From the Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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Previously we showed that activation of Erk in quiescent cells occurs in the caveolae fraction isolated from fibroblasts. Since the structure and function of caveolae is sensitive to the amount of cholesterol in the membrane, it might be that a direct link exists between the concentration of membrane cholesterol and mitogen-activated protein (MAP) kinase activation. We acutely lowered the cholesterol level of the caveolae fraction by incubating Rat-1 cells in the presence of either cyclodextrin or progesterone. Cholesterol-depleted caveolae had a reduced amount of several key protein components of the MAP kinase complex, including Ras, Grb2, Erk2, and Src. Incubation of these cells in the presence of epidermal growth factor (EGF) caused a rapid loss of EGF receptor from the caveolae fraction, but the usual recruitment of c-Raf was markedly inhibited. Despite the reduced amount of c-Raf and Erk2 in the cholesterol-depleted caveolae fraction, EGF caused a hyperactivation of the remaining caveolae Erk isoenzymes. This was followed by an increase in the amount of active Erk in the cytoplasm. The increased amount of activated Erk produced under these conditions was linked to a 2-fold higher level of EGF-stimulated DNA synthesis. Even cholesterol depletion by itself stimulated Erk activation and DNA synthesis. These results suggest that the MAP kinase pathway can connect the cholesterol level of caveolae membrane to the control of cell division.
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INTRODUCTION |
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Cholesterol is an important structural lipid that modulates the fluidity of biological membranes. Several new studies also indicate that membrane cholesterol has a direct role in cell signaling. The membrane-bound transcription factor SREBP1 controls cholesterol homeostasis (1). The release of the mature SREBP from the ER is regulated by SCAP, a protein containing up to 8 membrane-spanning regions. One of the transmembrane domains of SCAP appears to sense the level of cholesterol in the membrane and regulate the proteolytic cleavage of SREBP when cellular cholesterol gets low. Similar cholesterol "sensing" domains are present in the NPC1 (2) and PATCHED gene products (3). The former is a molecule involved in controlling cholesterol traffic from the lysosome whereas the later is a morphogen receptor. PATCHED is inhibited by the binding of Hh (hedgehog), a protein that is post-translationally modified in the ER by the covalent addition of cholesterol (4, 5). Cholesterol may anchor Hh to the membrane. These findings suggest that cholesterol-protein interactions can directly regulate the expression of genes important for cell behavior and development.
Another protein that interacts with cholesterol is caveolin-1 (6). This integral membrane protein is found principally in surface caveolae and the Golgi apparatus (7, 8) of many cells. Caveolin-1 appears to have a major function in carrying cholesterol between ER and caveolae membranes (9). This is accomplished using a novel membrane traffic route that shuttles caveolin-1 back and forth between the two compartments (10). Ordinarily the cholesterol-to-protein ratio of the caveolae fraction is 4-5 times higher than the surrounding plasma membrane (10). The maintenance of this level of cholesterol in the caveolae fraction of fibroblasts and transformed lymphocytes (10) appears to depend on the caveolin-1 shuttle. Experimentally lowering the cholesterol level of the caveolae fraction disrupts the molecular organization of the domain (11) and inhibits internalization of both molecular (11) and particulate material by potocytosis (12). Treatment of cells with cholesterol-binding drugs such as filipin appears to have a similar effect (13).
In addition to being a portal of entry into the cell, caveolae are the surface locations where multiple signaling pathways converge (9, 14). One of the pathways organized at this site links multiple receptor tyrosine kinases to MAP kinases (15, 16). Interactions between as many as 11 different molecules can take place during activation of a MAP kinase, and representatives of each are present in the caveolae fraction of quiescent cells (17). These molecules apparently are functionally linked to MAP kinase because incubation of fibroblasts in the presence of growth factors activates the Erk in the caveolae fraction, which is followed by the appearance of the active enzyme in the cytoplasm (17). Incubation of isolated caveolae fractions in the presence of platelet-derived growth factor also activates the resident Erk enzymes (17). The molecular machinery responsible for holding these molecules so they can optimally interact during signal transduction is not known. Based on what is known about caveolae membrane structure (9), however, cholesterol could be an important determinant in organizing the MAP kinase complex. We now report that depletion of cholesterol from the caveolae fraction results in a decline in several key proteins of the MAP kinase complex, an increase in the sensitivity of the resident Erk enzymes to stimulation, and an increase in mitogenesis.
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EXPERIMENTAL PROCEDURES |
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Materials
Fetal calf serum was from Hazleton Research Products, Inc.
(Lenexa, KS). Dulbecco's modified Eagle's medium (DMEM), glutamine, trypsin-EDTA, penicillin/streptomycin, and OptiPrep were from Life
Technologies, Inc. Percoll was from Amersham Pharmacia Biotech. EGF was
from Calbiochem. Immobilon transfer nylon (polyvinylidene difluoride)
was from Millipore. ECL Western blotting detection reagents were from
Amersham. 2-Hydroxypropyl--cyclodextrin and cholesterol were from
Sigma. [3H]Thymidine (86.2 Ci/mmol) was from NEN Life
Sciences. Antibodies were obtained from the following sources.
Anti-Raf-1 (mAb), anti-Ha-Ras (mAb), anti-Sos-1 IgG (mAb), anti-Erk2,
anti-Grb2 IgG (mAb), and anti-caveolin-1 (pAb) were from Transduction
Laboratories (Lexington, KY). Anti-pan Ras IgG (mAb) and anti-EGF
receptor IgG (mAb) were from Calbiochem. Anti-Src (mAb) and anti-Shc
(pAb) were from Upstate Biotechnology Inc. (Lake Placid, NY).
Anti-active Erk1 and -2 (pAb) were from Promega (Madison, WI).
Anti-G
i (pAb) and anti-G
(pAb) were gifts
from Dr. Susanne Mumby (The University of Texas Southwestern Medical
Center at Dallas). Both goat anti-rabbit IgG and goat anti-mouse IgG
conjugated to horseradish peroxidase were from Cappel (Durham, NC).
Methods
Cell Culture-- Two culture conditions were used. For all of the immunoblotting experiments, Rat-1 cells (5 × 105 cells) were seeded in 100-mm dishes and grown in 10 ml of DMEM supplemented with 10% (v/v) fetal calf serum for 2 days. Cells were then incubated for 24-40 h in serum-free DMEM before the indicated treatments. For all of the mitogenesis experiments, Rat-1 cells (105 cells) were seeded into 12-well plates and grown for 2 days in 1 ml of DMEM supplemented with 10% (v/v) fetal calf serum. Cells were washed once with 1 ml of DMEM and incubated further in DMEM without serum for 24 h at 37 °C.
Cholesterol Depletion-- Cyclodextrin (CD) was dissolved in DMEM and used directly. Cholesterol depletion was carried out by incubating cells in the presence of cyclodextrin for 1 h at 37 °C. Cells were repleted with cholesterol by incubating them in the presence of a cholesterol/CD mixture for 1 h at 37 °C. A stock solution of 0.4 mg/ml cholesterol and 10% cyclodextrin was prepared by vortexing at ~40 °C in 10 ml of 10% cyclodextrin with 200 µl of cholesterol (20 mg/ml in ethanol solution). Each solution was filtered through a 0.2-µm filter before use.
Isolation of Caveolae-- Detergent-free caveolae fractions were prepared using two OptiPrep gradients according to the method of Smart et al. (18). Fourteen fractions were collected from the first OptiPrep gradient. Fractions 1-7 were collected, mixed with 50% OptiPrep, and overlaid with 1 ml of 15% OptiPrep plus 0.5 ml of 5% OptiPrep and centrifuged at 52,000 × g for 90 min. An opaque band located between 15% and 5% interface was collected and designated as the caveolae fraction. Fractions 8 and 9 from OptiPrep 1 were pooled and designated the intermediate fractions while pooled fractions 10-14 were designated the heavy fractions.
Electrophoresis and Immunoblotting-- Proteins were separated by SDS-polyacrylamide gel electrophoresis (19), transferred to Immobilon-P membrane, and incubated in 5% nonfat milk dissolved in TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20). Primary antibodies were diluted in TBS plus 1% nonfat milk using the dilutions recommended by the suppliers. The membranes were incubated in the presence of the indicated primary antibody overnight at 4 °C and washed 4 times in TBS for 5 min at room temperature. The appropriate secondary antibody conjugated to horseradish peroxidase was diluted (from 1:5,000 to 1:30,000) in TBS plus 1% nonfat milk. The membranes were incubated in the presence of the second antibody at room temperature for 1-2 h. The membranes were then washed 5 times with TBS at room temperature. ECL was used to visualize the reactive proteins.
DNA Synthesis-- Cells were incubated in the presence of the indicated concentrations of either CD or cholesterol-CD complex for 1 h at 37 °C. The cells were then incubated in the presence or absence of EGF (50 ng/ml) for 21 h at 37 °C. [3H]Thymidine (1 µCi, 0.4 Ci/mmol) was added to each well, and the cells were incubated for an additional 3 h at 37 °C. The medium was removed, and the cells were washed twice with 1 ml of phosphate-buffered saline. Ice-cold 10% trichloroacetic acid (1 ml) was then added to the well for 30 min to precipitate the DNA and remove all soluble isotope. Finally, the precipitate was dissolved in 0.5 ml of 0.1 N NaOH, mixed with 10 ml of scintillation fluid, and counted in a Beckman LS 6000SC.
Other Methods-- Protein concentrations were determined using the Bradford assay (20). Cholesterol was measured according to the method of Gamble et al. (21).
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RESULTS |
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Modulation of Caveolae Cholesterol--
We used Rat-1 cells and
EGF to study the effects of cholesterol depletion on MAP kinase
activation. These cells synthesize very little cholesterol after
growing for 24-48 h in the absence of serum (data not shown).
Cholesterol was specifically (22) removed from the plasma membrane
(~65% decrease) by incubating the cells in the presence of 2%
cyclodextrin (2-hydroxypropyl--cyclodextrin) for 1 h at
37 °C. The membrane cholesterol levels returned to near normal when
the cells were subsequently incubated in the presence of a cholesterol
(16 µg/ml)-cyclodextrin (0.4%) complex for 1 h. No change
occurred, however, if the complex was omitted from the incubation
media.
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Disruption of MAP Kinase Complex following Cholesterol
Depletion--
Previously we showed that the major protein components
of the MAP kinase complex are concentrated in the caveolae fractions isolated from quiescent cells (17). We used immunoblotting to determine
whether cholesterol depletion had any effect on the concentration of
these proteins (Fig. 2). Equal amounts of
protein (5 µg/lane) from cholesterol-depleted (lane
2) and non-depleted (lane 1) caveolae
fractions were separated by gel electrophoresis and blotted with
specific antibodies. As we have shown in other studies (15-17, 23,
24), caveolae fractions from control cells were enriched in caveolin-1
(Cav), EGF receptors (A, EGF-R), all three isoforms of Shc (A, Shc), Grb2
(A, Grb2), both Gi (A, G
) and G
(B,
G
) heterotrimeric proteins, both Ki- and
Ha-Ras (A, K-Ras, H-Ras), and Src
(B, Src). In cholesterol-depleted caveolae, by
contrast, the amount of caveolin-1, Grb2, Ras, and Src was markedly
reduced. Shc isoforms and G
were decreased to a lesser
extent. EGF receptors and G
i were unchanged, suggesting
that cholesterol depletion does not alter the entire protein
composition of caveolae. The concentration of each of the depleted
proteins increased after cells were incubated in the presence of the
cholesterol-CD complex (lane 4), but not if the cells were
incubated in media alone (lane 3).
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Erk in Cholesterol-depleted Caveolae Is Hyper-responsive--
We
used an antibody that recognizes activated Erk1 and -2 to determine how
the cholesterol depletion of the caveolae fraction affected the ability
of EGF to activate these enzymes (Fig.
4). Two different incubation sequences
were used to look at early (0, 1, 3 min, lanes 1-6) and
late (0, 5, 10 min, lanes 7-12) effects of EGF. Cells were
preincubated in the presence (CD+) or absence (CD) of cyclodextrin
before the addition of EGF, and both the caveolae (Cav) and
cytoplasmic (Cyt) fractions were analyzed. The EGF receptor
(EGF-R) moved out of the cholesterol-depleted caveolae
fraction with the same rapid kinetics as it does from untreated
caveolae fractions (Cav, compare lanes 1-3 with
4-6). Cholesterol depletion also suppressed c-Raf
(c-Raf) recruitment and decreased the level of Erk2
(Erk2) without affecting the G
i (G
) concentration (Cav, compare lanes
1-3 with 4-6 and 9-11 with
12-14). By contrast, cholesterol depletion caused a marked increase in the amount of activated Erk (phoMAPK) in the
caveolae fraction Cav, compare lanes 3 with
6 and 9-11 with 12-14). Even cyclodextrin alone caused a significant increase in activated Erk
(Cav, compare lanes 1 with 4 and
9 with 12). Usually the appearance of activated
Erk in the cytoplasm lags a little behind the caveolae fraction (17),
but after 5 or 10 min in the presence of EGF we saw a similar dramatic
increase in the amount of active enzyme in the cytoplasmic
fraction of cholesterol-depleted cells (Cyt, compare
lanes 9-11 with 12-14. The cytoplasmic
concentrations of c-Raf and Erk2 were unaffected by cholesterol
depletion.
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DISCUSSION |
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Two experimental conditions that acutely lower the cholesterol level of the caveolae fraction cause a resident population of MAP kinase to become hyper-responsive to EGF. While cyclodextrin caused a general cholesterol depletion of the plasma membrane, progesterone removed cholesterol specifically from the caveolae fraction. The effects of cholesterol depletion on caveolae MAP kinase activity were blunted by incubating the cells in the presence of cyclodextrin plus cholesterol. These results suggest that the caveolae MAP kinase signaling pathway is influenced by how much cholesterol is in the membrane.
Cholesterol depletion caused a disruption in the molecular organization of the MAP kinase signaling complex. Lowering the cholesterol caused a decline in many of the key molecules in the complex, including Erk2. It also blocked EGF-stimulated c-Raf recruitment to caveolae, as well as the recruitment of other molecules in the complex. This loss of organization might have two potential effects. 1) It uncoupled a feedback loop between EGF receptors and Erk2, leading to the generation of higher than normal amounts of activated Erk2 per bound EGF. 2) The decreased concentration of Erk in caveolae caused a hyperphosphorylation of the enzyme by MEK (25). The MAP kinase activated under these circumstances could stimulate DNA synthesis nearly 2-fold above EGF alone. Even cholesterol depletion by itself activated Erk and stimulated DNA synthesis. Therefore, a direct connection exists between the downstream signaling activities of Erk and the cholesterol content of the plasma membrane.
Biophysical studies (26-28) suggest caveolae membrane lipids are in a
liquid-order phase (referred to as the phase) because of a high
concentration of glycosphingolipids, sphingomyelin, and cholesterol.
The
phase has characteristic physical properties, such as
resistance to solubilization by Triton X-100 (28) and a relatively low
lateral mobility of resident lipids (26). This special lipid
environment appears to be important for attracting and organizing
lipid-anchored proteins (glycosylphosphatidylinositol, acyl, and prenyl
anchors) in caveolae. Since cholesterol is a critical ingredient for
the
phase transition (28), decreasing the cholesterol content of
caveolae can directly affect the number and organization of
lipid-anchored proteins as a result of changing the phase properties of
the bilayer (11, 29). Any protein that interacts with lipid-anchored
proteins in caveolae will be similarly affected. Therefore, the loss of
components from the MAP kinase complex following cholesterol depletion
as well as the hyper-responsive Erk can be traced to a stringent
requirement for high cholesterol to maintain the proper organization of
caveolae membranes.
The maintenance of high cholesterol levels in caveolae is an active process. Newly synthesized cholesterol transported directly from the ER to the plasma membrane (30) initially appears in the caveolae membrane (10). Caveolin-1 may be necessary for caveolae to have high cholesterol levels (10), which suggests it is a component of the ER-to-caveolae shuttle. Moreover, caveolin-1 moves from caveolae through the ER to the Golgi apparatus when caveolae cholesterol becomes oxidized (31). Caveolae can also acquire cholesterol released from low density lipoproteins in lysosomes (32). Furthermore, the high density lipoprotein-binding protein SR-B1, which binds both normal and damaged lipoproteins (33), has been localized to caveolae (34) where it may facilitate the bidirectional transfer of cholesterol between lipoproteins and caveolae. The convergence of both cholesterol import and export at caveolae provides the cell with redundant mechanisms for delivering cholesterol to this membrane domain.
Several pathological conditions exist where the concentration of cholesterol in caveolae might influence the transduction of mitogenic signals. Caveolae cholesterol levels could either be too high or too low. Overaccumulation of cellular cholesterol is common in cells within atherosclerotic lesions. No one has studied the MAP kinase activity in caveolae of cholesterol-enriched cells, but our results predict the enzyme would be hyporesponsive to growth factors such as EGF. Recently, Pomerantz et al. (35) reported that cultured smooth muscle cells with elevated levels of cholesterol exhibit a reduced MAP kinase activity in response to platelet-derived growth factor. At the other extreme, transformed cells have below normal amounts of caveolae cholesterol, which is restored to normal after the cells are transfected with caveolin-1 (10). Loss of caveolin-1 often occurs in cells infected with oncogenic viruses (36), and expression of caveolin-1 in transformed cells can revert the cell to a normal phenotype (37). This suggests caveolin-1 acts as a tumor suppresser (38). Since the hyper-responsive MAP kinase in cholesterol-depleted caveolae is mitogenic (Fig. 7), the tumor-suppressing effect of caveolin-1 may be due to its ability to return the concentration of cholesterol in caveolae to normal.
The ability of cholesterol to directly influence an entire signal pathway suggests that the concentration of cholesterol in each membrane compartment within the cell must be tightly controlled. Otherwise signals originating from sources such as the EGF receptor would be unreliable. Therefore, in addition to balancing endogenous and exogenous sources of cholesterol (1), cells must regulate the supply of cholesterol to specific membrane compartments such as caveolae. This level of regulation should involve at least two components: a sensing device that detects how much cholesterol is present in the membrane of the compartment and a transporter that responds to the cholesterol sensor by delivering or removing cholesterol. Caveolin-1 is a candidate component of the transporter that supplies cholesterol for the caveolae compartment. So far no one has identified a protein in caveolae that contains a transmembrane domain homologous to those thought to be involved in detecting membrane cholesterol (1).
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ACKNOWLEDGEMENTS |
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We would like to thank William Donzell and Ann Horton for their valuable technical assistance and Stephanie Baldock for administrative assistance. Drs. Pingsheng Liu and Chieko Mineo provided valuable insights during the course of these studies.
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FOOTNOTES |
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* This work was supported by grants HL 20948 and GM 52016 from the National Institutes of Health and the Perot Family Foundation.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.
To whom correspondence should be addressed. Tel.: 214-648-2346;
Fax: 214-648-7577; E-mail: anders06{at}utsw.swmed.edu.
The abbreviations used are: SREBP, sterol regulatory element-binding protein; ER, endoplasmic reticulum; MAP, mitogen-activated protein; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; mAb, monoclonal antibody; pAb, polyclonal antibody; CD, cyclodextrin.
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REFERENCES |
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