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Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade

1998; Springer Nature; Volume: 17; Issue: 22 Linguagem: Inglês

10.1093/emboj/17.22.6633

1460-2075

Ferruccio Galbiati,

Protein Kinase Regulation and GTPase Signaling

Article16 November 1998free access Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade Ferruccio Galbiati Ferruccio Galbiati The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Daniela Volonté Daniela Volonté The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Jeffrey A. Engelman Jeffrey A. Engelman The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Genichi Watanabe Genichi Watanabe The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Departments of Developmental and Molecular Biology and Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Robert Burk Robert Burk The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Departments of Pediatrics, Microbiology and Immunology, and Epidemiology and Social Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Richard G. Pestell Richard G. Pestell The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Departments of Developmental and Molecular Biology and Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Michael P. Lisanti Corresponding Author Michael P. Lisanti The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Ferruccio Galbiati Ferruccio Galbiati The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Daniela Volonté Daniela Volonté The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Jeffrey A. Engelman Jeffrey A. Engelman The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Genichi Watanabe Genichi Watanabe The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Departments of Developmental and Molecular Biology and Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Robert Burk Robert Burk The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Departments of Pediatrics, Microbiology and Immunology, and Epidemiology and Social Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Richard G. Pestell Richard G. Pestell The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Departments of Developmental and Molecular Biology and Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Michael P. Lisanti Corresponding Author Michael P. Lisanti The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Author Information Ferruccio Galbiati1,2, Daniela Volonté1,2, Jeffrey A. Engelman1,2, Genichi Watanabe1,3, Robert Burk1,4, Richard G. Pestell1,3 and Michael P. Lisanti 1,2 1The Albert Einstein Cancer Center, 1300 Morris Park Avenue, Bronx, NY, 10461 USA 2Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA 3Departments of Developmental and Molecular Biology and Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA 4Departments of Pediatrics, Microbiology and Immunology, and Epidemiology and Social Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:6633-6648https://doi.org/10.1093/emboj/17.22.6633 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether downregulation of caveolin-1 is sufficient to mediate cell transformation or tumorigenicity. Here, we employ an antisense approach to derive stable NIH 3T3 cell lines that express dramatically reduced levels of caveolin-1 but contain normal amounts of caveolin-2. NIH 3T3 cells harboring antisense caveolin-1 exhibit anchorage-independent growth, form tumors in immunodeficient mice and show hyperactivation of the p42/44 MAP kinase cascade. Importantly, transformation induced by caveolin-1 downregulation is reversed when caveolin-1 protein levels are restored to normal by loss of the caveolin-1 antisense vector. In addition, we show that in normal NIH 3T3 cells, caveolin-1 expression levels are tightly regulated by specific growth factor stimuli and cell density. Our results suggest that upregulation of caveolin-1 may be important in mediating contact inhibition and negatively regulating the activation state of the p42/44 MAP kinase cascade. Introduction Caveolae are 50–100 nm vesicular invaginations of the plasma membrane (Severs, 1988). It has been proposed that caveolae participate in vesicular trafficking events and signal transduction processes (Lisanti et al., 1994a; Couet et al., 1997a; Okamoto et al., 1998). Caveolin, a 21–24 kDa integral membrane protein, is a principal component of caveolae membranes in vivo (Glenney, 1989, 1992; Glenney and Soppet, 1992; Kurzchalia et al., 1992; Rothberg et al., 1992). Caveolin is only the first member of a new gene family; as a consequence, caveolin has been re-termed caveolin-1 (Scherer et al., 1996). Caveolin-2 shows the same tissue distribution as caveolin-1 (Scherer et al., 1997), while caveolin-3 is only expressed in striated muscle cell types (cardiac and skeletal) (Way and Parton, 1995; Song et al., 1996b; Tang et al., 1996). It has been proposed that caveolin family members function as scaffolding proteins (Sargiacomo et al., 1995) to organize and concentrate specific lipids (cholesterol and glyco-sphingolipids; Fra et al., 1995a; Murata et al., 1995; Li et al., 1996b) and lipid-modified signaling molecules (Src-like kinases, H-Ras, eNOS and G-proteins; Li et al., 1995, 1996a,b; Garcia-Cardena et al., 1996; Shaul et al., 1996; Song et al., 1996a) within caveolae membranes. In support of this idea, caveolin-1 binding can functionally suppress the GTPase activity of hetero-trimeric G-proteins and inhibit the kinase activity of Src-family tyrosine kinases through a common caveolin domain, termed the caveolin-scaffolding domain (Li et al., 1995, 1996a; Song et al., 1996a). Since the identification of the caveolin-scaffolding domain and caveolin-binding sequence motifs, these observations have been extended to other caveolin-interacting proteins. Functional caveolin-binding motifs have been deduced in both tyrosine and serine/threonine kinases, as well as eNOS (Li et al., 1996a; Couet et al., 1997c; Garcia-Cardena et al., 1997; Ju et al., 1997; Michel et al., 1997; Oka et al., 1997; Engelman et al., 1998b). In all cases examined, the caveolin-binding motif is located within the enzymatically active catalytic domain of a given signaling molecule. For example, in the case of tyrosine and serine/threonine kinases, a kinase domain consists of 11 conserved subdomains (I–XI) (Couet et al., 1997c; Oka et al., 1997; Engelman et al., 1998b). The caveolin-binding motif is located within conserved kinase subdomain number IX, suggesting that caveolin could function as a ‘general kinase inhibitor’ (Okamoto et al., 1998). This hypothesis has been substantiated by the observation that the caveolin scaffolding domain inhibits Src family tyrosine kinases (c-Src/Fyn), EGF-R, Neu and PKC with similar potencies (Li et al., 1996a; Couet et al., 1997c; Oka et al., 1997; Engelman et al., 1998b). Sessa and colleagues have performed site-directed mutagenesis to modify the predicted caveolin-binding motif (from FSAAPFSGW to ASAAPASGA) within eNOS (Garcia-Cardena et al., 1997). It is known from in vitro studies that aromatic residues (W, F or Y) are required for the proper recognition of the caveolin-binding motif (Couet et al., 1997b). In their work, they show that mutation of the caveolin-binding motif within eNOS blocks the ability of caveolin-1 to inhibit eNOS activity in vivo (Garcia-Cardena et al., 1997). These findings provide the first demonstration that a caveolin-binding motif is relevant and functional in vivo. The direct interaction of caveolin with signaling molecules leads to their inactivation (Lisanti et al., 1994a; Couet et al., 1997a; Okamoto et al., 1998). Since many of these signaling molecules can cause cellular transformation when constitutively activated, it is reasonable to speculate that caveolin itself may possess transformation suppressor activity. Consistent with this hypothesis, both caveolae and caveolin are most abundantly expressed in terminally differentiated cells: adipocytes, endothelial cells and muscle cells (Bretscher and Whytock, 1977; Forbes et al., 1979; Fan et al., 1983; Simionescu and Simionsecu, 1983; Scherer et al., 1994, 1996). In addition, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes such as v-abl and H-ras (G12V); caveolae are absent from these cell lines (Koleske et al., 1995). The potential ‘transformation suppressor’ activity of caveolin-1 has recently been evaluated by using an inducible expression system to upregulate caveolin-1 expression in oncogenically transformed cells. Induction of caveolin-1 expression in v-Abl- and H-Ras (G12V)-transformed NIH 3T3 cells abrogated the anchorage-independent growth of these cells in soft agar and resulted in the de novo formation of caveolae (Engelman et al., 1997). Thus, downregulation of caveolin-1 expression and caveolae organelles may be critical for maintaining the transformed phenotype. However, it remains unknown whether a loss of caveolin-1 protein expression is sufficient to mediate cell transformation. Caveolae have also been implicated in signaling through the p42/44 MAP kinase pathway. Morphological studies have directly shown that ERK-1/2 is concentrated in plasma membrane caveolae in vivo using immunoelectron microscopy (Liu et al., 1997b). Evidence has been presented suggesting that other components of the p42/44 MAP kinase cascade are localized within caveolae membranes. These include receptor tyrosine kinases (EGF-R; PDGF-R; Ins-R) (Smart et al., 1995; Liu et al., 1996; Mineo et al., 1996; Liu et al., 1997a), H-Ras (Mineo et al., 1996; Song et al., 1996a), Raf kinase (Mineo et al., 1996), 14-3-3 proteins (Liu et al., 1996), ERK (Lisanti et al., 1994b; Liu et al., 1996), Shc (Liu et al., 1996), Grb-2 (Liu et al., 1996), mSos-1 (Liu et al., 1996) and Nck (Liu et al., 1996). Recently, we examined the functional role of caveolins in regulating signaling along the MAP kinase cascade (Engelman et al., 1998a). Co-expression with caveolin-1 dramatically inhibited signaling from EGF-R, Raf, MEK-1 and ERK-2 to the nucleus in vivo (Engelman et al., 1998a). Using a variety of caveolin-1 deletion mutants, we mapped this in vivo inhibitory activity to caveolin-1 residues 32–95. In addition, peptides derived from this region of caveolin-1 (i.e. the caveolin-scaffolding domain) also inhibited the in vitro kinase activity of purified MEK-1 and ERK-2 (Engelman et al., 1998a). Thus, caveolin-1 can inhibit signal transduction from the p42/44 MAP kinase cascade both in vitro and in vivo by acting as a natural endogenous inhibitor of both MEK and ERK. Conversely, a prediction of these findings would be that downregulation of caveolin-1 should lead to constitutive activation of the p42/44 MAP kinase pathway. As constitutive activation of the p42/44 MAP kinase cascade is sufficient to mediate cell transformation, another predicted consequence of caveolin-1 downregulation would be cell transformation. In order to test this hypothesis directly, we have employed an antisense approach to derive stable NIH 3T3 cell lines that express dramatically reduced levels of caveolin-1, but contain normal amounts of caveolin-2. Here, we show that antisense mediated reductions in caveolin-1 protein expression are sufficient to drive oncogenic transformation and constitutively activate the p42/44 MAP kinase cascade. In normal NIH 3T3 cells, we find that caveolin-1 expression levels are downregulated in rapidly dividing cells and dramatically upregulated at confluency. Our results suggest that upregulation of caveolin-1 expression levels may be important in mediating normal contact inhibition and in negatively regulating the activation state of the p42/44 MAP kinase cascade. This is the first demonstration that a loss of caveolin-1 expression is sufficient to mediate cellular transformation. In accordance with our current findings, we have recently localized the caveolin-1 gene to a suspected tumor suppressor locus in mice (6-A2) and humans (7q31) that is deleted in many forms of cancer (Engelman et al., 1998c,d). Results Targeted downregulation of caveolin-1 protein expression, but not caveolin-2, in NIH 3T3 cells harboring caveolin-1 antisense In order to selectively downregulate the expression of the caveolin-1 protein, we engineered two different expression vectors containing the full-length untagged murine caveolin-1 cDNA in the antisense orientation. These antisense constructs were first tested in transient transfection assays with Cos-7 cells and were found to significantly reduce the expression levels of the endogenous caveolin-1 protein, as compared with mock-transfected controls (not shown). Given the partial success of this approach in transient transfections, we decided to derive stable cell lines that harbor these caveolin-1 antisense constructs. For this purpose, we used the well-established murine NIH 3T3 cell line. These cells contain caveolae and express caveolins 1 and 2, as we have shown previously (Koleske et al., 1995; Engelman et al., 1997; Scherer et al., 1997). In addition, NIH 3T3 cells have been used by many investigators as a model system to study the tumorigenicity of a given activated oncogene. Figure 1A shows a Western blot analysis of the expression of caveolin-1 in Ras-transformed NIH 3T3 cells, parental NIH 3T3 cells and three independent NIH 3T3 clones harboring caveolin-1 antisense vector (termed A1, 25 and M). Note that caveolin-1 levels are effectively reduced in all three antisense clones tested. In addition, these three clones were derived using two independent vectors. Clones A1 and M were derived using pCAGGS (hygromycin resistance) and clone 25 was derived using pCB6 (G418 resistance) (see Materials and methods). Importantly, expression of caveolin-1 is reduced ∼15- to 20-fold, but is not eliminated. Two exposures are shown to better illustrate this point. In addition, caveolin-2 levels were not affected by the expression of caveolin-1 antisense, demonstrating that the expression of caveolin-1 antisense selectively downregulates the expression of the caveolin-1 protein. NIH 3T3 cells harboring vector alone did not show any changes in the expression of caveolin-1. Figure 1.Derivation of NIH 3T3 cells harboring caveolin-1 antisense. (A) Western blot analysis. Expression of caveolins 1 and 2 in normal NIH 3T3 cells and in NIH 3T3 cells harboring caveolin-1 antisense. Lysates were prepared from parental NIH 3T3 cells, Ras-transformed NIH 3T3 cells, and NIH 3T3 cells lines harboring antisense caveolin-1 (termed A1, 25, and M). After SDS–PAGE and transfer to nitrocellulose, immunoblotting was performed with mono-specific antibody probes that recognize only caveolin-1 (mAb 2297) or caveolin-2 (mAb 65). Note that only reductions in caveolin-1 protein expression were observed, while levels of caveolin-2 remain relatively constant in all the cell lines examined. Upper panel, caveolin-1 immunoblot; middle panel, caveolin-1 immunoblot (a longer exposure to show residual levels of caveolin-1); and lower panel, caveolin-2 immunoblot. Each lane contains equal amounts of total protein. (B–D) Morphological characterization. NIH 3T3 cells harboring caveolin-1 antisense spontaneously form foci in Petri dishes and show anchorage-independent growth in soft agar. Parental NIH 3T3 cells, Ras-transformed NIH 3T3 cells and NIH 3T3 cells lines harboring antisense caveolin-1 (termed A1, 2 5, and M) were compared for their ability to generate foci or to undergo anchorage-independent growth in soft agar. (B) Foci formation in plastic tissue culture dishes. (C) Growth and colony formation in soft agar. (D) Quantitation of growth in soft agar is shown. The number of colonies per field is as indicated. Note that the behavior of NIH 3T3 cells lines harboring antisense caveolin-1 is closest to that of Ras-transformed NIH 3T3 cells under these conditions. Download figure Download PowerPoint As expected, caveolae were also downregulated in these caveolin-1 antisense cell lines, as seen by transmission electron microscopy (data not shown). In support of this observation, we and others have previously shown that expression of caveolin-1, but not caveolin-2, is sufficient to drive the formation of caveolae or caveolae-like vesicles in heterologous expression systems (Fra et al., 1995b; Scherer et al., 1995; Engelman et al., 1997; Scherer et al., 1997; Li et al., 1998). NIH 3T3 cells harboring caveolin-1 antisense show a loss of contact inhibition and anchorage-independent growth, and appear morphologically transformed To investigate whether targeted downregulation of the caveolin-1 protein is sufficient to drive cell transformation, we next subjected these cell lines to assays that are routinely used to measure oncogenic potential. All three NIH 3T3 cell clones harboring caveolin-1 antisense spontaneously formed foci in Petri dishes and exhibited growth in soft agar (Figure 1B–D). Ras-transformed NIH 3T3 cells served as a positive control and parental NIH 3T3 cells served as a negative control for these assays; quantitation of these results is presented in Figure 1D. In addition, NIH 3T3 cells harboring vector alone did not show foci formation or growth in soft agar (see below). These results suggest that targeted downregulation of caveolin-1 protein expression disrupts contact inhibition and leads to anchorage-independent growth. Closer examination of NIH 3T3 cells harboring caveolin-1 antisense by scanning electron microscopy reveals that these cells also have a dramatically altered morphology (Figure 2). They contain an increased number of fine projections and lamellopodia, and exhibit a loss of contact inhibition, as compared with normal NIH 3T3 cell controls. Figure 2.NIH 3T3 cells harboring caveolin-1 antisense have increased cellular projections and appear to lose contact inhibition as seen by scanning electron microscopy. Samples were prepared for microscopy and examined using a JEOL scanning electron microscope. Each panel contains two images. The upper panel shows a view at a magnification of ×1000; the lower panel shows a higher magnification view of the same area (×3000). (A) NIH 3T3, (B) A1, (C) 25 and (D) M. Note that NIH 3T3 cells lines harboring antisense caveolin-1 (A1, 25 and M) appear crowded, suggesting a loss of contact inhibition. In addition, they appear to possess many more fine projections that are characteristic of the transformed phenotype. Download figure Download PowerPoint NIH 3T3 cells harboring caveolin-1 antisense are tumorigenic in immunodeficient strains of mice As NIH 3T3 cells harboring caveolin-1 antisense behaved as expected for a transformed cell line, we next assessed their ability to form tumors in immuno-deficient strains of mice (Figure 3). All three cell lines were tumorigenic in this assay system, forming tumors with high frequency (65–100%). After 2–3 weeks, these tumors reached masses of ∼2–4 g. Importantly, no tumors were observed with normal NIH 3T3 cells used for control injections. These results indicate that downregulation of caveolin-1 protein expression is sufficient to confer tumorigenicity. Figure 3.NIH 3T3 cells harboring caveolin-1 antisense form tumors in nude mice. (A) Immunodeficient mice were injected subcutaneously with ∼107 cells each in PBS. Mice injected with parental NIH 3T3 cells did not show the development of tumors. However, mice injected with NIH 3T3 cells lines harboring antisense caveolin-1 (A1, 25 and M) developed noticeable tumors between 1 and 2 weeks after injection. An arrow points to the injection site. Two examples are shown for mice injected with clone 25 at 14 days post-injection. (B) Graphical representation of tumor formation in nude mice showing frequency of tumor formation (upper), tumor area (middle) and tumor weight (lower). Quantitation was performed at 2.5–3 weeks post-injection. Download figure Download PowerPoint Activation of the p42/44 MAP kinase pathway, but not the p38 MAP kinase pathway, in NIH 3T3 cells harboring caveolin-1 antisense What is the mechanism by which downregulation of caveolin-1 promotes a loss of contact inhibition, anchorage-independent growth and tumorigenicity? One possibility is through the de-repression of signal transduction, as caveolin-1 has been suggested to function as a negative regulator of a variety of signaling events (Okamoto et al., 1998). To investigate this hypothesis, we employed a variety of phospho-specific antibodies that have been generated against the activated forms of well-known signal transducers. It has previously been shown that these antibodies can be used to detect activated forms of a given signaling molecule by Western blotting. Lysates from normal NIH 3T3 cells and NIH 3T3 cells harboring caveolin-1 antisense were first probed using antibodies directed against activated MEK or activated ERK. Blotting with MEK and ERK phospho-independent antibodies was performed as a control for equal loading. Figure 4A and B shows that both MEK and ERK are constitutively activated in all three caveolin-1 antisense clones. In further support of this conclusion, treatment with the well-characterized MEK inhibitor PD 98059 reduced levels of activated ERK to normal (Figure 4C). Similarly, treatment of caveolin-1 antisense clones with PD 98059 also blocked their ability to undergo anchorage-independent growth in soft agar, indicating that constitutive activation of the p42/44 MAP kinase pathway was necessary to maintain their transformed phenotype (Figure 4D). Immunoblot analysis of lysates from parental NIH 3T3 cells, Ras-transformed NIH 3T3 cells, and NIH 3T3 cells harboring caveolin-1 antisense with anti-MEK and anti-activated MEK is shown for comparison (Figure 4E). In support of these observations, we have previously shown that caveolin-1 is a natural endogenous inhibitor of both MEK and ERK (Engelman et al., 1998a). Thus, loss of caveolin-1 expression would be predicted to lead to constitutive activation of the p42/44 MAP kinase cascade. Figure 4.MEK and ERK are constitutively activated in NIH 3T3 cells harboring caveolin-1 antisense. Lysates were prepared from parental NIH 3T3 cells and NIH 3T3 cells lines harboring antisense caveolin-1 (termed A1, 25 and M). After SDS–PAGE and transfer to nitrocellulose, immunoblotting was performed with control and phosphospecific antibody probes. (A) Immunoblot analysis with anti-MEK and anti-activated MEK. (B and C) Immunoblot analysis with anti-ERK and anti-activated ERK. In (C), clone 25 was treated with and without a well-characterized inhibitor of MEK (PD 98059, 50 μM) for 24 h. Treatment with this specific inhibitor of MEK reduced the level of activated ERK to normal levels. Immunoblotting with anti-MEK and anti-ERK IgG served as an additional control for equal loading. Each lane contains equal amounts of total protein. (D) Treatment with the MEK inhibitor (PD98059, 50 μM; indicated as PD) blocked the ability to NIH 3T3 cells harboring caveolin-1 antisense to undergo anchorage-independent growth in soft agar. (E) Immunoblot analysis of lysates from parental NIH 3T3 cells, Ras-transformed NIH 3T3 cells, and NIH 3T3 cells harboring caveolin-1 antisense (clone A1) with anti-MEK and anti-activated MEK is shown for comparison. (F) Parental NIH 3T3 cells and NIH 3T3 cells harboring caveolin-1 antisense were lysed and separated into cellular (C) and nuclear (N) fractions. Immunoblot analysis was then performed using anti-ERK (upper) and anti-activated ERK IgG (middle and lower). Two exposures of the immunoblot with activated ERK are shown. Note that in all three caveolin-1 antisense clones the amount of total ERK is quantitatively shifted to the nucleus, while in parental NIH 3T3 cells it is predominantly excluded from the nucleus. Immunoblotting with antibodies directed against activated ERK confirmed that the nuclear fraction of ERK was indeed activated. In all panels, each lane contains equal amounts of total protein. Download figure Download PowerPoint How does targeted downregulation of caveolin-1 affect the cellular distribution of ERK? To answer this question, parental NIH 3T3 cells and NIH 3T3 cells harboring caveolin-1 antisense were lysed and separated into cellular (C) and nuclear (N) fractions using a standard protocol (see Materials and methods). Figure 4F (upper panel) shows that in all three caveolin-1 antisense clones the amount of total ERK is quantitatively shifted to the nucleus, while in parental NIH 3T3 cells it is predominantly excluded from the nucleus. Immunoblotting with antibodies directed against activated ERK confirmed that the nuclear fraction of ERK was indeed activated (Figure 4F, middle and lower panels). These results are as predicted based on previous studies showing that activated ERK translocates from the cytoplasm to the nucleus to phosphorylate its target substrates, such as the nuclear transcription factor Elk. Importantly, NIH 3T3 cells harboring vector alone did not show the ability to form foci or to undergo anchorage-independent growth in soft agar (Figure 5A). Up to 10 additional vector alone controls were analyzed, and none showed a transformed phenotype (not shown). In addition, these vector alone controls did not show elevated levels of either activated MEK or activated ERK (Figure 5B). These cells also did not show any changes in the expression of caveolin-1 (not shown). Figure 5.Analysis of the phenotype of NIH 3T3 cells harboring vector alone. (A) Morphological characterization. Left panels, foci formation; right panels, growth in soft agar. Note that NIH 3T3 cells harboring vector alone behaved as parental untransfected NIH 3T3 cells. They did not exhibit foci formation or show any anchorage-independent growth in soft agar. The results obtained with two representative clones are shown here. Up to 10 additional vector alone controls were analyzed and none showed a transformed phenotype. (B) Western blot analysis. Activation state of MEK and ERK using phosphospecific antibody probes. Each lane contains equal amounts of total protein. Note that NIH 3T3 cells harboring vector alone did not show any elevation in the levels of activated MEK or ERK, and behaved as parental untransfected NIH 3T3 cells. Download figure Download PowerPoint What about the SAPK/JNK or p38 MAPK pathways? Figure 6A and B shows that neither SEK or p38 MAPK are constitutively activated by targeted downregulation of caveolin-1. These results suggest that downregulation of caveolin-1 results in selective activation of the p42/44 MAPK cascade, but not the SAPK/JNK or p38 MAPK cascades. Are other cellular pathways affected by targeted downregulation of caveolin-1 protein expression? Figure 6C shows that downregulation of caveolin-1 protein expression also does not affect the amount of normal or activated CREB. Thus, downregulation of caveolin-1 only selectively affects a subset of signaling pathways. Figure 6.Analysis of the activation state of other established signaling pathways in NIH 3T3 cells harboring caveolin-1 antisense. (A and B) Components of the SAPK/ JNK and p38 MAP kinase pathways are not activated in NIH 3T3 cells harboring caveolin-1 antisense.