Division of Endocrinology (E.R.L.), VA Medical Center, Long Beach, Long Beach, California 90822; Departments of Medicine (M.R., A.P., E.R.L.) and Pharmacology (E.R.L.), University of California, Irvine, California 92717; and Sidney Kimmel Cancer Center (P.O., J.S.), La Jolla, California 92121
Address all correspondence and requests for reprints to: Dr. Ellis R. Levin, Medical Service (111-1), Long Beach VA Medical Center, 5901 East Seventh Street, Long Beach, California 90822. E-mail: ellis.levin{at}med.va.gov
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the plasma membrane ER has not been physically isolated and sequenced, it appears to be very similar to the nuclear protein. Both membrane and nuclear receptors can originate from a single transcript (6), and membrane ER can be identified by antibodies raised against various epitopes of the nuclear receptor (11). The process whereby the ER localizes to the membrane is not known. Also, examination of the nuclear ER sequence does not identify a motif that is homologous to a kinase or catalytic domain of growth factor receptors, binding proteins that are typically inserted into the plasma membrane. Thus, it is not clear how ER enacts signal transduction, but this probably occurs through physical interactions with proteins that functionally modulate signaling. As a mitogen, E2 activates signaling in some target cells (2, 3, 7) but also inhibits signaling and proliferation induced by vascular growth factors in other cells (12, 13). These dichotomous findings illustrate an important but unknown mechanism whereby cytokines can act either in a positive or negative fashion, depending upon the cellular context.
Signaling cascades are activated when a growth factor binds its
transmembrane receptor, causing the translocation of proximal signaling
molecules to the plasma membrane (14). This often results
in the localization of these molecules to subdomains within the
membrane bilayer, including rafts and caveolae. Caveolae are
-shaped, invaginated microstructures that are found in most
mammalian cell types and communicate with the cell surface while
residing within the membrane (15). They function in
vesicular transport, endocytosis, and transcytosis (16),
but also play a role in signal transduction (17). Caveolae
are predominantly structurally composed of a family of proteins, known
as caveolins. Caveolin 1 and 2 are found in many cells expressing
caveolae (18), whereas caveolin 3 is restricted to muscle
cells (19). Growth factor receptors are enriched within
the caveolae (20) where they bind to caveolin proteins
(17) and form complexes with signaling molecules localized
to this structure (21). Caveolin-1 contains a
cytosolic, N-terminal juxtamembrane domain (scaffolding domain), which
binds to signaling molecules and inhibits their usual activation after
growth factor ligation of receptors (17, 22). Thus, within
the caveolae, an ER interaction with other signaling molecules may be
important for the propagation of signal transduction. In the studies
reported here, we examined E2-ER interactions with caveolin proteins
and the implications for the rapid, nongenomic effects of estrogen.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The caveolae attached on the cytoplasmic side of the membranes opposite
to the silica coating were stripped by shearing and then were isolated
by sucrose density centrifugation to yield a low buoyant density
fraction of intact caveolar vesicles (V). This was well separated from
the membranes stripped of the caveolae (P-V). As show in Fig. 1B, ER
is enriched in V relative to P. It is also detected in P-V, but to a
lesser extent than in V. This result is consistent with the confocal
localization, where ER colocalized extensively but not completely with
caveolin-1. Note that the nuclear membrane proteins, transportin and
NTF2, are not detected in V. As we previously reported (15, 24, 25), V was enriched in caveolin-1 but not 5'NT. Little
caveolin-1 signal remained in P-V.
We also took advantage of another technique (albeit not our preferred
method) that does not use silica coating for caveolae isolation
(25). Here, the homogenized lung is subjected to percoll
gradient centrifugation to yield a plasma membrane-rich fraction (PM)
that contains the plasma membrane markers 5'NT and caveolin-1 but also,
unfortunately, ß-Cop and one of the two nuclear membrane proteins
(transportin, but not NTF2). Sonication of PM followed by flotation via
sucrose density centrifugation yielded a low buoyant density fraction
(AC) quite enriched in caveolin-1. 5'NT, transportin, and ß-Cop, are
readily detected in AC, whereas NTF2 is only found in the homogenate
(H) with little to no signal elsewhere. We subjected AC to further
subfractionation to isolate the caveolae more selectively by
immunoaffinity separation. 5'NT, transportin, and ß-Cop are all
detected in the unbound fraction (U) with little to no signal in the
immunoisolated, caveolin-coated caveolae bound to the magnetic beads
(B). Both ER and caveolin-1 are found enriched in the
immunoisolated, caveolin-coated caveolae bound to the beads (B). Thus,
ER
and caveolin-1 are in the same vesicles. Past work shows that B
and V appear equivalent in molecular composition and both methods agree
here. Thus, ER
is contained within low-density, caveolin-coated
plasmalemmal vesicles, namely caveolae.
Caveolin Proteins Differentially Associate with Membrane ERs in
MCF-7 and Vascular Smooth Muscle Cells (VSMC)
Caveolin proteins constitute an important structural component of
caveolae but are also found to a smaller extent in noncaveolar
fractions of the membrane. We next determined whether ER and
caveolin proteins could associate in the membrane. This is potentially
important to begin to understand the organization of signaling
molecules within this membrane domain. We turned to cell systems in
which we have previously characterized E2 signaling through the
membrane and the attendant effects on cell biology (9, 12). Thus, MCF-7 and VSMC serve as potential models by which to
investigate how E2 can differentially stimulate or inhibit signaling.
We first immunoprecipitated ER
in the membranes of both VSMC and
MCF-7 cells, followed by blotting against caveolins, and found that
ER
can associate with either caveolin-1 or -2 (Fig. 2
). Similar results were found by
immunoprecipitating caveolin, followed by ER blotting (data not shown).
Interestingly, ligation of ER with E2 for 30 min strongly alters the
above association, and this occurs differentially in the two cell
types. In VSMC, there was relatively little association of ER and
caveolin-1 in basal cells, but in response to E2, the association
increased 3-fold (Fig. 2A
, top, lanes 1 and 2). However, in
the basal MCF-7 cells, there was a relatively strong association of
these two proteins in the membrane, but E2 addition caused a 67%
down-regulation of this interaction (Fig. 2A
, top, lanes 3
and 4). Similar results were found for caveolin-2, although the E2
stimulation of ER-caveolin-2 association was not as strong (only 50%
increased) in the VSMC (Fig. 2B
). To support the purity of the sucrose
gradient-isolated plasma membranes, immunoblots for ER and caveolin-1,
5'NT (integral membrane protein), transportin, and NTF2 (nuclear
proteins), and the endosomal/Golgi marker ß-Cop were carried out. As
seen in Fig. 2A
(bottom), 5'NT and caveolin-1 were enriched
in the plasma membrane fraction (P). However, the other three proteins
were not found, despite their presence in whole cell homogenates. ER
was detected in both samples, as expected.
|
We then assessed the situation in VSMC. First, we determined the
ability of E2 to affect ERK activity in VSMC (Fig. 2D, left). The low basal ERK activity was inhibited 44% by 10
nM E2, and the specific ER antagonist, ICI
182,780, prevented this. Furthermore, serum-induced ERK activity was
almost completely blocked by E2. When we expressed an activated MEK
construct in the VSMC, and incubated the cells with 10
nM E2, the augmented ER-caveolin-1 association
seen with E2 alone was significantly prevented (Fig. 2D
, right). Thus, the ability of E2 to differentially modulate
ER-caveolin-1 association in the two cell types is consistent with its
differential effects on ERK activity. This, in turn, is consistent with
the known differential effects of E2 on cell proliferation, positive in
MCF-7 and negative in VSMC, where proliferation/antiproliferation is
related to the appropriate modulation of ERK activity (7, 12). We also determined that ICI 182,780 blocked the
association-modulating actions of E2 in both cell types (Fig. 2E
),
consistent with ICI 182,780 effects on E2-modulated ERK activity.
Overall, this indicates that E2 specifically acts through ER to signal,
and thus influence, the association of ER-caveolin.
E2 Differentially Modulates Caveolin Production in MCF-7 and
VSMC
E2 could modulate the chronic interactions of ER-caveolin in part
by affecting caveolin production. This would likely occur through the
actions of the nuclear receptor. We therefore determined whether E2
could regulate caveolin protein synthesis. In VSMC, E2 stimulated the
production of newly synthesized caveolin-1 and -2 proteins after 8
h incubation in a dose-responsive manner (Fig. 3A, upper). These effects were significant
at physiologically relevant concentrations of 1 and 10
nM E2. In contrast, E2 significantly inhibited
caveolin-1 and -2 new protein production in MCF-7 cells (Fig. 3A
, lower). These findings could have important implications for
the ability of E2 to signal, since caveolin often serves as an
inhibitory scaffold protein (17, 22).
|
Does the ability of E2 to inhibit ERK (see Fig. 2D, left)
lead to the up-regulation of the reporter fusion gene activity in
theses cells? We found that expression of active MEK-1 significantly
inhibited the stimulatory effect of E2 (Fig. 3C
). These results support
overall the ideas that E2 inhibition of ERK stimulates ER-caveolin
association (Fig. 2D
, right) and the transcriptional
transactivation of caveolin-1 in the VSMC.
We then asked whether E2 might also modulate the stability of the
caveolin-1 protein. To examine this, we carried out pulse-chase
labeling studies in both MCF-7 and VSMC, in the presence and absence of
E2 (Fig. 3D). In VSMC, there was a 35% degradation of newly formed
caveolin-1 protein over an 8-h period in the absence of E2. In
contrast, the steroid completely prevented this degradation. Thus, the
increased levels of VSMC caveolin-1 protein in the presence of E2
reflect both the stimulation of transcription/synthesis, as well as the
inhibition of protein loss. In MCF-7 cells, the loss of newly produced,
labeled caveolin-1 over 8 h was only 13% in the absence of E2
(Fig. 3D
, right). However, the sex steroid accelerated
caveolin degradation, yielding a 45% decrease during this same time
period. Therefore, E2 inhibits the concentration of caveolin-1 protein
in these cells through several mechanisms.
Caveolin Expression Inhibits E2-Induced ERK Activity
What might be the role of caveolin-ER interactions in the
membrane? In MCF-7 cells, E2 rapidly stimulates signaling to ERK
activation, which is necessary for DNA synthesis or cell survival
(7, 9). In contrast, E2 inhibits growth factor activation
of ERK and the proliferation of VSMC (12). We hypothesized
that the differential modulation of caveolin production/association
that we have shown here might explain these divergent actions in
different cell types.
To support this hypothesis, we transfected different amounts of
caveolin-expressing plasmid (pCB7Cav-1) and assessed the ability of E2
to stimulate ERK activity. In MCF-7 cells expressing the empty vector
(Fig. 4, lane 2), E2 caused a 3-fold
increase in MAPK activity (lane 3). Transfecting increasing quantities
of caveolin-1 plasmid led to a concentration-related decrease in the
ability of E2 to activate ERK. The maximal 83% inhibition was seen
with 10 µg of caveolin-1 plasmid. (lanes 6 and 7, compared with lanes
2 and 3). These results directly indicate that caveolin impairs E2
signaling through ERK, consistent with its reported negative signaling
function (17, 22). In VSMC, E2 stimulates caveolin
production and enhances ER-caveolin association. These findings provide
a mechanism for our previous observations that E2 inhibits ERK
activation and ERK-mediated, growth factor-induced cell proliferation
in vitro (12).
|
|
To further support the role of caveolin-1 to facilitate ER
localization at the membrane, we examined Caco-2 rat intestinal
epithelial cells. These are among the few mammalian cells that lack
caveolae and do not produce caveolin-1 protein (28). We
confirmed the lack of caveolin-1 by immunoblot studies in the native
cells. These cells are reported to produce a small population of ER
when confluent, which we confirmed by our binding studies (see below).
In the control or pcDNA3 transfected cells, there was virtually no
specific binding in the membrane, and a small amount of binding in the
nucleus, consistent with a modest expression of endogenous ER detected
in the latter location (Fig. 3C). Upon expression of ER
, specific
binding was clearly detectable but was somewhat modest at the
membrane (Fig. 3
C, left). In part, this reflected our
transfection efficiency in these cells, which was approximately 20%,
determined by using a green fluorescent protein-ER
construct. However, coexpression of caveolin-1 increased the
specific binding by labeled E2 at the membrane nearly 70%. Expression
of caveolin-1 in the absence of ER was similar to that of control (data
not shown). In the nucleus, there was much more specific binding after
ER transfection (Fig. 5C
, right). This likely reflected the
predominance of ER at this location, similar to what we have shown in
CHO cells (6). However, caveolin expression did not
further enhance the binding of E2 at this site, indicating that there
was no facilitation of ER moving to this location in this model. The
results from the nuclear fraction rule out an effect of caveolin to
enhance E2 binding through a mechanism apart from facilitating
receptor translocation/number. The differential effects at the
two sites also support the lack of perinuclear membranes contaminating
our plasma membrane fractions. These findings in a non-overexpression
model support our contention that caveolin significantly facilitates ER
translocation to the plasma membrane.
Role of the Scaffolding Domain in ER-Caveolin Interactions
If caveolin-1 facilitates ER translocation to the membrane,
then it is probably necessary for the two proteins to associate in the
cytoplasm. Furthermore, it is not clear whether the scaffolding domain
of caveolin-1 is needed for the protein-protein interaction with ER or
whether it serves to target the steroid receptor to the membrane. The
latter role would be consistent with a known function of the
scaffolding domain (17). To examine this, we transfected
full-length or scaffolding domain mutant caveolin-1 into MCF-7. We then
immunoprecipitated ER from cytosol, followed by blotting for
caveolin-1, and also carried this out in reverse order. As seen in Fig. 6A (upper), there is a strong
association of caveolin-1 with ER
, and the association is the same
whether expressing full-length or scaffolding domain-deleted
mutant caveolin-1. Upon transfection with either construct, Western
blot for caveolin-1 revealed comparable amounts of caveolin-1 protein
(lower bands). Furthermore, the same amount of endogenous ER
protein was expressed across the experimental conditions. We therefore
conclude that that these two proteins associate both in cytosolic
and membrane compartments of the cell, and the scaffolding domain is
not generally required for the physical association of caveolin-1 with
endogenous ER.
|
Targeted Expression of ER E Domain to the Plasma Membrane
Activates ERK
To this point, we have demonstrated the localization of ER in the
plasma membrane and have presented data to support the proposal that it
is the membrane, and not the nuclear ER, that participates to activate
ERK. Another approach to this latter, important issue is to target ER
to each of the two cell locales and determine the effects of E2.
Recently, Kousteni et al. (29) showed that E2
acts through the cell membrane ER to prevent etoposide-induced
osteoblast cell death. This occurred after E2 activated a signal
transduction cascade that ultimately resulted in ERK activation, and
this signaling was required for cell survival. These authors also
targeted just the E domain of ER to the plasma membrane and showed
that this was sufficient to prevent HeLa cell death. Signaling to ERK
was not determined in the targeting model.
We therefore asked whether targeting the E domain to the plasma
membrane could result in the ability of E2 to activate ERK. We found
that in CHO cells transfected to express this domain at the plasma
membrane (E-Mem-ECFP) (29), E2 strongly activated ERK
activity (Fig. 7). In contrast, when the
E domain was targeted to the nucleus (E-Nuc-ECFP), there was no
activation of this MAPK by E2. E2 also could not activate ERK in the
native CHO cells, which we previously showed do not express endogenous
ER (6). Expression of the membrane-targeted E domain did
not result in E2 activating the -3 kb caveolin-1/luciferase reporter
(data not shown). This was expected, since in the VSMC, expression of
active MEK (and hence ERK) suppressed E2-induced caveolin-1/luciferase
reporter activation. Thus, it is likely that, in response to steroid,
the E domain portion of the membrane ER
activates the signaling to
MAPK activation.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Here we report that ER is found in the plasma membrane. Immunocolocalization shown by confocal microscopy indicated extensive, but not complete, overlap of ER with caveolin-1 in the whole-cell plasma membrane. Regarding our immunoblot studies, we determined that ER exists in noncaveolar compartments of the membrane, but predominantly is associated with isolated caveolae vesicles. The latter finding is consistent with a recent report that ER can be localized to caveolar subfractions of the endothelial cell (EC) plasma membrane (34). We additionally illustrate novel findings that ER associates with caveolin proteins in the plasma membrane and that E2 modulates this association differentially, dependent upon cell context. Furthermore, the differential modulation by E2 occurs through signal transduction to ERK via ER. In the MCF-7 cells, association of ER and cav-1 is down-regulated due to ERK activation, whereas in the VSMC, E2 inhibits basal or growth factor-induced ERK and up-regulates ER/cav-1 association. We also show that E2 differentially modulates the production of caveolin, and that this can occur in part through effects on transcription. The latter finding implicates an action of the nuclear receptor. E2 also modulates the stability of caveolin-1, occurring in cell-specific fashion. Finally, we demonstrate that caveolin-1 expression down-regulates E2-induced signal transduction and facilitates ER translocation to the membrane. Both of these functions require the caveolin scaffolding domain for full efficiency.
Caveolin appears to organize the association of signaling molecules
within the caveolae (17), including Ras, Src, and PI3K.
These molecules move to the membrane from cytoplasm to be activated
(35) and are found in caveolae, where they complex and
attach to caveolin proteins. Growth factor receptors also localize to
the caveolae, where downstream signaling, e.g. to ERK, may
be facilitated (17). Caveolae dynamics have been
elucidated for the activation of endothelial nitric oxide synthase
(eNOS). Caveolin-1 attaches to eNOS, keeping the enzyme in a relatively
inactive state in the caveolae (36). Activators of eNOS
trigger a calcium- and calmodulin-dependent displacement of caveolin-1
from eNOS, leading to the increased activity of the enzyme
(37). Also as a result, caveolin translocates out of the
plasma membrane. Recently, Chambliss et al.
(38) reported that ER and eNOS exist together in EC
membrane caveolar fractions, and this facilitates eNOS activation by
E2. The results of Chambliss et al. seem to be at odds with
the aforementioned studies (36, 37), which show that
caveolin protein inhibits E2 activation of signaling. However, we
believe that the caveolin proteins have a dual purpose in this setting.
Serving as a scaffold protein, caveolin helps assemble and localize the
signaling molecules into a complex that is capable of being activated.
Nevertheless, caveolin itself can inhibit signal activation and may
need to dissociate from the assembled complex, as for instance, to
allow eNOS activation (36, 37). There is a precedent for
this idea with other scaffold proteins. The kinase suppressor of Ras
(ksr) purportedly acts as a scaffold protein, forming a complex with
MEK, 143-3 proteins, ERK, and heat shock proteins 70 and 90, but
itself inhibits Ras signaling (39). Similarly, the Jip
family of proteins assemble signal molecules in the c-Jun N-terminal
kinase (JNK) pathway, thereby modulating JNK activation in the
cytoplasm, yet they restrain JNK from translocating to the nucleus and
thus inhibit the function of this kinase (40, 41).
E2 has been previously shown to rapidly stimulate NO production in the caveolae (23) through the activation of ERK (10), although it is not clear how ERK participates. We show that blocking E2 activation of ERK prevents the dissociation of ER and caveolin-1 within the MCF-7 membranes. Thus, we propose that the ability of E2 to activate eNOS (10) may result from an ERK-dependent dissociation of ER and caveolin, leading to the activation of NO synthase, perhaps through the recently described activation of PI3K and AKT (42). We also note that, because E2 can regulate caveolin transcription/production, this could influence the longer term interaction of membrane ER and caveolin, promoting localization of ER at the membrane and modulating signal transduction. This likely represents an example of the coordinated cellular actions of the nuclear and membrane pools of ER, a general concept that has a precedent in other cell models (9, 43).
In our studies, transfection of caveolin-1 cDNA in MCF-7 cells prevented E2 activation of ERK. Overexpression of caveolin-1 inhibits ERK activation by epidermal growth factor (44) and breast cancer cell migration and anchorage-independent growth (45). Transformation of cells by oncogenes is associated with a reduction or loss of caveolin-1 expression (45). Furthermore, stable expression of caveolin-1 antisense in NIH 3T3 cells leads to transformation that is reversed by restoring caveolin-1 protein to normal levels. In this model, hyperactivation of p42/p44 isoforms of ERK resulted from caveolin down-regulation (46). In human breast cancer specimens, endogenous ERK activity is consistently hyperexpressed (47). ERK activation in response to E2/ER action at the membrane significantly contributes to breast cancer cell growth and survival in vitro (7, 9). Therefore, the ability of E2 to down-regulate caveolin synthesis and association with ER in MCF-7, leading to the activation of signaling molecules such as ERK, is likely to be important in this regard.
In these same cells, we found that expression of exogenous caveolin-1 caused the loss of ER in the cytosol and an increased amount of ER expressed in the membrane. Although the effects were moderate, this probably reflects the presence of endogenous caveolin protein in the MCF-7 that may have limited the functional effects of overexpression on ER translocation to the membrane. More importantly, the total membrane pool of endogenous ER is only approximately 3% (6, 12), and therefore a limited number of ERs are available to move to the membrane under most circumstances. In a non-overexpression model for ER and caveolin-1, we found binding of E2 at the cell surface and that expressing caveolin-1 into cells that normally do not produce this protein greatly increased this binding, but only at the membrane. Importantly, the lack of enhanced E2 binding to ER in the nuclear fraction of Caco-2 cells rules out an alternative effect of caveolin to enhance E2 binding, through a mechanism apart from facilitating receptor translocation. In all, these data support our proposal that caveolin-1 facilitates the translocation of ER to the membrane, after binding to this receptor in the cytoplasm. In MCF-7, our results also extend the recent findings of Schlegel et al. (48), who demonstrated that overexpression of caveolin-1 results in translocation of ER from the cytoplasm to the nucleus. In a similar model in prostate cells, Lu et al. (49) have recently shown that caveolin-1 interacts with the AR and facilitates androgen transcriptional activity. We also found translocation of ER from the cytosol to the nucleus after overexpression in MCF-7 (our unpublished results). However, at steady state, 90% of caveolin-1 is at the plasma membrane (23) and, therefore, endogenous caveolin may be most important to facilitate ER movement to this location. Supporting this idea, caveolin-1 enhances the transport of the caveolin-2 protein to the plasma membrane (50).
Recently, Schlegel and Lisanti (51) determined that the plasma membrane attachment and caveolae targeting regions of caveolin-1 reside within the scaffolding domain (residues 82101) and the first 16 nucleotides of the C-terminal region (135150). In fact, caveolin-1 that lacks the scaffolding domain is incapable of reaching the cell surface (27). We found that the scaffolding domain is an important contributor to the ability of exogenous caveolin-1 to promote ER translocation, which is consistent with the aforementioned studies. Furthermore, these two proteins associate in the cytoplasm, but this is not dependent upon the presence of this domain. Therefore, we propose that the scaffolding domain facilitates ER localization at the membrane and, therefore, the full-length caveolin protein (but not the scaffolding domain-deleted protein) inhibits ERK activation. Importantly, these data further support the idea that it is the membrane-localized ER that modulates signal transduction cascades to MAPK (see below). The scaffolding domain may also serve a second role to restrain signaling, in some way, at the membrane (52, 53). A detailed analysis of other caveolin, as well as ER, domains that contribute to this process, and the mechanisms facilitating ER translocation and modulation of signal transduction in caveolae, is underway.
A still controversial issue is whether the membrane ER is responsible
for the activation of signaling (for instance to ERK) after E2 ligation
of endogenous steroid receptors. Previous results suggest that the
membrane receptor is important. This is based upon 1) the rapid effects
of E2 to stimulate a variety of signaling pathways, 2) the activation
of these pathways by a membrane-impermeable E2-BSA compound, and 3) the
linkage of the membrane ER to G protein activation and subsequent
signaling, an event that is known to occur only at the plasma membrane.
Here, we have taken a different approach and found that targeting the
expression of the E domain of ER to the plasma membrane allowed the
activation of ERK but did not result in the transactivation of an
estrogen response element/luciferase reporter by E2. For comparison,
targeting of the E domain to the nucleus did not result in E2-induced
MAPK activation. These results suggest that the E domain of the native
membrane receptor is important to activate the signaling molecules at
the plasma membrane that result in the subsequent activation of ERK
(29).
One important finding is that E2 can differentially modulate the production of caveolins and association with ER in the membrane, depending upon cell type. It is well appreciated that a variety of growth-modulating cytokines, including E2, can stimulate proliferation in one cell while inhibiting this process in another. Although this is perhaps related to modulating different signaling cascades in cell autonomous fashion (54, 55, 56), the exact details of these responses to proteins such as TGFß, platelet-derived growth factor, or angiotensin II is unknown. We propose that, at least for E2, this occurs in part via the differential modulation of caveolin production and association/function. It has been demonstrated that endothelial cell proliferation factors, such as vascular endothelial growth factor and basic fibroblast growth factor, inhibit caveolin-1 synthesis in EC (57), consistent with the effects of E2 reported here. Our findings allow us to hypothesize that the positive or negative modulation of caveolin production and association with signaling molecule complexes may contribute to the differential actions of other growth-regulatory cytokines as well.
As mentioned, the down-regulation of caveolin, and the subsequent activation of signaling, could contribute significantly to E2-induced growth and survival of breast cancer (7, 9). Furthermore, E2-related inhibition of VSMC proliferation and migration has been demonstrated in vivo after acute vascular injury (58). This may result from the up-regulation of caveolin dynamics at the membrane, as shown here. Increased caveolin would inhibit growth factor signaling to ERK, providing a mechanism by which E2 inhibits VSMC proliferation (12), and the reactive hyperplasia that results in the injured vessel wall in vivo (58, 59).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Subcellular Fractionation of Rat Lung Homogenates to Isolate
Endothelial Cell Plasma Membranes and Caveolae
The luminal EC plasma membranes and caveolae were isolated
directly from rat lung tissue using an in situ
silica-coating procedure (15, 24). Briefly, the rat lungs
were perfused via the pulmonary artery with a colloidal silica solution
to coat the surface endothelium. This allowed selective isolation of
the EC plasma membranes from the lung homogenate by centrifugation. The
caveolae were separated from the membrane by shearing and then isolated
by sucrose density centrifugation in a low buoyant density fraction,
well separated from the membrane pellet stripped of caveolae.
For translocation and other membrane studies, cells were washed three times with PBS, then lysed in buffer A (50 mM Tris-HCl, pH 7.5; 5 mM EDTA; 100 nM NaCl; 50 mM NaF; 100 µM phenylmethylsulfonyl fluoride; protease inhibitor cocktail; and 0.2% Triton X-100) and sonicated, after which nuclear pellets were collected through low-speed centrifugation. The supernatants were centrifuged at 100,000 x g for 30 min to pellet cell membranes. The cell membranes were then further separated by sucrose gradient overlay, and [in accordance with our extensive past experience (15, 16, 24)], fractions 35 contained the buoyant membranes (with caveolae and rafts) that were pooled for experiments. Briefly, membrane samples were placed into a tube with an equal volume of 85% (wt/vol) sucrose/25 mM A-morpholine-ethanesulfonic acid and 0.15 M NaCl solution, then overlaid with 8.5 ml of 35% sucrose, topped up with 16% sucrose, and centrifuged at 35,000 rpm for 18 h at 4 C. Ten fractions (1 ml each) were obtained and further processed, or separated on SDS-PAGE followed by membrane transfer for immunoblotting. The membrane fractions were immunoblotted with antibodies to caveolin-1 and 5'NT (plasma membrane proteins), transportin and NTF-2 (nuclear proteins), and ß-COP (endosomal/Golgi marker protein). Only caveolin and 5'NT were enriched and found in the early pooled fractions.
Kinase Activity Assays
For ERK activity assays, the cells were synchronized for 24
h in serum, phenol red, and growth factor-free medium. The cells were
then exposed to E2, 10 nM, for 8 min with or without
additional substances, as previously described (12).
Immunoprecipitated kinases were then added to substrate myelin basic
protein for in vitro assays (6, 9).
In some studies, the E domain of ER was transiently expressed in CHO
cells, from plasmid vectors that targeted this portion of the receptor
to either the nucleus (E-Nuc-ECFP) or the plasma membrane (E-Mem-ECFP)
(29). After recovery, the cells were synchronized without
serum for 12 h, after which 10 nM E2 was
added to the cells for 8 min, and ERK activity was then determined.
Transient Transfections
VSMC (passage 12) or MCF-7 were grown to 5060% confluence,
and then transiently transfected with LipofectAMINE and 1.5 µg of
fusion plasmid when cells were cultured in each well of six-well plates
(luciferase reporter studies). For all other studies, 10 µg total
DNA/100-mm dishes of cells were used. For reporter assays, the plasmids
included pA3-Luc, and Pr-750bp, Pr-3kb, and Pr-3 kb and Int 1/pA3 Luc,
which contain various lengths of the caveolin-1 promoter, upstream of
the ATG site and cloned into a luciferase reporter (pA3-luciferase)
(26). In other experiments, 10 µg pCB7Cav-1 (full
length), which expresses canine caveolin-1, or pCB7Cav-1 ( 60100),
which is missing the scaffolding domain segment, were used, except for
ERK studies in which 5, 10, and 15 µg plasmid were used. The cells
were synchronized and incubated with E2 as previously described
(6, 12). Cell extract supernatants were assayed by the
dual-luciferase reporter assay system (Promega Corp.,
Madison, WI). To correct for transfection efficiency, cells were
cotransfected with 0.1 µg of pRL-SV40 expressing the Renilla
luciferase (Promega Corp.). In other experiments VSMC,
MCF-7, or CHO cells were transfected with plasmids expressing a
constituitively active MEK-1 (Upstate Biotechnology, Inc.,
Lake Placid, NY).
Caveolin Synthesis
Cells were serum deprived for 24 h and then incubated in
methionine-free DMEM with dialyzed 10% FBS for 1 h before
experimentation. The cells were then incubated in the absence of serum
or unlabeled methionine, but with 250 µCi of
35S-methionine in the presence or absence of E2
for 8 h. Caveolin-1 or -2 protein was immunoprecipitated from
lysed cells, and the proteins were denatured in SDS and resolved by
PAGE, followed by fluorography and autoradiography. In additional
pulse-chase studies, the cells were labeled for 1 h with
35S-methionine, and then the medium was replaced
with 10-fold excess unlabeled methionine, in the presence or absence of
10 nM E2. At intervals over 8 h, the cells were lysed,
and caveolin-1 was immunoprecipitated and resolved by PAGE.
Coimmunoprecipitation and Immunoblot Protein Analysis
Membrane and cytosolic fractions were incubated with protein
A-Sepharose for 1 h, after which supernatants were transferred to
fresh tubes containing protein A-Sepharose conjugated to caveolin
proteins and incubated for 4 h at 4 C. Immune complexes were
washed and boiled and then separated by SDS-PAGE. After transfer to
nitrocellulose, the proteins were washed with blocking solution and
incubated with primary antibody to ER for 2 h and then with
horseradish peroxidase-conjugated second antibody (Santa Cruz Biotechnology, Inc.). Bound IgGs were visualized using enhanced
chemiluminescence reagents (Amersham Pharmacia Biotech,
Arlington Heights, IL) and autoradiography. In other experiments, as
described previously (15, 24, 25), rat lung protein
subfractions were solubilized and separated by SDS-PAGE and transferred
to nitrocellulose filters, followed by immunoblotting. Primary antibody
(diluted from 1:500 to 1:5,000 in Blotto, Sigma, St.
Louis, MO), was followed by the appropriate horseradish
peroxidase-labeled reporter antibodies (diluted 1:1,000). Reactivity
was visualized using enhanced chemiluminescence and quantified
densitometrically using ImageQuant (Quantum Images, San Diego, CA).
Protein concentrations were measured using the micro BCA protein assay
kit with BSA as a standard following a protocol previously described
(24, 25).
Immunofluorescence Microscopy
Bovine aortic endothelial cells were grown on coverslips and
then methanol fixed before dual immunofluorescence confocal microscopy
was performed (60). Cells were then stained with
monoclonal antibody to caveolin (1:250 dilution of clone Z034;
Zymed Laboratories, Inc., South San Francisco, CA) and
polyclonal antibody to ER (1:250 dilution of MC20; Santa Cruz Biotechnology, Inc.). Binding of primary antibody was detected
by a reporter IgG conjugated to Texas Red (antimouse IgG) and Bodipy
(antirabbit IgG) (Molecular Probes, Inc., Eugene, OR).
Binding Studies
Caco-2 cells (rat intestinal epithelial cells from
ATCC) were grown on 100-mm petri dishes in DMEM-F12
without phenol red. Twenty four hours after transfection with 5 µg of
pcDNA3-ER (plus 5 µg of backbone vector), or with 5 µg each of
both ER
and caveolin-1 constructs, the cultures were washed, and
lysed in buffer A (50 mM Tris-HCl, pH 7.5, 5 mM
EDTA, 100 nM NaCl, 50 mM NaF, 100
µM phenylmethylsulfonyl fluoride, protease inhibitor
cocktail, and 0.2% Triton X-100). Nuclear pellets were collected
through low-speed centrifugation. The supernatants were centrifuged at
100,000 x g for 30 min to pellet cell membranes. Both
pellets were washed twice, once without detergent. Fifty microliters of
membrane proteins from the cells were incubated with/without 1
µM unlabeled E2 but always with 50 µl
3H-E2 (specific activity, 80 Ci/mmol, pH 7.5)
(Perkin-Elmer Corp., Norwalk, CT) (1.4 pmol of labeled
steroid) at 37 C for 45 min, as previously described by us
(6). The pellets were washed three times and then
quantified by ß-scintillation counting. The differences in the
presence and absence of unlabeled E2 constituted specific binding.
![]() |
ACKNOWLEDGMENTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: CHO, Chinese hamster ovary; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; 5'NT, 5'-nucleotidase; NTF2, nuclear transport factor 2; VSMC, vascular smooth muscle cells.
Received for publication May 8, 2001. Accepted for publication September 14, 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|