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Endocrinology, doi:10.1210/en.2007-1211
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Endocrinology Vol. 149, No. 10 5199-5208
Copyright © 2008 by The Endocrine Society

Modulation of Caveolin-1 Expression Can Affect Signalling through the Phosphatidylinositol 3-Kinase/Akt Pathway and Cellular Proliferation in Response to Insulin-Like Growth Factor I

Laura C. Matthews, Michael J. Taggart and Melissa Westwood

Endocrine Sciences (L.C.M.), Maternal and Fetal Health Research Group (M.J.T., M.W.), University of Manchester, Manchester M13 9PT, United Kingdom

Address all correspondence and requests for reprints to: Dr. Melissa Westwood, Maternal and Fetal Health Research Group, University of Manchester, St. Mary’s Hospital, Hathersage Road, Manchester M13 0JH, United Kingdom. E-mail: melissa.westwood{at}manchester.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The IGFs mediate their effects on cell function through the type I IGF receptor and numerous intracellular signalling molecules, including the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway. The type I IGF receptor also binds to the caveolae protein caveolin-1, but the impact of caveolae on IGF/PI-3K/Akt signalling remains controversial. We have examined the effect of complete (knockout) and partial (knockdown) caveolin-1 deficiency on cellular IGF effects mediated via the PI-3K/Akt pathway. Under basal conditions, caveolin-1-deficient mouse embryonic fibroblast cells [MF(–/–)] incorporated significantly more [3H]thymidine than wild-type mouse embryonic fibroblast cells [MF(+/+)]; however, small hairpin RNA-mediated knockdown of caveolin-1 (80% reduction) in 3T3L1 fibroblasts had no effect on basal proliferation. Interestingly, IGF-I induced proliferation was similar in MF(–/–) and MF(+/+) cells, whereas caveolin-1 knockdown promoted a hyperproliferative response to IGF-I [pkDCav3T3L1(80) 12.4 ± 0.4-fold; pkDShuffle3T3L1 4.3 ± 0.2-fold induction; P < 0.01]. Immunoblot analysis showed that caveolin-1 knockdown had no affect on Akt expression or activation. However, in MF(–/–) cells, IGF-I-stimulated phosphorylation of Akt was reduced despite up-regulated Akt levels. Further investigation demonstrated that caveolin knockout up-regulated Akt-2 and Akt-3 isoform expression, but Akt-1 expression was down-regulated; interestingly, coimmunoprecipitation studies revealed Akt-1 as the predominant isoform to be phosphorylated in response to IGF-I. In summary, caveolin-1 deficiency promotes a hyperproliferative response to IGF-I that is unrelated to Akt expression/activation. However, cells that lack caveolin are able to respond appropriately to IGF-I through compensatory changes in Akt isoform expression. These data posit caveolin-1 as a component of the IGF/PI-3K/Akt signalling modulus regulating cellular proliferation with implications for diseases, including cancers, which have altered caveolin expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IGFs ARE important regulators of cell function, with effects on growth and survival that are mediated primarily through the type I IGF receptor (IGF-IR) (1, 2, 3). Recently, we and others (4, 5, 6) have shown a direct interaction between IGF-IR and caveolin-1, an integral protein of caveolae, which are a subset of plasmalemmal lipid rafts.

Caveolin-1 modulates the function of a broad range of molecular species (7, 8, 9), and data from some studies have led to the suggestion that caveolae are important for coordinating the intracellular signaling molecules involved in mediating IGF-I induced cellular proliferation and survival (10, 11). Indeed, caveolin-1 is known to influence signal transduction along the MAPK cascade (12), and more recently, overexpression of caveolin-1 has been linked to enhanced activation of the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway (13). Furthermore, because caveolin-1 has been postulated as a potential tumor suppressor (14, 15), and the levels of caveolin expression alter with various cancers (16, 17, 18, 19), caveolar regulation of the IGF-IR, which is also implicated in cellular transformation (3, 20, 21), may be important in tumor onset and/or progression. If so, an elucidation of the adaptive responses to caveolin deficiency would be informative; therefore, in this study we have used two mouse cell models of altered caveolin expression, i.e. small hairpin RNA (shRNA)-mediated knockdown of caveolin-1 in a previously characterized 3T3L1 mouse fibroblast cell line and fibroblasts isolated from caveolin-1 knockout mice, to examine the impact of caveolae on IGF-I signaling and function. Our data suggest that the loss of caveolin can promote a hyperproliferative response to IGF-I unless accompanied by changes in Akt isoform expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All chemical reagents were purchased from Sigma (Dorset, UK) unless otherwise stated. The primary antibodies used in this study were: anti-tubulin (1:2000) from Sigma; anti-caveolin (1:2000) from BD Transduction Laboratories (Oxfordshire, UK); anti-IGF-IRβ (1:2000) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-PY162IRS-1 (no. 44–816; 1:1000) and anti-PY1162/1163-IGF-IRβ (no. 44–804; 1:1000) from BioSource Intl. (Camarillo, CA); anti-insulin receptor substrate (IRS-1) (1:1000) from Upstate (Milton Keynes, UK); and anti-total Akt (1:1000), anti-Akt-1 (no. 2967, 1:1000), anti-Akt-2 (no. 2964, 1:1000), anti-Akt-3 (no. 4059, 1:1000), anti-PS473-Akt (no. 9217; 1:1000), anti-PT308-Akt (1:1000), anti-MAPK (1:1000), and anti-PT202/PY204-MAPK (no. 9101; 1:1000) from Cell Signaling Technology, Inc. (Danvers, MA). The anti-total Akt antibody recognizes the residues surrounding the ser(473) phosphorylation site, regardless of phosphorylation state, and, therefore, detects all Akt isoforms. The anti-PS473-Akt and anti-PT308-Akt antibodies also recognizes all three Akt isoforms but only when activated, whereas the antibodies raised against the individual isoforms recognize protein regardless of phosphorylation state and do not cross-react with the other two isoforms. The secondary antibodies used were horseradish peroxidase (HRP)-conjugated antimouse (1:5000) and HRP-conjugated antirabbit (1:2000–1:5000) from GE Healthcare (Buckinghamshire, UK).

Generation of a cell line with stable caveolin-1 knockdown
Using a two-step cloning strategy, double-stranded oligonucleotides (VH Bio Ltd., Gateshead, UK), which were based on a published sequence for targeting caveolin-1 by shRNA (22), were ligated into pkD (Upstate) to generate pkDCav. A control plasmid, pkDShuffle, was generated using a scrambled version of the same oligonucleotide sequences. To establish a cell line model of caveolin-1 deficiency (pkDCav3T3L1) and an appropriate control (pkDShuffle3T3L1), each of the plasmids was cotransfected (fugene 6 reagent; Roche Diagnostics, Basel, Switzerland) with a construct conferring mammalian antibiotic resistance (pTk-Hyg; a generous gift of Professor Julian Davis, University of Manchester, Manchester, UK) into 3T3L1 mouse fibroblasts (European Collection of Cell Cultures, Wiltshire, UK), and after 48 h, cells were transferred to antibiotic selective media. Subsequently, five clones from each cell line were selected, expanded, and subjected to immunoblot analysis for caveolin-1 expression, and then single clonal cell lines of pkDCav3T3L1(80) (~80% cavolin-1 knockdown; Fig. 1Go, clone 3), pkDCav3T3L1(60) (~60% caveolin-1 knockdown; Fig. 1Go, clone 5), and pkDShuffle3T3L1 were chosen for use in all further experiments.


Figure 1
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  		FIG. 1. pkDCav3T3L1 and MF(–/–) cells have reduced caveolin-1 expression. 3T3L1 cells transfected with pkDCav (clones 1–5) or pkDShuffle (A) were lysed and immunoblotted for cavolin-1; tub was analyzed as a measure of equal loading (50 µg protein). Immunoblots (n = 3) were subjected to densitometric analysis using ImageJ software (Institutes of Health, Bethesda, MD), caveolin to tub ratios determined, and data represented graphically (bar indicates mean). A representative blot of the five pkDCav3T3L1 clones [clone 3, pkDCav3T3L1(80), and clone 5, pkDCav3T3L1(60), were selected for subsequent experiments] is also shown in A. B, The caveolin-1 status of the MF(+/+) and MF(–/–) cell lines was confirmed by immunoblotting, again using tub as a measure of equal loading (50 µg protein).

 
Caveolin-1 knockout cell line
Fibroblasts isolated from thymus of caveolin-1 gene knockout [MF(–/–)]; a generous gift of Professor Richard Anderson, University of Texas, TX) served as a model of complete caveolin knockdown. Fibroblasts isolated from the thymus of wild-type animals [MF(+/+); also donated by Professor Anderson] were used to provide control data.

Cell culture
MF cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS) (Pierce, Rockford, IL), 1 mM pyruvate, 4 mM glutamine, 1% penicillin, and 1% streptomycin (growth media). Hygromycin (0.01%) was added to the growth media for the culture of pkDCav3T3L1(80), pkDCav3T3L1(60), and pkDShuffle3T3L1 cells. All cells were cultured in a humidified atmosphere of 5% carbon dioxide at 37 C.

Sample preparation
Cell extracts.
Cells approaching 70% confluence were serum starved (growth media lacking 10% FCS) for 24 h and then treated with 5 nM human recombinant IGF-I for 15–60 min at 37 C, 5% CO2. Cell extracts were prepared by scraping cells washed with PBS (Oxoid, Hampshire, UK) into radioimmunoprecipitation assay lysis buffer [50 mM TrisHCl (pH 7.4), 1% NP40, 0.25% Na-deoxycholate, 150 mM NaCl, and 1 mM EDTA] containing protease (Calbiochem, San Diego, CA) and phosphatase inhibitors, before centrifugation for 30 min at 10,000 x g and 4 C. The supernatants were harvested, assayed for protein content (Bio-Rad Laboratories, Inc., Hercules, CA), and diluted in reducing loading buffer [0.125 M TrisCl (pH 6.8), 0.1% sodium dodecyl sulfate, 20% glycerol, 0.2% β-mercaptoethanol, and 0.001% bromophenol blue] before boiling for 5 min.

Immunoprecipitates.
Cell extracts (300 µg protein) were precleared with 50 µl (50% slurry) protein A-coated Sepharose beads (Zymed Laboratories, Inc., San Francisco, CA) for 30 min at room temperature. These beads were pelleted by centrifugation at 5000 x g for 20 sec and discarded. In the test samples, supernatant was incubated with 2 µg primary antibody and 50 µl protein A-coated Sepharose beads overnight at 4 C. The control sample supernatants were incubated with either 1 or 2 µg IgG and 50 µl protein A-coated Sepharose beads, or with 50 µl protein A-coated Sepharose beads alone. After this incubation, test and control samples were centrifuged (5000 x g for 20 sec) to pellet the beads, and the supernatants were collected, precipitated with 10% trichloracetic acid, and boiled in reducing loading buffer for 5 min. The pellets were washed three times in ice-cold PBS, boiled for 5 min in reducing loading buffer, and before electrophoresis, the beads were removed by centrifugation at 5000 x g for 20 sec. The precipitates harvested from test and control samples were analyzed by immunoblot to demonstrate that, in lysates known to contain the protein of interest, precipitation was dependent on the presence of the antibody.

Immunoblotting
Cell extracts (50 µg protein) or immunoprecipitates were electrophoresed (Protean II Xi; Bio-Rad Laboratories) on sodium dodecyl sulfate/acrylamide (10%) gels and transferred to 0.2 µM nitrocellulose membranes (Bio-Rad Laboratories). Membranes were blocked for 6 h (0.15 M NaCl, 1% dried milk, and 0.1% Tween 20) and incubated with primary antibodies (diluted in blocking buffer) overnight at 4 C. After three 10-min washes [88 mM Tris (pH 7.8), 0.25% dried milk, and 0.1% Tween 20], membranes were incubated with a species-specific HRP-conjugated secondary antibody (diluted in wash buffer) for 1 h at room temperature, then washed a further three times, each for 10 min. Immunoreactive proteins were visualized using enhanced chemiluminescence (Supersignal West Pico, West Femto Maximum Sensitivity Substrate; Pierce), and the protein molecular masses were established using calibrated full-range (7–201 kDa) kaleidoscope standards (Bio-Rad Laboratories).

[3H]Thymidine incorporation
Cells approaching 70% confluence were serum starved (growth media lacking 10% FCS) for 24 h and then incubated with 5 nM IGF-I; in some experiments, cells were pretreated with 100 nM LY294002 or 500 nM PD98059 before the addition of IGF-I. Twenty hours later, methyl-[3H]thymidine was added to a final concentration of 0.25 µCi/ml. After 4 h, cells were washed twice with PBS and then incubated with 10% trichloroacetic acid for a further 2 h at 4 C. After solubilization with 0.1 M NaOH, thymidine incorporation was assessed using a β-counter (Packard Tri-Carb LS counter; General Medical Instrumentation, Inc., Ramsey, MN) and Optiphase HiSafe liquid scintillant. Each condition was examined in triplicate; means of triplicate counts were calculated and values expressed as fold induction compared with serum-free control (mean ± SEM).

Apoptosis assays
Cells approaching 70% confluence were serum starved (growth media lacking 10% FCS) for 24 h and then incubated with 5 nM IGF-I. Twenty hours later, the cell culture medium (containing dead cells) was aspirated and combined with trypsinized (live) cells, which were then pelleted at 1400 x g and fixed in 3% paraformaldehyde for 2 h at 4 C. Cells were cytospun onto poly-L-lysine coated slides (5 min, 1400 x g), nuclear DNA was stained with 4',6-diamidino-2-phenylindole (Vectashield; Vector Laboratories, Burlingame, CA), and then, using a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany), more than 1000 single cells per replicate were scored using chromatin fragmentation as an indication of apoptosis. Each condition was assessed in triplicate; means of the triplicate counts were calculated, and values expressed as the number of apoptotic cells were compared with the serum-starved control (mean ± SEM).

Data analysis
Triplicate counts from three or four independent experiments were analyzed by the Student’s t test where P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
pkDCav3T3L1 and MF(–/–) cells have reduced caveolin-1 expression
3T3L1 cells were transfected with an shRNA plasmid containing a sequence known to target caveolin-1 (pkDCav) or a control shuffle sequence (pkDshuffle). Stable integration of pkDCav reduced caveolin-1 expression; however, levels of caveolin-1 were unaltered in 3T3L1 cells containing the control shuffle sequence (Fig. 1AGo, upper panel). pkDShuffle3T3L1 clone 3 (104 ± 8% wild-type 3T3L1 caveolin-1 expression) and pkDCav3T3L1 clone 3 [22 ± 6% wild-type 3T3L1 caveolin-1 expression; pkDCav3T3L1(80)]) were selected for further study (Fig. 1AGo, lower panel), though in some of the experiments to investigate the effect of altered caveolin-1 expression on IGF-I functional activity, pkDCav3T3L1 clone 5, pkDCav3T3L1(60), which expresses an intermediate level of caveolin-1 (36 ± 5% wild-type 3T3L1 caveolin 1 expression), was used also.

Immunoblot analysis of lysates of cells isolated from wild-type and caveolin-1 knockout mice confirmed that caveolin-1 was expressed by MF(+/+), but not MF(–/–) cells (Fig. 1BGo). The caveolin-1 knockdown and caveolin-1 knockout cells, along with their control counterparts, respectively, provide the models of partial and complete caveolin deficiency used for all subsequent experiments.

Neither partial nor complete deficiency of caveolin-1 affects IGF-I-mediated cell survival
Caveolin is reported to be an important regulator of cell survival (23, 24), and, therefore, the impact of caveolin-1 deficiency on basal and IGF-I-mediated cell survival was examined by morphological assessment of nuclear chromatin fragmentation. In comparison to control cells, neither partial caveolin-1 deficiency [pkDShuffle3T3L1, 18 ± 1.5% apoptosis, pkDCav3T3L1 (80) 16 ± 1.4% apoptosis, n = 4; P = not significant] nor complete caveolin deficiency [MF(+/+) 25 ± 0.6% apoptosis, MF (–/–) 24 ± 0.6% apoptosis, n = 4; P = not significant] altered the basal rate of apoptosis induced by serum withdrawal (Fig. 2Go, A and B). Furthermore, the ability of IGF-I to rescue cells from apoptosis was not impaired by modification of caveolin-1 expression [Fig. 2Go; pkDShuffle3T3L1 and pkDCav3T3L1 (80) cells, 40 ± 0.5% and 35 ± 0.3% reduction in apoptosis, respectively; MF(+/+) and MF(–/–) cells, 52 ± 0.1% and 61 ± 0.1% reduction in apoptosis, respectively].


Figure 2
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  		FIG. 2. Neither caveolin-1 knockdown nor caveolin-1 knockout affects IGF-I-mediated cell survival. Serum-starved pkDshuffle3T3L1 (A), pkDCav3T3L1(80) (A), MF(+/+) (B), and MF(–/–) cells were incubated in serum-free medium with or without the addition of 5 nM IGF-I for 20 h. Apoptosis was determined by assessing the nuclear morphology of 4',6-diamidino-2-phenylindole stained cells (n = 1000). Graphs depict mean ± SEM of four independent experiments completed in triplicate. The asterisk (*) denotes the significant difference from serum-starved controls.

 
Knockdown of caveolin-1 induces a dose-dependent hyperproliferative response to IGF-I, whereas cells with complete caveolin-1 deficiency respond similarly to wild-type cells
Other researchers have noted increased cellular growth in association with a reduction in caveolin levels (19, 25), and in the current study, we found that in comparison to MF(+/+) cells, MF(–/–) cells incorporated significantly more [3H]thymidine (Fig. 3AGo; P < 0.01). Both MF(+/+) and MF(–/–) cells proliferated in response to IGF-I (Fig. 3Go, A and B), and when the differences in basal proliferation were accounted for, it was apparent that IGF-I induced an increase in thymidine incorporation in MF(+/+) cells (Fig. 3BGo; 2.2 ± 0.1-fold increase vs. untreated cells; P < 0.01; n = 4) that was similar to the IGF-induced increase in thymidine incorporation of MF(–/–) cells (Fig. 3AGo; 2.4 ± 0.2-fold increase; P < 0.01 n = 4).


Figure 3
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  		FIG. 3. Caveolin-1 knockdown induces a hyperproliferative response to IGF-I, but caveolin-1 knockout cells behave similarly to wild-type cells. Serum-starved caveolin-1 knockout [A and B; MF(+/+) and MF(–/–)] and knockdown [C; pkDShuffle3T3L1, pkDCav3T3L1(60) and pkDCav3T3L1(80)] cells were treated with 5 nM IGF-I for 20 h, and cell proliferation was assessed by monitoring [3H]thymidine incorporation. Graphs depict the mean ± SEM of four independent experiments completed in triplicate. The asterisk (*) denotes a significant difference in comparison to serum-starved control, where P < 0.01. The double asterisks (**) denote a significant difference from pkDShuffle3T3L1 cells, where P < 0.01.

 
Caveolin-1 knockdown had no affect on basal 3T3L1 proliferation because the level of [3H]thymidine incorporated by pkDCav3T3L1(80), pkDCav3T3L1(60), and pkDshuffle3T3L1 cells was similar (Fig. 3CGo). However, caveolin-1 knockdown did affect the cellular response to IGF-I because both pkDCav3T3L1(80) and pkDCav3T3L1(60) cells were hyperproliferative in comparison to the pkDshuffle3T3L1 cells (12.4 ± 0.4-fold and 4.3 ± 0.2-fold increase in [3H]thymidine incorporation vs. untreated cells; P < 0.01, n = 4).

Knockdown of caveolin-1 does not alter expression of putative IGF signaling proteins, but the expression of IGF-IR, IRS-1, and Akt is up-regulated in cells completely deficient in caveolin-1
We have previously shown that IGF-IR activates both the PI-3K and MAPK pathways to promote proliferation of 3T3L1 cells (4), and experiments using the signaling inhibitors LY294002 and PD98059 (Fig. 4AGo) demonstrated that these pathways contribute to IGF-I-mediated proliferation in caveolin-1 knockdown and caveolin-1 knockout cells also. There is evidence in the literature to suggest that caveolin regulates the activity and expression of such intracellular signaling molecules (26, 27, 28), and, thus, it is possible that partial deficiency of caveolin-1 could affect IGF-I-stimulated mitogenesis differently to complete caveolin-1 deficiency through changes to the expression and,/or activation of IGF signaling proteins. To examine this, cells were lysed and subjected to immunoblot analysis for IGF-IR, IRS-1, p42/44 MAPK and Akt. As depicted in Fig. 4BGo, caveolin-1 knockdown did not impact on the expression of IGF-IR, IRS-1, p42/44 MAPK, or Akt. However, densitometric analysis of immunoblots confirmed that cells deficient in caveolin-1 have a significant increase (P < 0.01) in the expression of IGF-IR [IGF-IR to tubulin (tub) ratio: 0.65 ± 0.03], IRS-1 (IRS-1 to tub ratio: 0.31 ± 0.02), and Akt (Akt to tub ratio: 0.19 ± 0.02) (Fig. 4CGo) when compared with wild-type cells (signaling molecule to tub ratio: 0.42 ± 0.02, 0.16 ± 0.01, and 0.13 ± 0.05, respectively).


Figure 4
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  		FIG. 4. The MAPK and PI-3K/Akt signaling pathways are involved in mediating the proliferative response to IGF-I in both cavelin-1 knockdown and caveolin-1 knockout cells, but only caveolin-1 knockout affects the expression of IGF signaling proteins. A, Cells were serum starved for 24 h and then pretreated for 30 min with 100 nM LY294002 or 500 nM PD98059, and then incubated with 5 nm IGF-I for 20 h. Cell proliferation was assessed by monitoring [3H]thymidine incorporation. Graphs depict the mean ± SEM of four independent experiments completed in triplicate. The asterisk (*) denotes a significant difference in comparison to serum-starved control where P < 0.01. The double asterisks (**) denote a significant difference from control cells where P < 0.01. Lysates (50 µg protein) of caveolin-1 knockdown (pkDShuffle3T3L1 and pkDCav3T3L1(80) (B) and caveolin-1 knockout [MF(+/+) and MF(–/–)] (C) cells were subjected to immunoblot analysis for the IGF-IR, IRS-1, p42/44 MAPK, and Akt; data from three independent experiments are shown. Images in B and C were analyzed by densitometry and values normalized to tub.

 
Knockdown of caveolin-1 does not alter activation of putative IGF signaling proteins, but Akt activation is compromised in cells that lack caveolin-1
Next, we examined if caveolin-1 deficiency affected IGF-mediated phosphorylation of the IGF signaling proteins; serum-starved cells were treated with 5 nM IGF-I for 15–60 min, and then cell extracts were subjected to phospho-immunoblot analysis for activation of IGF-IR, IRS-1, p42/44 MAPK, and Akt.

In the caveolin-1 knockdown model (pkDCav3T3L1(80) cells), IGF-I promoted the sustained phosphorylation (up to 60 min) of IGF-IR, IRS-1, p42/44 MAPK, and Akt, and examination of the degree and timing of these phosphorylation events suggests that in comparison to pkDShuffle3T3L1 cells (Fig. 5AGo), there is no clear difference in the activity of any of these signaling molecules in response to IGF-I.


Figure 5
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  		FIG. 5. Caveolin-1 knockdown does not alter IGF-I-stimulated phosphorylation of intracellular signaling molecules, but IGF-I-stimulated phosphorylation of Akt is reduced in caveolin-1 knockout cells. Serum-starved pkDShuffle3T3L1, pkDCav3T3L1(80) (A), and serum-starved MF(+/+)and MF(–/–) (B) cells were treated with 5 nM IGF-I for 0–60 min, and then 50 µg protein from whole cell lysates was electrophoresed on the same gel. Samples were immunoblotted for phospho (P)-IGF-IR, phospho-IRS-1, phospho-p42/44 MAPK, and phospho-Akt [P-(ser473)Akt and P-(thr308)Akt in MF(+/+)and MF(–/–) cells]. Immunoblots were stripped and reprobed with antibodies against IGF-IR, IRS-1, p42/44 MAPK, and Akt to show equal loading. The immunoblots shown are representative of three independent experiments, each immunoblotted in duplicate. All immunoblots were analyzed by densitometry, and the data from analysis of MF(+/+)and MF(–/–) cells are shown in C. The asterisk (*) depicts significantly different from the serum-starved control, and the double asterisks (**) depict significantly different from wild-type cells, where P < 0.01.

 
IGF-I treatment of both MF(+/+) and MF(–/–) cells promoted an increase in the phosphorylation of all four signaling proteins (Fig. 5BGo). However, in the knockout model of caveolin-1 deficiency (MF(–/–) cells), IGF-I-mediated activation of IGF-IR and IRS-1 appeared to be increased, and the activation of Akt (P-(ser473)Akt and P-(thr308)Akt) was decreased, when compared with wild-type cells. In contrast, IGF-I-stimulated phosphorylation of MF(–/–) p42/44 MAPK was similar to that observed in MF(+/+) cells. Densitometric analysis (Fig. 5CGo) suggested that the apparent increase in IGF-I-stimulated phosphorylation of IGF-IR and IRS-1 in MF(–/–) cells most likely reflects the enhanced expression of these two signaling molecules. However, the decrease in IGF-I-induced phosphorylation of MF(–/–) Akt could not be explained by a reduction in Akt expression because the expression of total Akt was up-regulated in MF(–/–) cells; consequently, we next considered if the expression of the three Akt isoforms, Akt-1, -2, and -3, was altered in MF(–/–) cells.

Caveolin-1 knockout alters the expression of Akt isoforms
To clarify which of the Akt isoforms were expressed by MF(+/+) and MF(–/–) cells, lysates were subjected to immunoblot analysis using antibodies that specifically recognize Akt-1, Akt-2, or Akt-3. Figure 6AGo shows that in comparison to MF(+/+) cells, MF(–/–) expression of Akt-1 was reduced (Akt-1 to tub ratio: 2.07 ± 0.09 vs. 1.44 ± 0.11), though the expression of the Akt-2 (1.09 ± 0.06 vs. 2.02 ± 0.13) and Akt-3 (0.35 ± 0.01 vs. 0.60 ± 0.06) isoforms was increased. Consequently, the reduction in IGF-I-stimulated activation of Akt in MF(–/–) cells could be due either to reduced activation of the Akt-1 isoform or to less efficient activation of the more abundant Akt-2 or Akt-3 isoforms. To investigate these two possibilities, lysates of IGF-I-treated MF(+/+) and MF(–/–) cells were immunoprecipitated with antibodies specific for Akt-1, Akt-2, or Akt-3, and then the resulting precipitates were immunoblotted for P(ser473)Akt.


Figure 6
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  		FIG. 6. Caveolin-1 knockout alters the expression of Akt isoforms. A, Lysates (50 µg protein) of MF(+/+) and MF(–/–) cells were immunoblotted with antibodies against Akt-1, Akt-2, Akt-3, or total-Akt. B, Cell extracts (300 µg total protein) from unstimulated and IGF-I treated (5 nM; 15 min) MF(+/+) and MF(–/–) cells were immunoprecipitated (IP) with a phosphor(P)-Akt antibody, or an antibody specific for Akt-1, Akt-2, or Akt-3. Immunoprecipitates were then immunoblotted with a phosphor-Akt or total-Akt antibody. Samples immunoprecipitated without primary antibody were routinely included as a control (as shown in B and C), though to prove the specificity of the precipitating reaction, we also incubated samples with normal rabbit IgG and incubated the immunoprecipitating antibody with lysis buffer only, and these data are shown in C. D, In some experiments a phosphor-Akt antibody was used as the immunoprecipitating antibody, and immunoprecipitates were subjected to Western blot analysis for Akt-1 or phosphor-Akt. The images presented are representative of data from three independent experiments, and all immunoblots were subjected to densitometric analysis.

 
As depicted in Fig. 6BGo (upper panel), incubation of both MF(+/+) and MF(–/–) whole cell lysates with an antibody that specifically recognizes Akt-1 precipitated a 60-kDa protein that was also recognized by the P(ser473)Akt antibody used for immunoblot analysis. However, in comparison to the MF(+/+) cell lysates, the intensity of this band, which corresponds to P-Akt-1, was reduced in lysates of MF(–/–) cells. Proteins precipitated by either the anti-Akt-2 or anti-Akt-3 antibody (shown to be present by Western blotting with a total-Akt antibody; Fig. 6BGo, lower panel) demonstrated very weak or no immunoreactivity when the P(ser473)Akt antibody was used in the immunoblot analysis (Fig. 6BGo, upper panel). Control experiments (Fig. 6CGo) confirm the specificity of the reaction. Phosphorylation of Akt1 was also demonstrated after immunoprecipitation with a P(ser473)Akt antibody followed by an immunoblot for Akt1 (Fig. 6DGo). Collectively, these experiments suggest that IGF-I predominantly signals through the Akt-1 isoform of Akt in both caveolin-1-positive and caveolin-1 negative MF cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously demonstrated that through a direct association with caveolin, the IGF-IR is likely present within caveolae of the plasma membrane, but the functional significance of this observation has yet to be determined because our studies using the cholesterol-chelating agent, methyl-cyclodextrin, to disrupt caveolae suggested that these membrane domains are not essential for IGF-I-stimulated cellular proliferation and survival. However, in addition to caveolae, methyl-cyclodextrin affects other liquid-ordered domains within the plasma membrane, and, therefore, the current study used cellular models with reduced or ablated expression of caveolin-1 to investigate the impact of caveolae on IGF function.

Our data suggest that caveolin-1 is not a regulator of cellular survival because neither basal nor IGF-mediated survival was significantly altered in models of partial or complete caveolin-1 deficiency. This finding supports our previous work on methyl-cyclodextrin treatment of caveolae-positive (3T3L1) and caveolae-negative (HepG2) cells, which suggested that rather than caveolae, cholesterol and the broader class of lipid rafts are more important for survival signaling (4). In contrast, we have evidence to suggest that through modulation of Akt isoform expression and, consequently, IGF-I activation of the PI-3K/Akt pathway, caveolin-1 may regulate the proliferative response to IGF-I.

Cells in which caveolin-1 levels had been reduced to 80% of wild type via shRNA-mediated knockdown were hyperproliferative when stimulated with IGF-I. However, this functional effect could not be explained by changes in the expression or activity of intracellular kinases because the expression and IGF-I stimulated phosphorylation of IGF-IR, IRS-1, MAPK, and Akt in the pkDCav3T3L1 cell line were similar to that observed in pkDShuffle3T3L1 cells. Some studies, typically those using methyl-cyclodextrin, have found MAPK and/or Akt activity to be altered after caveolar disruption (27, 29, 30), however, the data from those using an RNA interference approach to knockdown caveolin-1 are varied; some suggest reduced activation of Akt, but not MAPK (31), whereas others, in accordance with our own findings, have reported that neither basal nor growth factor-stimulated activation of MAPK and Akt is affected (32, 33). It is possible that our observation of enhanced proliferation in response to IGF-I reflects altered phosphatase activity because studies that have assessed the consequence of caveolin-1 overexpression found that the activity of PDK1, Akt, and p42/44 MAPK was altered through inhibition of PP1 and PP2A (27). Alternatively, caveolin-1 knockdown may have altered flow through the various signaling pathways or, more likely, disrupted IGF-IR/downstream molecule compartmentalization such that nuclear signaling is more efficient (34).

Caveolin-1 knockdown did not affect basal cellular proliferation, though a recent in vivo study (35) suggests that a complete loss of caveolin-1 is necessary to impact on basal cellular function. Our data from experiments using cells isolated from caveolin-1 null mice agree with this finding because in this scenario, there was an enhanced basal proliferation. Consequently, we were surprised to find that in such cells, the proliferative response to IGF-I was similar to that observed in the cells obtained from wild-type mice. A possible explanation for these contrasting data from caveolin-1 knockdown and caveolin-1 knockout cells is that cells with complete, or prolonged, loss of caveolin-1 can respond appropriately to IGF-I due to compensatory changes in the expression and/or activity of key signaling molecules. Indeed, this hypothesis is supported by data demonstrating that in comparison to wild-type cells, the type and level of endothelin (ET)-1 receptor expressed by smooth muscle cells obtained from caveolin-1 knockout mice are altered [ET(A) decreased, ET(B) increased]), and as a result, the level of intracellular Ca2+-induced by ET-1 was enhanced (36).

In our study, the apparent increase in IGF-I-induced activation of the signaling molecules IGF-IR and IRS-1 could be explained by a proportionate increase in the level of their expression; in contrast, the expression of Akt, as determined by immunoblotting using an antibody that recognizes all three Akt isoforms, was increased in cells obtained from the caveolin-1 knockout mice, yet IGF-I-stimulated phosphorylation of Akt was significantly reduced. Further investigation revealed that wild-type and caveolin-1 knockout cells differentially express the three isoforms of Akt because the expression of Akt-1 was decreased, and that of Akt-2 and Akt-3 increased, in cells lacking caveolin-1. Our data demonstrate that in this cell model, Akt-2 and AKt-3 are only minimally phosphorylated in response to IGF-I, and therefore the reduced capacity for IGF-I signaling through the Akt pathway may explain why caveolin-1 knockout cells are not hyperproliferative in response to IGF-I. However, it is not clear whether the loss of Akt-1 promotes increased expression of Akt-2 and Akt-3, possibly as some form of compensation, or whether the loss of caveolin allows up-regulation of Akt-2 and Akt-3, which in turn down-regulates the expression of the Akt-1 isoform. In vitro studies have suggested that the Akt isoforms have distinct functions; Akt-2 was found to be more important than Akt-1 for insulin-stimulated translocation of glucose transporter 4 to the plasma membrane (37), and in mouse myoblasts, Akt-1 was needed for cellular proliferation, whereas Akt-2 was involved in regulating exit from the cell cycle (38). Evidence from Akt transgenic mice fits with the idea that Akt-1 is important for promoting cellular proliferation (39, 40), however, in vivo data also suggest that there may be functional redundancy between the isoforms because Akt-2 and Akt-3 have also been implicated in the mediation of this cellular event (41, 42). Future experiments to assess the impact of Akt isoform-specific knockdown (with small interfering RNA or morpholinos) upon IGF-I-mediated proliferation of wild-type cells will be required to test our speculation.

Nonetheless, it is possible that after the loss of caveolin, cells that do not switch Akt isoform expression are more responsive to proliferative signals from IGF-I, and other growth factor receptor kinases that mediate their effects through Akt1, which in turn, makes them more susceptible to transformation.

Studies have previously demonstrated that the targeted down-regulation of caveolin-1 is sufficient to drive cell transformation (43), and it is well known that hyperactivation or overexpression of IGF-IR can cause cellular transformation (44, 45, 46). Although Akt-1, Akt-2, and Akt-3 are frequently overexpressed or hyperactivated in human cancers (47, 48, 49, 50, 51, 52), further evidence suggests that cytoplasmic Akt-1, at least in prostate cancer, can be used as a marker of tumor aggression.

In summary, our data confirm that caveolin-1 is not essential for IGF signaling/function, however, they also suggest that changes in caveolin-1 expression can impact on cellular mitogenesis in response to IGF-I and, therefore, that caveolin-1 is important for fine-tuning signals through the IGF-IR/PI-3K/Akt pathway. Further study into the relationship between the IGF-IR, Akt, and caveolin is warranted because these proteins are all potential therapeutic targets in the treatment of various cancers.


   Acknowledgments
 
We thank Richard Anderson, Julian Davis, and David Ray for their kind donation of cells, plasmids, and antibodies, and also thank Andrew Berry, Helen Garside, Pingsheng Liu, and Rebecca Garside for technical assistance.


   Footnotes
 
The Biotechnology and Biological Sciences Research Council (UK) provided Ph.D. studentship for L.C.M.

Disclosure Statement: The authors have nothing to declare.

First Published Online June 26, 2008

Abbreviations: ET, Endothelin; FCS, fetal calf serum; HRP, horseradish peroxidase; IGF-IR, type I IGF receptor; IRS-1, insulin receptor substrate 1; PI-3K, phosphatidylinositol 3-kinase; shRNA, small hairpin RNA; tub, tubulin.

Received August 31, 2007.

Accepted for publication June 13, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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