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Commentaries |
vß3 Integrin Expression
From the Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa
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Introduction |
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 Top Introduction References |
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and ß subunit
transmembrane glycoproteins containing large extracellular domains,
short transmembrane domains, and carboxy-terminal cytoplasmic domains
of variable length.1-5
There are presently 17
subunits and eight ß subunits known, which occur in just over 20
integrins identified so far. However, these numbers may belie the added
complexity introduced by the alternately spliced cytoplasmic domains
observed in some variants of these subunits.1,6
The eight ß subunits share approximately 40 to 80% amino acid
sequence homology and are similar in size (90 to 110 kd) except for the
ß4 chain, which is almost twice as big because
of its large intracytoplasmic domain. The ß chains contain a fourfold
repeat of cystein-rich segments and a highly conserved cytoplasmic
domain with an Asp-X-Ser-X-Ser sequence (where X is any amino acid)
associated with cation-dependent ligand binding and with the metal
ion-dependent adhesion site motif.2,7
This
cytoplasmic tail region of the ß subunits has been implicated both in
cytoskeletal interactions and with signaling complexes. The
subunits, with molecular weights ranging between 120 and 180 kd,
tend to be more heterogeneous than the ß subunits. Furthermore, some
units contain light and heavy chains linked by a disulfide bridge
in the extracellular domain, whereas other subunits contain an
extra segment of approximately 180 amino acids called the
A-domain7
(or I
domain)1,2
inserted before the last five
homologous repeats, which contain a cation-binding domain. This
A-domain contains a sequence homologous to the collagen-binding
domains of von Willebrand factor, cartilage matrix protein,
and complement proteins. Only recently
has functional activity in recombinant versions of this domain
permitted the opportunity to study ligand binding; the fragment L
A-domain has been shown to bind the intercellular cell adhesion
molecule-2 (ICAM-2) and the fragment 1/2 A-domain has been shown
to bind to laminins.7
All subunits contain a
sevenfold repeat of a homologous segment with the last three or four
repeats containing the sequence
Asp-X-Asp-X-Asp-Gly-X-X-Asp1
(or related
sequence)2
motif. This motif is associated with
the divalent cation-binding EF-handlike
domains2,7
and contributes to cation-dependent
ligand binding to the integrin receptor. While divalent cations are
required for receptor function they can also, depending on the nature
of the cation, affect both the integrin's affinity and specificity for
ligands.1-5,7
Some integrins also require
divalent cations for their ß subunit
association.1,8,9
In general, whereas the
theoretical number of integrin heterodimers exceeds 100, the 20-plus
observed integrins fall into three basic groups based on similar chain
structures and/or the ability to recognize similar protein or adhesion
motifs. These three groups include integrins which contain the
ß1, ß2, and
ß3 or v subunits;
three ß integrins do not fall within these
groups.1,2
Many subunits can associate
with just one of the ß subunits, although some subunits can
associate with more than one ß subunit. In particular, the
v subunit appears to be one of the most
promiscuous of the subunits and can associate with at least five
different ß subunits, including the ß1 chain
(see below). Although originally identified as cell adhesion
molecules (both cell-extracellular matrix and cell-cell), integrins
have most recently been shown to play significant roles in signal
transduction events,1-7
10-27
gene
expression,12,25,28
cell
proliferation,12,13,15,17,26,27,29
regulation of
apoptosis30-33
and
anoikis,32
invasion and
metastasis,2,10-12,21,26,29,33-37
embryogenesis,38-41
tumor
progression,29
inflammation and
immunity,28
hemostasis,42
and angiogenesis.26,33,43-48
Recent studies have
also identified integrins as points of entry for certain infection
agents including hantaviruses, which appear to use the
ß3-containing integrins to gain entry into
cells,49
and Lyme disease spirochetes, whose
attachment to human cells is mediated by the
vß3 and
5ß1
integrins.50 Integrin function in normal and pathological processes in terms of ligand and adhesive specificity was initially determined using cell adhesion assays, monoclonal antibodies, and affinity chromatography.1,2 It became apparent that individual integrins can often bind to different ligands and that different ligands are recognized by more than one integrin. Integrins bind to extracellular matrix proteins and facilitate cell-substratum adhesion and, in the case of the ligand fibrinogen, can facilitate cell-cell aggregation. Some integrins can also recognize integral membrane proteins of the immunoglobulin superfamily (ICAM-1, ICAM-2, and VCAM-1) and thereby mediate direct cell-cell adhesion (ie, recognize and bind to a counterreceptor on other cells). The first defined integrin recognition site was the sequence Arg-Gly-Asp (RGD),1-5 found in fibronectin, vitronectin, and other adhesive proteins. Subsequent binding motifs identified include Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) in fibrinogen, Asp-Gly-Glu-Ala (DGEA) in type I collagen, Glu-Ile-Leu-Asp-Val (EILDV) in an alternatively spliced segment of fibronectin, and Gly-Pro-Arg-Pro (GPRP) in fibrinogen. Whereas the integrins that bind laminin appear to recognize specific parts of the laminin molecule, integrins that bind counterreceptors appear to recognize specific immunoglobulin-like domains.1,2
At present, integrins are described structurally as heterodimeric
glycoproteins that contain an extracellular ligand-binding site
composed of the N-terminal domains of the and ß
subunits. This region is connected by two stalks, one from each
subunit, to the membrane-spanning segments and ends at the and ß
subunit cytoplasmic domains. All current evidence indicates that these
cytoplasmic domains interact with cytoskeletal proteins and components.
The cytoplasmic tail of some ß subunits appears to direct integrin
receptors in a ligand-independent manner to focal adhesion sites where
the integrins become organized at the ends of actin filaments. These
focal contacts also contain the proteins vinculin, talin, and
-actinin, which are thought to mediate interactions between the
ligand-integrin structure outside the cell and the actin microfilaments
inside the cell.1,2,7
The cytoplasmic tail of
some subunits appear to convey ligand-specific signals to the cells
in response to the integrin binding its ligand.51
In contrast, the 6ß4
integrin is unique in that it becomes concentrated in epithelial cells
specifically at hemidesmosomes and is thought to interact with
the intermediate filaments characteristic of hemidesmosome
structure.1
We now know that individual cells can vary their adhesive properties by
selectively expressing different integrins and by modulating their
integrin specificity and affinity for ligands through a process known
as integrin activation and deactivation. The change in integrins'
activation/deactivation state as a conformation change in the
receptors' extracellular domains has been detected using both
immunohistochemical and biophysical techniques and could relate to the
degree of phosphorylation of the ß subunit (see
below)51
or result from interactions of the
integrins with lipid-derived mediators.1
Recently, it has been shown that integrin function is also subject to
modulation by interaction with other membrane proteins, including other
integrins.52-56
The
glycosyl-phosphatidylinositol (GPI)-linked cell surface protein
urokinase receptor (uPAR), which can function as an adhesion receptor
for vitronectin with distinct sites for binding both vitronectin and
urokinase,52,53
can interact with the active form
of ß1-containing integrins to form a stable
integrin-uPAR-caveolin complex that suppresses the cells' normal
ß1-dependent adhesion to fibronectin. The
result is that uPAR alters cells' ability to adhere and interact with
different extracellular matrices (and/or different environmental
signals) via the interactions it establishes through its
integrin-uPAR-caveolin connection with the
cytoskeleton.52,53
Other studies have identified
transmembrane adaptors such as the integrin-associated protein (IAP),
which is physically and functionally associated with the
vß3 and
IIbß3
integrins.57
IAP not only cooperates with
ß3 integrins in binding to
thrombospondin, but also appears to activate a heterotrimeric Gi
protein-dependent intracellular pathway leading to activation of
tyrosine kinase Syk and its association with FAK. As such, IAP seems to
work in concert with ß3 integrins to regulate
intracellular signaling in response to
thrombospondin.27,57
It is now clear that altered, modulated, or regulated integrin
interactions can change the way cells interact with their environment
with dramatic and far-reaching consequences for both normal and
pathological conditions. This is apparent from studies that have shown
that perturbation of certain integrins, either by ligation or by
treatment with certain anti-integrin antibodies, can result in the
generation of signals that cause an increase in intracellular
pH26,58,59
and Ca2+
levels,26,60
changes in inositol lipid
synthesis,26,61
tyrosine phosphorylation of
pp125FAK,11,12,26
and
activation of p34/cdc213,26
cyclin
A,26,62
protein kinase
C,26,63
mitogen-activated protein kinase
(MAPK),15,26
phosphatidylinositol
3-kinase,17,18,26
p21
Ras,20,26
and
NF-
B.20,21,26,64
Furthermore, recent studies
have shown that the
vß3 integrin on
melanoma cells can bind and localize proteolytically active MMP-2 on
the cell surface, facilitating cell-mediated collagen degradation and
directed cellular invasion.65
Integrins have also
been shown to act synergistically with growth factor receptors to
modulate cellular functions including cell
growth66
and migration.67
One of the most diversely functional integrins studied to date is the
vß3 integrin. This
integrin is the original, classic vitronectin receptor which recognizes
the RGD binding motif found in vitronectin.1,2,3
The v subunit is now known to associate with
at least five out of the eight different ß subunits, making it one of
the most promiscuous subunits studied so far. When associated with the
ß3 subunit, the
vß3 integrin can bind
to vitronectin and colocalize to focal adhesion sites with the proteins
vinculin, talin, and -actinin at the end of actin filaments. When
associated with the ß5 subunit, this homologous
integrin can bind vitronectin but remains randomly distributed over the
surface of the cells and does not localize to focal adhesion
sites.68
Recent work has shown that the
cytoplasmic tail of the ß3 chain undergoes
tyrosine phosphorylation upon adhesion of the
vß3 integrin to
vitronectin, antibody perturbation of the integrin, or incubation with
a manganese-containing buffer. This phosphorylation, which requires the
presence of the cytoplasmic tail of the v
subunit, does not occur in the ß5 subunit of
the vß5 integrin under
similar conditions.51
The ability of the vß3
integrin to cluster on the cell surface and colocalize with
cytoskeletal elements in response to specific stimuli can result in
polymerization of the actin cytoskeleton and lead to changes in cell
morphology and migratory ability. This permits the integrin to act as a
physical bridge between external stimuli and the cells' cytoskeleton,
and as a transducer of messages from outside the cell to its
internal signal processing pathways. Ultimately, changes in
adhesiveness of cells for their environment, through integrins like the
vß3 integrin, can lead
to gross changes in cellular morphology via the actin
cytoskeleton, which is directly involved in cell migration. In this
respect, high expression of the
vß3 integrin has been
associated with different motile cells in vivo including
neural crest cells, vascular endothelial cells, and malignant melanoma
cells.1,2,26
Recent work has identified the NPXY
motif within the ß3 subunit (comprising
residues 744–747) as essential for cell morphological and migratory
responses (cell attachment, spreading, and migration on a immobilized
ligand) mediated by the
vß3 integrin both
in vitro and in vivo.69
This work showed that the hamster cell line CS-1 could be transfected
with and express the wild-type
vß3 integrin, leading
to a change in the cells' migratory response to vitronectin and the
acquisition of an ability to form spontaneous pulmonary metastases in a
chick embryo grown on a chorioallantoic membrane. Mutations in the NPXY
region of transfected
vß3 integrin abrogated
the cells' metastatic ability and migratory response to vitronectin
but did not disrupt the integrin's ability to bind to
vitronectin.69
Other work has shown that
ligation of different integrins, including
vß3 on human melanoma
cells, can lead to altered protein expression associated with increased
invasive ability in vitro.70
More
specifically, as an extension of the work by Werb and
colleagues71
that demonstrated that the
5ß1 integrin could
transduce extracellular signals in rabbit synovial fibroblasts
resulting in a change in the cells' expression and extracellular
levels of collagenase and stromelysin, work with the human melanoma
cell line A375M showed that ligation of the
vß3 integrin with
vitronectin (either matrix bound or soluble) or an activating antibody
to the v subunit could increase the expression
and extracellular levels of the matrix-metalloproteinase-2 enzyme
(MMP-2, gelatinase A) coincident with an increase in the cells'
ability to invade in vitro.70
As a
whole, these results suggest a role for the
vß3 integrin in the
pathological progression of melanoma that is facilitated by specific
vß3 integrin-mediated
interactions of cells with their extracellular environment. These
interactions can then generate physical (possibly
mechanical)72-74
and biochemical signaling
events that contribute to a change in cell behavior resulting in an
aggressive, pathological phenotype.
Significant in light of these observations is the evidence presented by
Hsu and colleagues in this issue75
that the
vß3 integrin plays a
direct role in the progression of human primary cutaneous melanoma from
the nontumorigenic, nonmetastatic radial growth phase to the
tumorigenic, metastatically competent vertical growth phase. An
important aspect of this work was the approach that Hsu and colleagues
used to design and perform these studies. First, information about the
long-term clinical and histopathological observations of cutaneous
melanoma formed the basis for defining the disease and its progression
in patients and provided the touchstone for the results obtained in the
laboratory. Second, using these in vivo observations, Hsu
and colleagues isolated and propagated in vitro cells
derived from different stages of the disease as a model for primary
cutaneous melanoma progression as defined in vivo. Care was
taken to correlate the biological properties of the different isolated
cell types in culture with observed biological properties and stages
in vivo. Significant in the planning and execution of these
experiments was the authors' progression from the reductionist
in vitro cell culture model of the disease stages to the
more complex three-dimensional skin reconstruct model which recreates
the physiological milieu of the in vivo environment. Unlike
other traditional invasion assays, this model accounts for both tumor
cell-derived mechanisms and microenvironmental factors from stromal
cells during the invasive process. Therefore, differences between the
control and ß3 transfected cells could be
observed and the invasion by transfected melanoma cells deep into the
dermis without entering apoptosis could be clearly delineated from the
control cells, which did not invade but remained in the epidermis and
entered apoptosis. Ultimately, the experiments returned to the in
vivo SCID mouse model where tumorigenicity of early stage melanoma
cells was shown to increase after forced expression of the
ß3 subunit.
Central to this experimental approach was the clinical observation that
the onset of expression of the ß3 integrin was
one of the most specific markers for identifying the transition of
primary human cutaneous melanoma from the nontumorigenic, nonmetastatic
radial growth phase to the tumorigenic, metastatically competent
vertical growth phase. Hsu et al demonstrated that forced expression of
the ß3 integrin subunit (via
adenovirus gene transfer) results in the expression of a functional
vß3 integrin and in a
malignant phenotype that corresponds to the observed in vivo
progression of cells from radial growth phase to vertical growth phase
. This study not only contributes to the growing body of work that
clearly identifies the importance of integrin analysis in the diagnosis
and prognostic evaluation of diseases, but also is the first to extend
clinical observations about a key prognostic marker for cutaneous
melanoma progression to the demonstration of a specific functional role
for the ß3-containing integrin
vß3 in
vitro and in vivo, which directly correlates with the
progression of the disease as clinically defined. In conclusion, these
results clearly provide the rational basis for research that is just
now beginning to identify integrins as unique biological targets for
tumor therapy,34,76
as potential targets for
inhibiting or blocking the metastatic cascade,77
and as useful tools for targeted chemotherapy strategies in the
treatment of cancer based on the selective expression of integrin
receptors in the tumor vasculature.78
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Acknowledgements |
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Footnotes |
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Accepted for publication September 3, 1998.
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References |
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TopIntroductionReferences |
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5ß1 integrin-mediated cell spreading on fibronectin. J Cell Biol 1993, 268:21495-21462
B dependent gene expression. Mol Biol Cell 1995, 6:841-850[Abstract]
B mediates vß3 integrin-induced endothelial cell survival J Cell Biol 1998, 141:1083-1093vß3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994, 79:1157-1164[Medline]
4 integrins and tumor metastasis. Curr Topics Microbiol Immunol 1998, 231:125-141[Medline]
vß3 in melanoma metastasis. Cancer Metastas Rev 1995, 14:241-252[Medline]
vß3 for angiogenesis. Science 1994, 264:569-571vß3 complex on human microvessel endothelial cells. J Invest Dermatol 1994, 103:295-299[Medline]
v integrins. Science 1995, 27:1500-1502
vß3 blocks breast cancer growth, and angiogenesis in human skin J Clin Invest 1995, 96:1815-1822
vß3 prevents maturation of blood vessels during embryonic neovascularization. J Cell Sci 1995, 108:2655-2661[Abstract]
(v)ß(3), and apha(5)ß(1) mediate attachment of lyme disease spirochetes to human cells Infection Immunity 1998, 1952, 66:1946
vß3 integrins regulates 5ß1-mediated cell migration toward fibronectin. J Biol Chem 1997, 272:29380-29389IIbß3. J Biol Chem 1993, 123:485-496
vß3. Cell 1996, 85:683–693
vß3 integrin associates with activated insulin, and PDGFß receptors, and potentiates the biological activity of PDGF. EMBO J 1997, 16:5600-5607[Medline]
vß3 integrin and PDGF receptor increases cell migration. J Cell Science 1998, 111:469-478[Abstract]
vß3 and vß5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface J Cell Biol 1991, 113:919-929vß3 integrin in human melanoma cell invasion. Proc Natl Acad Sci USA 1992, 89:1557-1561(v) ß3 in cell adhesion, motility, and liver metastasis of murine RAW117 large cell lymphoma. Cancer Res 1996, 56:3103-3111This article has been cited by other articles:
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  L. Packer, S. Pavey, A. Parker, M. Stark, P. Johansson, B. Clarke, P. Pollock, M. Ringner, and N. Hayward Osteopontin is a downstream effector of the PI3-kinase pathway in melanomas that is inversely correlated with functional PTEN Carcinogenesis, September 1, 2006; 27(9): 1778 - 1786. [Abstract] [Full Text] [PDF]
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 D. E. Elder Pathology of melanoma. Clin. Cancer Res., April 1, 2006; 12(7): 2308s - 2311s. [Abstract] [Full Text] [PDF]
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A. Abdollahi, D. W. Griggs, H. Zieher, A. Roth, K. E. Lipson, R. Saffrich, H.-J. Grone, D. E. Hallahan, R. A. Reisfeld, J. Debus, et al. Inhibition of {alpha}v{beta}3 Integrin Survival Signaling Enhances Antiangiogenic and Antitumor Effects of Radiotherapy Clin. Cancer Res., September 1, 2005; 11(17): 6270 - 6279. [Abstract] [Full Text] [PDF]
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 A. Abdollahi, K. E. Lipson, X. Han, R. Krempien, T. Trinh, K. J. Weber, P. Hahnfeldt, L. Hlatky, J. Debus, A. R. Howlett, et al. SU5416 and SU6668 Attenuate the Angiogenic Effects of Radiation-induced Tumor Cell Growth Factor Production and Amplify the Direct Anti-endothelial Action of Radiation in Vitro Cancer Res., July 1, 2003; 63(13): 3755 - 3763. [Abstract] [Full Text] [PDF]
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E. Geissinger, C. Weisser, P. Fischer, M. Schartl, and C. Wellbrock Autocrine Stimulation by Osteopontin Contributes to Antiapoptotic Signalling of Melanocytes in Dermal Collagen Cancer Res., August 15, 2002; 62(16): 4820 - 4828. [Abstract] [Full Text] [PDF]
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R. A. Sturm, K. Satyamoorthy, F. Meier, B. B. Gardiner, D. J. Smit, B. Vaidya, and M. Herlyn Osteonectin/SPARC Induction by Ectopic {beta}3 Integrin in Human Radial Growth Phase Primary Melanoma Cells Cancer Res., January 1, 2002; 62(1): 226 - 232. [Abstract] [Full Text] [PDF]
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 S. Hapke, M. Gawaz, K. Dehne, J. Köhler, J. F. Marshall, H. Graeff, M. Schmitt, U. Reuning, and E. Lengyel {beta}3A-Integrin Downregulates the Urokinase-Type Plasminogen Activator Receptor (u-PAR) through a PEA3/ets Transcriptional Silencing Element in the u-PAR Promoter Mol. Cell. Biol., March 15, 2001; 21(6): 2118 - 2132. [Abstract] [Full Text]
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 S. C. Even-Ram, M. Maoz, E. Pokroy, R. Reich, B.-Z. Katz, P. Gutwein, P. Altevogt, and R. Bar-Shavit Tumor Cell Invasion Is Promoted by Activation of Protease Activated Receptor-1 in Cooperation with the alpha vbeta 5 Integrin J. Biol. Chem., March 30, 2001; 276(14): 10952 - 10962. [Abstract] [Full Text] [PDF]
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