From The Jerome Lipper Multiple Myeloma Center,
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard
Medical School, Boston, Massachusetts 02115, the
§ Department of Internal Medicine, University of Michigan,
Medical Center, Ann Arbor, Michigan 48109-0640, ¶ Leeds General
Infirmary, Leeds 3EX LS1, United Kingdom, and the
Veterans
Affairs Health Care Center, Brockton, Massachusetts 02401
Received for publication, August 22, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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Caveolae, specialized flask-shaped
lipid rafts on the cell surface, are composed of cholesterol,
sphingolipids, and structural proteins termed caveolins; functionally,
these plasma membrane microdomains have been implicated in signal
transduction and transmembrane transport. In the present study, we
examined the role of caveolin-1 in multiple myeloma cells. We show for
the first time that caveolin-1, which is usually absent in blood cells,
is expressed in multiple myeloma cells. Analysis of myeloma
cell-derived plasma membrane fractions shows that caveolin-1 is
co-localized with interleukin-6 receptor signal transducing chain gp130
and with insulin-like growth factor-I receptor. Cholesterol depletion
by Multiple myeloma (MM)1
is characterized by the clonal proliferation of malignant plasma cells
in the bone marrow (BM) associated with bone loss, renal disease, and
immunodeficiency. The annual incidence of MM is ~3.8 per 100,000 population (~14,400 new cases of MM/year), and it is almost twice as
common in the black versus Caucasian population (1). The
median age at diagnosis is ~62 years, and despite current therapeutic
approaches, the median survival remains at 3-4 years. New biologically
based targeted therapies are therefore urgently needed. Cholesterol is
an essential component of MM cell membranes, has been implicated in
disease pathogenesis, and represents one such target (2). Cholesterol and sphingolipids form lipid rafts, which represent dynamic assemblies in the otherwise homogeneous, phospholipid-rich two-dimensional lipid
layer of the plasma cell membrane. As "liquid-ordered" membranous microdomains, they function as platforms for membrane trafficking and
signal transduction, initiating complex protein-protein interactions between ligands, receptors, and kinases in response to intra- and
extracellular stimuli (3). However, the role of lipid rafts in MM cell
growth and survival in the BM milieu is not yet defined.
Caveolae ("little caves") are specialized lipid raft microdomains
forming 50-100 nm flask-shaped vesicular invaginations of the plasma
membrane, which serve as a scaffold for signaling molecules related to
cell adhesion, growth, and survival (4). They are composed of
caveolins, 22-24-kDa integral membrane proteins with cytoplasmic N and
C termini, and a central intramembrane domain, which forms an unusual
hairpin loop structure in the membrane (5, 6). Caveolins are
functionally and structurally highly conserved and initiate formation
of caveolae from raft-derived components. Specifically, caveolin-1
(Cav-1) is required for caveola formation, because caveolae are not
formed in the Cav-1 knockout mouse (7, 8); conversely,
caveolin-deficient lymphocytes acquire plasma membrane caveolae when
transfected with Cav-1 cDNA (9). Three distinct caveolin genes have
been identified: caveolin-1, caveolin-2, and caveolin-3; in addition,
alternate initiation of translation creates two isoforms of Cav-1,
Cav-1 Caveolae regulate signal transduction (13, 14, 20, 21); specifically,
Cav-1 interacts with lipid-anchored, integral membrane and soluble
signaling proteins including Src family tyrosine kinases, G-protein
subunit In the current study we demonstrate for the first time that Cav-1 is
present in human MM cells, and we suggest a functional model in which
caveolae play a pivotal role in interleukin-6 (IL-6)- and insulin-like
growth factor-I (IGF-I)-induced complex formation and activation of
downstream phosphatidylinositol 3-kinase (PI3-K)/Akt-1 signaling,
which enhances MM cell survival and confers resistance to dexamethasone
(27). These studies provide the basis for novel therapeutic strategies
targeting caveolae, Cav-1, and other structural components of the MM
cell membrane to improve patient outcome.
Materials--
Recombinant human IL-6 was purchased from
PeproTech, Inc. (Rocky Hill, NJ), and recombinant human IGF-I was from
R&D Systems (Minneapolis, MN).
4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d])pyrimidine (PP2), a Src family tyrosine kinase inhibitor, and lovastatin, an
irreversible inhibitor of HMG-CoA reductase, were obtained from
Calbiochem, and Cell Culture, IL-6 and IGF-I Stimulation--
All human MM
(ARP-1, Dox40, MM.1R, MM.1S) cell lines, as well as freshly isolated
patient plasma cell leukemia (PCL) cells, were cultured in RPMI 1640 media (Cellgro, Herndon, VA) and supplemented with 10%
heat-inactivated fetal bovine serum (FBS) (Harlan, Indianapolis, IN),
100 units/ml penicillin, 10 µg/ml streptomycin, and 2 mM L-glutamine (Cellgro). Prior to stimulation of cells with
IL-6 (100 ng/ml) or IGF-I (200 ng/ml), they were incubated overnight in
RPMI 1640 with 2% FBS and then for an additional 3 h in RPMI 1640 without FBS. Isolation of Tumor Cells from Patient--
After appropriate
informed consent, mononuclear cells were obtained by Ficoll-Paque
(Amersham Biosciences) from a patient with PCL. MM patient cells (96%
CD38+CD45RA Microarray Assay--
Total RNA was isolated from CD138+
purified cells from 8 newly diagnosed MM patients and 4 newly diagnosed
MGUS patients using TRIzol Reagent (Invitrogen). Double-stranded
cDNA was prepared from 5 µg of total RNA using Invitrogen
Superscript choice system and oligo(dT)-anchored T7 primer. Cell
viability was performed using trypan blue exclusion. Affymetrix huGene
U95AV2 arrays containing 12,600 genes (Santa Clara, CA) were used for
mRNA expression profiling, as described previously (30, 31).
Digitalized image data were processed using GeneChip Microarray Suite
software 4.0 (Affymetrix) and processed using the DNA Chip Analyzer
(32). Internal controls of housekeeping genes and a test chip were run
prior to test samples. Statistical significance of differences in Cav-1
expression in MGUS patients versus MM patients was
determined using an unpaired Student's t test.
Reverse Transcriptase-PCR Analyses of Caveolin-1--
Total RNA
was extracted from 2 × 106 cells using TRIzol Reagent
(Invitrogen). cDNA was synthesized by means of SuperScript One-step
RT-PCR system with Platinum Taq (Invitrogen). Primers (30 pg) used were caveolin-1-F, 5'-CTA CAA GCC CAA CAA CAA GGC-3', and
caveolin-1-R, 5'-AGG AAG CTC TTG ATG CAC GGT-3'. The expected size of
the amplification product was 340 bp (33). Expression of
glyceraldehyde-3-phosphate dehydrogenase was used as a control to
measure integrity of the RNA samples. To ensure that RNA samples were
not contaminated with DNA, purified RNA was incubated with the
appropriate primers and Taq polymerase, without reverse
transcriptase. PCR products were separated on a 1% agarose gel and photographed.
Cell Lysis, Immunoprecipitation, and Immunoblotting--
After
stimulation with IL-6 or IGF-I, cells were washed three times with PBS,
lysed with lysis buffer (10 mM Tris, 50 mM
NaCl, sodium pyrophosphate, 1% Triton X-100, 1 mM
Na3VO4, and 1× protease inhibitor mixture
(Roche Molecular Biochemicals)), and further processed for
immunoprecipitation and immunoblotting, as in previous studies (34). As
a control for immunoprecipitation, nonspecific protein binding and
detection were excluded by incubating protein A-Sepharose beads with
lysis buffer and specific antibody only.
Detergent-free Purification of Caveola-enriched Membrane
Fractions--
Low density, caveolin-enriched membrane fractions were
prepared by a detergent-free method, using discontinuous sucrose
density gradient centrifugation (35, 36). After collecting the
fractions from the top of the gradient, they were processed for
immunoblotting using the Abs indicated.
Thin Section Electron Microscopy--
1 × 106
MM.1S cells were grown for 2 days in 15-cm tissue culture plates,
washed three times with PBS, and fixed for 30 min at room temperature
(2.5% glutaraldehyde, 0.1% tannic acid in 0.1 M
cacodylate buffer, pH 7.4). After three rinses (0.1 M
cacodylate buffer) and fixation (1% osmium tetroxide in 0.1 M cacodylate buffer, 30-60 min at room temperature), the
cells were dehydrated through graded ethanol and flat-embedded in epoxy
resin (Agar 100). Thin sections (60-80 nm) were cut parallel to the
plane of the culture on a microtome (Reichert Ultracut-S microtome), mounted on copper grids, and contrasted with 1.5% uranyl acetate and
0.1% lead citrate. Specimens were examined and photographed using an
electron microscope (JEOL Ltd. 1200EX transmission electron microscope).
PI3-Kinase Activity Quantitation--
PI3-kinase assays were
performed as described previously (37). The radioactivity was
visualized by autoradiography (BIOMAX MR Film, Eastman Kodak).
Autoradiograms were scanned, and the images were analyzed with NIH
Image 1.62 software. The relative changes in
phosphatidylinositol-3,4,5-P3 produced in the PI3-kinase assays were calculated as a percentage of
phosphatidylinositol-3,4,5-P3 production.
MTT Colorimetric Survival Assay--
The inhibitory effect of
Cell Cycle Analysis--
MM.1S cells cultured for 48h in Increased Caveolin-1 Expression in MM Versus MGUS
Patients--
Recent studies have utilized cDNA microarray
methodology to improve diagnosis and classification of cancer (39, 40).
MM can be preceded by MGUS, characterized by monoclonal protein in low
amounts in otherwise normal individuals (41). To define target genes
associated with the transition from MGUS to MM, we first performed
cDNA microarray profiling of 12,600 genes in CD138+ cells isolated
from BM mononuclear cells from 8 untreated MM patients and 4 individuals with MGUS. As shown in Fig.
1a, Cav-1 was up-regulated in
MM (158 ± 36) versus MGUS (49 ± 24) samples,
suggesting a possible role for caveolae in the transition of MGUS to
MM.
Expression of Caveolin-1 in MM Cells--
To confirm the
expression of Cav-1 in MM cells, we performed RT-PCR analysis. As shown
in Fig. 1b, all MM cell lines expressed Cav-1, whereas
normal B-cells lacked Cav-1. NIH3T3 cells served as a positive control
for caveolin expression. Cav-1 expression was further confirmed by
Western blot analysis of the above MM cell lines and tumor cells from a
patient with plasma cell leukemia (PCL) (Fig. 1c). NIH3T3
cells and human endothelial cells served as positive controls for
expression of Cav-1, whereas normal B-cells were a negative control. To
confirm equal protein loading, the membrane was stripped and re-probed
with an Ab directed against actin.
IGF-IR, gp130, and p85, but Not ERK, Co-localize with Cav-1 in
Caveolae-enriched Microdomains--
The role of Cav-1 and caveolae in
initiating insulin receptor signal transduction is well characterized
(42-44), and a functional link between Cav-1 and IGF-I receptor
(IGF-IR) has been defined recently (45) whereby IGF-IR co-localizes
with Cav-1 in the lipid raft fraction, and IGF-I induces Cav-1
phosphorylation at Tyr-14. Moreover, a recent report (46) shows
that interleukin-6 receptor signal transducing chain gp130 (gp130),
Cav-1, and signal transducer and activator of transcription (STAT3) are
present in lipid raft fractions of human hepatoma Hep3B cells. Although the role of IL-6 and IGF-I in mediating MM cell growth and drug resistance via PI3-K/Akt-1 signaling is well established (27, 47-49),
whether caveolae and Cav-1 initiate IL-6- and IGF-I-triggered signaling
is unknown. Therefore, we next investigated whether IGF-IR and gp130
are co-localized with Cav-1 in MM cells. Caveolin-enriched membrane
fractions were prepared by detergent-free purification using
discontinuous sucrose density gradient centrifugation (35, 36); the
plasma membrane caveolae fraction resides mainly in the light buoyant
membranes (Fig. 2, fractions 4 and 5), as evidenced by detection of Cav-1 in unstimulated
(Fig. 2a) and stimulated (Fig. 2, b and
c) MM.1S cells. Western blot analysis for gp130, IGF-IR, and
Cav-1 demonstrated that Cav-1 co-migrated with gp130 and IGF-IR,
respectively, independent of stimulation with both IGF-I and IL-6.
Importantly, IGF-I and IL-6 increased PI3-K protein expression,
evidenced by immunoblotting with an anti-p85 Ab directed against the
regulatory p85 subunit of PI3-K in the caveolae fractions. In contrast,
IGF-I and IL-6 did not change the distribution of phosphorylated or
total extracellular signal-regulated kinase (ERK) in the caveolae
fractions (Fig. 2, a-c).
To confirm these results, cells were lysed in lysis buffer
containing Triton X-100, and immunoprecipitations were performed using
anti-IGF-IR, anti-gp130, and anti-Cav-1 Abs on cell lysates prepared
from control as well as IGF-I- and IL-6-treated MM cells. Fig.
3, a and b, shows
that Cav-1 and IGF-IR co-immunoprecipitate, indicating that they are
present in the same detergent-resistant membrane fragments independent
of the activation state of the IGF-IR. Moreover, gp130 and Cav-1 also
co-immunoprecipitate, also indicating their presence in the same
detergent-resistant membrane fragments independent of both
IL-6-triggered gp130 phosphorylation (data not shown) and formation of
IL-6 receptor Effect of Cav-1 Tyrosine Phosphorylation Induced by IL-6 and IGF-I Is
Associated with Src Family Tyrosine Kinase Activation and Intact
Caveolae Structure--
Other studies have shown that Cav-1 is
phosphorylated on Tyr-14 by Src, Fyn, and Abl in response to
insulin, angiotensin II, osmotic shock, oxidative stress, and IGF-I
(44, 45, 53-56). In MM cells, IL-6 stimulates Src and p59Fyn,
p56/59Hck, and p56Lyn activation (57). Because tyrosine phosphorylation
facilitates binding to Src homology 2 (SH2) domain-containing proteins
(58), we hypothesized that phosphorylation of Cav-1 may be an
intermediate step by which SH2 domain-containing proteins are recruited
to caveolae to initiate downstream signaling cascades. Therefore, we
next investigated the following: 1) whether Src associates with Cav-1;
2) whether IL-6- and IGF-I-induced tyrosine phosphorylation of Cav-1 is
Src-associated; and 3) whether depletion of cholesterol by Cholesterol Depletion Blocks IL-6-induced Formation of
gp130·SH-PTP2·PI3-K·Cav-1 Complexes at the Plasma
Membrane--
We next defined the role of caveolae on the activation
and compartmentalization of immediate downstream targets in IL-6 and IGF-I signaling pathways. To verify the role of caveolae in IL-6 signal
transduction, MM cells were next either pre-treated with Cholesterol Depletion Blocks IL-6-induced STAT3 Phosphorylation in
MM Cells--
A recent study (46) reports that IL-6-induced STAT3
activation is associated with caveolae, because it is blocked by
treatment with Cholesterol Depletion Blocks IGF-I-induced Phosphorylation of
IRS-1--
We next investigated the role of caveolae in IGF-I
signaling. Specifically, IRS proteins are phosphorylated by IGF-IRs
(60) and transduce downstream signals via interaction with SH2 domains on other proteins, e.g. the regulatory p85 subunit of PI3-K,
thereby leading to phosphorylation of PI3-K p85 (61). As noted above, we and others (48, 49, 62) have defined previously the role of
PI3-K/Akt-1 signaling in mediating IGF-I-induced MM cell growth, drug
resistance, and survival. As shown in Fig. 7b, IGF-I-induced IRS-1 phosphorylation is inhibited (70%) by pre-treatment with Effect of Cholesterol Depletion on IL-6- and IGF-I-induced PI3-K
and Akt-1 Activity in MM Cells--
By having shown that both IL-6 and
IGF-I trigger recruitment of PI3-K to caveolae fractions (Fig. 2),
which is required for activation and compartmentalization of immediate
downstream targets of their receptors, e.g. SH-PTP2 and
IRS-1 (Figs. 6 and 7b), we next examined the effect of
cholesterol depletion of MM cells on IL-6- and IGF-I-triggered
PI3-K/Akt-1 signaling (Fig. 8). Treatment with
Previous studies show that both IGF-I and IL-6 stimulate
phosphorylation of IGF-IR and gp130, which activates/phosphorylates IRS-1 and/or SH-PTP2 and leads to activation of PI3-K. PI3-K, in turn,
regulates two distinct pathways, one mediating inhibition of apoptosis
and the other proliferation. The anti-apoptotic effect in MM cells is
mediated via Akt-1 phosphorylation on Thr-308 and Ser-473 as well as
Bad; the mitogenic effect is mediated via the mitogen-activated protein
kinase signaling (27, 48, 62). Similar results have been reported (50)
in adipocytes, in which caveolae sort anti-apoptotic versus
mitogenic events. Our previous studies (49, 63) show IL-6- and
IGF-I-induced growth via the mitogen-activated protein kinase
versus survival/drug resistance via PI3-K/Akt-1 signaling
(27, 49). Taken together, these results suggest a role for caveolae in
MM cells in regulating IL-6- and IGF-I-mediated mitogenic events
versus anti-apoptosis/drug resistance.
Lovastatin Inhibits IL-6- and IGF-I-induced Activation of Akt-1 in
MM.1S and Patient PCL Cells--
LS is an irreversible inhibitor of
hydroxymethylglutaryl-CoA reductase, which blocks the production of
mevalonate, an intermediate product in cholesterol and isoprenoid
synthesis. Previous studies (64) have reported decreased viability of
MM cell lines and patient MM cells in the presence of LS, due to
induction of apoptosis and inhibition of proliferation. In the present
study we show that LS, like Effect of Cholesterol Depletion on MM Cell Survival and Cell Cycle
Profile in MM Cells--
Pre-treatment of MM cells with
Taken together, our results demonstrate the presence of caveolae in MM
cells and add an additional level of regulatory complexity to IL-6- and
IGF-I-triggered signaling cascades and their sequelae. These findings
may have important clinical implications, because circulating serum
cholesterol is a major source of plasma membrane cholesterol and is
associated with higher mortality in black versus Caucasian
men (65). The increased incidence of MM in African-Americans could, at
least in part, also be related to their increased serum cholesterol
levels (66). In other systems, lipid rafts and caveolae have been
linked to chemotherapeutic drug resistance, cholesterol regulation (67,
68), as well as cell adhesion and migration (69). Coupled with our
findings that caveolae modulate PI3-K/Akt-1 signaling and related
growth and survival in MM cells, these studies suggest caveolae and
other membrane components as novel therapeutic targets in MM.
-cyclodextrin results in the loss of caveola structure in myeloma
cells, as shown by transmission electron microscopy, and loss of
caveolin-1 function. Interleukin-6 and insulin-like growth factor-I,
growth and survival factors in multiple myeloma, induce caveolin-1
phosphorylation, which is abrogated by pre-treatment with
-cyclodextrin. Importantly, inhibition of caveolin-1 phosphorylation
blocks both interleukin-6-induced protein complex formation with
caveolin-1 and downstream activation of the phosphatidylinositol
3-kinase/Akt-1 pathway.
-Cyclodextrin also blocks insulin-like
growth factor-I-induced tyrosine phosphorylation of insulin-responsive
substrate-1 and downstream activation of the phosphatidylinositol
3-kinase/Akt-1 pathway. Therefore, cholesterol depletion by
-cyclodextrin abrogates both interleukin-6- and insulin-like growth
factor-I-triggered multiple myeloma cell survival via negative
regulation of caveolin-1. Taken together, this study identifies
caveolin-1 and other structural membrane components as potential new
therapeutic targets in multiple myeloma.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(containing residues 1-178) and Cav-1
(containing residues
32-178). Caveolin-1 and caveolin-2 are expressed in most cell types,
including adipocytes, endothelial cells, and muscle cells, whereas
caveolin-3 is found specifically in muscle cells. A number of
investigators have shown absence of caveolin isoforms in human
peripheral blood cells including myeloid, lymphoid, and erythroid cell
lines (9-11), but Cav-1 is expressed in human T cell leukemia Jurkat
cell lines (12). Importantly, diseases including cancer, diabetes,
Alzheimer's disease, atherosclerosis, and muscular dystrophy have also
been associated with caveolae (13, 14). Moreover, Cav-1 expression has
been associated with progression of human prostate cancer, primary and
metastatic human breast cancer (15, 16), progression of papillary
carcinoma of the thyroid (17), high grade bladder cancer (18), and
lymph node metastasis in esophageal squamous cell carcinoma (19). To
date, however, the expression as well as biologic and clinical
importance of caveolae and Cav-1 in MM is undefined.
, endothelial nitric-oxide synthetase, protein kinase C
,
Ca2+ pumps, and inositol 1,4,5-triphosphate receptor. By
binding signal transducers within a distinct region of the plasma
membrane, caveolin regulates the activation state of
caveolin-associated signaling molecules and integrates signal
transduction pathways into discrete modules. Conversely, malfunctioning
of caveolin leads to indiscriminate cross-talk among distinct pathways.
Importantly, Cav-1 directly binds cholesterol, a cofactor in formation
of caveolae (6, 22, 23), forming homo- and hetero-oligomers. The
threshold of plasma membrane cholesterol below which caveolae cannot be formed is 40 mol % (23); therefore, cholesterol-binding
substances like
-cyclodextrin (
-CD), lovastatin (LS), nystatin,
and filipin prevent caveolae formation, with the caveolin-containing
caveolar coat initially remaining as a precipitate on the flattened
membrane and then being lost from the membranes (6). Cav-1 mRNA
levels are up- and down-regulated by cholesterol and oxysterols,
respectively (24). Taken together, these data indicate that
protein-protein interactions and protein-lipid interactions modulate
caveolae formation and related initiation of signaling pathways. In MM cells, we have characterized growth, survival, drug resistance, and
apoptotic signaling cascades (25, 26), but the role of caveolae in
modulating these sequelae is unknown at present.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-cyclodextrin was from Sigma. Mouse monoclonal antibodies (Abs) raised against Cav-1, pCav-1, and Cav-2 were purchased
from BD Transduction Laboratories, Inc. (Lexington, KY). Abs raised
against pERK, ERK2, actin, Cav-1, PI3-kinase p110
, Src, IGF-IR
,
pSTAT3 (Tyr-705), and IRS-1 were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal Abs raised
against PI3-kinase p85 and gp130 were purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). Rabbit polyclonal Abs raised
against pAkt-1 (Ser-473) and Akt-1 were purchased from Cell Signaling
Technology (Beverly, MA). IGF-IR (Ab-1) monoclonal Ab was purchased
from Oncogene Research Products (Boston). Anti-pY (4G10) monoclonal Ab
was kindly provided by Dr. Tom Roberts (Dana-Farber Cancer Institute, Boston).
-Cyclodextrin (
-CD) was dissolved in RPMI and used
directly. Cholesterol depletion was carried out by incubating cells
with the indicated concentrations of
-CD for 1 or 48 h at
37 °C. For experiments using lovastatin, MM.1S cells and patient PCL
cells were cultured in cholesterol-free medium (Cellgro) and then
stimulated with IL-6 or IGF-I.
) were obtained from patient bone marrow samples by
Ab-mediated negative selection using RossetteSep (StemCell
Technologies, Vancouver, British Columbia, Canada), as described
previously (28). Normal B-cells were isolated from healthy donor bone
marrow, as described previously (29).
-CD on MM.1S cell growth was assessed using MTT assay, as described
previously (38). Cell survival was estimated as a percentage of the
value of untreated controls.
-CD
or control were harvested, washed in PBS, and analyzed for cell cycle
profiling, as in prior studies (38).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Expression of Cav-1. a,
expression of Cav-1 in 4 MGUS and 8 MM patients was evaluated using
gene microarray. Total cellular RNA was isolated and processed as
described under "Experimental Procedures." Data shown are the
graphical representation of cDNA microarray analysis. The relative
intensity indicates an average difference in hybridization signal
intensity of Cav-1 expression in MGUS versus MM patients.
Significantly increased (p < 0.0001) Cav-1 expression
was observed in MM patients versus MGUS patients.
b, RT-PCR analysis of Cav-1 transcripts in human MM
cell lines, bone marrow stromal cells, NIH3T3, and normal B-cells.
Equal amounts of RNA were reverse-transcribed to generate cDNA,
which was subjected to Cav-1-specific PCR amplification using paired
primers, as described under "Experimental Procedures." The quality
of RNA was confirmed by RT-PCR amplification of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
c, detection of Cav-1 by Western blotting
(IB). Equal aliquots of cell lysates derived from indicated
cell lines and patient cells were loaded and separated by SDS-PAGE.
NIH3T3 cells and human endothelial cells (EC) served as
positive controls for Cav-1 expression. Equal membrane loading was
confirmed by re-probing the membrane with Ab directed against
actin.
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Fig. 2.
Co-fractionation of gp130, IGF-IR, and
PI3-kinase in caveolae-enriched microdomains. Lysates of
un-stimulated (contr) (a), IGF-I-stimulated
(b), or IL-6-stimulated (c) cells were subjected
to discontinuous sucrose density gradient fractionation. The relative
enrichment in IGF-IR, gp130, p85, pERK, ERK, and Cav-1 was assessed by
Western blot (IB) analysis (left panels).
Quantitative densitometry reflects the distribution of Cav-1
(open box), IGF-IR (closed diamond), gp130
(open circle), PI3-K p85 (filled circle), and ERK
(filled triangle) throughout the gradient (right
panels).
-gp130 complexes (Fig. 3, d and
e). In contrast, neither phosphorylated ERK nor total ERK
was co-immunoprecipitated with Cav-1 after both IGF-I (Fig.
3c) or IL-6 (Fig. 3f) stimulation. Taken
together, these results show that gp130 and IGF-IR co-localize with
Cav-1, and suggest that Cav-1 may play a functional role in IL-6- and
IGF-I-induced downstream PI3-K, but not ERK, signaling in MM cells.
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Fig. 3.
IGF-IR and gp130, but not ERK,
co-immunoprecipitate with Cav-1. Serum-starved MM.1S cells were
stimulated with IL-6 or IGF-I for the indicated intervals. Equal
amounts of whole cell lysates were immunoprecipitated with anti-IGF-IR,
anti-Cav-1, and anti-gp130 Abs as described under "Experimental
Procedures" and then immunoblotted with the indicated Abs.
a and b, co-immunoprecipitation of IGF-IR
and Cav-1. d and e, co-immunoprecipitation of
gp130 and Cav-1. c and f, absence of
phosphorylated and total ERK in Cav-1 immunoprecipitations.
IP, immunoprecipitation; C, IP control;
IB, immunoblot; WCL, whole cell lysate;
Ig, heavy chains of Abs.
-Cyclodextrin on Caveolae Structure in Membranes of MM
Cells--
Cav-1 directly binds cholesterol, the most important
cofactor in caveolae morphogenesis (6, 22, 23); conversely, the importance of membrane rafts in cell signaling can be inferred from the
blocking effects of cholesterol depletion and membrane fractionation.
Specifically, the physical association of receptors and signaling
molecules with raft domains can be confirmed by using substrates to
modulate cholesterol content; for example, studies use
-CD to
disrupt caveolae-like structures by binding to and sequestering
cholesterol from the plasma membrane of intact cells (42, 50, 51). One
previous study (2) has demonstrated an absolute requirement for
cholesterol for growth in mouse MM cells, and in this study we
similarly characterized the role of cholesterol in human MM cells. As
shown by transmission electron microscopy, morphologically recognizable
caveolae (closed arrowheads) are present in MM cells
adjacent to coated vesicles containing electron-dense contents
(open arrowheads) (Fig. 4,
a-f). Fig. 4e additionally shows perinuclear
cisterns and the Golgi apparatus. Smooth surface vesicles originating
from the outer margin of the perinuclear cistern associated with
distended Golgi sacs have been described previously in MM cells (52);
however, the function of these vesicles has not been defined.
Importantly, our studies show morphologically recognizable caveolae at
an ultrastructural level in untreated MM.1S cells (Fig. 4,
a-f) but not in MM cells treated with
-CD (1 h) (Fig. 4,
g and h). Pre-treatment of MM cells with
-CD
changes the distribution of Cav-1 within a discontinuous sucrose
density gradient, confirming the absence of caveolae structures. Consequently, neither IGF-I nor IL-6 can trigger increased PI3-K p85 in
the light buoyant fractions. In contrast, no changes of the
distribution of total (data not shown) and phosphorylated ERK could be
observed in
-CD pre-treated (Fig. 4i) versus
non-pretreated MM cells (Fig. 2). Taken together, these data show that
caveolae formation in MM cells is dependent on the presence of
cholesterol. They further indicate that intact caveolae play a
functional role in IL-6- and IGF-I-induced downstream PI3-K, but not
ERK, signaling.
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Fig. 4.
Effect of -CD on
caveolae structure in membranes of MM cells. MM.1S cells were
grown for 3 days in 15-cm cell culture plates. After treatment with 10 mM
-CD or control for 1 h, cells were fixed and
processed for transmission electron microscopy, as described under
"Experimental Procedures." Electron micrographs of non-treated
(a-f) and treated (g and h) MM.1S
cells are shown. a, whole MM.1S cell (magnification ×5000);
b-f show caveolae specimens with different morphological
appearances at higher magnification as follows: b, typical
flask-shaped caveola (left) and caveola with open stomata
(right) (magnification ×40,000); c,
caveola with long neck (magnification ×40,000); d,
typical flask-shaped caveola and a coated vesicle with electron-dense
content (magnification ×30,000); e, flask-shaped
caveola (magnification ×30,000); f, flask-shaped
caveola with elongated and narrow neck, sharply bent rims, and a coated
vesicle (magnification ×40,000). g, whole MM.1S cell
(magnification ×5000); h, representative membrane area of a
pre-treated cell (magnification ×40,000). Closed
arrowheads, caveolae; open arrowheads, coated
vesicles. i, after pre-treatment with
-CD (10 mM, 1 h), lysates of un-stimulated (contr
+
-CD), IGF-I stimulated (IGF-I +
-CD), or IL-6
stimulated (IL-6 +
-CD) cells were subjected to
discontinuous sucrose density gradient fractionation. The relative
enrichment in PI3-K p85, pERK, and Cav-1 was assessed by Western blot
(IB) analysis.
-CD
affects IL-6- and IGF-I-induced Cav-1 tyrosine phosphorylation in MM
cells. Our results demonstrate that Src co-immunoprecipitates with
Cav-1 in MM cells (Fig. 5a)
and that both IL-6 and IGF-I can trigger significant phosphorylation of
Cav-1, as determined with a specific pCav-1 Ab and analyzed by
densitometry analysis. Furthermore, inhibition of Src family tyrosine
kinases by PP2 abrogates IL-6- and IGF-I-induced Cav-1 phosphorylation,
suggesting that Src family tyrosine kinases contribute to Cav-1-related
signaling (Fig. 5, b and c). Importantly, Cav-1
phosphorylation triggered by both IL-6 and IGF-I stimulation is also
blocked after cholesterol depletion using
-CD (Fig. 5, d
and e).
View larger version (39K):
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Fig. 5.
Cav-1 tyrosine phosphorylation induced by
IL-6 and IGF-I is associated with Src family tyrosine kinase activation
and intact caveolae structure. a,
co-immunoprecipitation of Src and Cav-1. Immunoprecipitation of whole
cell lysates (lys) was performed as described under
"Experimental Procedures." Whole cell lysates of human umbilical
vein endothelial cells (HUVEC) served as control;
b and c, serum-starved MM.1S cells were
pre-treated with either PP2 (5 µM, 30 min) or
Me2SO (DMSO) or with -CD (d and
e) and then stimulated with IL-6 (b and
d) or IGF-I (c and e). Immunoblotting
was performed with either anti-Cav-1 or anti-pCav-1 Abs. Data shown are
representative of three separate experiments. IP,
immunoprecipitation; Ig, immunoglobulin, heavy chains;
IB, immunoblot; C, immunoprecipitation
control.
-CD or left
untreated, prior to stimulation with IL-6 for the indicated intervals.
Cav-1 and PI3-K catalytic subunit p110 immunoprecipitations were then
performed on whole cell lysates. As shown in Fig.
6a, tyrosine phosphorylation
of Cav-1 is required for the formation of protein complexes containing
SH2 domain-containing protein tyrosine phosphatase 2 (SH-PTP2) and
PI3-K, because inhibition of Cav-1 phosphorylation by pre-treatment of
MM cells with
-CD also inhibits the formation of this protein
complex. Conversely, immunoprecipitation with anti-p110 Ab shows that
IL-6 triggers the association of p110 with gp130 (Fig. 6b)
and thereby with Cav-1; as expected, this association is blocked by
pre-treatment with
-CD. Taken together, these data show that an
intact caveolae structure is necessary for complex formation of gp130,
SH-PTP2, and PI3-K.
View larger version (38K):
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Fig. 6.
Cholesterol depletion blocks IL-6-induced
formation of gp130·SH-PTP2·PI3-K·Cav-1 complexes at the plasma
membrane. a, IL-6-induced phosphorylation of Cav-1 is
required for SH-PTP2/PI3-K-containing complex formation at the plasma
membrane. Serum-starved MM.1S cells were incubated with or without 10 mM -CD for 1 h and then stimulated with IL-6 for
the indicated intervals. Equal amounts of whole cell lysates were
immunoprecipitated with anti-Cav-1 Ab, as described under
"Experimental Procedures," and then immunoblotted with the
indicated Abs. b, IL-6 triggers association of p110
with gp130. Serum-starved MM.1S cells were incubated with or without 10 mM
-CD for 1 h and then stimulated with IL-6 for
the indicated intervals. Equal amounts of whole cell lysates were
immunoprecipitated with anti-p110 Ab, as described under
"Experimental Procedures," and immunoblotted with either anti-gp130
or anti-p110 Abs. The data shown are representative of three separate
experiments. IP, immunoprecipitation; C,
immunoprecipitation control.
-CD. In U266 MM cells, STAT3 mediates IL-6-induced
survival (59), and we therefore next investigated whether
-CD (10 mM, 1 h) modulates IL-6-triggered phosphorylation of
STAT3 in MM.1S cells. As shown in Fig.
7a, IL-6-induced STAT3
activation is inhibited by pre-treatment with
-CD. This blockade of
STAT3 activation is observed by immunoblotting with a phospho-specific
STAT3 Ab and is not associated with changes in total cellular levels of STAT3 protein.
View larger version (33K):
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Fig. 7.
Cholesterol depletion blocks IL-6-induced
STAT3 phosphorylation and IGF-I-induced phosphorylation of IRS-1.
a, cholesterol depletion blocks IL-6-mediated STAT3
phosphorylation. Starved MM.1S cells were preincubated with or without
10 mM -CD for 1 h and then stimulated with IL-6 for
the indicated intervals. STAT3 tyrosine phosphorylation was detected by
immunoblot analysis using both anti-pSTAT3 Ab. Immunoblotting for STAT3
confirmed equal protein loading. b, effect of
cholesterol depletion on IGF-I-induced IRS-1 phosphorylation.
Serum-starved MM.1S cells were incubated with or without 10 mM
-CD for 1 h and then stimulated with IGF-I for
the indicated intervals. Whole cell lysates were immunoprecipitated
with anti-IRS-1 Ab, as described under "Experimental Procedures,"
and immunoblotted with either anti-pY (4G10) Ab or anti-IRS-1 Ab. The
data shown are representative of three separate experiments.
IP, immunoprecipitation; C, immunoprecipitation
control; IB, immunoblot.
-CD,
confirming that intact caveolae are required for optimal signaling from
IGF-IR to IRS-1.
-CD blocked IL-6- and IGF-I-induced PI3-K (Fig. 8,
a-d) and Akt-1 (Fig. 8, e and f)
activation in MM.1S cells, as well as in tumor cells isolated from a
patient with PCL (Fig. 8, g and h). In contrast,
cholesterol depletion of MM.1S cells did not alter ERK phosphorylation
(Fig. 8, e and f), confirming that Cav-1 is
required only for PI3-K/Akt-1-mediating signaling.
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Fig. 8.
Effect of cholesterol depletion on IL-6- and
IGF-I-induced PI3-kinase and Akt-1 activity in MM and PCL cells.
Serum-starved MM.1S cells were incubated with or without 10 mM -CD for 1 h and then stimulated with IL-6
(a, c, and e) or IGF-I
(b, d, and f) for the indicated
intervals. Lysates were either used for in vitro PI3-K
assays (a and b and c and
d) or subjected to SDS-PAGE and immunoblotting with
indicated Abs (e and f). For the PI3-K assay,
equal amounts of lysates were immunoprecipitated with anti-pY (4G10)
Ab, and immunocomplexes were assayed for the ability to phosphorylate
phosphatidylinositol 4,5-biphosphonate as a substrate. Migration of
phosphatidylinositol 3,4,5-triphosphonate (PIP3), as
determined by iodine staining of non-radioactive standards, is
indicated. Phosphatidylinositol 3,4,5-triphosphonate production was
visualized by autoradiography and analyzed by scanning densitometry.
The relative changes in phosphatidylinositol 3,4,5-triphosphonate
produced in the PI3-K assays were calculated, and the data were plotted
as fold activation; data from three separate autoradiograms are
presented as means ± S.D. (c and d).
Control (C) (a and b) indicates PI3-K
assay performed with protein A-Sepharose alone. g and
h, effect of cholesterol depletion on IL-6- and
IGF-I-induced Akt-1 activity in patient PCL cells. Patient
PCL cells were incubated for 2 h in RPMI without FBS and then for
an additional 1 h with or without 10 mM
-CD. Cells
were then stimulated with IL-6 (g) or IGF-I (h)
for the indicated intervals. Lysates were subjected to SDS-PAGE and
then immunoblotted for pAkt-1 or Akt-1. i and
j, lovastatin inhibits IL-6- and IGF-I-induced
activation of Akt-1 in MM.1S and patient PCL cells. MM.1S
(i) or patient PCL (j) cells were incubated in
cholesterol-free medium with or without 30 µM LS for
24 h and then stimulated with IL-6 or IGF-I for the indicated
intervals. Lysates were subjected to SDS-PAGE and then immunoblotted
for indicated proteins. IB, immunoblot; C,
stimulation of non pre-treated cells.
-CD, decreases IL-6- and IGF-I-induced
activation of Akt-1 in MM.1S and patient PCL cells (Fig. 8,
i and j), confirming the requirement for an
intact plasma membrane structure in MM cells to mediate PI3-K/Akt-1 signaling.
-CD for
1 h resulted in
-CD concentration-dependent
decreased Akt-1 phosphorylation induced by IL-6 (Fig.
9a) or IGF-I (Fig.
9b). By having demonstrated that cholesterol depletion for
1 h inhibits Akt-1 activation even at low concentrations, we next
investigated the effect of several
-CD concentrations on survival of
MM cells cultured for 48 h with IL-6 or IGF-I. As expected from
our demonstration that IL-6- and IGF-I- mediated survival signaling in
MM cells depends upon the presence of intact caveolae,
-CD induces
dose-dependent cell death of MM cells, as quantified by MTT
assay, which is not overcome by IL-6 or IGF-I (Fig. 9c).
Because we have reported recently (27) that inhibition of IL-6-induced
PI3-K/Akt-1 activity by the PI3-K inhibitor LY294002 induces
G1 arrest and blocks IL-6-induced G1/S phase
transition in MM cells, we further investigated whether
-CD
similarly modulates the cell cycle profile. As seen in Fig. 9d,
-CD also induces G1 growth arrest in a
dose-dependent fashion, further supporting the functional
importance of intact caveolae in MM cells.
View larger version (27K):
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Fig. 9.
Effect of cholesterol depletion on MM cell
survival and on MM cell cycle profile. a and
b, -CD induces concentration-dependent
inhibition of IL-6- and IGF-I-triggered Akt-1 phosphorylation.
Serum-starved MM.1S cells were incubated with the indicated
concentrations of
-CD for 1 h and then stimulated with IL-6
(a) or IGF-I (b) for 10 min. Lysates were
subjected to SDS-PAGE and immunoblotted with antibodies directed
against pAkt-1 or Akt-1. IB, immunoblot. c,
-CD induces dose-dependent cell death of MM cells as
quantified by MTT assay, which is not overcome by IL-6 or IGF-I. Data
shown (mean ± S.D.) are representative of 3 experiments.
d, MM cells were pre-treated with indicated
concentrations of
-CD, harvested, and subjected to propidium
iodide staining for cell cycle analysis, as described under
"Experimental Procedures." One experiment representative of three
experiments is shown.
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ACKNOWLEDGEMENTS |
---|
We thank A. Dring, S. Shah, and J. Ryoo for their technical assistance; M. Ericsson and L. Trakimas (Harvard Medical School Electron Microscopy Facility, Boston) for expert assistance with electron microscopy; and Dr. M. Lu (Brigham and Women's Hospital, Harvard Medical School, Boston) and Dr. H. Ludwig (Wilhelminenspital, Vienna, Austria) for helpful discussions.
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FOOTNOTES |
---|
* This work was supported by an IMF/Brian D. Novis/Benson Klein research grant award (to K. P.), National Institutes of Health Grant PO-1 78378, and the Doris Duke Distinguished Clinical Research Scientist award (to K. C. A.).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 and reprint requests should be addressed: Dana-Farber Cancer Institute, Dept. of Medical Oncology, Jerome Lipper Multiple Myeloma Center, 44 Binney St., Boston, MA 02115. Tel.: 617-632-2144; Fax: 617-632-2140; E-mail: Kenneth_Anderson@dfci.harvard.edu.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M208636200
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ABBREVIATIONS |
---|
The abbreviations used are:
MM, multiple
myeloma;
BM, bone marrow;
Cav-1, caveolin-1;
-CD,
-cyclodextrin;
LS, lovastatin;
IL-6, interleukin-6;
IGF-I, insulin-like growth factor
I;
PI3-K, phosphatidylinositol 3-kinase;
Ab, antibody;
PCL, plasma
cell leukemia;
MGUS, monoclonal gammopathy of undetermined
significance;
IGF-IR, insulin-like growth factor I receptor;
gp130, interleukin-6 receptor signal transducing chain gp130;
STAT3, signal
transducer and activator of transcription 3;
ERK, extracellular
signal-regulated kinase;
SH2, Src homology 2;
SH-PTP2, SH2-domain
containing protein tyrosine phosphatase 2;
IRS, insulin-responsive
substrate;
FBS, fetal bovine serum;
RT, reverse transcriptase;
PBS, phosphate-buffered saline;
PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d])pyrimidine;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide.
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