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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Methods Mol Biol. Author manuscript; available in PMC 2012 January 1.
Published in final edited form as:
PMCID: PMC3248401
NIHMSID: NIHMS344484

Overview-studying integrins in vivo

Clifford A. Lowell, M.D., Ph.D.* and Tanya N. Mayadas, Ph.D.

Introduction

Integrins are expressed in all nucleated cells of multicellular animals and are essential for cell-matrix adhesion and, in vertebrates, cell-cell interactions. Since their recognition in 1987, they have been one of the most widely studied family of cell adhesion receptors. Integrins contribute to development, hemostasis, the immune response as well as diseases such as cancer and autoimmunity. In addition to their role as mechanical links, integrins are important conduits of bidirectional signaling in cells that influence processes from cytoskeletal arrangement and growth factor signaling to gene transcription (1). This overview will highlight a number of the major physiologic functions of integrins in both higher and lower organisms. Use of genetic approaches (i.e. gene knockouts) has provided a wealth of information about integrin function and will be emphasized in this chapter; given the significant published literature in this area we will focus on those examples in which specific functions have been revealed.

Integrin genes in vertebrates and invertebrates

Integrins are αβ heterodimeric, transmembrane proteins that are restricted to metazoa. Caenorhabditis elegans (C-elegans) has one β and two α subunits forming two integrins, Drosophila melanogaster also has orthologs of these primordial integrins referred to as PS1 and PS2. These two specialize in adhesion to basement membrane laminins, and recognition of Arg-Gly-Asp (RGD) peptides present in extracellular molecules such as fibronectin and vitronectin, respectively (1). Mammalian integrin α subunits can be classified into four subfamilies (Figure 1). One group (α3, α6, and α7), which is related to Drosophila PS1, pairs mainly with the β1 subunit to form the major laminin receptors in mammals (Table 1). Another subgroup (α IIb, αv, α5 and α8), which is related to the Drosophila PS2 proteins, pairs mainly with β1 and β3 subunits to form the primary receptors for RGD-containing extracelluar matrix (ECM) proteins. In mammals, this group of integrins plays a particularly important role in early embryonic development. In mammals, the RGD motif is also present in non-ECM proteins (e.g. fibrinogen, latent TGFβ), thus expanding the repertoire of ligands recognized by this integrin subfamily to allow development of novel cell-specific functions. This is evident in the PS2 subgroup whose members perform more specialized functions in bone, kidney and platelets. For example, α IIbβ3 integrin on platelets recognizes a motif in fibrinogen similar to RGD to promote platelet adhesion. Although the PS1 and PS2 groups of integrins are present in many invertebrates, including C-elegans, the PS3-PS5 groups characterized in Drosophila seem to be specific for insects and may align with the laminin group when homology across the whole protein is compared. The third mammalian subgroup, the α4/α9 cluster is present only in chordates. These α subunits pair with β1 or β7 to form integrins that recognize a host of ECM proteins as well as certain plasma proteins, cell-associated counter-receptors of the IgG superfamily such as VCAM-1 and vascular endothelial cell growth factors (2).

Figure 1
The integrin family
Table 1
The major ligands of human integrins

A structurally and functionally unique subgroup of integrins arose in chordates from the incorporation of an “inserted” domain or “αI domain”, which is not found in invertebrates. This protein motif is also referred to as the “A” domain as it has structural similarity to a domain present in von Willebrand factor. The I (A) domain is present in proteins that are components of multiprotein complexes and promote cell adhesion frequently requiring divalent cations (3). The I domain contains a metal ion dependent adhesive site (MIDAS) that comprises the ligand binding domain of the integrin. Four of these integrins (α1, α2, α10 and α11, all of which pair with β1) are collagen receptors while the remaining five (αD, αE, αL, αM and αX, which pair with β1 and β7) are integrins expressed on leukocytes. Counter-receptors for the leukocyte integrins are present mainly on vascular endothelial cells and include members of the IgG superfamily, (ICAMs and VCAM-1) as well as the unrelated protein E-cadherin. The leukocyte integrins also recognize plasma proteins such as complement component iC3b and fibrinogen.

The integrin β subunits can also be classified into three major phylogenetic branches (Figure 2). Two are represented in vertebrates (group A and B) while the third contains only invertebrate sequences. Group A contains β subunits β1, β2 and β7 that are associated with most of the α-subunits from the PS1, PS2 and I domain subgroups. Of these, β1 is the most widely expressed in many cell types, primarily because it associates with α1-α11 and αv subunits. The β2 and β7 subunits are restricted to hematopoietic cells. The β integrin subunits within Group B (β3, β4, β5, β6, and β8 proteins) tend to have a more restricted tissue distribution and associate with fewer α chains (Table 1). It is important to note that the production of α and β chains within any given cell type may not be balanced; often the widely expressed subunits such as αv or β1, which are promiscuous in their pairing with β and α subunits, respectively, are overproduced relative to other subunits. However, only the intact heterodimeric αβ integrins are found on the cell surface – unpaired α or β chains are retained in the endoplasmic reticulum and degraded. This restricts the combination of α and β subunits and thus the integrin repertoire on the surface (2). The repertoire of integrins can switch at the cell surface as a result of gene transcription that is in some cases regulated by signaling events elicited by ligated integrins themselves. This in turn has effects on cell fate as distinct ECM components initiate integrin signaling events to promote proliferation versus differentiation (4). The fact that only specific α and β chains pair in defined patterns suggests that these chains must have co-evolved, even though the genes encoding them are very different and are distributed throughout the genome. This co-evolution must have occurred with the development of novel major functional systems in which the integrins are required; for example the development of the adaptive immune system necessitated co-evolution of αL, αM, αX and β2 integrins to form the major receptors needed for guiding immune cells during host defense and inflammatory reactions (2).

Figure 2
Generalized organization of the integrin β subunits

Integrin signaling events – inside out and outside in

The ability of integrins to bind their ligands (whether ECM proteins or counter-receptors on other cell types) is dynamically regulated. This allows cells to carefully control their ability to adhere to matrix or establish connections with other cell types. This property is obviously important during cell migration in development and on circulating immune cells that need to migrate out of the vasculature at sites of infection or injury to mediate host defense. The processes regulating integrin affinity changes are referred to as “inside-out” signaling (Figure 3). Such events are best recognized for β2 and β3 integrins but are probably common to all integrin types. The leukocyte β2 integrins and platelet integrin αIIbβ3, reside in a resting state (bent/closed conformation) and are rapidly activated (extended/open conformation) to bind ligand when the cells that express these integrins receive activation signals. In addition to conformational changes, inside-out signals control integrin clustering and accumulation at specific regions of the plasma membrane. In leukocytes, these activating signals can be transduced by chemokine and other inflammatory receptors as well as receptors for members of the selectin family of adhesion molecules. In platelets, αIIbβ3 activation is triggered by G-protein coupled receptors, by the von Willebrand factor receptor or by collagen receptors (such as GPVI) (1). Major insights into integrin activation have come from studies of integrin structure obtained by crystalization of the ligand binding A domains. Additionally, the identification of various intracellular signaling proteins that associate with the integrin cytoplasmic tails have helped define some of the steps in integrin activation during inside-out signaling responses (1). Although integrin activation is best described in circulating immune cells, as it serves to localize the accumulation of cells to sites of inflammation, it is likely that dynamic regulation of integrin affinity plays a central role in processes such as vascular development and neurite outgrowth (5).

Figure 3
Integrin signaling

Integrins are unique among transmembrane receptors in their ability to transmit bidirectional signals. Ligation of integrins by extracellular matrix proteins leads to the assembly of a number of cytoplasmic proteins that link the integrin to the actin and microtubule cytoskeletal network (Figure 3). Active integrins in turn participate in the regulation of the cytoskeleton and reorganization of the extracellular matrix. Ligation of integrins also leads to the transmission of intracellular signals that modulate many aspects of cell behavior. This includes stimulation of cell cycle via ERK and cyclin D1 and others, inhibition of apoptosis via PI3-kinase, Akt and NFkB, changes in shape, polarity and motility via protein tyrosine kinases, phosphatases and members of the Ras and Rho family of small GTPases. The activation of these signaling pathways, which are referred to as “outside-in” signal transduction events, also leads to the modulation of gene expression (1). Anchorage dependent signaling through integrins is, in some cases, required for cellular responses to growth factors and other receptors. For example, in immune cells, αLβ2 integrin mediated T cell adhesion to antigen presenting cells is required for induction of T-cell receptor-specific T cell responses (6).

Integrins in lower organisms

Integrins are widely expressed in embryonic tissue of Drosophila and C. elegans and studies in these organisms have defined the minimal set of integrins required for fundamental processes in development. The two integrins identified in C. elegans correspond to the Drosophila PS1 (laminin integrin) and PS2 (RGD integrin). The two alpha subunits, αpat-1 and αpat-2 pair with a single β subunit, pat-3. Drosophila has 5 α-subunits PS1 through PS5, and two β subunits, βPS and Bv. βPS is most similar to both the C. elegans β subunit and β subunits found in vertebrates (7). The basement membranes in fruit flies are composed principally of four proteins, type IV collagen, laminin, nidogen/entactin and the heparan sulfate proteoglycan perlecan. These have homologues in vertebrates suggesting that they form the basis for an early basement membrane. Other well known vertebrate ECM proteins are absent including fibronectin, vitronectin, osteopontin, fibrinogen, and von Willebrand factor. These ECM proteins, important in vascular development and adhesion functions of blood cells, may have appeared later during the evolution of vertebrates (8).

Loss of function mutations of integrins or their extracellular matrix ligands in fruit flies and nematode worm leads to lethality that is attributed to defects in muscle attachment and contraction, cell migration, gut morphogenesis and adhesion between epithelial cell layers. Distinct roles for the RGD and laminin binding integrins have been documented in some of these cases. For example, RGD-binding integrins are required for muscle contraction by organizing the actin-myosin contractile structure into sarcomeres in C.elegans, and attachment of the ends of the muscle to the ECM in Drosophila. Laminin binding integrins promote differentiation of endodermal cells required for gut morphogenesis, with integrin deletion leading to misshapen pharynx in the mutant C.elegan larvae, and midgut in Drosophila mutant larvae (7). These examples also illustrate the point that despite the large differences between these two simple model organisms, the role of integrin/extracellular matrix interactions for fundamental processes during development are conserved. Roles of both RGD and laminin binding integrins in post-embryonic development have been identified by generating organisms that are mosaic for homozygous mutant cells. Drosophila PS1 and PS2 are present in the developing wing which is made of two layers of epidermal cells with PS1 on the dorsal and PS2 on the ventral side. Both integrins are required for providing adhesion between the basal surfaces of the dorsal and ventral epithelia. The complementary expression of integrins may be required to insure asymmetric adhesion and thus prevent wrinkling due to adhesion within the same layer of cells (7). In addition to maintaining structural integrity in animal tissues, laminin integrin anchoring to the ECM in Drosophila is essential for establishing a stem cell niche that in turn is important for the retention of a stable stem cell population. This integrin mediated function, first established in Drosophila, has been shown to be conserved in vertebrates (9). Finally, studies in both flies and worms suggests that axon guidance is dependent on the laminin-binding integrins (10). The role of the other Drosophilia integrins, PS3-PS5 are not well described. Mutations in PS3 affect short-term olfactory memory (11) while the function of PS4 and PS5 remains unknown.

Focal contacts link integrins to the cytoskeleton in mammalian systems and contain cytoskeletal/adaptor/linker and signal transduction molecules such as talin, α-actinin, vinculin, paxillin, FAK, ILK, p130 and others. These proteins are well conserved in Drosophila and possibly C. elegans (8). Genetic deletion of several of these proteins has revealed their relative importance in integrin dependent functions. For example, talin deficiency mimics integrin deficiency whereas deletion of ILK results in a much weaker defect (12). The conservation of integrins, their cytoskeletal associated proteins and extracellular matrix ligands in flies, worms and people makes these genetically tractable lower organisms attractive for analysis of ECM-integrin-cytoskeleton connections. Also the presence of the aforementioned signaling molecules as single genes in flies and worms reduces the possibility of redundancy and thus functional overlap in the system (8). However, it is also evident that vertebrates have evolved proteins with more specialized functions than those found in flies and worms. This is particularly evident in vascular biology, some aspects of neurobiology and likely other uniquely vertebrate functions requiring multicellular adhesive interactions. The advent of genetic engineering in mice has provided important insights into the specialized, non-redundant roles that integrins and their interacting proteins, have developed in higher organisms.

Integrins in mammalian development and pathophysiology

All the individual integrin subunits have now been deleted in mice and the reported phenotypes highlight the non-redundant, specialized functions that integrins play in vivo (13). Embryonic lethality upon genetic deletion of some of the integrins may have been predicted from the known expression pattern of the integrin during development. Additional information about the role of these integrins in adult mice will be undoubtedly forthcoming as more conditional knock-out animals are generated – we have seen this especially with β1 integrins as outlined below. The phenotypes of knock-out mice lacking α or β subunits are grouped according to the organ system most effected by their deletion.

Vasculature

A number of integrins play central roles in vascular formation during fetal and embryonic development as well as regulating blood vessel integrity in adult animals (14, 15). This function is served through both binding of ECM proteins by vascular endothelial precursors (which provides “outside-in” signals to the cells to guide their migration during vascular branching) and through the ability of integrins to specifically bind a number of growth factors (in particular vascular endothelial growth factors – VEGFs) essential in blood vessel development. Integrin-mediated adhesion events also play a critical function in pathologic angiogenesis, for example in response to tumor development or chronic inflammation. Most of the integrins involved in vascular development and integrity are members of the PS2 family that recognize RGD peptide motifs in ECM proteins. Vasculogenesis is completely dependent on α5β1 and its ligand fibronectin (16). Mutation of α5 leads to early embryonic lethality characterized by mesodermal defects and poor vascularization of both the yolk sac and the embryo itself (17). This phenotype is similar, but not as severe as that observed in mice lacking fibronectin (18). Mutation of β1 has an even more profound effect, as expected due to the loss of many integrins, which manifests as gastrulation defects and pre-implantation mortality (19). Interestingly, the phenotype of Tie2-Cre; β1flox/flox mutants, in which all β1-integrins are deleted in endothelial cells only, closely resembles the phenotype of α5 mutants, suggesting that endothelial-expressed α5β1 is the dominant integrin that recognizes fibronectin during vascular morphogenesis (20). In contrast, during pathologic vascularization αvβ3 integrin appears to play a central role. This integrin is highly expressed in blood vessels at sites of tumor growth and wound healing (21). Indeed, treatment of animals with anti-αvβ3 mAbs blocks neovascularization in tumor models, (22). In this role it appears that it is the ability of αvβ3 to bind VEGF and even to associate with the VEGF receptor that mediates its role in pathogenic neovascularization. In tumor cells, expression of αvβ3 correlates with overproduction of VEGF (23) while, probably more importantly, in endothelial cells αvβ3 directly associates, and synergizes with VEGF receptor signaling to promote enhanced responses to this growth factor (24, 25).

The physiologic association of integrins with vascular growth factors is also revealed in the phenotype of α9 mutant mice. Surprisingly, deficiency of α9 produces a lymphatic vascular defect that results in disordered lymphatic development and perinatal lethality due to accumulation of lymph fluid in the thorax (26). Lineage specific ablation of α9 in blood vessels (using α9flox/flox; VE-cadhrin-Cre mice) revealed that α9β1 plays a critical role in assembly of fibronectin bundles in the valves of developing lymphatic vessels, which is requited to prevent leakage of lymph fluid (27). However, integrin α9β1 is also the major binding protein for VEGF-C and VEGF-D, which together are the two primary growth factors for lymphatic vessels (28). Hence, much of the lymphatic phenotype of α9 deficient mice can be ascribed to loss of VEGF-C and VEGF-D signaling events in the lymphatic precursors.

Deletion of another major group of integrins, those containing the αv susubnit, reveals the importance of integrins in organizing the parenchyma surrounding the vasculature. Mice lacking αv are embryonic lethal due to widespread vascular malformations and hemorrhage. However these arise later in development (after e10.5) and affect primarily the brain in the embryo (29) due to defective interactions of cerebral vessels with surrounding brain parenchyma (30). The specific deletion of αv in endothelium has no effect on vascular development, which is consistent with its relative absence in this microvasculature, whereas its deletion in neural cells, particularly glia leads to cerebral hemorrhage(31). Of the αv integrins, it appears that αvβ8 is the primary heterodimer involved in this process, since mice lacking β8 also have cranial vascular defects and intracerebral hemorrhage (32). Indeed, targeted deletion of β8 within neuroepithelium (β8flox/flox; nestin-Cre mice) is sufficient to produce the late developmental cranial hemorrhage phenotype (33). Disruption of α4 also has a later vascular phenotype resulting from failed fusion of the allantois with the chorion during placentation, as well as impairments in cardiac development (34, 35). In this case the ligand is likely VCAM-1, as mice lacking this member of the IgG superfamily exhibit a similar phenotype to the α4 deficient mice(36).

Ocular/CNS

A number of studies have demonstrated the critical function of integrins in ocular and CNS development and hemostasis. Within the eye, lens development is best studied, since this tissue consists of relatively simple assemblies of fibronectin, collagen and laminin (37). Integrins that recognize these ECM proteins have all been implicated. Of them, clearly the β1 containing heterodimers are most important, since mice lacking β1 specifically in the lens (β1flox/flox; MLR10-Cre animals) are microphthalmic due to apoptosis of the lens epithelium and disintegration of the lens fibers (38). However, whether this major defect is due to loss of fibronectin receptors (such as α5β1) versus collagen receptors (α1β1) or laminin receptors (α3β1) remains to be seen and will only be sorted out using additional lineage specific mutagenesis methods. In lens development, there is also clear evidence of compensation by different integrins within the same class. For example, absence of the α3 or α6 integrins (which when paired with β1 form the major laminin receptors in the developing lense) by themselves have little effect on lens development. In contrast, α3/α6 double mutant mice show severe dysmorphogenesis of the developing lens (39).

The involvement of integrins in retinal development more closely mirrors their overall contribution to CNS formation, mainly as related to their role in guiding the migration of neuronal precursor cells (40). Though early studies (using blocking mAbs) had suggested members of the β1 integrins play a dominant role in guiding neuronal migration along glial fibers in the developing brain, more modern approaches with conditionally deficient mice have revealed more subtle functions. Specifically mice lacking the β1 subunit in neurons only (β1flox/flox; nestin-Cre animals) actually show relatively normal neuronal cell migration but have a completely disorganized cortex as a result of a defect in association between cortical structures and the developing meningeal basement membrane (41). This phenotype resembles the CNS cortical defects seen in mice lacking the α2 chain of laminin (42), suggesting that a laminin β1 integrin receptor (such as those in the PS1 family) is involved. Integrins and other cell adhesive molecules also play a major role in stabilizing neuronal synapses (43). Loss of this stabilizing function, as evidenced primarily by β3 integrin blocking studies, results in neuronal dysfunction that manifest as defects in long-term potentiation necessary for learning and memory (40). It is likely that other axonal guidance molecules, such as the netrins, semaphorins, and ephrins play the dominant role in guiding neuron migration during development (44).

In contrast to these findings in the CNS, there is better evidence for the involvement of integrins (in particular β1 family members) in neural crest migration during development of the peripheral nervous system. Mice with conditional deletion of β1 integrins in neural crest cells (β1flox/flox; Ht-PA-Cre animals) exhibit severe perturbations of the peripheral nervous system, including failure of normal nerve arborization, delay in Schwann cell migration, and defective neuromuscular junction differentiation, all of which can be attributed to defective migration of these cells through the embryonic ECM (45). The β1 integrins also directly affect peripheral nervous system Schwann cells, as conditional deletion of β1 in these cells (β1flox/flox; P0-Cre mice) results in reduced survival, proliferation and differentiation of Schwann cell precursors (46).

The RGD binding integrin αvβ5 plays a critical role in maintenance of retinal pigment epithelial cells in the eye. Mice lacking the β5 integrin slowly develop age related blindness due to progressive accumulation of shed pigment from retinal cells. The retinal pigment epithelial cells use the αvβ5 integrin as a phagocytic receptor to mediate uptake of shed photoreceptor particles, which occurs as a natural dinural process in the rods and cones of the retina (47).

Integrins clearly play a role in a number of neuroinflammatory diseases, such as multiple sclerosis. This likely reflects their major function in regulating immune cell trafficking in the body. There is also suggestive evidence that neuronal survival in degenerative diseases such as Parkinson’s disease or Alzheimer’s disease may be influenced by integrin signaling (44). This has raised interest in integrin targeted therapeutics as a means to treat neurologic disease.

Immune

The contribution of integrins to immune system (and general hematologic) function has been extensively studied and in fact serves as the paradigm for our understanding of many facets of integrin biology. The involvement of integrins in regulating neutrophil, monocyte and lymphocyte trafficking through out the immune system has been well studied both in vitro and more recently in vivo using a variety of intravital microscopy methods. Furthermore, the overall structural changes that take place during integrin activation have been best characterized in the leukocyte and platelet integrins, allowing a direct, but as yet incomplete understanding of how the protein associations involved in the “inside-out” signaling response mediate integrin subunit unfolding. The primary integrins expressed by immune cells are α4β1, α5β1, all of the β2 integrins, αvβ3 and αEβ7. The primary platelet integrin αIIbβ3 is also found on some immune cells.

The most well appreciated role for integrins in the immune system is their function in guiding leukocyte exit from the vasculature into the tissues, either within lymph nodes or directly into sites of tissue injury or inflammation (Figure 4). The process of leukocyte diapedesis from the vasculature is termed the leukocyte adhesion cascade (48). Broadly, this cascade involves three general classes of molecules – the selectins, chemokines and integrins. The selectins are carbohydrate recognizing receptors, present both on the leukocyte and vascular endothelium, that allow initial tethering but not full adhesion of the blood cells to endothelial cells. As a result, selectin interactions result in rolling of the leukocyte along the vascular wall. During this process, the leukocytes are exposed to chemokines and other inflammatory mediators, which induces “inside-out” signaling responses leading to integrin affinity modulation and binding to ligands on the surface of activated endothelial cells. This promotes cell arrest. The regional chemokines faciliate the directed, local egress of these cells into tissues. This simple three step model – rolling mediated by selectins, activation mediated by chemokines and full arrest mediated by integrins – is an oversimplification, as there are examples of leukocyte integrins (such as α4β1) that can mediate rolling (49) or which reduce the velocity of selectin dependent rolling. Similarly, there are many additional steps following firm adhesion, such as adhesion strengthening and signaling events (50) leading to leukocyte shape change that are mediated by integrins. Despite this complexity, the overall role of integrins in guiding leukocyte trafficking out of the vasculature is extremely well supported by the consequences of deficiency of leukocyte integrins. Mice deficient in β2 show profound defects in migration of all leukocyte types, which results in profound T-cell defects, impaired neutrophil recruitment in response to infection and resulting immunodeficiency (51). As expected, these mice show significantly reduced responses to a number of inflammatory stimuli and are protected from immune mediated tissue damage in a number of disease models, such as cardiac reperfusion injury (52). The susceptibility these mice manifest to infection is similar to that seen in human patients lacking the β2 integrin – a disease referred to as Leukocyte Adhesion Deficiency syndrome (discussed below). Lack of β1 integrin on blood cells effects their migratory capacity, resulting in a failure of fetal hematopoietic stem cells to colonize the fetal liver or spleen during development (53). In mature cells, β1 plays a critical role in guiding lymphocyte migration to the skin, lung, peritoneum and liver, all primarily through the actions of α4β1 (54). Finally, lack of the β3 integrin results primarily in a loss of platelet function (with subsequent hemorrhage), mainly through absence of the αIIbβ3 family member (55). This phenotype also mirrors what is seen in humans lacking this integrin, which develop a disease referred to as Glanzmann’s thrombasthenia (discussed below).

Figure 4
Integrin dependent leukocyte recruitment

Though there is clear evidence that integrins play a role in tissue specific recruitment of immune cells during inflammatory or disease processes – for example the specific involvement of αEβ7 integrin in the migration of T-cells to epithelial tissues such as the skin and gut (56) – it is too simplistic to assume that integrins alone play the dominant role in all immune cell trafficking. Indeed, the relative role of integrins in leukocyte migration in some inflammatory responses may be quite minor. This extremely surprising suggestion has emanated from studies of mice lacking ALL the major leukocytes integrins -- generated by conditional and inducible deletion of β1, αV combined with complete deletion of β2 and β7 in adult mice (57). Dendritic cells lacking all these adhesion molecules migrate normally into lymph nodes where they present antigens to stimulate immune responses. This indicates that migration through interstitial spaces may be less dependent on integrins than was originally envisioned and instead these molecules may have a dominant role only in allowing leukocyte exit from the vasculature. Whether this general conclusion applies to other immune cells besides dendritic cells remains to be examined. However, it is provocative studies such as this that continue to redefine our understanding of the role of integrins in the immune system.

Beyond their role in guiding cell migration, it is also clear that leukocyte integrins play a central role in activating the effector functions of immune cells through “outside-in” signaling (50). Often this signaling works with other receptors engaged on the cell. For example, co-stimulation of T-cells through their antigen receptors (TCR) and integrins (for example, when the cells are plated on a matrix-coated surface) leads to much more robust proliferative responses that stimulation through the TCR alone (58). Similarly, chemokine activation of neutrophils is dramatically enhanced in cells that are also stimulated through their integrin receptors (59). In some cases, the activation function of the leukocyte integrin is relatively more important than its homing/migration function. For example, in a model of thrombohemorrhagic vasculitis caused by endotoxin and cytokine exposure, deficiency of αMβ2 integrin does not affect the ability of leukocytes to be recruited to the site of vascular inflammation, but once there the cells cease to become activated because they do not recognize complement deposits that form along the vessel walls. As a result αMβ2 deficient neutrophils fail to release proteases that otherwise would cause vascular damage and hemorrhage(60). This clear role for “outside-in” signaling events is also seen in other physiologic conditions, such as keratinocyte development as outlined below.

Cutaneous

A number of integrins play a key role in development and maintenance of epidermal structures. Their primary function, along with other adhesion molecules, is to link keratinocytes to the underlying basement membrane (61). The primary integrins expressed in the epidermis are the laminin receptors (α6β4 and α3β1), the collagen receptor (α2β1) and less abundantly the vitronectin receptor αvβ5 (62). Most of these integrins are localized in focal adhesion contact sites in keratinocytes, where they link the extracellular matrix to the actin cytoskeleton of the keratinocyte to provide firm adhesion. The α6β4 integrin is localized to hemidesmosomes that help link keratinocytes to each other directly.

As in other organ systems, the role these individual integrins play in epidermal biology is best revealed in knockout mouse models. Conditional deletion of all β1 integrins in the epidermis (β1flox/flox; Keratin5 or Keratin14-Cre mice) leads to incomplete perinatal mortality due to separation of the epidermis from the underlying dermis (63, 64). Some mice survive up to 6 weeks after birth but show an absence of hair follicles, poor keratinocyte proliferation with fibrous dermal deposition associated with chronic skin inflammation. Interestingly, deletion of the α2β1 integrin or the α3β1 integrin alone (through deletion of the α subunits) has very little effect on skin biology – the loss of α3 produces mild foot blisters – suggesting that the epidermal function of these receptors is largely redundant (65, 66). Most interesting, however, is when the β1 integrin is deleted in adult epidermis, through use of an inducible K14-cre strain. In this case, if the skin is already formed, loss of β1 integrins results in an enhanced proliferative responses of keratinocytes to spontaneous wounding. In addition, the number of melanocytes in the interfollicular epidermis is greatly increased (62). This observation highlights the role of integrin “outside-in” signaling in regulating keratinocyte growth, in this case in a suppressive fashion. Consistent with these observations, knockdown of β1 integrins in mouse keratinocytes in culture leads to increased proliferation and differentiation (67). These results also illuminate the issue of secondary effects of these deletions, such as the cutaneous inflammation that occurs in newborn mice lacking epidermal β1 integrins, which may complicate interpretations of primary effects on the skin. Such is the case in mice lacking the α6β4 integrin, which develop epidermal and dermal separation, similar to that observed in humans born with β4 deficiency (referred to as junctional epidermolysis bullosa syndrome, see below). In this case alterations in TGFβ responsiveness in the absence of the these integrins may contribute to inflammatory responses in the skin that contribute to the epidermal/dermal separation. However, unlike the β1 integrins, it seems that α6β4 does not directly regulate keratinocyte differentiation (68).

Pulmonary

In terms of development, many of the same integrins that contribute to cutaneous epithelial cell morphogenesis are involved in formation of the pulmonary epithelial layer. This is particularly true for the α3β1 laminin receptor. Integrin α3 deficient mice die at birth due to renal and lung defects associated with disorganized subepithelial basement lamina and poor adhesion of epithelia to the basement lamina (69). This phenotype is in a sense a more profound manifestation of epithelial dysfunction than the modest skin blistering in these animals. Combinatorial mutants that more broadly affect laminin binding, such as double mutant α3/α6 mice have even a more severe epithelial phenotype that results in disordered limb development and severe pulmonary hypoplasia (39).

However, it is loss of the RGD binding integrin functions, in particular that of αvβ6 and to a lesser extent αvβ5 that result in the progressive pulmonary abnormalities, which have taught us the most about the intersection of integrins and TGFβ regulation. The αvβ6 integrin is expressed primarily in epithelial cells of lung, skin and kidney where it recognizes both fibronectin, but more importantly the latency associated peptide (LAP) that is non-covalently associated with newly secreted TGFβ and keeps TGFβ from binding its receptor (70). The LAP of both TGFβ1 and TGFβ3 has an RGD binding motif, which is recognized by αvβ6 and αvβ8. When these integrins bind the TGFβLAP, they cause dissociation of LAP/TGFβ complex allowing TGFβ to interact with its receptors present on epithelial cells and resident pulmonary macrophages. Binding of TGFβ serves as a powerful anti-inflammatory stimulus in both cell types – the presence of activated TGFβ6 in the lung helps suppress inappropriate alvelolar macrophage activation that would lead to lung injury (71). This amazing biology was revealed by study of the β6 knockout mice. These animals develop progressive pulmonary (and skin) inflammation that is strikingly similar to that seen in TGFβ mutant mice themselves (72). By age 6 months, β6 deficient mice develop emphysema, which is due to overproduction of the alveolar macrophage protease MMP12, as a result of the chronic inflammation present in these mice (73). Re-expression of either β6 or an activated version of TGFβ as a transgene in alveolar and bronchiolar epithelial cells of β6 mutants, rescues the pulmonary inflammation (73). Integrin regulation of TGFβ is also essential in limiting MMP12 release after bleomycin or irradiation mediated lung damage (74). These phenotypes point to the importance of αvβ6 mediated TGFβ activation in the lung to restrict the inflammatory properties of resident alveolar macrophages. Interestingly, this modulatory effect of TGFβ on macrophages also affects their phagocytic function. Alveolar macrophages play a major role in recycling pulmonary surfactant, however this function is lost in both β6 and TGFβ deficient mice, which is manifest as a progressive accumulation of surfactant material in addition to the inflammation that these mice develop (75). Hence, as seen in other macrophage types, the transition of alvelolar macrophages to the inflammatory state (leading to release of MMP12 and other mediators) correlates with the loss of the ability to phagocytose and degrade normal resident proteins.

Pulmonary activation of TGFβ is also mediated by αvβ8 integrin. This may play more of an effect on regulating inflammatory and proliferative responses of airway epithelial cells, versus macrophages, as determined in cell or organ culture models using functional blocking β8 mAbs (76). Complete loss of β8 results in embryonic lethality due to early vascular malformations; a function that is likely not related to the ability of this integrin to activate TGFβ(32).

Studies of αvβ5 in pulmonary vascular permeability during lung injury have also revealed novel functions for this integrin in the lung. Mice lacking this integrin (or normal animals treated with a β5 blocking mAb) manifest significant reduction in pulmonary edema and vascular permeability following either ischemia-reperfusion or hyperventilation (77). The effect was traced to the ability of αvβ5 to bind and activate VEGF, which in turn directly stimulated the vascular permeability. Indeed, direct treatment of pulmonary epithelial cells with VEGF will induce actin stress fiber formation and cell retraction – a phenotype not observed in αvβ5 mutant cells. Hence, as in vascular development, the ability of integrins to bind and activate vascular growth factors reflects an important function beyond just cell adhesion.

Musculoskeletal

As in the fruit fly and the worm, a number of RGD, collagen and laminin binding integrins serve critical roles in formation of muscle structures. In developing myofibrils, organization of integrin subunits into distinct focal adhesion sites in the sarcolemma allows for nucleation of actin filaments that help organize the actin/myosin structures (78). However, the individual integrins that contribute to this phenotype are less well defined, since loss of specific collagen receptors (α1β1 or α2β1) result in mild musculoskeletal defects. In addition to it’s role in vascular development, the RGD-binding integrin α5β1 also plays a role in maintenance of muscle structure, as determined by examination of embryonic chimeric mice containing cells derived from both normal and α5 deficient stem cells. These mice develop a progressive skeletal muscular dystrophy phenotype, characterized by diffuse muscle cell degeneration and apoptosis (79). This phenotype seems not to be due to a developmental disorganization of the actin/myosin structure of the muscle cell, but instead to a loss of adhesive signaling between muscle cells and the ECM, which results in poor myoblast survival over time. This illustrates another well-accepted role for integrins in promoting cell survival. Indeed disruption of integrin mediated anchorage to the ECM leads to apoptosis, a process referred to as “anoikis”. Integrin “outside-in” signaling to the PI3K-Akt pathway may be a major regulator of anoikis, with a breakdown of this process in epithelial cells contributing to neoplasia and metastasis (80).

Of the laminin binding integrins α7β1 clearly has the dominant role in orchestrating normal muscle development. Dystrophin, utrophin, and α7β1 are the major laminin receptors in skeletal muscle. Each of these receptors links laminin in the extracellular matrix and basal lamina to the myocyte actin cytoskeletal network (81). Loss of dystrophin produces human Duchenne’s muscular dystrophy, which is characterized by progressive myocyte degeneration that leads eventually to mortality. Mutations in α7 also produce myopathy in humans, though not nearly as severe as Duchenne’s dystrophy (82). Mice that lack α7 integrin also develop myopathy, characertized by poor force transmission, compliance and viscoelasticityin diaphragm muscle (83, 84). In addition, α7β1 plays an important role in maintaining the neuromuscular junctions and muscle-tendon junctions as these structures specifically degenerate in the α7 deficient mice (85). Interestingly, in both Duchenne’s patients and the mouse model of this disease (the mdx mouse), the levels of integrin α7β1 increase 2 – 3 fold, suggesting that these laminin receptors can functionally compensate for each other. Indeed, in combinatorial mice that lack dystrophin and α7 or utrophin and α7, the myopathy phenotype is dramatically accelerated (86)(87). These observations clearly indicate that these laminin binding receptors are dispensible for muscle development, but play a critical role in maintaining the structural integrity and functional capacity of skeletal muscle, primarily by linking myocytes to the ECM.

A number of both RGD and collagen binding integrins play major roles in skeletal development and homeostasis. The most extensively studied is the integrin αvβ3, which is abundantly expressed in osteoclasts and plays a central role in the ability of these cells to adhere to bone matrix and regulate bone morphogenesis. In normal bone, the primary RGD containing ligands recognized by osteoclast expressed αvβ3 are osteopontin and bone sialoprotein (88). Adhesion of osteoclasts to these surfaces through αvβ3 causes actin cytoskeletal rearrangement in the osteoclast, spreading and activates the cell to release proteases into a defined vacuolar space between the osteoclast and the bone, thus causing bone resorption and remodeling. In mice lacking the β3 integrin, the loss of osteoclast function results in bone overgrowth (osteopetrosis), however in humans lackingβ3 (ie those with Glanzmann’s thrombasthenia) bone density is unaffected. This may be due to increased expression of compensating β1 integrins in the human patients (89). In the mouse, it is also clear that loss of the “outside-in” signaling events that are elicited by engagement of osteoclast αvβ3 produce the functional equivalent of loss of the receptor. Hence deficiency of members of the Src-family of tyrosine kinases, Syk kinase, or downstream adapter proteins such as DAP12, or SLP76 or a guanine exchange factor for Rac GTPases, Vav3 all result in varying degrees of osteoclast dysfunction and, in the case of Src kinase mutant mice, severe osteopetrosis resulting in lethality (88, 90, 91).

The collagen binding integrins α10β1 and α11β1 also play specific, and rather limited, functional roles in musculoskeletal development. Mice lacking α10 develop growth retardation of the long bones in the limbs due to disorganized cartilage formation particularly at the growth plates (92). The chondrocytes in these mice manifest a disordered columnar arrangement, an abnormal shape and reduced proliferative capacity. As a result the collagen fibrillar network in the growth plates of α10 deficient animals is disorganized, leading to the growth plate failure and shortened bones. Integrin α11 plays a specific role in dental development through its regulation of periodontal ligament fibroblasts. The α11 deficient mice show severe defects in incisor formation characterized by disorganized periodontal ligaments leading to a block in tooth eruption (93). Embryonic fibroblasts from the α11 deficient mice show reduced cell adhesion and spreading on collagen I as well as reduced cell proliferation. This suggests that α11β1 is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate the forces needed for axial tooth movement.

Renal

A number of β1 integrins have been implicated as having specific roles in renal development and kidney disease. These functions range from roles in the development of the entire kidney to specific effects on the epithelial structures within the collecting system or in responses of the glomerulus to injury. Studies of α8β1 have revealed its unique role in renal development. Loss of α8 results in a high frequency of complete renal agenesis due to a failure of epithelial-mesenchymal interactions; epithelial-type cells referred to as ureteric bud cells (UB) fail to invade into surrounding mesenchyme to form the metanephric kidney (94). Although α8β1 recognizes a number of RGD containing proteins, such as fibronectin and tenascin C, in fact the ligand present in the developing kidney which activates α8 function is a novel ECM protein referred to as nephronectin (95). Nephronectin is expressed by the invading UB cells and induces expression of α8β1 in the surrounding mesenchymal cells. Indeed, genetic disruption of nephronectin results in the same type of renal agenesis as seen in α8 mutant animals (96). In this system, it is clear that the nephronectin/α8β1 interactions not only provide adhesive structures to guide invading kidney epithelia, but also are involved in induction of growth factors, specifically glial cell line-derived neurotrophic factor (GDNF), a member of the TGFβ superfamily. GDNF binds to its receptor, the RET tyrosine kinase on the epithelium, to provide growth and proliferative signals to the developing renal epithelial cells (97). Loss of any of these molecules also produces a renal agenesis phenotype similar to α8β1 deficiency. This system of interactions of different cell types in the developing kidney highlights the importance of integrin/ECM interactions in controlling organ morphogenesis by regulating cell growth, migration and adhesion.

Integrin α3β1 also has a dominant role in renal development. Mice lacking the α3 subunit have a reduced number of collecting ducts in the renal papilla suggesting that this integrin plays a role in the subsequent steps of branching morphogenesis to form the renal tubular collecting system (69). When α3 is specificallydeleted in the UB cells (α3flox/flox; HoxB7-Cre mice), the kidney papillae are either absent orabnormal (98). As in the earlier steps of renal development, it is clear that α3 interactions in the developing papilla also induce expression of growth factors required for organogenesis. In this case, elaboration of the growth factor Wnt7B following α3 engagement helps regulate epithelial cell survival in the developing tubules to allow formation of the mature renal papilla.

Beyond α3β1 and α8β1 it is likely that other laminin, collagen and RGD binding integrins play supportive roles in kidney development. This conclusion derives from deletion of β1 specifically in the UB cells (β1flox/flox; HoxB7-Cre mice). In this case, a severe defect in branching morphogenesis is observed, worse than just the α8 effect alone (99). Interestingly, when β1 is lost later in renal development (β1flox/flox; Aqp2-Cre mice) kidney formation is normal, however the mutant animals develop more severe injury and poor collecting duct healing following ureteric obstruction. In this case, it is likely that the injured renal collecting tubular cells fail to produce GDNF and other growth factors that would support repair of kidney structures following injury (99). Again, this highlights the cooperation of integrins in growth factor responses within the kidney.

It is also clear that β1 integrins play important roles in the development and maintenance of the glomerular structures of the kidney. In the glomerulus, the interactions between mesangial cells and podocytes with the glomerular basement membrane present in the fine vascular capillaries of the glomerulus forms the basic filtration structure of the kidney. These interactions are dependent on β1 integrins, since specific depletion of β1 in podocytes (β1flox/flox; podocin-Cre mice) leads to severe malformations of the glomerular basement membrane and effacement of podocyte/membrane contacts (100). As a result, the mice are born with massive proteinuria and rapidly progress to death. In the normal glomerulus, continued expression of β1 integrins is important in maintaining the filtration function of the kidney. During glomerular injury, for example as a result of immune complex deposition, β1 integrins of all types are induced in mesangial cells and podocytes (101). These interactions lead to elaboration of growth factors that can contribute to mesangial cell proliferation during glomerular injury. Indeed, mice lacking α1β1 integrin show protection from glomerular injury and reduced mesengial cell proliferation in a mouse model of Alport’s syndrome (102).

Human integrin deficiencies

Clear support for the importance of integrins and their activation have come from “experiments of nature”. Human mutations in integrins within 3 of the 5 mammalian subgroups have been shown to cause disease (103). These include mutations in either the α or β subunits of integrins α6β4 (PS1 cluster, laminin receptors) or αIIbβ3 (PS2 cluster, RGD receptors) or in the β2 integrin subunit (with the I-domain containing leukocyte receptors). In the case of the β2 and β3 mutations, multiple integrins are affected, which contributes to the variability in the disease phenotype from patient to patient.

Reduced or absent expression of α6β4 leads to the syndrome of junctional Epidermolysis Bullosa (EB) which presents as extreme blistering and fragility of the skin. The skin blistering is caused by the inability of epidermal cells to adhere to basement membranes present in the dermis, leading to epidemeral/dermal separation. The EB syndrome is accompanied by an occlusion of the intestine, termed pyloric atresia, which is often postnatally lethal. The occluded oesophageal, urethral and pyloric tissues may result from the detachment and subsequent fusion of epithelial linings (103, 104). Mice lacking either α6 or β4 have a very similar epidermal phenotype and suffer perinatal lethality.

Genetic mutations in αIIb or β3 produce a syndrome referred to as Glanzmann’s Thrombasthenia. These patients present with a range of problems, the most serious of which is a severe bleeding disorder due to platelet adhesion defects (105). Individuals with β3 mutations will often have bone cysts or other skeletal abnormalities, likely due to loss of αvβ3 function. Mutations in β3 can lead to little synthesis of the protein or allow full expression but no activity of integrin. The mice lacking β3 or αIIb serve as good models of Glanzmann’s thrombasthenia (106).

Leukocyte adhesion deficiency I (LADI) syndrome is caused by genetic mutations within the β2 subunit, resulting primarily in deletion or reduction in surface expression of multiple leukocyte integrins or in some cases, leading to near normal levels of integrins but defects in integrin function due to mutations in the ligand binding domain. These patients present with a variety of immune deficits, ranging from life threatening soft tissue infections, to wound healing defects and variable forms of infectious gingivitis. The classical clinical hallmark of LAD is the presence of tissue infections without formation of pus. This is due to the fact that impairment of β2 expression leads to reduced leukocyte recruitment and migration to sites of tissue infection because immune cells are unable to adhere to vascular endothelium. All mutations are found in the β2 subunit gene located on chromosome 21. Mutations that effect CD18 expression at the surface can lead to <0.3% to 2.5–31% β2 integrin expression, which correlates with presentation of severe to more moderate disease, respectively. Point mutations within both the ligand binding domain leading to near normal surface expression but poor ligand recognition have also been described {Roos, 2001 #122}. Mice with β2 deficiency also manifest a variety of immune defects due to poor leukocyte adhesion and hence are good models of LAD {Kakkar, 2004 #57}.

Leukocyte adhesion deficiency syndrome can also result from mutations in proteins involved in the inside-out pathway of integrin activation. Recent studies have identified a set of patients with bleeding, immune cell accumulation defects and an osteopetrosis-like bone defect, that may result from failure to activate β1, β2 and β3 integrins to high affinity ligand binding states. This defect in inside-out signaling has been mapped to mutations in the protein Kindlin-3, which binds to specific domains within the cytoplasmic tail of β subunits, leading to separation of the cytoplasmic tails that is required as part of the unfolding of the integrin to form the ligand binding conformation (Abram and Lowell, ICB 87:440, 2009, Svensson et al Nat. Med, 15:306, 2009 Malinin et al Nat Med 15: 313, 2009). In these patients, their clinical presentation would suggest mainly β2 (immune cell recruitment) and β3 (platelet and osteoclast) functional defects. Indeed, Kindlin-3 expression is restricted to mainly hematopoietic cells. Mutations in ubiquitously expressed Kindlin-1 produce Kindler’s Syndrome, which presents as a skin and mucosal blistering disease, due to defects in keritinocyte adhesion probably due to failure to activate β1 integrins in these cells. As in the other integrin deficiencies, mice lacking either Kindlin-1 or Kindlin-3 model the respective human disease, though the Kindlin-3 knockouts tend to have a less severe bleeding disorder (Moser et al Nat Med 15: 300, 2009). Mice lacking Kindlin-2, which is also widely expressed, die early in gestation due to implantation defects, potentially as a result of poor cell/cell adhesion (Montanez et al Genes Dev 15:1325, 2008).

Summary

Integrins are adhesive proteins that have evolved to allow cell-cell and cell-matrix communication that is indispensable for development and post-natal physiology. Despite their widespread expression, the genetic deletion of specific integrin family members in lower organisms as well as mammals leads to relatively distinct abnormalities. Many processes in which integrins participate, have in common a requirement for strong adhesion coincident with times of mechanical stress. As an example in Drosophila, the absence of specific integrins leads to detachment of muscle from the gut and body wall and separation of the two epithelial layers in the wing. In mice and humans, a deletion of either subunit of the laminin binding integrin, α6β4 leads to severe skin blistering and defects in other epithelial layers due to problems in formation and stabilization of hemidesmosomes, which anchor epithelial cells to the basement membrane and are necessary for the skin to withstand frictional forces. However, integrins have also evolved to serve more subspecialized roles. These range from the establishment of a stem cell niche in Drosophila and mammals, to the regulation of pathogenic tumor vascularization, platelet adhesion and leukocyte transmigration in mammalian systems. On the other hand, some cells seem to function normally in the absence of all integrins, as revealed by the very surprising finding that deletion of all the major integrin types on dendritic cells of mice has no effect on the ability of these cells to migrate within the interstitium of the skin and enter into lymphatics. In addition to serving as transmembrane mechanical links, integrins in vertebrates synergize with other receptors including growth factor receptors. This leads to the activation of a large signaling network that affects cell proliferation and differentiation, cell shape and migration. The ability of integrins to directly bind and activate growth factors and interact with their receptors as is the case for VEGF, or modulate growth factor activity as is the case for TGFβ, has major implications for adult homeostasis. In the latter case, the results from knockout mice may aid in the interpretation of observations in patients. That is, β6 deletion in mice leads to a failure in activation of TGFβ, which provides tonic suppression of inflammatory cells, and this may provide an explanation for the observed inflammation in patients with β6 deficiency. These types of in vivo studies, in lower organisms, knock-out models as well as in human conditions together have provided important insights into how this major, primordial family of adhesion receptors have remained true to their name “integrins” as their diverse functions have in common the ability to integrate extracellular stimuli and transmit signals into cells that affect cell behavior.

Some of the challenges in the next decade are to understand how these receptors signal their diverse functions, how the same integrin can specialize to promote specific functions that are dependent on the environment (cell and tissue in which it exists) and how integrins collaborate with other integrins and receptors within a network to deliver cues responsible for cell processes that range from migration to differentiation. This type of knowledge will allow integrin-targeted therapeutic modalities that inhibit pathologic processes in which integrins have been heavily implicated including tumor angiogenesis, inflammation and thrombosis.

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