in the plasma membrane play a
critical role in shear-dependent activation of ERK but not
in the JNK pathway.
Cell Culture and Drug Treatment--
BAEC harvested from
descending thoracic aortas were maintained (37 °C, 5%
CO2) in a growth medium (DMEM (1 g/liter glucose, Life
Technologies, Inc.) containing 20% fetal calf serum (FCS, Atlanta
Biologicals) without antibiotics) (34). Cells used in this study were
between passages 5 and 10. For shear experiments, 1 million cells per
glass slide (75 × 38 mm, Fisher) were seeded in growth medium.
The next day, the medium was changed to a starvation medium (phenol
red-free DMEM containing 0.5% FCS and 25 mM HEPES) and
incubated for 18 h. Stock solutions (1 mg/ml each freshly prepared
in 95% ethanol) of filipin, digitonin, nystatin, xylazine (Sigma) and
up to 0.5% of ethanol as a vehicle control were added to the
starvation medium 10 min before subjecting cells to shear stress.
Methyl- Membrane Integrity Assay Using Calcein AM--
BAEC were
incubated with 0.5 µg/ml calcein AM (Molecular Probe) for 30 min at
37° in DMEM, 0.5% FCS. The calcein-loaded cells were washed three
times with HEPES-buffered saline (MediaTech) followed by incubation
with 0-0.1% Triton X-100 in DMEM containing 0.5% FCS for 10 min at
37 °C. Then, the medium was collected, and cells were harvested in
phosphate-buffered saline (PBS). The amount of calcein that leaked out
from the cells was determined in a Perkin-Elmer fluorimeter (excitation
at 488 nm and emission at 530 nm) as described (37). Aliquots of cell
extracts were assayed by DC protein assay (Bio-Rad).
Shear Stress Studies--
The glass slide containing a BAEC
monolayer was assembled into a parallel plate shear chamber forming a
flow channel (220 µm height × 25 mm width × 62 mm length)
between the monolayer and fabricated polycarbonate plate as described
(38). Non-pulsatile, laminar shear stress was controlled by changing
the flow rate of the starvation medium containing drugs
(e.g. filipin or vehicle) delivered to the cells using the
constant head flow-loop or a syringe pump (KD Scientific) as described
(15, 38).
Preparation of Cell Lysates--
Following shear exposure, cells
were washed in ice-cold PBS, scraped in 0.25 ml of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1.0 mM vanadate, 1 mM dithiothreitol, 1.0% Triton
X-100, and 0.1 mM phenylmethylsulfonyl fluoride),
solubilized for 15 min, and centrifuged at 20,000 × g
for 15 min to prepare Triton-soluble lysate as described (15). Entire
fractionation or solubilization procedures were performed at 4 °C.
The protein content of solubilized cell lysates was measured by using a
Bio-Rad DC assay kit (Bio-Rad).
Western Blot Analysis of ERK Activation--
Soluble lysates (10 µg each) were resolved by 10% SDS-PAGE, transferred to a
polyvinylidene difluoride membrane (Millipore), and probed with
antibodies specific to ERK1/2 or phosphorylated forms of ERK1/2
(pERK1/2) (New England Biolabs) as described (15). Goat anti-rabbit
IgG-conjugated to alkaline phosphatase was used as a secondary
antibody, and the membrane was developed by a chemiluminescent detection method (15).
Immune Complex JNK Assay--
Activity of JNK1 was measured by
an immune complex kinase assay using an antibody specific for JNK1
(PharMingen, clone number G151-333) and c-Jun (amino acids 5-89) fused
to glutathione S-transferase (GST-c-Jun) as the substrate as
described (15). Briefly, Triton-soluble cell lysates (100 µg each)
were incubated with JNK1 antibody (0.25 µg) for 1 h at 4 °C,
followed by an additional 1-h incubation with protein G-agarose. The
immune complex was washed four times in the lysis buffer and twice in
buffer A (20 mM HEPES, pH 7.6, 20 mM
MgCl2, 20 mM Cytochemical Staining of Cholesterol--
Filipin is fluorescent
and has been widely used to localize cellular cholesterol (40).
Briefly, BAEC grown on glass slides were washed with ice-cold PBS,
fixed with 1% glutaraldehyde on ice for 15 min, rinsed with PBS, and
incubated with filipin (50 µg/ml) for 30 min at room temperature. The
cells were then washed in PBS followed by distilled H2O,
mounted in Slow-fade (Molecular Probe), and examined by epi-fluorescent
microscopy (Olympus AH-3) using a UV filter.
Transmission Electron Microscopy (TEM)--
To allow thin
sectioning for TEM studies, BAEC were grown on a plastic Mylar sheet
(1000A, DuPont) instead of glass slides. Cells were preincubated with
vehicle or filipin (5 µg/ml for 0- and 5-min shear groups and 1 µg/ml for 1-h group). Filipin was present during shear. Following
shear, cells were fixed for 1 h at 4 °C on ice with 1.6%
paraformaldehyde and 3% glutaraldehyde in 0.1 M sodium
cacodylate buffer, pH 7.3, washed with 0.1 M sodium cacodylate and 3.5% sucrose buffer, pH 7.3, and then post-fixed for
1 h with 1% Palade's OsO4. Cells were stained
en bloc with Kellenberger's uranyl acetate, dehydrated,
embedded in epoxy resin, and sectioned. Ultra-thin sections were
examined by TEM, and 26-67 different random fields (each field
containing part of 1 or 2 cells) were photographed. As suggested by
Schinitzer et al. (23), only distinctly flask-shaped,
non-coated vesicles (50-100 nm in diameter) found on or within 100 nm
of both apical and basal plasma membranes were scored as caveolae.
Filipin Does Not Inhibit Shear-dependent Activation of
JNK1--
To examine the role of cholesterol in the
shear-dependent activation of JNK, BAEC were pretreated
with increasing concentrations of filipin or vehicle prior to shear
exposure. Treatment of BAEC with filipin did not have any significant
effect on static (no shear controls) or shear-dependent
activation of JNK (Fig. 1, B
and C). In some experiments, cells that were pretreated with filipin were subjected to shear stress in the shear medium containing no filipin. The presence or absence of filipin (1 µg/ml) in the shear
medium during a period of 1 h shear had no effect on
shear-dependent activation of JNK (data not shown). As
shown previously (15), exposure of BAEC to shear stress for 1 h
activated JNK1 by 6.5-fold as measured by phosphorylation of GST-c-Jun
using JNK1 immunocomplex (Fig. 1B).
Filipin Inhibits Shear Stress-dependent Activation of
ERK--
Unlike the case for JNK, pretreatment of cells with filipin
(0-1 µg/ml for 10 min) inhibited shear-dependent
activation of ERK in a concentration-dependent manner (Fig.
2B and closed
circles in C). As shown previously (15), exposure of
BAEC to shear stress (10 dyn/cm2) for 5 min stimulated ERK
activity by 5-6-fold over the no shear (static) control as measured by
Western blot analysis with an antibody specific to phospho-ERK (pERK)
(Fig. 2B). Filipin had minor effects reaching up to 2-fold
activation of ERK over vehicle control in the static control cells
(p = 0.08 to 0.2, paired Student's t tests,
see also Figs. 3 and 4).
The Inhibitory Effect of Filipin on Shear Activation of ERK Is
Reversible--
Next, we tested whether the inhibitory effect of
filipin was reversible. BAEC that were pretreated with filipin (1 µg/ml for 10 min) were washed and incubated in a filipin-free
starvation medium for up to 1 h before exposure to shear stress.
After 60 min incubation in filipin-free media, shear-stimulated
activity of ERK was restored to ~70% of control (filipin-untreated
group) (Fig. 3).
Other Cholesterol-binding Reagents Inhibit Shear-stimulated
Activity of ERK--
Two other membrane-permeable cholesterol-binding
agents, digitonin and nystatin, and a lipid-binding agent, xylazine (as
a control), were used to examine the specific functional role of cholesterol in the shear-dependent activation of ERK.
Digitonin, a cardiac glycoside (42), inhibited
shear-dependent activation of ERK, while raising basal ERK
phosphorylation (Fig. 4A).
Nystatin, a polyene macrolide antibiotic (43), partially inhibited (by 42%) shear-dependent activation of ERK while having no
effect on the basal activity. In contrast, xylazine enhanced the
shear-dependent activation of ERK by 45% without any
significant effect on the basal activity (Fig. 4A). Next,
the effects of these agents on JNK activity were also examined. Unlike
ERK activity, digitonin, nystatin, and xylazine did not have any
significant effect on shear-activated JNK activity (Fig.
4B). Nystatin and xylazine also had little effect on basal
JNK activity, whereas digitonin increased it somewhat (Fig.
4B). At present, the underlying mechanisms for the
differential effects of each cholesterol-binding compound on basal ERK
activity are not known. Nevertheless, these results demonstrate that
removal of cholesterol has specific inhibitory effects on ERK
activation, but not JNK, in response to shear stress.
Permeabilization of Membrane Alone Does Not Lead to Inhibition of
Shear-dependent ERK Activation--
Filipin, digitonin,
and nystatin can create transmembrane pores and alter membrane
permeability. To determine whether the effect of these drugs was due to
their nonspecific effect on the membrane permeability, we chose to use
a detergent, Triton X-100, to permeabilize the cell membrane in a
non-cholesterol-dependent manner. As demonstrated in Fig.
5B, treatment of BAEC with
0.001-0.01% Triton X-100 effectively increased the leakiness of
calcein without significantly depleting the total cellular protein
level. Under similar conditions, total ERK levels did not change under
static conditions (lanes 1-3, Fig. 5A) unless
the Triton-treated cells were further subjected to shear stress
(lane 7, Fig. 5A). The Triton treatment did not
inhibit the shear-dependent activation of ERK, even when
there was a significant loss of ERK1/2 (lane 7, Fig.
5A). Although it is relatively minor compared with the robust fold stimulation of ERK by shear stress, basal ERK activity was
somewhat increased by Triton permeabilization of cells (Figs. 5A and 6B).
Next, we examined the effect of Triton permeabilization on JNK
activity. Similar to the results of ERK, the detergent treatment did
not have any effect on JNK activation induced by shear stress while
raising basal JNK activity somewhat (Fig.
6C). Again, the effect of
Triton on basal JNK activity was relatively minor compared with the
robust stimulation of JNK by shear stress (Fig. 6C). Taken
together, these results suggested that nonspecific alteration of
membrane permeability alone could not account for the inhibitory effect
of the cholesterol-binding drugs on shear-dependent ERK activation.
Plasma Membrane Cholesterol Is a Key Component in
Shear-dependent Activation of ERK--
To examine whether
cholesterol in the plasma membrane plays an essential role in the
shear-dependent ERK signaling pathway, a
membrane-impermeable cholesterol-binding drug cyclodextrin was used.
Cyclodextrin (10 mM) treatment of BAEC prevented the
shear-dependent activation of ERK (Fig. 6A).
This inhibitory effect was prevented if cholesterol was added back to
the incubation medium during the cyclodextrin treatment (Fig.
6A). Time course studies using 5-10 mM
cyclodextrin showed that it was necessary to incubate cells with the
drug for 1 h (data not shown) suggesting that the cholesterol
targeted by cyclodextrin may not initially be in the exofacial plasma
membrane where it could be readily accessible by the drug. This slow
kinetics could be explained if cholesterol affected by cyclodextrin was
present in the cytofacial plasma membrane which translocates slowly to
the exofacial side, where it could be removed out of the membrane by
binding to cyclodextrin as proposed (44). Treatment of BAEC with 5 mM cyclodextrin also substantially inhibited
shear-dependent activation of ERK, although its inhibitory
effect was lower than that induced by 10 mM (compare Fig.
6, A and B). However, if cyclodextrin was
delivered to the interior of the cell after the cells were
Triton-permeabilized, then 5 mM cyclodextrin completely
prevented the shear-dependent activation of ERK.
Next, we examined whether the JNK pathway is inhibited by removal of
membrane cholesterol using cyclodextrin. Identical to the result
obtained with filipin (Fig. 1), cyclodextrin treatment in the absence
or presence of Triton X-100 had no significant effect on
shear-activated JNK activity (Fig. 6C). Taken together these
results shown in Fig. 6 clearly demonstrated that removal of
cholesterol by cyclodextrin inhibits shear-dependent
activation of ERK but not JNK.
To study the effect of cyclodextrin on plasma membrane cholesterol, the
fluorogenic characteristic of filipin (used at 50 µg/ml) was used to
cytochemically stain cholesterol and examined by epi-fluorescent
microscopy (40). In control cells (Fig.
7A), cholesterol on the cell
surface appeared as fine lines, especially visible at borders between
cells (marked with arrowheads). Treatment with cyclodextrin
eliminated cholesterol staining in the plasma membrane as indicated by
arrowheads placed at the border lines between the cells
(Fig. 7B). If cholesterol was added during the cyclodextrin
treatment, however, the plasma membrane cholesterol was clearly visible
(arrowheads) again as in control cells (Fig. 7C).
When cells were permeabilized by Triton X-100 and subsequently incubated with cyclodextrin, then the cholesterol staining was no
longer visible (Fig. 7D). This complete chelation of
cellular cholesterol by cyclodextrin + Triton was not due to the loss
of cells as BAEC were still intact (see the phase photomicroscope, Fig.
7E). Moreover, the treatment of BAEC with 0.01% Triton
X-100 alone did not alter the cholesterol-staining pattern (data not shown). The results of the cytochemical cholesterol staining studies support that cyclodextrin is membrane-impermeable and its effect is
mediated by chelating cholesterol in the plasma membrane. Combined with
the results shown in Fig. 6, these findings clearly indicate that
cholesterol in the plasma membrane is the target of cyclodextrin and a
key element in the shear-dependent ERK signaling
pathway.
Effect of Filipin and Shear Stress on the Number of
"Invaginated" Caveolae--
Based on the previous reports in
bovine lung microvascular endothelial cells and fibroblasts (19, 23),
we speculated that the inhibitory effect of filipin on
shear-dependent activation of ERK would be due to loss of
"invaginated" caveolae. Consistent with previous reports (23, 45),
caveolae were found as single as well as clusters of non-coated
vesicles (see arrowheads in Fig.
8A) on or near the apical and
basal plasma membranes of BAEC. There were ~0.15 caveolae/µm in
control cells as quantified by counting the number of "invaginated"
caveolae seen by TEM using the morphological criteria suggested by
Schnitzer and colleagues (23, 45). To our surprise, however, filipin
treatment (up to 5 µg/ml for up to 1 h at 37 °C) did not
reduce the number of invaginated caveolae in static controls and
shear-exposed groups (Fig. 8B). On the other hand, exposure
of BAEC to shear stress for 1 h increased invaginated caveolae by
3-fold (Fig. 8B).
Early atherosclerotic plaques tend to develop in curved and
branched arterial regions, which are associated with disturbed and/or
low shear stress conditions, whereas areas exposed to stable and high
shear forces are relatively well protected (46, 47). Similarly, various
shear regimens (laminar, turbulent, step, and gradual changes) induce
different endothelial responses including gene expression, NO release,
and activation of MAP kinases over different time courses (1, 3, 9, 15,
38, 48). How do endothelial cells recognize the differences in shear
stress and respond accordingly? The results presented in this study
suggest that endothelial cells contain cholesterol-sensitive structures and/or signaling modules that may render signaling specificity leading
to selective activation of ERK without affecting that of JNK pathway in
response to shear stress.
Chelation of Cholesterol Prevents Shear-dependent
Activation of ERK but Not JNK--
In this study, we presented several
lines of evidence supporting the concept that the removal of
cholesterol in the plasma membrane results in selective inhibition of
the shear-sensitive ERK pathway. First, filipin treatment only blocked
activation of ERK, but not JNK, in response to shear stress (Figs. 1
and 2). Second, other membrane-permeable cholesterol-binding drugs (digitonin and nystatin), but not a lipid-binding drug (xylazine), inhibited activation of ERK, but not JNK, by shear stress (Fig. 4).
Third, the specific nature of these drugs was also supported by
demonstrating the reversibility of filipin effect (Fig. 3). Fourth, the
lack of Triton's ability to inhibit the shear-sensitive ERK activation
makes it unlikely that the inhibitory effects of filipin, digitonin,
and nystatin could be solely attributed to their characteristic as
detergents (Fig. 5). Finally, cyclodextrin, a cell-impermeant agent
that sequesters plasma membrane cholesterol by inducing its efflux,
selectively inhibited the shear-dependent activation of ERK
but not JNK. Moreover, the inhibitory effect of cyclodextrin was
reversed by adding exogenous cholesterol (Figs. 6 and 7).
Plasma Membrane Cholesterol Is Essential to the
Shear-dependent Activation of ERK--
It has been shown
that ~85% of free cholesterol is present within the plasma membrane,
and the majority (75-97%) of these cholesterol molecules are found in
the cytofacial leaflet in mammalian cells (44, 49). Fielding and
Fielding (44) proposed a classification of the cellular free
cholesterol into three pools as follows: (a) the "fast
pool" present in the exofacial plasma membrane, (b) an
"intermediate pool" representing the cytofacial free cholesterol in
transit toward the exofacial plasma membrane, and (c) a
"slow pool" that is found intracellularly. During our study, we
observed that the inhibitory effects of cyclodextrin (5 to 10 mM) were observed only after a relatively long incubation
period (30-60 min). The exofacial cholesterol (3-25% of total
cholesterol in the plasma membrane) is directly accessible by
cyclodextrin and is expected to be removed quickly (fast pool) (44,
50). However, we have not been able to block the shear-sensitive ERK by
less than 30 min cyclodextrin treatment (data not shown) suggesting that the exofacial cholesterol is not likely to be a key component in
this shear-signaling pathway. It seems more likely, however, that the
slow time course of cyclodextrin action could be explained by the slow
movement of the cytofacial cholesterol to the exofacial plasma
membrane. Therefore, the cholesterol or cholesterol-enriched compartments present in the cytofacial plasma membrane could be responsible for the regulation of shear-sensitive ERK activation.
A specific role for cholesterol in the proper functioning of receptors
and other membrane proteins, including G-proteins, has been reported.
For example, receptors for 5-methyltetrahydrofolate, rhodopsin,
oxytocin, cholecystokinin, transferrin, and acetylcholine have all been
shown to require cholesterol for optimal functioning (36, 51-54). As
early as 1975, Limbird and Lefkowitz (55) showed that the proper
coupling of
One question raised by the present study is whether
cholesterol-sensitive structures such as caveolae or
sphingolipid-cholesterol rafts play a role in the
shear-dependent activation of ERK. In support of the role
for these membrane domains, Anderson and co-workers (32) have shown
that ERK is activated in caveolae in intact cells. In addition, they
demonstrated that isolated caveolae contain the functionally intact,
signaling module that was able to stimulate ERK upon platelet-derived
growth factor addition (57). The results presented in our current study
are also consistent with a role for the cholesterol-sensitive domains
in activation of ERK by shear stress.
Number of Invaginated Caveolae Is Not a Critical Factor in ERK
Activation by Shear Stress--
Exposure of BAEC to shear for 1 h
increased the number of invaginated caveolae by 3-fold (Fig. 8). This
result suggests that the presence or absence of blood flow (shear
stress) may be a major determinant of the density of invaginated
caveolae. Although the number of invaginated caveolae is dynamically
controlled by shear stress, it does not seem to play a critical role in
shear-dependent activation of ERK (Fig. 8). Treatment of
fibroblasts or bovine lung microvascular endothelial cells with filipin
has been shown to decrease the number of invaginated caveolae (19, 23).
However, our study using endothelial cells of aortic origin (BAEC) show that the effect of filipin on ERK activation by shear stress is not due
to a decrease in the number of invaginated caveolae (Fig. 8). The
reasons for these discrepancies are not clear at this time. However, it
is well known that endothelial cells of different vascular origins
differ in function and morphology including the number of caveolae
(45, 58, 59). Therefore, it is not surprising to find that the
secondary effect of the cholesterol-binding drug, filipin, on the
number of invaginated caveolae is dependent upon cell types. The
current study indicates that cholesterol-binding drugs can inhibit
cellular functions (such as ERK activation) without apparent
morphological changes in BAEC. Since cholesterol binds directly to
caveolin-1 (27, 56), a potential mechanism of filipin action may
include the disruption of cholesterol-caveolin interaction.
Physiological Implications of Membrane Cholesterol in Differential
Regulation of MAP Kinases--
Previously, we showed (15) that shear
stress rapidly and transiently activates ERK in shear
force-dependent manner (maximum activation by 10 dyn/cm2 for 5 min), whereas JNK is activated over a slower
time course in a low threshold-dependent manner (maximum
activation by shear force as low as 0.5 dyn/cm2 for 1 h). This differential activation of ERK and JNK involves two separate
signaling pathways that depend on two different heterotrimeric G-proteins and some common signaling molecules. For example, we showed
that shear stress stimulates ERK by mechanisms involving Gi2
Taken together, accumulating evidence strongly indicates that
Gi2 We thank Drs. A. Kraft and C. Franklin for a
GST-c-Jun plasmid and a rabbit JNK antibody and Dr. V. Darley-Usmar for
critically reading this manuscript.
*
This work was supported by a National Institutes of Health
First Award HL53601 and an American Heart Association Grant-in-aid AL-G-960035 (to H. J.), National Institutes of Health Grant DK52483 (to D.R.A.), and National Institutes of Health FIRST Award GM-50443, a
grant from the G. Harold and Leila Y. Mathers Charitable Foundation, and a Scholarship in the Medical Sciences from the Charles E. Culpeper
Foundation (to M. P. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
MAP kinase, mitogen-activated protein kinase; BAEC, bovine aortic endothelial
cells; ERK, extracellular signal regulated kinase; GST-c-Jun, c-Jun
(amino acids 5-89) fused to glutathione S-transferase; JNK, NH2-terminal Jun kinase; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FAK, focal adhesion kinase; TEM, transmission electron microscopy.
Plasma Membrane Cholesterol Is a Key Molecule in Shear
Stress-dependent Activation of Extracellular
Signal-regulated Kinase*
,,,
Pathology and
§ Cell Biology, University of Alabama at Birmingham,
Birmingham, Alabama 35294 and the ¶ Department of Molecular
Pharmacology, Albert Einstein College of Medicine, Bronx, New
York 10461
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-1, suggesting its broad implication in
shear-dependent regulation of gene expressions (6, 8).
Shear stress also transiently activates nuclear factor-
B, immediate
early response genes, and transcription factors that are likely to be
involved in the regulation of shear-dependent gene
expression (8, 11).
i2/tyrosine kinase(s)/Ras and G/
/tyrosine
kinase(s)/Ras are upstream regulators of shear-dependent activation of ERK and JNK, respectively (15). How do endothelial cells
control activation of specific signaling pathways leading to
differential activation of ERK and JNK in response to various shear
regimens? We hypothesized that there may be a spatial and/or temporal
compartmentalization of signaling complexes in microdomains, which
enable an orderly and efficient regulation of signaling cascades in
response to shear stress. Cholesterol-enriched compartments such as
caveolae and "sphingolipid-cholesterol rafts" (also called as
"caveolae-like domains" or "pre-caveolae") are candidate
micro-signaling domains (19-22).
,
Src-like kinases, Ras, Raf, and even ERK itself (20, 28-33).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-cyclodextrin (prepared in distilled H2O, Sigma) was added to the starvation medium containing lipoprotein-free FCS (35)
and incubated with cells for 60 min. In some studies, 1.3 mM cholesterol (Avanti) was added during the 1-h
cyclodextrin treatment to block chelation of cholesterol from cell
membrane (36). To test whether the effect of filipin on
shear-dependent activation of ERK is reversible, cells were
first preincubated with filipin (1 µg/ml) for 10 min, washed in
starvation medium, and then incubated in the fresh medium without
filipin for up to another hour before subjecting cells to shear stress.
-glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM
vanadate, and 2 mM dithiothreitol). The washed
immunocomplexes were incubated in 20 µl of buffer A containing
GST-c-Jun (5 µg each), 20 µM ATP, and 5 µCi of
[-32P]ATP for 20 min at 30 °C. The reaction was
terminated by boiling in 5× Laemmli sample buffer, resolved by 10%
SDS-PAGE, and electrotransferred to a polyvinylidene difluoride
membrane. Autoradiograms were obtained from the dried blot, and
radioactivity incorporated into GST-c-Jun was quantitated by cutting
and counting each band in a scintillation counter. Quantitative
immunoprecipitation of JNK1 was examined by probing the same blot with
a polyclonal JNK antibody (39) (kindly provided by Drs. C. C. Franklin and A. S. Kraft at the University of Colorado).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (41K):
[in a new window]
Fig. 1.
Filipin does not inhibit
shear-dependent activation of JNK1. BAEC were
preincubated for 10 min with filipin (0-5 µg/ml) and further
incubated under no flow condition (Static) or exposed to shear stress
(5 dyn/cm2) for 1 h. Cell lysates were incubated with
a JNK1 antibody (PharMingen) and protein G-agarose, and the immune
complex was used to phosphorylate GST-c-Jun (15). Phosphorylated
proteins were detected by 10% SDS-PAGE followed by electrotransfer to
a polyvinylidene difluoride membrane and autoradiography
(B). The same membrane was probed with a rabbit JNK antibody
to demonstrate that similar amounts of JNK1 were used in each lane
(A). The radioactivity of phosphorylated GST-c-Jun was
quantified by cutting and counting each band (C). A
representative blot showing HA-JNK1 (~46 kDa) (A) and an
autoradiogram of GST-c-Jun phosphorylation (B) are shown.
The line graph shown in C is the mean ± S.E. of results obtained from 3 to 6 independent experiments. When
cells were preincubated with 5 µg/ml filipin and subsequently exposed
to shear stress for 1 h, all cells detached from the glass plate
so that JNK activity could not be measured. Filipin treatment did not
have any statistically significant effect on the
shear-dependent activation of JNK.
View larger version (47K):
[in a new window]
Fig. 2.
Filipin inhibits shear-dependent
activation of ERK. BAEC were pretreated with filipin (0 to 1 µg/ml for 10 min) followed by exposure to no shear (static) or shear
stress (10 dyn/cm2 for 5 min) and preparation of cell
lysates. The lysates (10 µg per lane) were resolved by 10% SDS-PAGE
and Western-blotted with antibodies specific to ERK1/2 (A,
to demonstrate the equal loading of proteins) or phospho-ERK1/2
(pERK1/2, B, to reveal activation of ERK) (both antibodies
from New England Biolabs). Densitometry was performed on both pERK1 and
pERK2 bands and showed essentially the same results. Shown in
C is the mean ± S.E. of the quantitation of pERK1
bands obtained from three independent experiments.
View larger version (34K):
[in a new window]
Fig. 3.
The inhibitory effect of filipin on
shear-dependent activation of ERK is reversible. After
preincubation of BAEC with or without 1 µg/ml of filipin for 10 min,
cells were washed and incubated again in filipin-free medium (DMEM
containing 0.5% FCS) for 0, 10, 30, or 60 min (recovery time). Cells
were then subjected to shear stress (10dyn/cm2 for 0 or 5 min) and lysed. The lysates were analyzed by Western blot with a
pERK1/2 antibody and densitometry as described in Fig. 2. The 1st
two lanes and the corresponding bar graph
below show the control experiment (static and shear groups
without filipin treatment). The line graph shows the percent
of recovery (mean ± S.E., n = 3) compared with
control (vehicle and shear-treated group) as a function of incubation
time in filipin-free medium. Note that the inhibitory effect of filipin
on ERK was not observed any more if filipin-treated cells were
incubated in a filipin-free medium for 1 h before subjecting cells
to shear stress.
View larger version (23K):
[in a new window]
Fig. 4.
Effects of other cholesterol-binding agents
on shear-dependent activation of ERK. BAEC pretreated
for 10 min with 5 µg/ml digitonin, nystatin, or xylazine were
subjected to shear stress (A, 10 dyn/cm2 for 5 min and B, 5 dyn/cm2 for 1 h).
A, phosphorylation of ERK1/2 in the cell lysates was
determined by Western blotting using a pERK1/2 antibody and quantitated
as described in the legend of Fig. 2. B, JNK activity was
determined by using JNK1 immunoprecipitates and GST-c-Jun
phosphorylation as described in the legend of Fig. 1. The bar
graphs show the mean ± S.E. (n = 3 each).
View larger version (35K):
[in a new window]
Fig. 5.
Membrane permeabilization by Triton X-100
does not inhibit shear-dependent ERK activity.
A, BAEC that were pretreated with 0-0.01% Triton X-100 for
10 min at 37 °C before subjecting cells to shear stress in fresh
shear medium without Triton X-100. Cell lysates were analyzed by
Western blotting using antibodies specific to ERK1/2 (top
panel) and pERK1/2 (middle panel). The line
graph shows the mean ± S.E. (n = 4) obtained
from densitometric quantitation of pERK1 bands. B, BAEC that
were pre-loaded with calcein AM were incubated with 0-0.1% Triton
X-100 as above before collecting the incubation media and cells. Shown
are the means ± S.E. (n = 3) of the levels of
calcein ( 
) that remained within the cells after the detergent
incubation and total protein (
) measured using the treated cells.
The results obtained by measuring the amount of calcein collected in
the incubation media were confirmatory (data not shown).
View larger version (34K):
[in a new window]
Fig. 6.
Sequestration of cholesterol inhibits
shear-dependent activation of ERK but not JNK.
A, BAEC were pretreated without or with 10 mM
methyl- -cyclodextrin, 10 mM methyl--cyclodextrin + 1.3 mM cholesterol for 1 h at 37 °C in DMEM
containing 0.5% lipoprotein-deficient serum. B, BAEC were
first incubated with or without 0.005% Triton X-100 for 10 min at
37 °C, followed by an additional 1 h incubation without or with
5 mM methyl--cyclodextrin in fresh DMEM containing 0.5%
lipoprotein-deficient serum. Treated cells were then subjected to shear
stress (10 dyn/cm2 for 0 or 5 min) in DMEM containing 0.5%
lipoprotein-deficient serum. Western blots for ERK1/2 and pERK1/2 and
quantitation were performed as described in Fig. 2. The bar
graph shows the mean ± S.E. (n = 3).
C, BAEC treated with or without 0.005% Triton X-100 and/or
5 mM methyl--cyclodextrin as described in B
were subjected to shear stress (10 dyn/cm2 for 0 or 1 h), and JNK1 activity was determined as described in Fig. 1. The
bar graphs show mean ± S.E. (n = 3).
View larger version (110K):
[in a new window]
Fig. 7.
Methyl- -cyclodextrin sequesters
cell-surface cholesterol. BAEC were pretreated without
(A and F) or with 10 mM
methyl--cyclodextrin (B), 10 mM
methyl--cyclodextrin +1.3 mM cholesterol (C),
and 0.005% Triton X-100 followed by 5 mM -cyclodextrin
(D and E) as described in Fig. 6. After
treatment, cells were washed in PBS, fixed in 1% glutaraldehyde, and
incubated with PBS (F) or 50 µg/ml filipin
(A-E) for 30 min. After washing in PBS, slides were mounted
in an anti-fade mounting medium and examined by fluorescent microscopy
(× 3,300 magnification) using a UV filter except for E
(phase microscopic image of D, × 500 magnification). The
surface cholesterol surrounding the cell boundary in control
(A) and CD + cholesterol (C)-treated cells are
marked with arrowheads. B, arrowheads
are used to illustrate the loss of surface cholesterol in cell surface
(compare with arrowheads in A and C)
by treatment of cells with methyl--cyclodextrin.
View larger version (72K):
[in a new window]
Fig. 8.
Filipin does not decrease the number of
invaginated caveolae, but shear stress increases their numbers in
BAEC. BAEC were preincubated with vehicle or filipin (5 µg/ml
for 0- and 5-min shear groups and 1 µg/ml for 1-h group). Following
shear, cells were thin sectioned and examined by electron microscopy as
described under "Materials and Methods." Two to four independently
prepared endothelial monolayers were used to photograph 26-62
different randomly chosen fields from each group. Only distinctly
invaginated vesicles (50-100 nm diameter) found within 100 nm of both
apical and basal plasma membranes were counted. Shown in A
are examples of caveolae (arrowheads) obtained from control
BAEC, and shown in B are average caveolae numbers per
µm ± S.E. (*, p < 0.05; **, p < 0.02; ***, p < 0.001 as determined by Student's
t tests). Note the 3-fold increase in caveolae numbers after
60 min shear stress over static control. Some portions of monolayers
that were saved before fixing them for EM studies were used for Western
blot analysis with a pERK1/2 antibody and confirmed that filipin
treatment inhibited shear activation of ERK as shown in Fig. 2.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-adrenergic receptors to adenylate cyclase was uncoupled
by removing cholesterol. The role of cholesterol could also be to
regulate membrane fluidity or rigidity (36). Since cholesterol directly
binds to caveolin-1, cholesterol could directly influence the function
of caveolin-1, which interacts and regulates many signaling molecules
including G-proteins, Src, and Ras (20, 27, 56).
, tyrosine kinase(s), and Ras, whereas JNK activation
by shear stress involves non-pertussis toxin-sensitive G, G/,
tyrosine kinase(s), and Ras (15). More recently, we found that the
G-protein-dependent phosphatidylinositol 3-kinase is rapidly and transiently activated by shear stress (maximum
activation by 15 s which returns to basal by 1 min shear exposure)
and that phosphatidylinositol 3-kinase selectively mediates shear
activation of JNK but not ERK (60). In contrast, Shyy, Chien, and their
colleagues (14, 61) have shown that focal adhesion kinase (FAK) and Src
are upstream signaling molecules required to activate both ERK and JNK
by shear stress in BAEC, although the role of FAK in shear activation
of ERK appears to be cell type-specific (62).
, PKC
, Src, FAK, and Ras are upstream
signaling molecules for the shear activation of ERK (13-15, 61) at
least in BAEC. In contrast, a pertussis toxin-insensitive
heterotrimeric G, /, phosphatidylinositol 3-kinase , Src,
FAK, and Ras regulate the shear-dependent activation of JNK
(14, 15, 60, 61). The molecular basis for the differential activation
of ERK and JNK by involving two different G-proteins as well as the
common signaling molecules (Src, FAK, and Ras) is then likely to
require a spatial and/or temporal compartmentalization of signaling
modules/molecules. Cholesterol-sensitive microdomains in the plasma
membrane, such as caveolae-like domains, provide a plausible means of
spatial and/or temporal sequestration of signaling units for the
differential activation of MAP kinases. Determining the underlying
mechanisms and morphologic structures by which cholesterol or
cholesterol-sensitive microdomains segregate signaling molecules and
render selective regulation of MAP kinases will be an important area of
future investigation.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: University of
Alabama at Birmingham, Division of Molecular and Cellular Pathology, Dept. of Pathology, G019C Volker Hall, Birmingham, AL 35294. Tel.: 205-975-8041; Fax: 205-934-1775; E-mail: Jo{at}path.uab.edu.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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