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Department of Anesthesiology Research, Cleveland Clinic Foundation; and
* Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA
1 Correspondence: Department of Anesthesiology Research, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland OH 44122, USA. E-mail: paratm{at}ccf.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONCYTOSKELETON AND PLASMA MEMBRANE...ROLE OF CAVEOLIN AND...EFFECT OF CAVEOLAE MANIPULATION...UNANSWERED QUESTIONS AND FUTURE...CONCLUDING REMARKSREFERENCES |
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Key Words: angiogenesis • caveolin-1 • endothelial cell • motility
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONCYTOSKELETON AND PLASMA MEMBRANE...ROLE OF CAVEOLIN AND...EFFECT OF CAVEOLAE MANIPULATION...UNANSWERED QUESTIONS AND FUTURE...CONCLUDING REMARKSREFERENCES |
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and later named as structures resembling little caves (2)
, caveolae have been shown to be specialized plasma membrane subdomains capable of vesicular transport and sophisticated compartmentalization of cell signaling (3)
. Numerous cell functions, including cell type-specific functions, involve caveolae and require caveolin-1, the major protein component of these organelles. Caveolae are particularly abundant in endothelial cells and participate in endothelial transcytosis, vascular permeability, vasomotor tone control, and vascular reactivity. The importance of caveolae in endothelial function has been reviewed recently by others (4
5
6)
. This review summarizes the current available data showing that caveolae and their key protein caveolin-1 play an essential role in cell migration, with special emphasis on endothelial cell migration. |
CYTOSKELETON AND PLASMA MEMBRANE ARE TWO MAJOR PLAYERS IN CELL MIGRATION |
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TOPABSTRACTINTRODUCTIONCYTOSKELETON AND PLASMA MEMBRANE...ROLE OF CAVEOLIN AND...EFFECT OF CAVEOLAE MANIPULATION...UNANSWERED QUESTIONS AND FUTURE...CONCLUDING REMARKSREFERENCES |
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Much effort to understand the molecular machinery that drives the movement of the cell has focused on the nature of cytoskeletal structures and their integrin-mediated interactions with the extracellular matrix. Indeed, the cytoskeleton is involved in every step of the migration process. Extension of the membrane at the front of the cell is coupled with controlled, polarized polymerization of actin and cross-linking of these filaments into bundles and networks. Actin bundles anchor and become focal contacts, then focal adhesions. The forward flow of cytoplasm, nucleus, and organelles involves myosin-dependent cortical contraction. De-adhesion of the rear of the cell is believed to involve contraction of stress fibers in the tail (7)
. Members of the Rho family of small GTPases (Rho, Rac, Cdc42) play a key role in regulating actin organization in the process of cell migration. Cdc42 regulates filopodia protrusion, Rac triggers membrane ruffling and lamellipodia formation, and they both control the formation of focal complexes associated with these extensions. Rho regulates the assembly of contractile actin-myosin filaments to form stress fibers and the formation and maintenance of focal adhesions (8
9
10
11)
.
The involvement of the plasma membrane in migration has been the focus of recent attention (12
, 13)
. Biophysical studies show that the lamellipodial extension rate is inversely correlated with the apparent membrane tension (14)
and that plasma membrane microviscosity is polarized during endothelial cell migration (15)
. Anterograde secretion pathway from the trans-Golgi network to the plasma membrane is required for locomotion (12)
. Moreover, endocytosis from anywhere on the cell surface coupled to exocytosis at the advancing edge is believed to provide a fresh source of cell surface material to accommodate the extension and to recycle integrins to provide new potential attachments at the front (16)
. Whether polarized endo- and exocytosis also generate a membrane flow that might propel the cell has been highly controversial (17
, 18)
. Ruffling membranes, a common characteristic of an advancing edge, form within 1–2 min after the cell is stimulated by addition of a growth factor. This process is mediated by Rac, which redirects exocytosis to those sites on the cell surface where ruffles form (19)
. Coordination of membrane transport and actin dynamics is postulated to be regulated by small GTPases (20
, 21)
.
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ROLE OF CAVEOLIN AND CAVEOLAE IN CELL DYNAMICS AND MIGRATION |
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TOPABSTRACTINTRODUCTIONCYTOSKELETON AND PLASMA MEMBRANE...ROLE OF CAVEOLIN AND...EFFECT OF CAVEOLAE MANIPULATION...UNANSWERED QUESTIONS AND FUTURE...CONCLUDING REMARKSREFERENCES |
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) are plasmalemmal caveolin-coated invaginations that carry out signaling and trafficking functions. They can assume a variety of other shapes (such as closed vesicles or tubular structures) due to their dynamic properties. They represent a subset of membrane lipid domains enriched in glycosphingolipids, cholesterol, and lipid-anchored proteins. Caveolae functions rely on caveolin-1, their major protein. Caveolin-1 also has a structural role, and oligomers of caveolin-1 constitute the filaments that decorate the cytosolic surface of caveolae (22)
. Caveolin-1 exists as two isoforms (caveolin-1-
and -ß) differing by the length of their N terminus. Two other gene products are part of the caveolin family: caveolin-3 is muscle-specific and shares the greatest amino acid identity with caveolin-1; caveolin-2 is coexpressed and hetero-oligomerizes with caveolin-1 in most cell types. Caveolin-1 is triply palmitoylated on residues 133, 143, and 156 in the C terminus and is phosphorylated on serine 80, and possibly other serine residues (23)
. Caveolin-1- is subject to tyrosine phosphorylation on residue 14, which is absent from caveolin-1-ß (Fig. 2
, upper panel). Its unusual topology, with both N and C termini facing the cytosol and separated by a short hydrophobic domain inserted in the plasma membrane, is depicted in Fig. 2
(lower panel). A specific motif in caveolin-1 N terminus, called the scaffolding domain, functions to recruit proteins to caveolae and regulates their activity.
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Caveolin-1 drives the formation of plasma membrane caveolae and anchors them to the actin cytoskeleton, modulates cell interaction with the extracellular matrix, pulls together and regulates signaling molecules, and transports cholesterol (3
, 24
25
26)
. Via these functions, caveolin-1 could play an important role in cell movement through control of cell membrane composition and membrane surface expansion, polarization of signaling molecules, and/or cytoskeleton remodeling. There are several indications that caveolae and their major component caveolin-1 are involved in the process of migration.
Caveolae play a role at the interface of cytoskeleton and extracellular matrix
A connection between caveolae and the cytoskeleton has long been described (27
28
29
30
31)
. Caveolin-1 collects at the margins of cells and in patches aligned with actin-rich stress fibers (24
, 32)
. Caveolin-1 binds to and colocalizes with the actin cross-linking protein filamin. Structures positive for caveolin-1 and filamin coalign with the stress fibers induced by Rho stimulation (33)
. There may be a link between caveolin-1 Tyr14 phosphorylation and the cytoskeleton: hyperosmotic shock induces the phosphorylation of caveolin-1 on Tyr14, and this induction is potentiated by cytoskeleton disruption (34)
.
Even though integrins normally are not in caveolae, binding of Triton X100-soluble (noncaveolar) caveolin-1 to integrins has been reported (25
, 35)
. Caveolin-1 and the GPI-anchored urokinase receptor uPAR interact with ß-1 integrins in a complex that regulates adhesion and signaling through Src family kinases and focal adhesion kinase (FAK). The formation of such functional units contributes to cell adhesive process, migration, and invasion (36)
. Caveolin-1 depletion results in the loss of focal adhesion sites, FAK phosphorylation, and adhesion (37)
. Caveolin-1 therefore is considered an adaptor protein regulating integrin function (25
, 38)
.
Furthermore, caveolin-1 is thought to modulate the effect of endostatin, the potent anti-angiogenic C-terminal fragment of collagen XVIII. Endostatin binds to 5ß1 integrin, caveolin-1, and a heparan sulfate proteoglycan at the endothelial cell surface, inducing rapid clustering of 5ß1 integrin associated with actin stress fibers and its concomitant localization with caveolin-1. The proposed signaling is that caveolin-associated Src is then activated and phosphorylates p190RhoGAP, resulting in down-regulation of RhoA activity and in disassembly of actin stress fibers and focal adhesions (39
, 40)
. Similarly, fibronectin exogenously expressed in fibronectin null cells cofractionates with caveolin-1-enriched membrane domains and colocalizes with caveolin-1 (as shown by immunofluorescence) by a mechanism that requires heparan sulfate proteoglycans. This association with caveolae occurs when soluble FN is polymerized or remodeled within the extracellular matrix (41)
.
Another level of involvement of caveolae in cell interaction with the extracellular matrix is through regulation of matrix degradation, an essential process in the invasiveness of both normal and neoplastic cells. In addition to promoting efficient cell surface plasminogen activation (42)
, caveolae host metalloproteases (MMPs), including MT1-MMP and MMP-2 (43
44
45)
, and, in cancer cells, the cysteine protease cathepsin B (46)
. Caveolar localization of these proteases might focus matrix proteolysis to a limited cell surface compartment. Recent evidence suggests that caveolin-1 also indirectly regulates MMP-1 by interacting with CD147 and diminishing CD147 MMP-inducing activity (47)
.
Caveolae compartmentalize signaling molecules relevant to migration
Caveolae are thought to be critical for compartmentalization of signaling molecules because caveolin-1 oligomers form a scaffold for assembly of signaling molecules, including receptors, signal transducers, and effectors, and regulate the activation state of these signaling complexes (3
, 48)
. Thus, it is likely that caveolae may represent functional platforms for the organization and coordination of signaling pathways involved in cell migration (49
, 50)
.
Caveolae play a key role in calcium signaling, as shown by experiments in which caveolae disruption impairs specific calcium transduction steps (51
, 52)
. Initiation of Ca2+ waves can be detected at caveolae-rich regions of the plasma membrane in static or migrating cells (49
, 51)
. In migrating cells, these waves originate from the rear of the cell, where caveolae accumulate (49)
. Several proteins directly involved in Ca2+ translocation have been located in caveolae (reviewed in ref 51
), including an IP3 receptor-like protein, a Ca2+-ATPase, and transient receptor potential channels Trp1 and Trp3. Store-operated Ca2+ entry, also called capacitative Ca2+ entry (CCE), present in all nonexcitable cells, is activated by an as yet unknown mechanism when internal Ca2+ stores are depleted. There is increasing evidence for a role of caveolae and caveolin-1 in CCE. The signaling machinery involved in CCE is organized in caveolae (52)
into complexes that are functional in living ECs and can lead to the local activation of endothelial nitric oxide synthase (eNOS) (50)
. In fact, it has been shown that eNOS activation requires CCE (53
, 54)
. Caveolae could play a role in CCE through their compartmentalization capabilities via caveolin-1 scaffolding domain (55)
and their endocytic properties as a link between the plasma membrane and the endoplasmic reticulum (50)
.
Nitric oxide synthases are regulated by caveolin-1 (and caveolin-3 in muscle cells) (56)
. Nitric oxide is involved in endothelial cell proliferation, migration, protease release, and increased vascular permeability, each important for angiogenesis. eNOS plays a central role in endothelial cell migration, as shown by the impaired angiogenesis and wound healing of eNOS-deficient mice (57)
. By its interaction with caveolin-1 scaffolding domain, eNOS is maintained in an inactive state in close proximity to the CCE machinery and receptors for agonists (3)
and growth factors (58)
that are able to regulate NO production. Upon stimulation and local [Ca2+] increase, eNOS dissociates from caveolin-1 and binds positive regulatory proteins, including calmodulin and hsp90 (59
, 60)
(Fig. 3
).Caveolae are therefore regulatory platforms for the cascade of events resulting in NO production. Recent evidence further suggests that caveolae host downstream NO signaling (61)
.
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Several growth factor receptors are enriched in caveolae. Of particular interest is the recent finding that the VEGF receptor-2, also called Flk-1/KDR, is localized in endothelial caveolae where it is negatively regulated by association with caveolin-1. Proper localization of Flk-1 in caveolae seems to be necessary for VEGF-induced downstream signaling and cell migration (58)
, which is mediated by eNOS activation (62
, 63)
. Accordingly, in EC devoid of caveolae, VEGF-induced eNOS activation is defective (64)
.
Rho family small GTPases play a key role in controlling cell migration. Caveolin-1 might interact with small GTPases since RhoA, Rac1, and Cdc42 cofractionate with caveolin-1, although a direct interaction with caveolin-1 has been proved only for RhoA (65
, 66)
. Upon stimulation, RhoA and Rac-1 can be recruited to caveolae (65
, 67)
. Integrin-mediated recruitment of Rac to the plasma membrane has recently been shown to occur preferentially by Rac targeting to specific plasma membrane lipid domains that might be caveolae (67
68
69)
. In addition, it has been suggested that Rho may regulate caveolae formation (70)
.
Several other proteins involved in the control of cell migration such as prostacyclin synthase (71)
, Nogo-B (72)
, or CD36 (73)
have been localized to caveolae (71
, 72
, 74)
. In cells with no caveolin-1, such as lymphocytes, or with severely reduced expression of caveolin-1, such as MCF7 cells, this function of compartmentalization of signaling molecules (e.g., chemokine receptors) is performed by lipid rafts. Whether the negative regulation by the caveolin-1 scaffolding domain is played in these cells by other proteins is not known.
Caveolae transport cholesterol and may participate in membrane recycling
Caveolae are highly enriched in cholesterol. Caveolin-1 is a cholesterol binding protein (75)
containing a cholesterol binding amino acid consensus sequence (76)
overlapping with the scaffolding domain. Caveolin-1 is involved in transport of newly synthesized cholesterol from endoplasmic reticulum to plasma membrane (26)
. This process is mediated at least in part by a cytosolic complex containing chaperones, cholesterol, and caveolin-1 (77)
. Palmitoylation of two cysteines of the C terminus of caveolin-1 is required for formation of this complex and for the transport of cholesterol to caveolae (78)
. Caveolin-1 could therefore be involved in dynamics of plasma membrane composition through cytosolic transport and membrane delivery of cholesterol. HMG-CoA reductase inhibitors, which reduce caveolin expression and block de novo cholesterol synthesis, affect cell migration (79
80
81
82)
with a biphasic dose dependence.
Caveolae may play a role in cell migration by internalization and oriented delivery to the expanding edge of the cell. The extent to which caveolae participate in constitutive endocytic processes in the cell is still unclear (83)
. Early literature on endothelial caveolae showed the involvement of caveolae in transcytosis (reviewed in ref 4
). Caveolae contain the molecular machinery for fission, docking and fusion (84
85
86)
. The internalization of albumin (87)
and GPI-anchored proteins (28)
through caveolae has been reported. Internalization of caveolae seems to require local actin polimerization (88)
in nonmigrating cells. Whether the same requirement applies to caveolae internalization in migrating cells is not known. It is accepted that endocytic cycle initiated at clathrin-coated pits results in polarized exocytosis in migrating cells. Similarly, endocytosis initiated by caveolae may contribute substantially to the total pool of recycling membrane (89)
.
Caveolin-1 and caveolae are polarized in migrating cells
Whether the cell is randomly migrating or in the process of chemotaxis (migrating directionally toward an attractant), a precise spatial and functional asymmetry must occur with adhesion and detachment taking place at opposite edges of the cell (90
, 91)
. Several structures and events important in the migration process have been shown to be polarized, including integrin-cytoskeletal interactions (92)
, matrix metalloproteinases (93)
, actin binding proteins, actin polymerization, ß-actin mRNA, which localizes to the leading edge of migrating fibroblasts (94)
, and myosin II (95)
, which is involved in polarization of the locomotive protrusions (96)
. The Golgi apparatus and the microtubule organizing center localize in front of the nucleus (12
, 97)
toward the leading edge. Microtubules align along the axis of cell movement in most motile cell types and display a polarized dynamic instability, with microtubule growth predominating at the leading edge and microtubule shortening predominating at the trailing edge. In addition to controlling actin cytoskeleton reorganization, Rho family GTPases are responsible for regulation of polarization (98
99
100)
.
Another level of polarization is found in directional sensing, which is used by cells for chemotaxis. Whether chemoattractant receptors are polarized is still controversial. They might be uniformly distributed along the plasma membrane and the activity of the signal transduction pathway be increased on the side of the cell facing the higher concentration of chemoattractant, presumably due to a difference in receptor occupancy between leading and trailing edge (101
102
103)
.
It is becoming evident that lipids are polarized in cell migration. Cholesterol-enriched membrane lipid domains (membrane rafts) redistribute to different poles of migrating cells, with cell- and raft-type specificity (104
105
106
107)
. Cold triton X-100 extraction of polarized migrating neutrophils selectively removes proteins at the leading edge while sparing the cell body and the trailing edge, suggesting that rafts are polarized toward the rear (108)
. Raft-associated proteins are polarized when cells migrate (104
, 107)
; modification of these proteins, so that they are no longer located in rafts, inhibits their asymmetric redistribution (106)
. Moreover, raft disruption by cholesterol depletion impedes signaling, acquisition of a polarized phenotype, and chemotaxis (106
, 109)
.
Caveolae may mediate shear stress-induced endothelial cell migration. Shear stress regulates the density and localization of caveolae (110
, 111)
where shear stress-induced signaling originates (110
, 112
, 113)
. Caveolae have therefore been proposed to act as mechanosensors or mechanotransducers. Shear stress-induced cell migration in the direction of the flow is accompanied by translocation of caveolin-1 and caveolae to the rear of the cells (32
, 114)
.
Polarization of caveolin-1 and caveolae in migrating cells, which is in favor of a role for caveolae in cell migration, has been confirmed in other migration models. Caveolae accumulation at the rear of migrating endothelial cells is a feature common to migration induced by laminar shear stress, monolayer wounding, and both chemotaxis and random migration across a porous membrane (49
, 115)
. However, whereas in 2-dimensional migration caveolin colocalizes at the trailing edge of polarized cells, in 3-dimensional migration (across a membrane) caveolin-1 concentrates in the leading cell extension in a seemingly soluble, cytoplasmic form, whereas caveolae devoid of caveolin-1 remain concentrated at the rear.
Caveolin-1 as well as caveolae could well be an important link reconciling the cytoskeleton-driven and the membrane-driven cell movement hypotheses. This organelle and its defining protein are connected to the two main components of the migration machinery, the cytoskeleton and the plasma membrane, which are dynamically polarized during cell movement (Fig. 4
).
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EFFECT OF CAVEOLAE MANIPULATION ON CELL MIGRATION AND ANGIOGENESIS |
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TOPABSTRACTINTRODUCTIONCYTOSKELETON AND PLASMA MEMBRANE...ROLE OF CAVEOLIN AND...EFFECT OF CAVEOLAE MANIPULATION...UNANSWERED QUESTIONS AND FUTURE...CONCLUDING REMARKSREFERENCES |
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Several studies have shown that manipulating caveolin-1 expression or caveolae formation affects cell migration and/or angiogenesis in a complex way. Most studies point to a positive role for caveolin-1 in cell migration/angiogenesis. However, there are indications of caveolin-1 negatively regulating these processes (Table 1
). One must keep in mind that angiogenesis is a multistep process in which endothelial cell migration represents a critical step but that also involves increased vascular permeability, extracellular matrix degradation, endothelial cell proliferation, and differentiation, all processes regulated by caveolin-1. It has been proposed that caveolin-1 is down-regulated during the proliferation phase of angiogenesis and up-regulated during the differentiation phase, which might reflect different functions for this protein at different stages (116)
. In addition, caveolin-1 often plays paradoxical functions in cell signaling, because it binds and inhibits several receptors or enzymes, which nevertheless need to be in caveolae to become activated.
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Altering membrane cholesterol is a common pharmacological approach to assess the involvement of caveolae in a given function. Caveolae are exquisitely sensitive to cholesterol removal by using cholesterol binding or sequestering drugs (117)
. However, the effect of these drugs is not specific for caveolae and will affect all cholesterol-enriched lipid domains. For example, cyclodextrin or filipin treatment impairs HUVECs migration in a transwell assay and capillary-like tube formation on matrigel (118)
.
Down-regulation of caveolin-1 expression is commonly assumed to be correlated with a reduced amount of caveolae at the cell surface. It is experimentally achieved in vitro using antisense oligonucleotides, antisense adenovirus, or siRNA. Caveolin-1 down-regulation reduces the number of capillary-like tube formation in vitro using human microvascular endothelial cells (116)
or HUVECs (118
, 119)
and inhibits blood vessel development in chick embryo chorioallantoic membrane (119)
. Antisense deletion of caveolin-1 in kidney 293 cells results in the loss of focal adhesion sites, FAK phosphorylation, and cell adhesion (37)
. Caveolin-1 reduction by small interfering RNA suppresses astrocytes’ chemotactic response to the chemokine MCP-1 (120) and decreases HUVECs migration (118)
. Confirmation of these in vitro experiments had to be obtained in vivo. The phenotype of caveolin-1 gene-disrupted mice seems to be more complex than initially thought. Although they are viable and fertile, they show diverse abnormalities resulting in a reduced life span (121)
. As far as their vascular system is concerned, these mice appear to have a normal vasculogenesis. Only recently have these mice been challenged with bFGF-supplemented matrigel plugs or tumor cell line implants, and they showed an impaired angiogenic response (122)
. In a model of adaptive angiogenesis after artery resection, these mice also displayed impaired angiogenesis (64)
.
Overexpression of caveolin-1 via adenoviral gene delivery system in human microvascular endothelial cells accelerates and increases in vitro capillary tube formation (116)
. By contrast, restoration of caveolin-1 expression in MTLn3 metastatic cells reduces EGF-stimulated lamellipod extension and cell migration (123)
, and caveolin-1 overexpression in CHO or ECV304 cells transfected with Flk-1 reduces VEGF-induced signaling to the nuclear transcription factor Elk-1 as measured by a luciferase reporter system (124)
. Caveolin-1 interacts via a consensus binding motif (the scaffolding domain, amino acids 82-101) with numerous signaling proteins that are usually maintained in an inactive state by the interaction (125)
. A synthetic peptide corresponding to that sequence linked to a cellular internalization peptide can be used in vitro and in vivo to mimic or enhance the regulatory effects of caveolin-1. Delivery of the scaffolding domain into the cytoplasm of cultured microvascular endothelial cells enhances capillary-like tubule formation in vitro (116)
. In vivo, this peptide has the ability to reduce NO synthesis and NO-dependent inflammation and vascular leakage (126)
. By blocking microvascular hyperpermeability at tumor sites, this peptide prevents tumor development despite its absence of direct cytostatic or anti-angiogenic effects (127)
.
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UNANSWERED QUESTIONS AND FUTURE DIRECTIONS |
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TOPABSTRACTINTRODUCTIONCYTOSKELETON AND PLASMA MEMBRANE...ROLE OF CAVEOLIN AND...EFFECT OF CAVEOLAE MANIPULATION...UNANSWERED QUESTIONS AND FUTURE...CONCLUDING REMARKSREFERENCES |
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Another important issue, which should be solved in the near future by biophysical studies, is the role of caveolin-1 expression and caveolae presence on plasma membrane properties such as membrane tension and viscosity, which regulate cell migration. Finally, because caveolae are capable of vesicular transport and are anchored to the cytoskeleton through caveolin-1 and because soluble caveolin is capable of lipid transport, the study of the role of caveolae in migrating cells might shed new light on the respective roles of plasma membrane and filamentous actin in propelling the cell.
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CONCLUDING REMARKS |
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TOPABSTRACTINTRODUCTIONCYTOSKELETON AND PLASMA MEMBRANE...ROLE OF CAVEOLIN AND...EFFECT OF CAVEOLAE MANIPULATION...UNANSWERED QUESTIONS AND FUTURE...CONCLUDING REMARKSREFERENCES |
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ACKNOWLEDGMENTS |
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Received for publication June 28, 2004. Accepted for publication August 8, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONCYTOSKELETON AND PLASMA MEMBRANE...ROLE OF CAVEOLIN AND...EFFECT OF CAVEOLAE MANIPULATION...UNANSWERED QUESTIONS AND FUTURE...CONCLUDING REMARKSREFERENCES |
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