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Integrins and Development: Integrins in Skeletal Cell Function and Development

* and .

* Corresponding Author: Bone and Mineral Centre, Department of Medicine, Rayne Building, 5 University St, London WC1E 6JJ, U.K. Email: m.horton@ucl.ac.uk

Genes for 18 αv and 8 β integrin subunits have been identified in vertebrates, their protein products associating into 24 different heterodimeric membrane receptors. Skeletal cells—osteoclasts, osteoblasts and chondrocytes—exhibit characteristic integrin profiles and for some there is good evidence for a functional role. This is best exemplified by the osteoclast, the ‘bone’ resorbing cell of the skeleton which expresses high levels of an RGD peptide-sensitive integrin, the αvβ3 vitronectin receptor, that is intimately involved in recognition and adhesion to mineralised matrix and its degradation; this property has been utilised in the development of novel drugs that inhibit αvβ3 function for use in osteoporosis and arthritis. This article will review the expression profiles of integrins in the major cell types of bone and cartilage, evidence for integrin function therein, and their role in skeletal development where known.

Introduction

Connective tissue cells in general, and bone and cartilage cells in particular, are surrounded by an abundance of extra-cellular matrix. Chondroblasts and osteoblasts are responsible for the synthesis of the majority of the organic components of skeletal matrix, which subsequently calcifies, whereas osteoclasts degrade the matrix and dissolve mineral components (Fig. 1). The function of bone and cartilage cells reflects the matrix in which they are embedded and, conversely, the composition of the matrix is highly dependent upon the cellular function of skeletal cells. Cell-matrix and cell-cell interactions are mediated by adhesion molecules and in recent years much information has become available regarding the expression and role of integrin adhesion receptors in the skeleton. Adhesion molecules not only play a part in the function of fully differentiated cells in the skeleton, but they are also increasingly implicated in their developmental pathways. Skeletal biology is well reviewed in1 and cell adhesion in the bone and cartilage in.2-4 In this chapter, we will review the expression and function of integrins in bone and cartilage cells and discuss results from gene deletion studies.

Figure 1. I) Osteoblasts and chondroblasts are derived from a common precursor, the mesenchymal stem cell.

Figure 1

I) Osteoblasts and chondroblasts are derived from a common precursor, the mesenchymal stem cell. Osteoblasts differentiate to a committed preosteoblast and then on to a matrix synthesizing osteoblast. When bone formation switches off, osteoblasts can (more...)

Scope of the Chapter

This review has been restricted in scope to describing the integrin phenotype and function of the different cell types in the skeleton and the impact upon the skeleton of integrin gene deletion. Due to space limitations, we have decided not to extend our discussion, whilst it could be considered informative, to the role of associated matrix protein ligands for the different expressed integrins, cytoskeletal molecules and the numerous signaling pathways downstream of integrin receptors, unless crucial to the discussion. Readers can easily identify relevant publications on the web: for example, the fibronectin gene when deleted produces a phenotype like deletion of α5;5 many clinical drug targets for osteoporosis modify intracellular F-actin cytoskeleton6 and inhibition of c-src function leads to osteopetrosis due to defective osteoclast resorptive function.7

The Integrin Phenotype of Skeletal Cells

The Osteoblast Lineage

Osteoblasts secrete bone matrix proteins (as osteoid), which subsequently become mineralized to form the mechanically stable skeleton. They line bone surfaces and become entrapped in osteoid as it is deposited, when they are named osteocytes—in this capacity they act as sensors of mechanical forces in the skeleton and hence regulate bone turnover, though a comprehensive role for cell adhesion receptors in this process is still lacking.8 Osteoblasts from normal human bone have been reported to express β1 and β5 integrins9-11 but there are controversies regarding the a subunits associated with β1 and concerning expression of αvβ3.4 β2, β4 and β6 integrins have not been reported in osteoblastic cells. There are also differences reported on the integrin repertoire expressed by osteoblasts in situ, cultured human osteoblasts, or osteoblastic cell lines and these are most likely due to the heterogeneity of the cell population under study and the different culture conditions employed. Moreover, information is beginning to accrue regarding the effect of osteoblast activation status (i.e., synthetic osteoblast versus bone lining cell, see (Fig. 1), anatomical site (e.g., endochondral versus intramembranous bone, (see Fig. 1) or disease on adhesion molecule expression,12,13 though no definitive studies specifically on integrin expression have been published. For example, it has been clearly demonstrated that, in vitro, the substrate on which cells are cultured directly influences the pattern of integrin expression by osteoblasts.14,15 Generally, cultured osteoblasts express a wider integrin repertoire than osteoblasts in situ and in particular increased expression of the collagen binding integrin α2β1, the fibronectin receptor α5β1 and the vitronectin receptor αvβ3 is seen.4 As yet it is unclear whether increased expression of these integrins occurs in bone pathology, since osteoblast integrin expression in bone disease is relatively unexplored. Integrin expression in osteocytes has not been studied extensively, but there is some data from immuno-cytochemical analysis of human bone sections and from functional studies in the chick. In the chick, where isolation procedures for osteocytes have been developed, osteocytes bound to a range of matrix proteins in vitro in a β1- and partially RGD- dependent way16 and expression of β1 integrin has been confirmed in mammalian osteocytes.10,17 However, there are few definitive reports where integrin expression has been followed throughout osteoblastic differentiation to osteocytes within the same species. Difficulties in interpretation of immuno-cytochemical staining in bone sections, in particular for cells embedded within matrix, have been reported by many authors and this may well have contributed to the current controversies in integrin phenotype. From the limited reports on isolated cells at different stages of differentiation, it has become clear that the fibronectin-receptor α5β1 is the most abundantly expressed integrin throughout osteoblastic differentiation and that, maybe surprisingly, the collagen-receptor α2β1 is expressed at much lower levels, in particular in more differentiated cells.18

Functional studies (see Table 1) on osteoblast integrins have included adhesion assays to a variety of extra-cellular matrix molecules and inhibition by RGD-containing peptides. In keeping with their extensive repertoire of integrins, osteoblasts have been found to adhere to osteopontin, bone sialoprotein, vitronectin and fibronectin in an RGD-dependent way, whereas binding to type I collagen and thrombospondin was less inhibited by RGD peptides.9,19,20 In organ cultures of mineralizing fetal rat parietal bone, RGD peptides decreased bone formation accompanied by a decrease in α2 and β1 expression and disruption of the organization in the osteoblast layer.21 Functional studies on osteoblast integrins include reports on their role in osteoblast differentiation. First Moursi et al22,23 demonstrated convincingly that osteoblast-fibronectin interaction was a critical event in the differentiation of fetal rat osteoblasts in an in vitro model and involved interaction between α5β1 and the central binding domain of fibronectin. These results confirmed earlier work suggesting a role for α5β1 in differentiation of the human osteoblastic cell line MG63.24-26 Later, Ganta et al27 found that ascorbic acid deficiency, which leads to under-hydroxylation of type I collagen, resulted in down-regulation of α2β1 in osteoblasts and dysregulation of differentiation and mineralization in cultures of rat calvaria. Jikko et al28 showed that α1 and α2 integrins are mainly important in early stages of osteoblast differentiation to mediate differentiation signals, such as from BMP-2 and Mizuno et al29 demonstrated a role for α2β1and collagen in osteoblast differentiation from early progenitors. These findings correlate well with the reduced levels of expression for collagen binding integrins in more differentiated cells, as mentioned above.18 Even though mature osteoblasts adhere to collagen, it is likely that this is complemented by a nonintegrin-dependent mechanism. Thus, in addition to providing an anchor for osteoblasts to the bone matrix, integrin-ligand interactions play a role in the differentiation of osteoblasts and via yet undefined signaling pathways in the expression of differentiation-associated genes.

Table 1. Functional integrin receptor-ligand interactions in the skeleton.

Table 1

Functional integrin receptor-ligand interactions in the skeleton.

Chondrocytes

Chondroblasts secrete the specialized extra-cellular matrix of cartilage and mature into chondrocytes that become entrapped therein (for a comprensive discussion of this complex tissue type see 1). As for cells of the osteoblast lineage, the integrin phenotype of chondrocytes is complex, with inconsistency between publications30-34 (for a recent review see ref. 35). A synthesis of the literature shows that human chondrocytes express the β1 integrins α1, α2, α3, α5 and α6, but not α4. β2, β4 and β6 are absent; analysis of β7-9 and CD11 has not been reported. Some studies have shown high expression of αv integrin and, as with osteoblasts, this is mainly as αvβ5 and not the αvβ3 dimer; however, a subpopulation of superficial articular chondrocytes has been found to express αvβ3.33 These complexities could well relate in part to variation in sampling site, use of fetal versus adult material, species differences, culture artifacts, or influences of disease on phenotypes. Indeed the first possibility is born out by the study of Salter et al32 where the distribution of integrin is clearly different by site (human articular, epiphyseal and growth plate chondrocytes were studied) and changes have been reported in cultured chondrocytes.36,37

Two relatively newly discovered integrins, α1038,39 and α11,40,41 have been shown to be collagen receptors that are expressed in cartilage. α10 has a cellular distribution that differs from α1 and α2 and is the dominant integrin during embryonic development.39 α10 preferentially binds basement membrane collagens, as α1, whereas a11 resembles α2 showing specificity for fibril forming collagens.42

The functional role of cell adhesion molecules in cartilage is beginning to be elucidated (see Table 1). Examples include roles in cartilage differentiation during fetal development; response to mechanical forces (for example in articular cartilage, menisci); maintenance of tissue architecture and integrity including by matrix synthesis and assembly and cell adhesion; and regulation of chondrocyte gene expression. Additionally, there is likely to be a role of cell adhesion molecules in responses in cartilage to injury and disease.43-45 For example, up-regulation of β2 and α4 subunit expression in osteoarthritic versus normal and cartilage were reported by Ostergaard et al.44 Changes in distribution of both integrin and matrix proteins28 in different zones of cartilage suggest a role in chondrocyte differentiation from mesenchymal precursors46,47 and/or interaction with matrix. Evidence is also accruing for a specialised function for integrins in responses of chondrocytes to mechanical stresses48-50 (reviewed in ref. 51) that is mediated by α5β1 integrin in osteoarthritis cartilage.52

Extensive studies have been performed to address the role of α5β1 in chondrocyte interaction with fibronectin.30,31,53-58 Function blocking antibodies and RGD peptides have been shown to inhibit cell adhesion to fibronectin and its fragments, thus modifying chondrocyte behaviour and cartilage function. Likewise, chondrocyte recognition of collagen (including types I, II and VI collagen) has been studied in vitro30,31,53,56,59-61 and shown to be mediated via β1 integrins.

There is also increasing evidence for a connection between chondrocyte adhesion to extra-cellular matrix proteins, especially fibronectin, chondrocyte-synovial cell interaction,62 chondrocyte cell signaling63 and survival,45,58,60 regulation of integrin expression and function by cytokines such as IL1, TGFβ and IGF1, and the release of matrix metalloproteinases64 and hence cartilage breakdown.55,61,65,66 The key role for downstream signaling following integrin interaction with matrix ligands is underscored by the phenotype of integrin-linked kinase (ILK) knockout mice.67 ILK acts as a pleiotropic adapter interfacing beta integrin cytoplasmic domains into the PKB/Akt signaling pathway; mice that lack ILK have chondrodysplasia and isolated chondrocytes have matrix adhesion defects. Such events are likely to be involved in the pathogenesis of the cartilage destruction seen in osteoarthritis and rheumatoid arthritis.

Osteoclasts

Osteoclasts are bone cells derived from the monocyte-macrophage cell lineage under the influence of a membrane-bound osteoclast differentiation factor of the TNFα family, Receptor Activator of NFκB Ligand (RANKL).1 They are specialized to recognize, adhere to and degrade the proteinaceous and mineral phases of skeletal tissues1 and utilize integrin cell adhesion receptors in these processes (Table 1); (reviewed in refs. 2, 3 but first recognized by Beckstead et al, 68). The principle integrin of the osteoclast is the αvβ3 vitronectin receptor2,3,69 which is present at high copy number in osteoclasts. αvβ3 binds promiscuously to several noncollagenous bone protein ligands in an RGD-dependent manner.2,3 Other αv integrins, particularly αvβ5, are not conspicuously expressed by mature osteoclasts.69 Recognition of native collagen is mediated by another integrin, alpha;2β1.70 Other integrins have not been found consistently in osteoclasts in extensive analyses of several species (reviewed in refs. 2, 3).

Inhibition of osteoclast αvβ3 (reviewed in refs. 2, 3) and α2β1 integrins70 leads to reduced bone resorption in vitro and in a range of in vivo animal models. These features have encouraged the pharmaceutical industry to develop nonpeptidic RGD analogue drugs for use as inhibitors of osteoclast function in treating skeletal diseases, such as osteoporosis or bone cancer,1,71 and are now in clinical trial.72-75 These developments have emphasised the need to establish the impact of chronic inhibition or lack of αvβ3 on osteoclast function, but, despite this, the integrin phenotype of osteoclasts from animals treated long-term with αvβ3 antagonist drugs has not been reported. The question, thus, still remains unanswered as to whether a compensatory response will occur in the osteoclast to counteract the effects of chronic down-regulation or inhibition of αvβ3–ligand interactions, such as might occur via an up-regulation of an existing or ‘new’ integrin dimer (see discussion below in section on β3 null osteoclast function).

Detailed localization studies have tried to answer the question whether integrins are present in, and possibly responsible for maintenance of, the tight sealing zone, the specialized F-actin rich membrane domain seen in actively resorbing osteoclasts, but the data so far is conflicting. Although some studies have excluded presence of αvβ3, reviewed in2,76,77 and α2β178 in the sealing zone, others have reported the presence of αv and/or β3 subunits at this site.79-81 It is clear, however that none of the integrin receptors are enriched in the clear zone of resorbing osteoclasts and that they are in fact most abundant on the opposite, basolateral membrane, where they may act as true receptors, coupled to signal transduction pathways.78,82,83 There is also high expression of integrins on the ruffled border membrane in resorbing osteoclasts,78,82 suggesting that cell-matrix interactions at that site may be involved in the progression, or cessation, of resorption.

The role of the vitronectin receptor αvβ3 in osteoclast biology has been extensively studied and gene knockout studies where β3 integrin has been deleted in the mouse84,85 have underscored the central role of αvβ3 integrin in osteoclast biology (vide infra). It has become clear that mammalian osteoclasts can adhere to a wide range of extracellular matrix proteins known to be expressed in bone and bone marrow using this receptor (Table 1) and that an RGD peptide sequence forms the recognition site for the receptor in all these ligands. Which protein constitutes the natural ligand of osteoclasts in bone remains to be determined. Colocalization studies have suggested that osteopontin is a candidate,86 since it was found to be enriched underneath the clear zones of resorbing osteoclasts. However, since osteoclasts actively synthesize osteopontin87 this finding should be interpreted with some reservation. Osteopontin also has been suggested to have an important role in osteoclast motility, an effect mediated through the αvβ3 receptor.88 RGD-containing peptides and snake venoms (echistatin and kistrin) have been shown to inhibit osteoclast polarization, activity and formation in vitro81,89-91 and they, as with vitronectin receptor antibodies, also block bone resorption in various rodent models in vivo,92-95 reviewed in ref. 2. In vitro, occupation of the vitronectin receptor by antibodies or RGD peptides causes osteoclasts to retract and detach from matrix, similar to the shape changes observed after administration of the potent anti-resorptive peptide hormone calcitonin. In mammalian osteoclasts this effect is preceded by a rise in intracellular calcium,96,97 localized predominantly to the nucleus,96 linked to the extensive downstream signaling pathways evoked by ligand engaging with integrin receptors (reviewed in refs. 98, 99). The impressive effects of RGD peptide analogues developed by the pharmaceutical industry75 on bone resorption (both in vitro assays and more recently in vivo animal models) (reviewed in ref. 2) has led to their introduction into clinical trails for bone diseases associated with excessive bone resorption such as osteoporosis.

The function of α2β1 in mammalian osteoclasts is predominantly as a receptor for type I collagen. In contrast to other cell types, adhesion of osteoclast to collagen appears to be RGD-dependent,78,81 although this could possibly be explained as a dominant-negative effect of RGD occupation of the abundant αvβ3 receptors on osteoclasts. Antibodies to α2 and β1 integrin inhibit resorption by human osteoclasts in vitro, but not to the same extent as anti-vitronectin receptor antibodies. Avian osteoclasts express abundant β1 integrin, but do not adhere to collagen.100 Avian osteoclasts probably use β1 integrin, predominantly in association with α5, as a fibronectin-binding receptor.

The role of αvβ1 on osteoclasts has not been explored in functional assays since no receptor complex-specific antibodies are available at present. This receptor is far less abundant than αvβ3 and α2β1 in osteoclasts.69 By analogy with other cell types it is likely that αvβ1 functions as a receptor for collagen or fibronectin in osteoclasts.

Integrin Knockouts and Skeletal Cell Function

The best evidence for a role for integrins in skeletal development comes from gene knockout studies in mice,101,102 complemented by studies of human diseases such as Glanzmann thrombasthenia (GT) that carry mutations in integrin genes.

αv and β3 Mouse Knockouts

Gene deletion of the key osteoclast integrin, αv, has been performed in mice.103 The deletion of αv leads to a complex phenotype: 80% embryonic lethality is observed with the surviving pups dying in the perinatal period of cerebral and other vascular malformations. The skeleton appeared grossly normal and no obvious bone or cartilage cellular defects were observed, though cell function was not specifically addressed. This model only points to a redundant role for αv in skeletal modeling during development and cannot be used to study remodelling processes that might produce a skeletal phenotype on ageing.

The skeletal phenotype of β3 integrin knockout mice has recently been reported.84,85 The skeleton at birth was relatively unaffected by the lack of β3 integrin, but mild osteosclerosis developed by adulthood. Some information on the function of mature β3 null osteoclasts has come from these murine studies—there is altered osteoclast adhesion to matrix and a reduction in bone resorption observed in vitro.84,85 However, the fact that normal multinucleated osteoclasts form in the absence of β3 excluded an essential role of this integrin in osteoclast development and precursor fusion.

Mice from both deletions have shown normal skeletal development and/or patterning, contrary to the prediction that bone modeling would have been significantly affected. This suggests that other integrins may compensate in the process of bone recognition by osteoclasts. Indeed, this appears to be true as, in an equivalent study in humans using peripheral blood cultures, patients with GT carrying an integrin β3 null mutation104 have upregulated α2β1 collagen-binding receptor that enables bone resorption to proceed, albeit to a reduced amount (vide infra). The main abnormality seen in β3 null osteoclasts is in the appearance of the ruffled border, which has wide and blunt folds in the knockout, rather than the numerous narrow membrane protrusions penetrating deep into the bone matrix, which are characteristic of normal osteoclasts. These deficiencies are seen both in vivo and in vitro and indicate that β3 plays an important role in normal ruffled border formation, possibly through a specific adhesive interaction helping the membrane to penetrate into the bone, which cannot be compensated for by other integrins. Inadequate ruffled border formation is known to affect bone resorption and is a well known phenomenon in osteopetrosis.105

Osteoclast Function in Glanzmann Thrombasthenia: A Human β3 Loss of Function Mutation

We have recently exploited the ability to generate osteoclasts from human peripheral blood mononuclear cells, under the influence of RANK,106 to study genotype-phenotype relationships in human ‘experiments of nature’, the equivalent of mouse knockouts. GT is an autosomal recessive bleeding disorder defined by defective or quantitatively abnormal platelet αIIbβ3 receptors for fibrinogen; diagnosis is based upon the clinical syndrome of mucocutaneous bleeding in association with platelet aggregation abnormalities in response to physiological stimuli. Earlier biochemical and molecular genetic characterization of the αIIb and β3 integrin subunits and their genes in patients with GT has shed considerable light on the aetiology of this genetic disorder. One particular form is the extreme β3 null phenotype observed in the Iraqi-Jewish population in Israel.107-109 Patients from this genetic isolate110,111 have no platelet αIIbβ3 due to the complete absence of the integrin β3 protein caused by an 11 bp deletion in exon 12 in the β3 (CD61/gpIIIa) gene, the mutation producing a frame-shift leading to premature termination of translation amino-terminal to the trans-membrane domain of the β3 protein. This form of GT has been used by our group to derive and analyse osteoclasts, lacking the integrin β3 protein, by in vitro culture;104 results have been compared with those reported for murine β3 -/- osteoclasts.84,85 Deficiency of β3 leads to complete absence of the osteoclast integrin, αvβ3, that shares its β chain with αIIbβ3 of platelets (Fig. 2), (Table 2a). Other αv integrins, for example αvβ5 that is normally at low levels in mature osteoclasts,69 are not up-regulated to compensate for lack of αvβ3, despite a similar but not identical range of ligand recognition. In contrast, the integrin α2β1 collagen receptor, normally expressed at an intermediate level in osteoclasts, is significantly up-regulated (Fig. 2), (Table 2a). β3 null osteoclasts polarised in vitro on bone substrate as with control cells and osteoclastic bone substrate resorption proceeded at approximately 50% of control levels, assessed by pit number and depth (Table 2b). The αv and β3 mouse knockout models aimed to address the same question, though, due to the absence of good reagents, integrin expression studies are more difficult in mice. Control levels of β5 integrin were seen on Northern analysis of bone marrow macrophages84 suggesting, as in our studies, that αvβ5 is not up-regulated to compensate for the absence of β3. Bone resorption is compromised in β3 knockout mice,84,85 as with human GT osteoclasts studied in vitro. There is, though, one difference evident between the data from the human and mouse models. In the mouse system there was a more marked effect upon cell adhesion and spreading. It could be that the role of α2β1 is less prominent in rodents, though our previous studies of rat osteoclasts would suggest otherwise;70 or this effect could relate to experimental differences.

Table 2. Alterations in integrin expression and osteoclast function in Glanzmann thrombastenia.

Table 2

Alterations in integrin expression and osteoclast function in Glanzmann thrombastenia.

Interestingly, osteoclast numbers are increased in vivo in the bones of β3 knockout mice. This effect is due to the marked hypocalcemia in the animals, leading to increased levels of parathyroid hormone and through this, increased levels of M-CSF in the bone microenvironment. 112 It is not known if a similar increase in osteoclast number is found in the β3 null GT due to the impossibility of performing skeletal biopsies in the face of a severe hemorrhagic diathesis in humans. Increased osteoclast numbers in absence of β3 goes against earlier in vivo studies in oophorectomised rats treated with a small molecule αvβ3 integrin antagonist drug where osteoclast numbers were reduced in chronically in treated animals.95 These latter data might point to fundamental differences between the dependence upon αvβ3 integrins during osteoclast development in the absence of αvβ3 and chronic exposure to ‘antagonist’ drugs that might have complex effects on cell function other than inhibition of cell adhesion. For example, direct effects of such drugs might induce signal transduction events and downstream cellular responses, unrelated to inhibition of cell adhesion (reviewed by ref. 98).

The Impact of Chronic αvβ3 Integrin ‘Deficiency’ on Skeletal Integrity: Implications of Integrin Antagonist Therapies

The establishment of the role of αvβ3 vitronectin receptor in osteoclast physiology (vide supra) led to its identification by the pharmaceutical industry as a potential drug development target as an osteoclast functional inhibitor for osteoporosis.72-74 This has now been realised and at least one drug has remained in the clinic for this indication.75

What are the implications for skeletal function and calcium metabolism in β3 null GT patients—that is, do GT patients develop an age-dependent osteosclerosis as in mice lacking the β3 integrin? A preliminary study of bone mineral density in five Iraqi-Jewish women with GT showed values for spine and femur lying within the normal range.111 More recently, there has been a single case report of the association of GT with osteopetrosis113 —whether this was a chance association or the result of a single pathogenetic mechanism awaits further study. Thus, these data support the concept that a lifelong, genetic lack of αvβ3 integrin may not be especially deleterious to the skeleton and, in support, bone disease is not a generally reported feature of GT. It would still be of considerable interest to investigate bone and calcium metabolism in this group of β3 null GT patients in more detail as it could provide significant information upon the effects of chronic treatment with αvβ3 integrin antagonists in humans.

Does the information provided by our studies impact upon the clinical development of integrin antagonist drugs? If chronic inhibition of αvβ3 resulted in a significant compensatory up-regulation of other osteoclast adhesion receptors in vivo, such as of αvβ5 expressed by osteoclast precursors or the α2β1 collagen-binding integrin of mature osteoclasts, then only a transient therapeutic benefit in bone might be expected. The lack of αvβ5 up-regulation, if also seen in other tissues, might result in the inhibitory effect being preserved in clinical situations where αvβ5 plays a more prominent role, for example in vascular restenosis.72,73 In contrast, the observed up-regulation of α2β1, an integrin that would not be expected to be inhibited by an RGD analogue drug, may result in significant loss of therapeutic response in target tissues such as bone. Thus, if data in mouse and human β3 gene deletion translates to αvβ3 antagonist drugs, then we can suggest that chronic exposure to αvβ3–selective antagonist drugs is unlikely to lead to total ablation of bone resorption. If similar compensatory mechanisms operate in normal osteoclasts at times of skeletal maturity, rather than during development as occurs in GT or knockout mice, then this response could curtail clinical efficacy. However, such a controlled inhibitory effect in the skeleton actually could be advantageous, since complete inhibition of bone turnover is deleterious as it leads to accumulation of microfactures in bone matrix. Early work with chronic dosing in ovariectomised rodent models, though, predicts that these issues may not be a major consideration in the clinical development of this new class of bone-active drugs.

The β1 Deletion Has an Embryonic Lethal Phenotype

Deletion of the β1 gene in mice leads to early embryonic lethality114,115 before skeletal elements are formed and hence is uninformative. α5 knockout mice have a similar phenotype. 116 The finding of a role for β1-fibronectin interaction in osteoblast development22,23 and studies on β1 integrins in osteoclast function (Horton, unpublished data) suggest that it would be informative to examining bone cell function in tissue-specific conditional β1 knockout mice; these have yet to be created with the appropriate targeting characteristics, though the Faessler group have created haemopoietic lineage-specific knockouts117 suggesting that it should be possible to generate osteoclasts from β1 null myeloid precursors. An alternative strategy has been used by the Damsky group; they expressed a dominant negative form of β1 under the control of an osteoblast ‘specific’ osteocalcin promoter;118 these mice had defects in osteoblast proliferation and matrix synthesis leading to low bone mass.

α1β1, α2β1 and Other Collagen-Binding Integrins of Osteoclasts

Despite extensive expression in mesenchymal cells;α1 knockout mice show normal skeletal development119 following fracture, though, there is reduced callus formation compatible with an effect on mesenchymal precursors of chondrocytes and osteoblasts. Zemyo et al120 have reported on the distribution of α1 in chondrocytes and have found that knockout mice have a propensity to develop significant age-related cartilage degeneration, similar to osteoarthritis, when compared with age-matched, wild-type litter mates. This implies that α1 might be a potential drug target for osteoarthritis, an area of unmet clinical need.

Of the four collagen-binding integrins found in vertebrates,42 α2β1 is known to be expressed by osteoclasts at levels somewhat less that αvβ3. α1β1 is not expressed by osteoclasts and α10β1 and α11β1 have not been examined though they are known to be found in skeletal tissues. The biological function of α2β1 in osteoclasts has been studied but its role in osteoblasts is less well established. However, α2β1 integrin is involved in 3-D matrix remodeling in collagen gels including osteoblasts, compatible with a function in bone modelling.29

Recently, two groups have described the phenotype of α2 knockout mice.121,122 As in the β3 null mouse, the skeleton is grossly normal at birth, though a detailed analysis of changes on skeletal maturation or of osteoclast or osteoblast function have not yet been reported. Given our results with β3 null GT patients, such studies should be undertaken to further elucidate the role of α2 integrin in bone and, by inference, its interplay with αvβ3.

Two new integrins, α10 and α11, collagen receptors have been identified recently and both are expressed in skeletal tissues, especially cartilage.42 Little is known of function other than by implication—no knockout mice have been reported in the literature.

Other Integrin Knockouts and the Skeleton

Faessler has reviewed the impact of integrin gene deletion in a number of tissues including the skeleton;123 of the other integrins that have been deleted in mice, two are informative. Amongst other features, the α4 knockout mouse124 exhibits defective closure of cranial sutures, though whether this is due to a local osteoblast, or other cell type, defect has not been studied. Combined deletion of α3 and α6125 results in severe skeletal abnormalities involving both the axial and peripheral skeleton that is not seen in single gene deletion animals; this phenotype is reminiscent of that seen on deletion of laminin α5 gene.

Osteoclasts develop from ‘monocytic’ precursor cells that express CD11/CD18 (LFA and β2 integrins).106 Interestingly, given the severe myeloid functional defects seen in knockout mice and patients with leukocyte adhesion deficiency (LAD I), no bone (osteoclast) functional or development defect has been observed in vivo. There has, though, been a single report126 showing an in vitro defect in osteoclast generation from LFA-1 deficient precursors at the level of interaction with the osteoblast counter-receptor, ICAM-1, for LFA. This is also true for mice deficient in the nonintegrin cell adhesion receptor, selectin, and combination knockouts where multiple adhesion proteins are deleted and major alterations in inflammatory responses are seen; the reason for the lack of a bone phenotype is not apparent.

Summary

There is extensive data on integrin expression in skeletal cells and increasing information on possible functions at the cellular level. The role of integrins in skeletal development, in contrast, is less well understood. Gene deletion studies have provided some indication as to redundant processes in cell-matrix and intercellular interactions that may be crucial for cell function in maturity, but little on whether integrins play a role in patterning and bone modeling in embryogenesis.

Acknowledgements

MAH is in receipt of a programme grant from The Wellcome Trust and MHH is supported by the Arthritis Research Campaign.

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