Caveolin-1 expression sensitizes fibroblastic and epithelial cells to apoptotic stimulation

Jun Liu1, Peiyee Lee2, Ferruccio Galbiati1, Richard N. Kitsis2,3, and Michael P. Lisanti1,3

Departments of 1 Molecular Pharmacology and 2 Medicine and Cell Biology and 3 Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The potential role of caveolin-1 in apoptosis remains controversial. Here, we investigate whether caveolin-1 expression is proapoptotic or antiapoptotic using a well-defined antisense approach. We show that NIH/3T3 cells harboring antisense caveolin-1 are resistant to staurosporine-induced apoptosis, as assessed using cell morphology, DNA content, caspase 3 activation, and focal adhesion kinase cleavage. Importantly, sensitivity to apoptosis is recovered when caveolin-1 levels are restored. Conversely, recombinant stable expression of caveolin-1 in T24 bladder carcinoma cells sensitizes these cells to caspase 3 activation. Consistent with the observations using NIH/3T3 cells, downregulation of caveolin-1 in T24 cells substantially diminishes caspase 3-like activity. Loss of sensitivity to apoptotic stimulation is recovered by inhibition of the phosphatidylinositol 3-kinase pathway using LY-294002, suggesting a possible mechanism for the sensitizing effect of caveolin-1. Thus our results suggest that caveolin-1 may act as a coupling or sensitizing factor in signaling apoptotic cell death in both fibroblastic (NIH/3T3) and epithelial (T24) cells.

caveolae; caveolin; signaling


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CAVEOLINS, a family of 21- to 24-kDa integral membrane proteins, are the principal structural components of caveolae membrane domains. Recent in vivo and in vitro studies have shown that the caveolins associate and directly interact with a variety of signaling molecules via a modular protein domain, termed the caveolin-scaffolding domain (21). More specifically, the caveolin-scaffolding domain recognizes a specific caveolin-binding motif within a given signaling protein (42, 49).

Interestingly, caveolin binding via the scaffolding domain inhibits the activity of both tyrosine and serine/threonine kinases, including Src family tyrosine kinases, epidermal growth factor receptor (EGFR), Neu, mitogen-activated protein kinase (MEK1), extracellularly regulated kinase 2 (ERK2), protein kinase C (PKC), and protein kinase A (11, 17, 34, 44, 49). Functional studies revealed that overexpression of caveolin-1 dramatically inhibits the p42/44 mitogen-activated protein kinase signaling cascade (17, 39). Hence, these results suggest that caveolin may function as a general inhibitor of protein kinases.

Although caveolin-1 is expressed in most cell types, caveolin-1 expression is downregulated or absent in oncogenically transformed NIH/3T3 cells, as well as human cancer cells (19, 32, 33, 43). Conversely, overexpression of caveolin-1 blocks anchorage-independent growth of oncogenically transformed cells (19, 33), indicating that caveolin-1 may act as a suppressor of oncogenic transformation (42). More recently, we have shown that an antisense-mediated reduction in caveolin-1 protein expression in NIH/3T3 cells is sufficient to drive oncogenic transformation and constitutively activate the p42/44 mitogen-activated protein (MAP) kinase cascade (27). Finally, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (D7S522; 7q31.1), a known fragile site (FRA7G) that is deleted in many forms of cancer (20-23). Thus downregulation of caveolin-1 expression and caveolae organelles may be critical for maintaining the transformed phenotype.

Apoptosis plays a pivotal role both in embryonic development and in maintaining homeostasis in adult tissues. Morphologically, cells undergoing apoptosis exhibit cytoplasmic shrinkage, nuclear condensation, internucleosomal DNA cleavage, and cellular fragmentation (13). A central component of the death machinery is a novel family of cysteine proteases, termed caspases (53). Caspases are expressed as proenzymes that contain an NH2-terminal prodomain, a large subunit (~20 kDa) and a small subunit (~10 kDa). Upon the receipt of "death signals", caspases become activated by proteolytic processing between their domains. The resulting active enzyme complex is a heterotetramer of two large and two small subunits. Activated caspases subsequently cleave multiple cellular proteins, including focal adhesion kinase (FAK), nuclear lamins, PKCdelta , DFF45/ICAD, Bcl-2, and poly (ADP-ribose) polymerase (PARP), etc., leading to the demise of the cell (12).

The potential role of caveolin-1 in apoptosis remains controversial. Using ceramide as a stimulus, Zundel and colleagues (56) observed that caveolin-1 expression in Rat-1 fibroblasts acts as a facilitator of ceramide-induced cell death. However, these authors used only cell morphology as a criterion for cell death, and they did not directly show that these effects were due to apoptosis; as a consequence, they did not use the word "apoptosis" to characterize the cell death that they observed (56). In contrast, Timme and colleagues (54) have now shown that caveolin-1 is a suppressor of c-myc-induced apoptosis in LNCaP cells, a human epithelial prostate cancer-derived cell line. Similarly, this group used only morphological criteria to assess cell death. Thus contradictory evidence has now been presented that caveolin-1 is both a facilitator and a suppressor of cell death in different contexts.

To address this discrepancy, we used a third independent apoptotic stimulus (staurosporine treatment, rather than ceramide or c-myc) to assess the potential role of caveolin-1 in apoptosis in two distinct cell lines, NIH/3T3 fibroblasts and T24 bladder carcinoma cells. In addition to morphological criteria, we used a variety of quantitative biochemical assays to directly assess apoptosis, such as DNA content, caspase 3 activity, and FAK cleavage. Our results are consistent with that idea that 1) caveolin-1 generally plays a proapoptotic role in both fibroblasts (NIH/3T3) and epithelial cells (T24), and 2) the antiapoptotic effects of caveolin-1 that were previously observed may be relevant only for myc-induced apoptosis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. A monoclonal antibody directed against caveolin-1 (MAb clone 2297; recognizing residues 61-71; Ref. 48) was kindly provided by Dr. Roberto Campos-Gonzalez (Transduction Laboratories, Lexington, KY). Reagents and other supplies were obtained from the following commercial sources: RNase A and anti-beta -actin MAb (Sigma Chemical, St. Louis, MO); FAK MAb (Transduction Laboratories); anti-Akt and anti-phospho-Akt rabbit polyclonal antibodies (New England Biolabs, Beverly, MA); staurosporine (Biomol Research Laboratories, Plymouth Meeting, PA); ApoAlert caspase 3 assay kit (Clontech Laboratories, Palo Alto, CA); KT-5720 (Alexis Biochemicals, San Diego, CA); hygromycin B and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk; CalBiochem, La Jolla, CA); propidium iodide (PI) and diamidinophenylindole (DAPI; Molecular Probes, Eugene, OR); and bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL).

Cell culture and generation of stable cell lines. Normal NIH/3T3 cells were grown in DMEM (GIBCO BRL) with 10% heat-inactivated donor bovine calf serum (normal growth medium). NIH/3T3 cells harboring antisense caveolin-1 (Cav-1-AS) were generated by cotransfection of NIH/3T3 cells with an expression vector, pCAGGS, encoding the murine caveolin-1 inserted in the antisense orientation, and a plasmid, pCB7, containing hygromycin resistance gene, as we described previously (27). Cav-1-AS cells were selected in normal growth medium supplemented with 200 µg/ml hygromycin B (selection medium). A revertant of NIH/3T3 cells harboring Cav-1 AS (Rev-Cav-1-AS) was derived after Cav-1-AS cells were cultured in the absence of selection medium for four to six passages (27). T24 (human bladder carcinoma) cells were grown in medium 199 with 10% fetal bovine serum. Stable T24 cell lines overexpressing caveolin-1 (T24-Cav-1) were generated by transfection of T24 cells with an expression vector, pCB7, encoding caveolin-1. T24 cells harboring Cav-1-AS (T24-Cav-1-AS) were generated as described above. Cell lines harboring empty vector alone (pCB7) were also generated and served as controls. The levels of caveolin-1 expression were examined by both Western and Northern blotting, as we carefully documented previously (27).

Induction of apoptosis. Staurosporine exposure has been used as a reliable model to induce apoptosis in a variety of cell types (3, 4, 10, 28, 29, 45). A stock solution was made by dissolving staurosporine in dimethyl sulfoxide (DMSO) at a concentration of 2 mM and with storage at -20°C. For induction of apoptosis, NIH/3T3, Cav-1-AS, and Rev-Cav-1-AS cells, as well as T24, T24-pCB7, T24-Cav-1, and T24-Cav-1-AS cells, were incubated with staurosporine for a period of time as indicated in RESULTS. Controls were treated with vehicle alone (0.05% DMSO).

Immunoblot analysis. The expression levels of caveolin-1 and FAK were determined by Western blotting. Briefly, cells were solubilized with SDS-sample buffer that contained 0.125 M Tris · HCl (pH 6.8), 2% (wt/vol) SDS, 5% (vol/vol) 2-mercaptoethanol, and 10% (vol/vol) glycerol in double distilled water. After being boiled for 4 min, proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes for immunoblotting using enhanced chemiluminescence. Before loading, the protein concentration of the samples was measured using the BCA method with bovine serum albumin as the standard.

Caspase 3-like activity assay. Caspase 3-like activity was assessed using a slightly modified ApoAlert CPP32/caspase 3 fluorescent assay. Briefly, after treatment with staurosporine, incubation medium was removed and the cells were lysed for 10 min in ice-cold lysis buffer that contained 20 mM Tris · HCl (pH 7.4), 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 µg/ml aprotinin, 1 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Total cell lysates (10 µg each) were incubated at 37°C for 60 min in enzyme assay buffer that contained 20 mM HEPES (pH 7.5), 10% glycerol, 2 mM dithiothreitol, 1 mM PMSF, and 100 µM caspase 3 substrate, N-acetyl-Asp-Glu-Val-Asp (DEVD)-7-amino-4-methyl coumarin (AMC). The reaction was terminated by adding 1 ml of deionized H2O. Fluorescence was measured with an LS50B luminescence spectrometer (Perkin Elmer, Norwalk, CT) equipped with a 380-nm excitation filter and a 460-nm emission filter. To assess specific caspase 3-like activity, a parallel set of cell lysates was preincubated with 30 µM caspase 3 inhibitor, DEVD-aldehyde (CHO), at 37°C for 30 min and then incubated with caspase 3 substrate for 60 min.

Determination of DNA content by flow cytometry. After treatment with staurosporine, 5 × 105 cells were isolated using trypsin-EDTA and then fixed with 70% ethanol at -20°C overnight. Cells were pelleted by centrifugation at 200 g for 5 min, and the 70% ethanol was discarded. Cells were then resuspended in DNA extraction buffer (0.2 M phosphate citrate buffer, pH 7.8) for 30 min at 37°C, followed by centrifugation at 1,500 g for 10 min. Cells were then resuspended in a PI solution (50 µg/ml in PBS) that contained 50 µg/ml RNase A and 0.1% (vol/vol) Triton X-100 and incubated in the dark at room temperature for 30 min. The DNA content of the cells was then analyzed using the FACScan immunocytometry system (Becton Dickinson, San Jose, CA) (7).

Nuclear morphology visualized by DAPI staining. NIH/3T3, Cav-1-AS, and Rev-Cav-1-AS cells (pretreated with or without staurosporine) were carefully washed once with PBS, fixed with 3.7% formaldehyde at room temperature for 5 min, and then stained with DAPI (1 µg/ml in PBS) for 10 min. Nuclear morphology was examined with an Olympus IX70 fluorescent microscope equipped with a photometric charge-coupled device camera that contained a DAPI channel.

Electron microscopy. Transmission electron microscopy was performed as described previously by our laboratory. Samples were fixed with glutaraldehyde, postfixed with osmium tetroxide, and stained with uranyl acetate and lead citrate (as detailed in Refs. 36 and 46). Samples were examined under the Philips 410 transmission electron microscope.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Staurosporine-induced apoptosis in NIH/3T3 cells. Staurosporine, a potent inhibitor of diverse protein kinases, is an extraordinarily potent antiproliferative agent and a commonly used apoptotic inducer. To assess a potential role for caveolin-1 in promoting apoptosis, we first developed an apoptotic model system employing staurosporine-treated NIH/3T3 cells. Normal NIH/3T3 cells contain numerous caveolae and endogenously express both caveolins-1 and -2 (32, 47).

As shown in Fig. 1A, treatment of NIH/3T3 cells with staurosporine (1 µM) from 0 to 24 h resulted in a time-dependent change in cell shape and size, an early event in apoptosis (13). Shrinkage first appeared in the cell periphery after a 30-min treatment and became more evident after 2 h (Fig. 1A, b and d). After 6 h of treatment, all cells appeared round in shape with spider-like projections, and, by 24 h, some cells began to detach from the plate (Fig. 1A, e and f). The plasma membrane remained intact until 24 h of treatment, as assessed by trypan blue exclusion. Interestingly, focal adhesion sites remained unchanged until after 1 h of treatment and disappeared after 2 h of incubation with staurosporine (Fig. 1A, b and c), when proteolytic cleavage of FAK started to occur (Fig. 2B).


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Fig. 1.   Staurosporine-induced apoptosis in parental NIH/3T3 cells. A: cellular morphology. NIH/3T3 cells were incubated with staurosporine (1 µM; b-f) in normal growth medium for 0.5-24 h. Control cells were treated with vehicle alone (0.05% DMSO; a) for 24 h. In the absence of staurosporine, NIH/3T3 cells had a flattened morphology (a). Note that staurosporine induced rapid cell shrinkage, which was observed first at the cell periphery after a 30-min treatment (b; arrow), without affecting focal adhesion sites until 1 h of treatment (b and c; arrowheads). Time-dependent cell shrinkage was observed after 30 min (b), 1 h (c), 2 h (d), and 6 h (e) of treatment with staurosporine. Some cells appeared to completely detach after 24 h of treatment (f; arrow). B: nuclear morphology. NIH/3T3 cells were incubated in the absence (a) or presence (b and c) of staurosporine for 10 h and then stained with diamidinophenylindole (DAPI) to visualize nuclear morphology. In d, cells were pretreated with N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk; 50 µM) for 1 h and then incubated with staurosporine for 10 h. Arrows point to nuclear condensation (b) and fragmentation (c). C: DNA content. NIH/3T3 cells were incubated in the presence (bottom) or absence (top) of staurosporine for 10 h and then stained with propidium iodide (PI) for flow cytometry analysis. The arrow (bottom) points to a subpopulation of cells with hypodiploid DNA, due to DNA loss. In B and C, note that staurosporine induces nuclear condensation/fragmentation and DNA loss.



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Fig. 2.   Staurosporine-induced activation of caspase 3 and focal adhesion kinase (FAK) cleavage in parental NIH/3T3 cells. A: caspase 3 activation. NIH/3T3 cells were incubated with staurosporine (1 µM) in normal growth medium for a period of 0, 2, 4, 6, 10, and 24 h. Caspase 3-like proteolytic activity was assessed with the ApoAlert CPP32/caspase 3 fluorescent assay (the same method for assessing caspase 3-like proteolytic activity was used in C and D, as well as in Figs. 5A, 7A, 8, B and C, and 9, A and B). The data shown are means ± SD of 1 representative experiment from 3 experiments. In each experiment, each time point was performed in triplicate. B: FAK cleavage. NIH/3T3 cells were incubated with staurosporine (1 µM) in normal growth medium for a period of 0, 2, 4, 6, 10, and 24 h. The proteolytic cleavage of FAK, an endogenous caspase 3 substrate, was monitored by Western blotting with a specific monoclonal antibody (MAb) directed against residues 354-533 of FAK (top). Arrow points to the expected proteolytic fragments of FAK (~77-85 kDa). The same membrane was reprobed with anti-beta -actin IgG (bottom) as a control for equal protein loading. C: effect of z-VAD-fmk. NIH/3T3 cells were incubated with vehicle alone (0.05% DMSO), with staurosporine (1 µM) in normal growth medium for 6 h, or preincubated with z-VAD-fmk (50 µM) for 1 h and then incubated with staurosporine [1 µM; staurosporine (STS)] in normal growth medium for 6 h. D: effect of KT-5720. NIH/3T3 cells were incubated with vehicle alone (0.05% DMSO), with staurosporine (1 µM) in normal growth medium for 6 h, or with KT-5720 (10 µM) in normal growth medium for 6 h. In C and D, the data shown are means ± SD of 1 representative experiment from 2 experiments. In each experiment, each condition was performed in triplicate.

Staurosporine-induced apoptosis was also apparent by nuclear morphology (DAPI staining). In control cells, the nuclei appeared round or oval in shape with scattered punctate staining (Fig. 1B, a). In contrast, incubation with staurosporine resulted in nuclear condensation and fragmentation (Fig. 1B, b and c). Preincubation of cells with a peptide caspase inhibitor (z-VAD-fmk; 50 µM) did not affect staurosporine-induced cell shrinkage but clearly blocked nuclear condensation and fragmentation (Fig. 1B, d), suggesting that the later event was mediated through the activation of caspases.

Quantitative analysis using flow cytometry showed that >10% of the cell nuclei exhibited hypodiploid DNA content when cells were treated with staurosporine for 10 h (Fig. 1C).

Time-dependent activation of caspase 3-like activity by staurosporine. Figure 2 shows that treatment of NIH/3T3 cells with staurosporine resulted in time-dependent activation of caspase 3-like activity. Caspase 3-like activity increased up to 2-fold after 2 h of treatment and became maximal after 6 h of treatment, resulting in ~5.8-fold activation over basal activity. The enzyme activity decreased by 10 h of treatment and returned to basal levels after 24 h of treatment (Fig. 2A). Importantly, pretreatment of cells with z-VAD-fmk (50 µM) abolished staurosporine-induced activation of caspase 3-like activity (Fig. 2C).

Activation of caspases results in proteolytic cleavage of a variety of substrates. These include FAK, nuclear lamins, PKCdelta , DFF45/ICAD, Bcl-2, and PARP (12). Coincident with the changes in caspase 3-like activity, treatment with staurosporine produced a time-dependent cleavage of FAK (125 kDa) to its proteolytic product (77-85 kDa; Fig. 2B, arrow). Proteolytic cleavage of FAK appeared after 2 h of treatment, reached maximal levels after 4-6 h, and declined after 10 h. Because maximal activation of caspase 3-like activity and FAK cleavage occurred after 6 h of staurosporine treatment (Fig. 2, A and B), we chose to use 6 h of staurosporine treatment in the following experiments.

Among various protein kinase and phosphatase inhibitors, staurosporine appears to be unique in its ability to induce apoptosis in a variety of cell types (4). KT-5720 is a semi-synthetic derivative of staurosporine and a specific inhibitor for protein kinase A (4, 30). Consistent with these observations, treatment of NIH/3T3 cells with KT-5720 (10 µM) for up to 10 h did not affect caspase 3-like activity (Fig. 2D). Similarly, treatment with KT-5720 did not cause cell shrinkage or DNA fragmentation (not shown). It is important to note that in all experiments described above, incubation of the cells with vehicle alone (0.05% DMSO) did not affect cell and nuclear morphology or caspase activity.

Downregulation of caveolin-1 inhibits apoptosis in NIH/3T3 cells. To investigate if caveolin-1 expression is required for induction of apoptosis in NIH/3T3 cells, we employed an antisense approach to generate an NIH/3T3 cell line that expresses dramatically reduced levels of caveolin-1 (see MATERIALS AND METHODS). If caveolin-1 expression is critical for mediating apoptosis, we would predict that downregulation of caveolin-1 expression in NIH/3T3 cells would block or blunt the response to apoptotic stimuli, such as staurosporine.

As we previously reported (27), NIH/3T3 cells harboring Cav-1-AS expressed substantially reduced levels of caveolin-1 (Fig. 3A). As expected, caveolae were also downregulated in these caveolin-1 antisense cells, as seen by transmission electron microscopy (Fig. 3B).


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Fig. 3.   Downregulation of caveolin-1 expression and caveolae organelles in NIH/3T3 cells harboring antisense caveolin-1. A: caveolin-1 expression. Total cellular proteins were harvested from normal parental NIH/3T3 cells, NIH/3T3 cells harboring antisense caveolin-1 (Cav-1-AS), and the corresponding NIH/3T3 revertant cell line (Rev-Cav-1-AS; see MATERIALS AND METHODS). An equal amount of protein (30 µg/lane) was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblot analysis with an MAb directed against caveolin-1 (MAb clone 2297) (top). Note that compared with parental NIH/3T3 cells, Cav-1-AS cells express little or no caveolin-1 (an ~15- to 20-fold reduction), as we have previously shown (27). In Rev-Cav-1-AS cells, caveolin-1 levels are ~70-80% of that seen in parental NIH/3T3 cells. The same membrane was reprobed with anti-beta -actin IgG (bottom) as a control for equal protein loading. B: caveolae organelles. Top: normal parental NIH/3T3 cell. Arrowheads point at numerous caveolae. Bottom: NIH/3T3 cell harboring Cav-1-AS. Note that caveolae are morphologically downregulated in Cav-1-AS, as observed by transmission electron microscopy.

Consistent with our above observations, treatment of normal NIH/3T3 cells with staurosporine (1 µM) resulted in nuclear condensation and fragmentation (Fig. 4A) as early as at 6 h of treatment (Fig. 4A, d). In contrast, the nuclei in NIH/3T3 cells harboring Cav-1-AS remained morphologically unchanged, even after 10 h of treatment (Fig. 4A, e and h).


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Fig. 4.   Downregulation of caveolin-1 expression confers resistance to an apoptotic stimulus. A: nuclear morphology. Normal parental NIH/3T3 cells (a, d, and g), Cav-1-AS cells (b, e, and h), and Rev-Cav-1-AS cells (c, f, and i) were treated with vehicle alone (0.05% DMSO) for 10 h (a-c) or with staurosporine (1 µM) for 6 h (d-f) or 10 h (g-i). Cells were then stained with DAPI to visualize their nuclear morphology. Arrows point to nuclear condensation (in d, f, g, and i). Arrowheads point to apoptotic bodies (g) and DNA fragmentation (i). Note that normal parental NIH/3T3 cells and Rev-Cav-1-AS cells undergo nuclear changes that are hallmarks of apoptosis, whereas Cav-1-AS cells do not show these changes, even at 10 h of exposure. B: DNA content. Normal parental NIH/3T3 cells, Cav-1-AS cells, and Rev-Cav-1-AS cells were incubated with vehicle alone (0.05% DMSO; open bars) or staurosporine (1 µM; filled bars) for 10 h and then stained with PI for flow cytometry analysis. The percentage of cells with hypodiploid DNA content (DNA loss) is indicated. Data are means ± SE from 3 independent experiments.

Flow cytometric analysis revealed that the number of normal NIH/3T3 cells undergoing apoptosis (as measured by hypodiploid DNA content) in the absence of staurosporine was less than ~3% of the total cell population. This number reached up to 11% after treatment with staurosporine for 10 h (Fig. 4B). In sharp contrast, staurosporine caused little change in DNA content in NIH/3T3 cells harboring Cav-1-AS, which increased from 1.7% (control) to 2.2% (staurosporine treated; Fig. 4B). Under these conditions, the number of Cav-1-AS cells with hypodiploid DNA content reached approximately one-fifth of that seen in normal NIH/3T3 cells (P < 0.05, unpaired t-test) and was even less in untreated control NIH/3T3 cells. Thus these results strongly suggest that downregulation of caveolin-1 expression blocks apoptosis in NIH/3T3 cells.

Inhibition of apoptosis by downregulation of caveolin-1 is reversible. After NIH/3T3 cells that harbor Cav-1-AS were cultured in the absence of selection media for four passages, caveolin-1 expression was restored (approaching ~70% of normal levels), due to loss of the caveolin-1 antisense vector (Fig. 3A; see Ref. 27). These results indicate that downregulation of caveolin-1 in this system is reversible.

Thus we next evaluated the ability of caveolin-1 antisense revertants to undergo apoptosis. Interestingly, the ability to undergo staurosporine-induced apoptosis was clearly restored in these cells (Rev-Cav-1-AS; Fig. 4, A and B). Treatment of the Cav-1-AS revertant cells with staurosporine resulted in nuclear condensation and fragmentation as early as 6 h (Fig. 4A). Similarly, flow cytometry analysis showed that the number of staurosporine-induced apoptotic Rev-Cav-1-AS cells increased from <3% to 6%, an ~2.8-fold increase over that which was observed in Cav-1-AS cells (P < 0.05, unpaired t-test; Fig. 4B).

Downregulation of caveolin-1 inhibits caspase 3-like activity and FAK cleavage. Figure 5A shows that treatment of normal NIH/3T3 cells with staurosporine dramatically increased caspase 3-like activity by ~7.7-fold over basal levels. In contrast, there was little or no increase in caspase 3-like activity in response to staurosporine in NIH/3T3 cells harboring Cav-1-AS (Fig. 5A). Importantly, activation of caspase 3-like activity was recovered when caveolin-1 expression was restored. More specifically, treatment of Rev-Cav-1-AS cells with staurosporine caused up to an ~3.5-fold increase in caspase 3-like activity (Fig. 5A). Virtually identical results were observed when cleavage of FAK was monitored by Western blot analysis (Fig. 5B).


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Fig. 5.   Downregulation of caveolin-1 expression blocks caspase 3 activation. Normal parental NIH/3T3 cells, Cav-1-AS cells, and Rev-Cav-1-AS cells were incubated with vehicle alone (0.05% DMSO; open bars) or staurosporine (1 µM; filled bars) for 6 h. A: caspase 3 activation. The data shown are means ± SD of 1 representative experiment from 3 experiments. In each experiment, each time point was performed in triplicate. B: FAK cleavage. The proteolytic cleavage of FAK, an endogenous caspase 3 substrate, was monitored by Western blotting with a specific MAb directed against residues 354- 533 of FAK (top). Arrow points to the expected proteolytic fragments of FAK (~77-85 kDa). The same membrane was reprobed with anti-beta -actin IgG (bottom) as a control for equal protein loading. Note that normal parental NIH/3T3 cells and Rev-Cav-1-AS cells show robust activation of caspase 3-like activity and FAK cleavage, whereas both apoptotic responses are blocked or blunted in Cav-1-AS cells.

Recombinant expression of caveolin-1 enhances caspase 3-like activity in human T24 bladder carcinoma cells. It is now generally recognized that caveolin-1 levels are downregulated during cell transformation in a variety of cell types (49). As a consequence, we chose to recombinantly express caveolin-1 in human T24 bladder carcinoma cells because they express low levels of endogenous caveolin-1. In accordance with these findings, T24 cells contain a copy of activated H-Ras (G12V) (52), and expression of activated H-Ras (G12V) is sufficient to downregulate caveolin-1 mRNA and protein levels in NIH/3T3 cells via transcriptional control (24). With this approach, we derived stable cell lines that recombinantly express caveolin-1 (T24-Cav-1) or control cells harboring vector alone (T24-pCB7). To further confirm our previous observations that downregulation of caveolin-1 desensitizes NIH/3T3 cells to apoptosis, we also derived T24-Cav-1-AS cells.

Figure 6 shows a Western blot analysis of lysates derived from parental T24, T24-Cav-1, T24-Cav-1-AS, and T24-pCB7 cells. Note that recombinant caveolin-1 was expressed at much higher levels in T24-Cav-1 cells (~4-fold over endogenous levels) compared with T24-pCB7 cells. By comparison, the level of caveolin-1 in T24-Cav-1-AS cells was reduced by approximately fivefold compared with T24 or T24-pCB7 cells.


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Fig. 6.   Recombinant expression of caveolin-1 in the human T24 bladder carcinoma cell line. We derived stable T24 cell lines that recombinantly overexpress caveolin-1 (T24-Cav-1), cells harboring anti-sense caveolin-1 (T24-Cav-1-AS), or control cells harboring vector alone (T24-pCB7). Western blot analyses of lysates derived from these cells are shown. Note that recombinant caveolin-1 is expressed at much higher levels in T24-Cav-1 cells (~4-fold increase over endogenous levels) and much lower levels in T24-Cav-1-AS cells (~5-fold reduction) compared with parental T24 and T24-pCB7 cells. Each lane contains an equivalent amount of total protein. The levels of beta -actin remain constant in these cells. This serves as an additional control for equal loading.

Treatment of T24 or T24-pCB7 cells with 0.8 µM staurosporine increased caspase 3-like activity by ~2.5- to 2.7-fold over basal levels (Fig. 7A). In striking contrast, in T24-Cav-1 cells, treatment with 0.8 µM staurosporine increased caspase 3-like activity by approximately sixfold over basal levels. Also, note that basal levels of caspase 3-like activity are elevated by ~1.5-fold in T24-Cav-1 cells, relative to T24 or T24-pCB7 cells. Thus, relative to unstimulated control (T24 or T24-pCB7) cells, staurosporine-treated T24-Cav-1 cells show ~10-fold more caspase 3-like activity.


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Fig. 7.   Recombinant expression of caveolin-1 enhances activation of caspase 3-like activity and apoptosis in human T24 bladder carcinoma cells. A: caspase 3 activation. Human T24 bladder carcinoma cells stably transfected with either T24-Cav-1 or T24-pCB7 were incubated with vehicle alone (0.05% DMSO; open bars) or staurosporine (0.8 µM; filled bars) for 6 h. Note that treatment of T24 cells and T24-pCB7 with staurosporine increased caspase 3-like activity by ~2.7-fold over basal levels. In striking contrast, in cells recombinantly expressing T24-Cav-1, treatment with staurosporine increased caspase 3-like activity by ~6-fold over basal levels. Also, basal levels of caspase 3-like activity are elevated by ~1.5- to 2-fold in T24-Cav-1 cells, relative to T24 and T24-pCB7 cells. The data shown are means ± SD of 1 representative experiment from 3 different experiments; each condition was performed in triplicate. B: DNA content. Parental T24 cells and T24-Cav-1 cells were incubated with vehicle alone (0.05% DMSO; open bars) or staurosporine (0.8 µM; filled bars) for 10 h and then stained with PI for flow cytometry analysis. The percentage of cells with hypodiploid DNA content (DNA loss) is indicated. Data are means ± SE from 3 independent experiments.

Flow cytometric analysis showed that in the absence of staurosporine, the number of T24-Cav-1 cells undergoing apoptosis was <0.58% of the total cell population. This number reached up to 30.8% after treatment with staurosporine for 10 h (Fig. 7B). The number of staurosporine-induced apoptotic cells in the T24-Cav-1 cell population was approximately threefold that of parental T24 cells, which increased from 0.37% (control) to 10.7% (staurosporine treated; Fig. 7B). Thus, consistent with our observations using NIH/3T3 cells, these results strongly suggest that upregulation of caveolin-1 expression sensitizes T24 cells to apoptotic stimulation.

Inhibition of the phosphatidylinositol 3-kinase signaling pathway restores the sensitivity of cells harboring antisense caveolin-1 to apoptotic stimulation. Caveolin-1 interacts with and inactivates a number of protein kinases involved in cell growth and proliferation, such as receptor tyrosine kinases [EGFR, Neu, and platelet-derived growth factor receptor (PDGFR) (18, 19, 55)], as well as components of the p42/44 MAP kinase cascade (MEK and ERK) (19, 27). As a consequence, we reasoned that caveolin-1 may sensitize cells toward apoptosis by inactivation of kinases that participate in antiapoptotic signaling pathways, such as phosphatidylinositol 3-kinase (PI 3-kinase)/Akt/protein kinase B (PKB).

To assess whether LY-294002 was able to block the PI 3-kinase signaling pathway in T24 cells, we first treated T24 cells with PDGF in the presence or absence of LY-294002. Figure 8A shows that inhibition of PI 3-kinase by LY-294002 blocked PDGF-induced activation of Akt/PKB as assessed by Akt/PKB phosphorylation, demonstrating that treatment of T24 cells with LY-294002 was able to block the PI 3-kinase/Akt/PKB antiapoptotic signaling pathway.


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Fig. 8.   Inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway restores the sensitivity of cells harboring antisense caveolin-1 to apoptotic stimulation. A: inhibition of platelet-derived growth factor (PDGF)/PI 3-kinase/Akt/protein kinase B (PKB) signaling by LY-294002. Human T24 bladder carcinoma cells were pretreated with LY-294002 (20 µM) for 1 h and then treated with or without 10 ng/ml PDGF (BB) in the presence or absence of LY-294002 for 10 min. Total cellular proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblot analysis with anti-phospho-Akt IgG (top). The same membrane was reprobed with anti-beta -actin IgG (bottom) as a control for equal protein loading. B: caspase 3 activity in T24-Cav-1-AS cells. Human T24 bladder carcinoma cells stably transfected with antisense caveolin-1 (T24-Cav-1-AS) were pretreated with LY-294002 (20 µM) for 1 h and then treated with 0.3 µM staurosporine for 5 h. Parental T24 cells were treated in parallel and served as a positive control. The data shown are means ± SD of 1 representative experiment from 3 experiments. C: caspase 3 activity in NIH/3T3 Cav-1-AS cells. NIH/3T3 Cav-1-AS cells were pretreated with LY-294002 (20 µM) for 1 h and then treated with 0.6 µM staurosporine for 5 h. Parental NIH/3T3 cells were treated in parallel and served as a positive control. The data shown are means ± SD of 1 representative experiment from 3 experiments. D: inhibition of Akt/PKB activity by recombinant expression of caveolin-1. Human T24 bladder carcinoma cells stably transfected with caveolin-1 (T24-Cav-1) or pCB7 vector alone (T24-pCB7) were prestarved for 3 h in medium 199 and then treated with 0, 1, or 3 ng/ml PDGF for 10 min. Total cellular proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblot analysis with anti-phospho-Akt IgG (top). The same membrane was reprobed with anti-beta -actin IgG (bottom) as a control for equal protein loading.

Consistent with our previous observations using NIH/3T3 cells, downregulation of caveolin-1 expression inhibited staurosporine-induced caspase 3-like activity in T24-Cav-1-AS cells (Fig. 8B). These results clearly suggest a proapoptotic role for caveolin-1 in staurosporine-induced apoptosis in both fibroblasts (NIH/3T3) and epithelial cells (T24).

To assess the mechanisms underlying caveolin-1-mediated sensitization to apoptosis, we pretreated T24 and T24-Cav-1-AS cells with the PI 3-kinase inhibitor, LY-294002, followed by staurosporine. Pretreatment of these cells with LY-294002 alone for 1 h caused little change in cell shape (not shown) and only small increases in caspase 3-like activity in T24 cells (1.2-fold over untreated cells) as well as in T24-Cav-1-AS cells (1.3-fold over untreated cells).

Significantly, pretreatment of T24-Cav-1-AS cells with LY-294002 restored the sensitivity of these cells to apoptotic stimulation and enhanced staurosporine-induced caspase 3-like activity by 3.2-fold over basal levels (Fig. 8B). By comparison, pretreatment of T24 cells with LY-294002 had little effect on staurosporine-induced caspase 3-like activity. Consistent with our results employing T24-Cav-1-AS cells, pretreatment of NIH/3T3 Cav-1-AS cells with LY-294002 increased their sensitivity to staurosporine stimulation by ~4.4-fold over basal activity (Fig. 8C). Thus loss of sensitivity to apoptotic stimulation caused by downregulation of caveolin-1 is recovered by inhibition of the PI 3-kinase/Akt/PKB signaling both in T24-Cav-1-AS and NIH/3T3 Cav-1-AS cells.

In support of these findings, human T24-Cav-1 cells show a dramatic decrease in basal and PDGF-stimulated Akt/PKB activity (Fig. 8D) compared with T24-pCB7 cells. Akt/PKB activation was assessed by immunoblot analysis with anti-phospho-Akt IgG that only recognizes the activated form of the enzyme.

Inhibition of the p42/44 MAP kinase pathway does not restore the sensitivity of cells harboring Cav-1-AS to apoptotic stimulation. To evaluate whether loss of sensitivity to apoptosis induced by downregulated caveolin-1 resulted from its action on the p42/44 MAP kinase signaling pathway (19, 27), NIH/3T3 Cav-1-AS cells were pretreated with a specific MEK1 inhibitor, PD-98059, for 1 h, followed by staurosporine treatment. Consistent with our above results, Fig. 9A shows that inhibition of PI 3-kinase by LY-294002 substantially increased caspase 3-like activity by 2.8-fold over control in response to staurosporine stimulation in NIH/3T3 Cav-1-AS cells. By comparison, inhibition of MEK1 by PD-98059 had little or no effect on staurosporine-induced caspase 3-like activity (1.1-fold over control).


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Fig. 9.   Inhibition of the p42/44 mitogen-activated protein kinase cascade does not restore the sensitivity of cells harboring antisense caveolin-1 to apoptotic stimulation. A: caspase 3 activity in NIH/3T3 Cav-1-AS cells. NIH/3T3 Cav-1-AS cells were pretreated with either LY-294002 (20 µM) or PD-98059 (40 µM) for 1 h and then treated with 0.6 µM staurosporine for 5 h. The data shown are means ± SD of 1 representative experiment from 3 experiments. B: caspase 3 activity in T24-Cav-1-AS cells. T24-Cav-1-AS cells were pretreated with either LY-294002 (20 µM) or PD-98059 (40 µM) for 1 h and then treated with 0.3 µM staurosporine for 5 h. The data shown are means ± SD of 1 representative experiment from 3 experiments.

Similarly, pretreatment of T24-Cav-1-AS cells with LY-294002 dramatically increased capase-3-like activity by 3.2-fold over control in response to staurosporine stimulation (Fig. 9B). In contrast, pretreatment with PD-98059 caused only a small increase in staurosporine-induced caspase 3-like activity (1.5-fold over control). These results suggest that the proapoptotic activity of caveolin-1 results from its inhibition of the PI 3-kinase/Akt/PKB signaling pathway, rather than the p42/44 MAP kinase signaling pathway.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The decision to survive and proliferate or to commit to apoptosis is essential for normal embryonic development and maintaining tissue homeostasis. This process is determined by the net balance between anti- and proapoptotic signals that are linked in a tightly regulated manner (25). Unless provided with specific survival signals, the default pathway may lead to apoptosis.

A large body of evidence suggests that caveolin-1, a putative tumor suppressor, interacts with and inactivates a number of signaling molecules along survival/proliferation pathways, such as EGFR (19), PDGFR (55), Neu (18), Src (34), Raf (19), MEK1 (19, 27), and Erk (19, 27). These results imply that caveolin-1 expression may sensitize cells toward apoptosis. However, this hypothesis remains untested.

In the present study, we have clearly demonstrated that caveolin-1 plays a critical role in regulating apoptosis. NIH/3T3 fibroblasts that express substantially reduced levels of caveolin-1 are highly resistant to apoptosis, as judged by nuclear morphology, flow cytometry, and caspase 3-like activity. Importantly, the sensitivity of these NIH/3T3 cells to proapoptotic stimulation is recovered when caveolin-1 levels are restored. In addition, the proapoptotic activity of caveolin-1 was also observed with the use an epithelial cell line, namely, T24 human bladder carcinoma cells. Recombinant expression of caveolin-1 in these cells substantially enhanced their sensitivity to apoptotic stimulation. Thus our results suggest that caveolin-1 expression may provide a coupling or sensitizing function in signaling cell death.

The PDGF-activated PI 3-kinase/PKB/Akt signaling cascade is a well-characterized growth factor pathway that promotes cell survival and inhibits cell death (14). In this survival cascade, PI 3-kinase and Akt are critical factors; suppression of Akt activity by recombinant expression of dominant-negative Akt or of a mutant Akt consisting only of the pleckstrin homology domain abrogates growth factor-induced survival signals (16, 51). Several recent reports have demonstrated that proteins involved in the apoptotic process, including BAD (15) and caspase 9 (8), are targets of Akt. Phosphorylation and inactivation of these proteins by Akt protects cells from apoptosis.

What is the mechanism by which caveolin-1 can promote apoptosis? Caveolin-mediated inactivation of growth/proliferation signals is an attractive explanation. Here, we observed that blocking PI 3-kinase-mediated survival signals restores sensitivity to proapoptotic stimulation in cells harboring the caveolin-1 antisense vector. These results suggest that the proapoptotic function of caveolin-1 may result from its inhibitory action on PI 3-kinase. Indeed, consistent with our observations, Zundel and colleagues (56) have recently shown that ceramide (a known proapoptotic inducer) inactivates PI 3-kinase by recruiting caveolin-1 into receptor tyrosine kinase/PI 3-kinase complexes and that overexpression of caveolin-1 can inhibit the activity of PI 3-kinase. These results suggest that the proapoptotic activity of caveolin-1 expression is mediated via suppression of growth factor-stimulated survival pathways. In support of these observations, we have previously shown that caveolin-1 expression inhibits the growth factor-activated p42/44 MAP kinase signaling cascade in vivo (17, 39).

Another explanation may be the effect of caveolin-1 on the organization and assembly of anti- and proapoptotic signaling molecules at the cell surface. It has been proposed that caveolae function as message centers that compartmentalize signaling molecules at the cell surface (1, 35). A variety of signaling molecules that mediate anti- and proapoptotic signals are dramatically enriched in caveolae or caveolin-enriched microdomains (31, 36, 38, 40, 41, 50). Disruption of caveolae also affects proliferative signal transduction (26, 38).

More recent evidence has demonstrated that proapoptotic signaling molecules [such as tumor necrosis factor (TNF) receptor 1 and CD36] reside within caveolae-related membrane domains and that disruption of these organelles (via cholesterol-binding drugs) specifically blocks TNF-alpha -induced apoptosis (31). These results are consistent with our current findings that disruption of caveolae organelles via antisense ablation of caveolin-1 expression blocks staurosporine-induced apoptosis.

Nerve growth factor-mediated activation of p75 neurotrophin receptor signaling in cultured oligodendrocytes leads to apoptosis (9). In addition, the role of p75 neurotrophin receptor in cellular apoptosis has been critically reevaluated by using genetically engineered mice that lack p75 neurotrophin receptor or neurotrophin brain-derived neurotrophic factor, or both (2). The results provided firm evidence that the p75 neurotrophin receptor mediates apoptosis in vivo. Recent studies have revealed that the p75 neurotrophin receptor is located within caveolae and caveolae-related domains and forms a physical complex with caveolin-1 in a heterologous expression system (5, 6). Upon ligand binding, it is likely that the p75 neurotrophin receptor utilizes caveolar sphingomyelin as a substrate to generate ceramide and induces apoptosis. Interleukin-1 receptor seems to adopt similar apoptotic signaling via caveolae (37). Thus caveolae may play a proapoptic role by providing a common organized platform for death receptors to induce cell apoptosis.


    ACKNOWLEDGEMENTS

We thank Dr. Roberto Campos-Gonzalez for generously providing caveolin-1-specific monoclonal antibodies and Dr. Michael Cammer and Carolyn Marks for assistance with cell and nuclear morphology studies.


    FOOTNOTES

This work was supported by grants from the National Institutes of Health, the Muscular Dystrophy Association, the American Heart Association, and the Komen Breast Cancer Foundation (to M. P. Lisanti). P. Lee and R. N. Kitsis were supported by National Heart, Lung, and Blood Institute Grants R01-HL-60665 and R01-HL-61550 (to R. N. Kitsis).

M. P. Lisanti is the recipient of a Hirschl/Weil-Caulier Career Scientist Award.

Address for reprint requests and other correspondence: M. P. Lisanti, Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: lisanti{at}aecom.yu.edu).

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.

Received 14 August 2000; accepted in final form 27 October 2000.


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