Essential Role of Caveolae in Interleukin-6- and Insulin-like Growth Factor I-triggered Akt-1-mediated Survival of Multiple Myeloma Cells*

Klaus PodarDagger , Yu-Tzu TaiDagger , Craig E. ColeDagger §, Teru HideshimaDagger , Martin SattlerDagger , Angela HamblinDagger , Nicholas MitsiadesDagger , Robert L. SchlossmanDagger , Faith E. Davies, Gareth J. Morgan, Nikhil C. MunshiDagger ||, Dharminder ChauhanDagger , and Kenneth C. AndersonDagger **

From Dagger  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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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 beta -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 beta -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. beta -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 beta -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.

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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-1alpha (containing residues 1-178) and Cav-1beta (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.

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 alpha , endothelial nitric-oxide synthetase, protein kinase C alpha , 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 beta -cyclodextrin (beta -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.

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.

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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 beta -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 p110alpha , Src, IGF-IRbeta , 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).

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. beta -Cyclodextrin (beta -CD) was dissolved in RPMI and used directly. Cholesterol depletion was carried out by incubating cells with the indicated concentrations of beta -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.

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-) 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).

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 beta -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.

Cell Cycle Analysis-- MM.1S cells cultured for 48h in beta -CD or control were harvested, washed in PBS, and analyzed for cell cycle profiling, as in prior studies (38).

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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.


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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.

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).


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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).

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 alpha -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.

Effect of beta -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 beta -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 beta -CD (1 h) (Fig. 4, g and h). Pre-treatment of MM cells with beta -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 beta -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 beta -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 beta -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 beta -CD (10 mM, 1 h), lysates of un-stimulated (contr +beta -CD), IGF-I stimulated (IGF-I +beta -CD), or IL-6 stimulated (IL-6 +beta -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.

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 beta -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 beta -CD (Fig. 5, d and e).


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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 beta -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.

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 beta -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 beta -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 beta -CD. Taken together, these data show that an intact caveolae structure is necessary for complex formation of gp130, SH-PTP2, and PI3-K.


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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 beta -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 beta -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.

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 beta -CD. In U266 MM cells, STAT3 mediates IL-6-induced survival (59), and we therefore next investigated whether beta -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 beta -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.


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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 beta -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 beta -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.

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 beta -CD, confirming that intact caveolae are required for optimal signaling from IGF-IR to IRS-1.

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 beta -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 beta -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 beta -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.

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 beta -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.

Effect of Cholesterol Depletion on MM Cell Survival and Cell Cycle Profile in MM Cells-- Pre-treatment of MM cells with beta -CD for 1 h resulted in beta -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 beta -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, beta -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 beta -CD similarly modulates the cell cycle profile. As seen in Fig. 9d, beta -CD also induces G1 growth arrest in a dose-dependent fashion, further supporting the functional importance of intact caveolae in MM cells.


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Fig. 9.   Effect of cholesterol depletion on MM cell survival and on MM cell cycle profile. a and b, beta -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 beta -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, beta -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 beta -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.

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.

    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.

    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

    ABBREVIATIONS

The abbreviations used are: MM, multiple myeloma; BM, bone marrow; Cav-1, caveolin-1; beta -CD, beta -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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