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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.35 no.2 Santiago  2002

http://dx.doi.org/10.4067/S0716-97602002000200006 

Biol Res 35: 139-150, 2002

Caveolae and caveolae-like membrane domains in cellular
signaling and disease: Identification of downstream targets
for the tumor suppressor protein caveolin-1

FLORENT BENDER2*, MARGARITA MONTOYA1*, VIRGINIA MONARDES1,
LISETTE LEYTON1, ANDREW F.G.QUEST1

1Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 7, CHILE
2 University of Pennsylvania, School of Dental Medicine, Department of Microbiology, 218, Levy Building, 4010 Locust Street, Philadelphia. PA 19104, USA
* Both authors contributed equally

ABSTRACT

Caveolae are small, flask-shaped invaginations of the plasma membrane present on a large number of mammalian cells. Recent results obtained with knock-out mice for the gene caveolin-1 demonstrate that expression of caveolin-1 protein is essential for caveolae formation in vivo. Caveolae are implicated in a wide variety of cellular events including transcytosis, cholesterol trafficking and as cellular centers important in coordinating signalling events. Caveolae share this role and the property of detergent insolubility with plasma membrane assemblies rich in glycosphingolipids and cholesterol, often called lipid rafts, but preferably referred to here as caveolae-like membrane domains. Due to such widespread presence and usage in cellular function, caveolae and related domains are implicated in human diseases, including cancer. In particular, the protein caveolin-1 is suggested to function as a tumor suppressor protein. Evidence demonstrating such a role for caveolin-1 in human colon carcinoma cells will be discussed together with data from microarray experiments seeking to identify caveolin-1 target genes responsible for such behavior.

Key terms: caveolae, caveolae-like domains, lipid rafts, caveolin-1, tumor suppressor, colon carcinoma cells, microarray analysis

PLASMA MEMBRANE MICRODOMAINS IMPORTANT FOR CELL SIGNALING

In recent years, a number of observations converged to suggest the importance of specialized plasma membrane regions in the biochemical mechanism of signal transduction (Anderson, 1998; Okamoto et al., 1998). These domains characterized by their high content in sphingolipids and cholesterol have been isolated from the bulk of the plasma membrane based on their resistance to non-ionic detergent dispersion and are therefore referred as detergent-resistant membranes (DRMs) (Brown and London, 1998) detergent insoluble glycolipid-enriched membranes (DIGs) (Simons and Ikonen, 1997) or lipid rafts (Simons and Toomre, 2000)

A number of molecules known to be involved in the early steps of signal transduction have been shown to be associated with these domains. On the one hand, lipids such as sphingomyelin (SM) and PIP2 are preferentially associated with DRMs that can serve as substrates for enzymes that release the second messengers ceramide, diacylglycerol and IP3 (Liu and Anderson, 1995; Hope and Pike, 1996; Pike and Casey, 1996; see also Magee et al., 2002; Faroudi et al., 2002). On the other hand, many proteins with functions related to cellular signaling are localized in these particular membrane domains. Several methods demonstrated the enrichment of the endothelial nitric oxide synthase (eNOS) in these microdomains, suggesting that DRMs could represent the site of production of nitric oxide (NO), another type of second messenger (Garcia-Cardena et al., 1996b; Liu et al., 1996a; Shaul et al., 1996; reviewed in Shaul, 2002). Furthermore, heterotrimeric G protein subunits (Sargiacomo et al., 1993; Chang et al., 1994; Smart et al., 1995a; Smart et al., 1995b), G-protein coupled receptors (Dupree et al., 1993; Feron et al., 1997) and effectors such as adenylate cyclase (Huang et al., 1997) are mostly found in DIGs, as well as Ras (Mineo et al., 1996; Song et al., 1996) and proteins of the MAPK pathway (Lisanti et al., 1994; Mineo et al., 1996). Finally, all purification methods have shown that receptor and non-receptor tyrosine kinases (PTK) were substantially enriched in DIGs from a variety of cells and tissues, in particular members of src-family kinases lck, fyn and src (Bohuslav et al., 1993; Gorodinsky and Harris, 1995), as well as EGF and PDGF receptors (Smart et al., 1995a; Liu et al., 1996b; Wu et al., 1997). Taken together, the enrichment of signaling molecules in defined microdomains of the plasma membrane has led to the suggestion that these domains are crucial to organize and interconnect different signal transduction pathways.

In addition to signaling molecules, proteins associated to the membrane via a glycosylphosphatidylinositol (GPI) anchor are also enriched in microdomain preparations (Chang et al., 1994; Simons and Ikonen, 1997). Partitioning of GPI-anchored proteins in these domains requires the GPI-anchor (Brown and Rose, 1992; Rodgers et al., 1994; Liu and Anderson, 1995; Smart et al., 1996a). Association of GPI-anchored proteins in DRMs, together with glycolipids was previously described to happen in the Golgi, where formation of lipid-protein complexes, or rafts, are required for the apical sorting of GPI-anchored proteins (Brown and Rose, 1992; Zurzolo et al., 1994). Hydrogen bonding between glycosphingolipids (GSL) causes formation of GSL-rich membrane domains in the Golgi apparatus (van Meer and Simons, 1988) which are then transported to the cell surface, where structure and detergent insolubility are maintained. Thus, what have been referred to as DIGs, DRMs and now rafts all represent a similar plasma membrane compartment, whose main characteristic is detergent insolubility. Based on their particular composition, rafts might contribute to the regulation of signaling by their physical ability to recruit or sequester a number of molecules involved in the early steps of signal transduction, in particular proteins with specific lipid modifications (see Magee et al., 2002; Patterson, 2002).

Caveolae

The biochemical properties of rafts are reminiscent of the characteristics of caveolae (see Fig. 1), earlier described organelles involved in transcytosis, lipid trafficking and more recently signal transduction (Parton, 1996; Anderson, 1998; Okamoto et al., 1998). However, these structures are not static inpocketings but rather very dynamic organelles that can pinch off the plasma membrane in a process that requires the hydrolysis of GTP (Severs, 1988; Parton and Simons, 1995; Schnitzer et al., 1996). Initially, these vesicles were shown to mediate trans-epithelial transport of small molecules across the cell by fusing together to form trans-cellular channels (Simionescu et al., 1973). Later, caveolae were shown to mediate the uptake of particular molecules and ions from the exterior and then redistribute these compounds in intracellular compartment through a process called potocytosis. The best-characterized example is the GPI-anchored folate receptor, which binds to its ligand 5-methyl-tetrahydrofolate at the cell surface, and then is internalized via this mechanism. In response to an acidic environment, the ligand dissociates from the receptor and diffuses directly into the cytoplasm of the cell (reviewed in Anderson et al., 1992). Finally, caveolae were shown to cycle between the plasma membrane and the ER for delivery of molecules inside the cell. This mechanism of internalization is suggested to require PKC activation and cytoskeleton integrity (Parton et al., 1994; Conrad et al., 1995; Stang et al., 1997), while membrane recycling from ER to the plasma membrane is controlled by phosphatases. A schematic of caveolae, highlighting some possible protein and lipid components, is provided (Fig. 1).

Fig. 1. Caveolae and Caveolae-like membrane microdomains
Caveolae are small invaginations of the plasma membrane (see inlet scheme) with a well-defined size (50-100 nm) and a particular lipid content (see Anderson, 1998 and main figure). They appear enriched in glycosphingolipids, cholesterol and proteins generally associated with the membrane via lipid anchors, such as glycosylphosphatidylinositol (GPI)-anchored proteins and non-receptor tyrosine kinases of the src-family. Also many receptors (not shown) and cytosolic signaling proteins that do not require lipid modifications to associate with membranes, such as PKCa, are reportedly found in caveolae. In addition, members of the caveolin family (here represented by caveolin-1) are important for the structure of caveolae, thanks to their ability to oligomerize and bind cholesterol. Additionally, caveolin-1 may play a role as negative regulator of signaling by interacting with and inhibiting many proteins via the scaffolding motif, which overlaps at least partially with the indicated element required for dimerization of caveolin molecules. Alternatively, src kinase mediated tyrosine phosphorylation at position 14 promotes association of Grb7, anchorage-independent growth and cell migration. Membrane microdomains also exist in the absence of caveolins, as morphologically less well defined, detergent-resistent membranes, thereby giving rise to the general concept of caveolae-like membrane microdomains (see text).

Caveolae-like domains

A key advance in the field came with the description of several methods for the purification of plasma membrane caveolae (Anderson, 1998; Simons and Toomre, 2000). The common feature of these domains are light buoyant density, a glycoshingolipid/shingomyelin/cholesterol core in the liquid-ordered phase (Brown and London, 1998), a high concentration of lipid-anchored proteins and signaling molecules, and a well-defined discrete size. Thus, membranes microdomains refered to as DRMs, DIGs or rafts, depending on whether those domains were characterized by detergent insolubility, association with glycolipids, or involvement in trafficking, respectively, represent flat, morphologically inconspicuous variants of the lipid-ordered organelle. Here, it is important to mention more recent experimental data indicating that these two types of microdomains (caveolae and non-caveolae) may be present in the same cells (see for instance Tkachenko and Simons, 2002; as well as Stuermer et al., 2001 discussed later). To insert all these concepts under a more generic terminology, the nomenclature caveolae-like domain has been proposed (Anderson, 1998) and will be preferentially used in this manuscript.

Isolation of caveolae-like membrane microdomains

While purification schemes have helped tremendously in advancing our understanding of the importance of caveolae and caveolae-like domains in many aspects of cell biology, caution is required in the choice of methodology for their isolation. This is illustrated here by the comparison of two methods described in the literature, one using hypertonic sodium carbonate buffer at pH11 (Song et al., 1996), the other a buffer containing 1% Triton X-100 (Sargiacomo et al., 1993). An MDCK cell line which stably expresses a myc-tagged transferrin receptor (Hunziker and Mellman, 1989) as well as caveolin-1 and the mouse thymoma cell line EL-4 which lack caveolin-1 but abundantly express the GPI-anchored glycoprotein Thy-1 (Bender and Quest, unpublished data), were employed as model cells to isolate caveolae and caveolae-like domains, respectively.

When MDCK-TfR cells were lyzed in sodium carbonate buffer at pH 11 in the absence of detergent (Fig. 2B), enrichment of caveolin-1 in the low-density fractions (4-6) was observed. A weaker signal was also detectable in heavier fractions (7-12) but not in the pellet. Moreover, actin was only detectable in the high-density fractions. Surprisingly, the TfR, a classical marker for non-raft membranes (Harder et al., 1998), was predominantly recovered in fractions together with caveolin-1. In contrast, when MDCK-TfR cells were lyzed in the presence of Triton X-100, the TfR was essentially excluded from the light fractions containing caveolin-1 (Fig. 2A), whereas actin was present in heavy fractions and pellet.

When EL-4 cells were fractionated on a sucrose gradient, using either the detergent or the sodium carbonate methods (Figure 2C and D, respectively), Thy-1 was particularly enriched in low-density fractions. Smaller amounts of the molecule were also recovered in high-density fractions. In addition, when cell extraction was performed in the presence of detergent, a significant amount of Thy-1, as well as actin, was found in the pellet after centrifugation, while this was not the case for the sodium carbonate method (compare Figure 2C and D, fraction P). A notable proportion of the tyrosine kinase Fyn, a dually acylated protein with myristate and palmitate residues (Peters et al., 1990; Shenoy-Scaria et al., 1993; see also Felley-Bosco et al., 2002; Magee et al., 2002; Patterson, 2002), is associated with low-density microdomains enriched in Thy-1, independent of the extraction method employed. The T cell receptor (TCR) z-chain was found only in the high-density fractions, containing detergent-soluble membrane elements in agreement with several reports (Montixi et al., 1998; Xavier et al., 1998; see also Faroudi et al., 2002). By contrast, when the sodium carbonate fractionation technique was used, most of the TCR z-chain was recovered in the low-density fractions, as was also observed for the TfR in MDCK cells. Taken together the results shown here for MDCK and EL-4 cells strongly suggest that cell lysis using sodium carbonate buffer in the absence of detergent did not permit separation of specialized microdomains (caveolae or caveolae-like) from the rest of the membrane.

As an additional means of characterizing membrane fractions, particularly in the absence of protein markers like caveolin-1, cells were labeled with 14C-cholesterol following protocols previously described (Oram, 1986; Rothblat et al., 1986). A comparison of the cholesterol distribution between gradient fractions obtained by the sodium carbonate method revealed that about 71% of the labeled cholesterol incorporated was recovered in low-density fractions and only 28% in the heavy fractions of MDCK cells (see Table I). By contrast, the distribution observed using the detergent fractionation technique showed that only 20% of total cholesterol incorporated was associated with the light fractions, 50% was recovered in heavy fractions and 30% was found in the pellet (Table I). Similar results were also obtained by characterizing in a similar fashion EL4 cells (see Table I). Segregation of the cholesterol into both low and high-density fractions is consistent with the notion that only part of the membrane cholesterol was detergent resistent. Since most of the cellular cholesterol is known to be plasma membrane associated (80-90% in human fibroblasts, Lange et al., 1989), but only a fraction thereof is present in caveolae or caveolae-like fractions (Magee et al., 2002) it is reasonable to conclude, in conjuction with the aforementioned protein distribution data, that the sodium carbonate procedure does not permit separation of cholesterol-rich caveolae-like domains from the rest of the plasma membrane, while the detergent extraction procedure employing Triton X-100 is more successful in this respect. For these reasons, fractionation studies from this laboratory employ predominantly the above-characterized detergent fractionation scheme for caveolae-like fractions from human colon carcinoma cells (Felley-Bosco et al., 2000). Similar methodology is also employed in the preparation of such fractions from synaptosomes and growth cones of neuronal cells (Patterson, 2002).

Fig. 2. Isolation of caveolae-like membrane fractions from MDCK cells and EL-4 cells. Caveolae-like domains were isolated either in a high pH sodium carbonate buffer (B and D) or in the presence of the detergent TritonX-100 (A and C) from MDCK cells stably transfected with the transferrin receptor (TfR) (A and B) or the thymoma EL-4 cells (C and D). Sucrose gradient fractions (1-13) were analyzed by Western blotting for the presence of the TfR (anti-myc monoclonal hybridoma supernatant 1-9E10.2; Evan et al., 1985), caveolin-1 (C13630, Transduction Laboratories, Lexington, KY) actin (Bioscience, Seikagu Corp., Tokyo, Japan), Thy-1 (rabbit R287, provided by C. Bron, University of Lausanne), TCR-z (Hamster monoclonal H146 hybridoma supernant (Golstein et al., 1982)), Fyn (Gift from Morris White, Harvard Medical School). Migration positions of molecular weight marker proteins are indicated to the left in kDa. To isolate caveolae-like fractions in the absence of detergent, the cells were homogenized in hypertonic sodium carbonate buffer at pH 11, sonicated and cell components were then separated on a discontinous 5-45% sucrose gradient by centrifugation overnight at 200000 x g, essentially as described (Song et al., 1996). Alternatively, to isolate such microdomains in the presence of detergent, cells were solubilized in a buffer containing 1% Triton X-100, homogenized very gently in a Dounce homogenizer and cell components were then also separated by centrifugation on a discontinuous sucrose gradient, essentially as described (Sargiacomo et al., 1993; Rodgers and Rose, 1996; Felley-Bosco et al., 2000).


TABLE I

[14C]-cholesterol distribution in sucrose gradient fractions from MDCK and EL4 cells


Homogenization method

Fractions 1-6
Fractions 7-12
Pellet
Cells

Sod. carb (n=5)

71 ± 10%
28 ± 10%
1 ± 0.5
MDCK

TX-100 (n=5)

20 ± 2%
50 ± 20%
30 ± 20%

Sod. carb (n=2)

80%
19%
1%
EL4

TX-100 (n=3)

40 ± 13%
38 ± 4%
20 ± 8%

[14C]-cholesterol distribution between fractions obtained either by the sodium carbonate or TritonX-100 method for MDCK (expressing caveolin-1) and EL4 (lacking caveolin-1) cells are summarized. Note the large discrepancy between [14C]-cholesterol levels detected in fractions 1-6 for the two fractionation procedures (for details, see text).

Caveolins

Proteins called caveolins have been shown to represent major components of the caveolar coat. This family of proteins includes three mammalian and two isoforms of C. elegans that share a relatively high degree of homology (Tang et al., 1997; Okamoto et al., 1998). Caveolin-1 and 2 have a similar tissue distribution, being mainly expressed in endothelial, epithelial and muscle cells, whereas caveolin-3 expression is limited to muscle cells. Functionally speaking and in terms of sequence homology, caveolin-1 and caveolin-3 are much more alike (Okamoto et al., 1998). The following discussion will focus predominantly on caveolin-1.

Two forms of caveolin-1, refered to as caveolin-1a and caveolin-1b, are generated by alternative initiation from the same mRNA or alternative splicing (Kogo and Fujimoto, 2000). In contrast to the other acylated proteins, caveolin mainly associates with DIGs via an unusual membrane loop, rather than through their three C-terminally attached palmitate residues, although such modifications do alter caveolin-1 localization and function (Dietzen et al., 1995; Schlegel and Lisanti, 2000). In particular, the ability of caveolin-1 to bind and transport cholesterol has been linked to palmitoylation. Furthermore, palmitoylation at cysteine 156 was shown to be essential for src-dependent phosphorylation of caveolin-1 at tyrosine 14 (Lee et al., 2001) where Grb7 binding is thought to link such phosphorylation to the focal adhesion machinery (Lee et al., 2000). The N-terminal domain of the protein is well conserved and mediates caveolin dimerisation as well as association with many other proteins, as discussed subsequently. Contacts between the COOH-terminal regions of caveolin-1 crosslink caveolin oligomers to yield large protein aggregates (see review Schlegel and Lisanti, 2001; see also Fig. 1).

Expression of caveolin-1 in cells devoid of caveolae and caveolin expression such as Jurkat T cells, FRT-cells and insect Sf9 cells, led to the formation of typical flask-shaped invaginations resembling caveolae, suggesting that caveolin-1 plays an important role in caveolae formation (Fra et al., 1995; Li et al., 1996c; Lipardi et al., 1998). Indeed, knock-out mice devoid of caveolin-1 completely lack detectable caveolae (Drab et al., 2001; Razani et al., 2001). Here again caveolins 1 and 3 are similar in that both are individually sufficient to promote formation of morphologically distinguishable caveolae membranes, whereas caveolin-2 lacks this ability (reviewed in Schlegel and Lisanti, 2001).

On the other hand, cholesterol is another essential component of caveolae, since drugs that sequester cholesterol such as filipin or depletion of intracellular cholesterol cause the coat to disassemble and caveolae to disappear (Chang et al., 1992). Also given that caveolin-1 binds cholesterol and cholesterol stabilizes caveolin oligomers, sterols and caveolin-1 must work together to form the caveolar coat (Murata et al., 1995; Li et al., 1996c; Monier et al., 1996).

Recently, another family consisting of two 47 kDa integral membrane proteins that may contribute to the structural organization of caveolae-like membranes was described. These proteins, identified independently by two groups, were called reggie-1 and reggie-2 (Schulte et al., 1997) or flotillin-2 and flotillin-1 (Bickel et al., 1997; Volonte et al., 1999), respectively. Reggie-1/flotillin-2 is enriched in detergent insoluble (TritonX-100) membrane fractions, co-localizes with activated GPI-linked proteins and fyn in neurons and T cells, and thus apparently participates in the assembly of protein complexes essential for signal transduction. (Lang et al., 1998; Stuermer et al., 2001). A similar function was attributed to Reggie-1/flotillin-2 in B cells (Solomon et al., 2002). When caveolins and reggie/flotillin are co expressed in A498 kidney cells, they apparently form stable hetero-oligomeric "caveolar complexes" as suggested by co-immunoprecipitation experiments (Volonte et al., 1999). Moreover, heterologous expression of murine flotillin-1 in Sf21 insect cells using baculovirus-based expression vectors is sufficient to drive the formation of slightly larger caveolae-like vesicles (50-200 nm). On the other hand, recent analysis in astrocytes using immunogold electron microscopy and confocal microscopy suggest that reggie/flotillin define plasma membrane microdomains distinct from caveolae (Lang et al., 1998; Stuermer et al., 2001). Thus while reggie/flotillins appear to share with caveolin-1 the ability to stucturally organize caveolae-like membrane domains, the experiments in astrocytes suggest that these two groups of proteins do not asociate with the same microdomain structures.

Caveolins in signaling

A variety of observations have implicated caveolin-1 as an adaptor molecule or scaffolding protein in signal transduction (Okamoto et al., 1998). In particular, several reports suggest that caveolin-1 interacts directly with and inhibits the function of many key signaling molecules via a motif referred to as the scaffolding domain (see also Fig. 1, Felley-Bosco et al. 2002). Direct interaction with the scaffolding domain of caveolin-1 promotes the sequestration of inactive Ha-Ras (Mineo et al., 1996; Song et al., 1996) and c-Src within caveolae (Li et al., 1996a; Li et al., 1996b). Two related caveolin-1 binding motifs were shown to mediate the interaction of caveolin-1 with different Ga subunits and Src (Couet et al., 1997a). As many other proteins including Src-family kinases, eNOS, PKCa, MAPK, EGF receptor, insulin receptor and the PDGF receptor, all contain caveolin-1 binding motifs (Okamoto et al., 1998), this may represent a general mechanism for caveolin-1 mediated sequestration and incorporation of a diverse group of signaling molecules within caveolae for regulated activation by receptor ligands. In essence, caveolin-1 may act as an adaptor protein to nucleate the formation of signal transduction complexes and/or help to maintain molecules in the inactive state (Anderson, 1998; Okamoto et al., 1998).

This interpretation should, however, be viewed with caution. Thus far, in vivo data underscoring the physiological relevance is essentially only available for interactions between caveolin-1 and eNOS (Drab et al., 2001; Razani et al., 2001). In addition, caveolin-1 not only perturbs the function of signaling pathways by blocking the function of key components, but also controls turnover of proteins like iNOS via proteasome-mediated degradation (Felley-Bosco et al., 2000). In this respect, caveolin-1 appears again functionally similar to caveolin-3, where the presence of a novel WW-like domain was shown to block b-dystroglycan/dystrophin interaction and thereby promote degradation of the latter via the proteosome pathway (Sotgia et al., 2000). Finally, caveolin-1 phosphorylation on tyrosine 14 reportedly favors association of Grb7 and activation of the MAPK pathway (Lee et al., 2000; reviewed by (Schlegel and Lisanti, 2001) as well as recruitment of the C-terminal src kinase (csk) which promotes inactivation of src-family kinases (Cao et al., 2002). Thus, caveolin-1 employs a variety of mechanisms to modulate cellular signaling, but not always does so in an inhibitory fashion.

Caveolae in endocytosis

Caveolae are also important vesicles implicated in endocytosis through a mechanism that differs significantly from that described for coated pits (Goldstein et al., 1985). Additionally, caveolae function as transcellular transport vesicles, or in a process called potocytosis, responsible for intracellular delivery of small molecules (Anderson et al., 1992). Thus abnormal expression of caveolin-1 is expected to lead to malfunction of the caveolae system and as a consequence would result in improper coordination of signal transduction events, a block in internalization processes linked to caveolae and impaired cholesterol homeostasis (Parton and Simons, 1995; Kurzchalia and Parton, 1996; Roy et al., 1999). More recent data from experiments employing caveolin-1 knockout mice demonstrated clearly that caveolin-1 presence is required for caveolae formation (Drab et al., 2001; Razani et al., 2001); however, the issue whether caveolae are essential for transcytosis was not resolved. On the one hand, the albumin concentration in cerebrospinal fluid, that was thought to depend on transcytosis via caveolae of capillary endothelial cells, was the same in knock-out and wild type mice (Drab et al., 2001). Yet, on the other hand, caveolin-1 deficient mouse embryonic fibroblasts (MEFs) from knock-out mice clearly showed defects in the endocytosis of albumin while uptake of transferrin was not affected (Razani et al., 2001). Thus, the interpretation of results with knock-out mice are ambiguous in this respect.

Caveolae and caveolins in human disease

Evidence is accumulating suggesting that a number of viruses, parasites and bacteria utilize caveolae (or caveolae-like domains) as an alternative route to enter cells. The classic pathway via endocytosis depends on clathrin-coated pits that bud from the cell surface and merge with lysosomes where vesicle contents are degraded. Clearly, the ability to avoid such a potentially hazardous entry route appears beneficial and has been exploited by a number of pathogens including mycobacteria, simian virus 40 (SV40), the malaria parasites Toxoplasma gonadii and Plasmodium falciparum, and toxins of Vibrio cholerae and Heliobacter pylori bacteria (see Shin and Abraham, 2001 and references therein). In addition, specific roles have been ascribed to individual caveolin isoforms in different diseases. For instance, caveolin-3 is dramatically upregulated in astroglial cells surrounding senile plaques in brains from patient's with Alzheimer's disease. Since caveolin-3 colocalizes with the amyloid precursor protein (APP) and overexpression promotes APP processing, increased expression of caveolin-3 and presenilins in reactive astrocytes is suggested to favor formation of toxic APP-derived metabolites (reviewed in Smart et al., 1999). Also, mutations in the caveolin-3 gene are linked to hereditary forms of muscle dystrophy (reviewed in Schlegel and Lisanti, 2001). Alternatively, caveolin-1 is suggested to function as a tumor suppressor and is thereby implicated in the development of cancer (see following discussion).

Caveolin-1 in cell transformation and cancer

Since its identification (Glenney and Soppet, 1992; Rothberg et al., 1992), caveolin-1 has been implicated in the process of cell transformation. Initially, caveolin-1 was described as the major substrate for phosphorylation on tyrosine upon cell transformation by the Rous Sarcoma virus (Glenney and Soppet, 1992), suggesting that caveolin-1 may represent a critical target protein during that process. Then, caveolin-1 mRNA and protein levels, as well as caveolae numbers, were shown to be reduced in NIH-3T3 fibroblasts transformed by several oncogenes, indicating that reduction of caveolin-1 expression and/or alteration of caveolae structure could favor cell transformation (Koleske et al., 1995). Furthermore, absence of caveolin-1 correlates with forms of cell transformation that can be reverted by re-expression of caveolin-1 (Engelman et al., 1997; Racine et al., 1999). Indeed, downregulation of caveolin-1 expression using antisense oligos was sufficient to induce transformation of NIH3T3 fibroblasts (Galbiati et al., 1998). Finally, reduced caveolin-1 levels have been observed in a variety of cancer cell lines, including human mammary (Lee et al., 1998), lung (Racine et al., 1999), colon (Bender et al., 2000) and ovarian carcinomas (Wiechen et al., 2001a), as well as human sarcomas (Wiechen et al., 2001b). Taken together, these results suggest that reduced caveolin-1 expression may represent a general characteristic or even a requirement of transformed cells, and that caveolin-1 could play a central role as an inhibitor of tumor formation.

Consistent with the notion that caveolin-1 may play an important role in the control of cell proliferation, MEFs from knock-out mice displayed an increase in cell proliferation and a decrease of cells in G0/G1 of the cell cycle (Razani et al., 2001). Also, pathomorphological defects of lung alveolar architecture, including thickened alveolar septa as well as hypercellularity were detected and linked to exercise intolerance. However, although caveolin-1 loss does promote cellular proliferation, it is not sufficient to induce immortalization of primary cells, including fibroblasts (Drab et al., 2001; Razani et al., 2001).

Caveolin-1 functions as a tumor suppressor in human colon carcinoma cells

Colorectal cancer is a leading cause of morbidity and mortality with about 300,000 new cases and 200,000 deaths in Europe and the USA per year (Midgley and Kerr, 1999). During progression from a normal epithelium to invasive or metastatic cancer, cells accumulate a combination of defects, including mutational activation of oncogenes such as Ras or Myc, and inactivation of tumor-suppressors genes like p53 and Adenomatous polyposis coli (Fearon and Vogelstein, 1990; Bishop, 1991). As a general consequence, several signal transduction pathways become constitutively activated, leading to enhanced cell proliferation, loss of adhesion and a transformed phenotype coupled with insensitivity to apoptosis (Vogelstein et al., 1988; King and Cidlowski, 1998; Lengauer et al., 1998). The number of both oncogenes and tumor suppressors identified is increasing constantly, but the mechanisms by which they regulate tumor formation and progression has been characterized for only few of them. Among the proteins potentially relevant to such events is caveolin-1 which, as mentioned above, has been proposed to act as a tumor suppressor protein. Studies from this laboratory have focused on investigating the possible role of caveolin-1 in the development of colon cancer.

In brief, the experimental data from our laboratory showed the following: 1) caveolin-1 protein levels are reduced in colon tumors from human patients; 2) colon carcinoma cell lines have low levels of caveolin-1 mRNA and protein; 3) expression of caveolin-1 in the colon carcinoma cell lines HT-29 and DLD-1 blocks or delays tumor formation in nude mice (see Fig. 3); 4) the ability of HT29-cav-1 (and also NIH-3T3 cells) to form tumors in nude mice, despite initial caveolin-1 presence, was linked to a selection process favoring proliferation of those cells with reduced basal caveolin-1 levels. Taken together, our results demonstrate that caveolin-1 functions as a tumor suppressor protein upon expression in human colon carcinoma cells (Bender et al., 2000).

Despite such striking effects, it is important to bear in mind that caveolin-1 expression levels neither are reduced in all cancer types nor need they remain low in all stages of a given cancer. For instance, elevated caveolin-1 levels are associated with the development of prostate cancer in both human and mouse model systems (Yang et al., 1998). Also, while caveolin-1 levels are generally low in colon and breast carcinoma lines, increases in caveolin-1 levels were observed in multidrug resistant HT29 colon carcinoma cells (Bender et al., 2000) and MCF7 breast carcinoma cells (Yang et al., 1998) as well as some colon carcinoma cells with higher metastatic potential (Bender et al., 2000). Thus, caveolin-1 appears to modulate a variety of pathways in such ways that either absence or presence of this protein may serve the needs of a tumor cell depending on the stage of progression. Clearly, a better understanding of proteins affected by the expression of caveolin-1 in cells is required.

Fig. 3. Tumorigenicity in nude mice of HT29 cells transfected with caveolin-1. Athymic immunodeficient nude mice were injected subcutaneously both on their (arrows) left and right sides with 106 cells. Mice photographed after 6 weeks are shown (A-D). Arrows point to the injection sites of control parental (A and D) or mock-transfected (B and C) HT29 cells, while arrow heads indicate sites where caveolin-1-transfected HT29 clones 14 (B and D) and 16 (A and C) were injected. All mice eventually developed noticable tumors on both sides, but tumor size was in most cases either significantly reduced or essentially absent for the HT29 clones expressing caveolin-1. Tumor formation assays were performed as described (Bender et al., 2000).

Mechanisms underlying the tumor suppressor function of caveolin-1

The precise mechanism by which reduced levels of caveolin-1 expression in epithelium would promote initial steps towards carcinoma formation is not clear. Several reports indicate that caveolin-1 possesses a scaffolding domain, which binds to and inhibits the activity of a number of proteins involved in signal transduction, including hetero-trimeric G-proteins (Li et al., 1995), Src family tyrosine kinases (Li et al., 1996a), endothelial nitric oxide synthase (eNOS) (Garcia-Cardena et al., 1996a; Ju et al., 1997; Michel et al., 1997; Ghosh et al., 1998), Neu tyrosine kinase (Engelman et al., 1998), EGF-receptor (Couet et al., 1997b) and PKCa (Oka et al., 1997). Thus reduced levels of caveolin-1 would prolong cell stimulation linked to one of these numerous signal transduction pathways. In particular, targeted down-regulation of caveolin-1 in NIH-3T3 cells led to specific hyper-activation of the p42/p44 MAP kinase pathway and as a consequence cell transformation (Galbiati et al., 1998) although this effect was not detectable in MEFs from knock-out mice (Razani et al., 2001).

Caveolin-1 is also involved in signal transduction events mediated by several integrins upon binding to extracellular matrix proteins. There, caveolin-1 plays a key role by linking integrins to Fyn activation, which in turn was responsible for Shc recruitment, regulation of Ras-Erk signaling and cell cycle progression (Wary et al., 1998). Thus anchorage-independent growth, observed in transformed cells upon down-regulation of caveolin-1, could be explained by this particular aspect of caveolin-1 function. However, more recent experiments suggest that tyrosine phosphorylation at position 14 may promote anchorage-independent growth and cell migration via a pathway involving recruitment of Grb7. In this particular scenario, caveolin-1 presence is predicted to facilitate anchorage-independent growth and cell migration (Lee et al., 2001; Volonte et al., 2001).

Caveolin-1 is also important as a protein involved in the organization of cholesterol-rich plasma membrane microdomains (Anderson, 1998), since it was demonstrated that caveolin-1 directly binds cholesterol (Murata et al., 1995) and plays a role as sensor or transporter of membrane cholesterol (Smart et al., 1996b; Fielding and Fielding, 1997; Hailstones et al., 1998). Disassembly of such organized membrane microdomains by drugs that sequester cholesterol (filipin, nystatin and methyl-ß-cyclodextrin) and thereby affect integrity of caveolae-like domains have been reported to block several signaling pathways. In T cells, alteration of cholesterol levels modulated their ability to respond upon cell stimulation (Stulnig et al., 1997; Viola et al., 1999; see also Magee et al. 2002). In fibroblasts, cholesterol sequestration reduced PDGF-stimulated tyrosine phosphorylation of plasma membrane proteins (Liu et al., 1997), as well as presence of the EGF receptor, ras and MAP kinase in caveolae fractions. The latter observation correlated with decreased MAP kinase activity (Furuchi and Anderson, 1998). Also, caveolin-3 expression has been shown to inhibit H-Ras mediated signaling by disrupting cholesterol-rich plasma membrane domains (Roy et al., 1999).

Thus, an alternative interpretation to the direct involvement of caveolin-1 as a negative regulator of signaling proteins via interactions in the scaffolding domain could be an indirect effect through its role as a modulator of cholesterol distribution and microdomain organization at the plasma membrane. However, our data show that expression of caveolin-1 in HT29 cells neither modified cholesterol distribution in the plasma membrane, nor its concentration in caveolae-like domains as determined by sucrose gradient fractionation of cells labeled with [14C]-cholesterol (Fig. 4). Enrichment of cholesterol in these so-called light fractions from HT29 cells, did not change significantly upon expression of caveolin-1. Thus, alteration in the distribution of cholesterol between raft and non-raft plasma membrane subdomains are not likely to be responsible for the effects observed in HT29 cells expressing caveolin-1.

Fig. 4. Characterization of HT29 cells by sucrose gradient fractionation.

MDCK and stably transfected HT29 clones (C16 or mock) were labeled with [14C]-cholesterol, lyzed in 1% Triton X-100 and fractionated on a sucrose gradient as described (Oram, 1986; Rothblat et al., 1986). Samples (10µl) of each fraction were separated by SDS-PAGE, transferred to nitrocellulose and analyzed for the presence of caveolin-1 as described (Bender et al., 2000). Proteins from MDCK cells (5µg) were included on each gel as positive controls (c) for caveolin-1 immunodetection. (A) The typical pattern of total protein distribution in a representative gradient from HT29 cells is shown after Ponceau Red S staining. (B) Caveolin-1 distribution in sucrose gradient fractions from the transfected HT29 clone C16 is compared to that of MDCK cells. Migration positions of molecular weight marker proteins are indicated to the left in kDa. Note that enrichment of caveolin-1 in fraction 4 was similar for C16 and MDCK cells, whereas no caveolin-1 could be detected in sucrose gradient fractions in the mock-tranfected HT29 clone. (C) Distribution of [14C]-cholesterol in sucrose gradient fractions from HT29 clones (mock, C16) and MDCK cells. The distribution of [14C]-cholesterol (cpm/fraction) in sucrose gradient fractions obtained from HT29 clones (mock, C16) and MDCK cells is shown. A similar pattern of [14C]-cholesterol distribution was obtained for all three cell types analyzed. In particular, enrichment of caveolin-1 in fraction 4 from transfected HT29 cells (see panel B), coincided with a peak of [14C]-cholesterol. Furthermore, [14C]-cholesterol was highly enriched in this fraction in both mock and caveolin-1 transfected clones.

Taken together, most of the available experimental data does not yield conclusive mechanistic insights as to how caveolin-1 presence modulates tumor cell function. To identify potential targets that may explain the tumor suppressor function of caveolin-1 in colon carcinoma cells, this laboratory is employing two fundamentally distinct approaches. On the one hand, we are investigating whether molecules generally implicated in the genesis of colon cancer may be functionally modulated by inducible expression of caveolin-1 in colon carcinoma cells. This approach led to the identification of inducible nitric oxide synthase (iNOS) as a potential target since caveolin-1 presence in HT29 and DLD-1 cells promotes iNOS protein turnover via the proteasome pathway (Felley-Bosco et al, 2002). A similar mechanism might be employed to regulate other molecules known to interact with caveolin-1, such as PKCa. Preliminary results in HT29 cells suggest that TPA-induced PKCa degradation is accelerated by caveolin-1 expression (Monardes et al., 2001).

Alternatively, as a more general approach, studies were initiated to identify genes that alter their expression levels in response to caveolin-1 presence. This preliminary analysis has led to the identification of a number of genes that alter their expression pattern. Interestingly, two trends important to cancer biology were detectable, namely modulation of the expression of proteins linked to apoptosis on the one hand and metastasis on the other (Hetz et al., 2001; Montoya and Quest, unpublished data). These observations underscore the complexity of caveolin-1 linked regulation and provide a mechanistic basis to understanding how caveolin-1 might in fact play opposing roles at different stages of tumor cell development or in different types of cancers, as discussed above. These results will be highlighted in our discussion of caveolin-1 function.

ACKNOWLEDGEMENTS

Work discussed from this laboratory was supported by FONDECYT (CHILE) regular grant awards #1990893 and #1020585; FONDAP 15010006 and ICGEB grant CRP/CH100-05 (to A.F.G.Q), Conicyt Ph. D. fellowship award (to M. M.).

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Corresponding author: Andrew F.G. Quest, Ph.D. Laboratory of Cellular Communication. Program of Cell and Molecular Biology, ICBM, Faculty of Medicine. University of Chile, Independencia 1027, Santiago, CHILE. Fax/phone 56-2-7382015. e-mail: aquest@machi.med.uchile.cl

Received: June 10, 2002. In revised form: July 04, 2002. Accepted: July 14, 2002

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