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Genetic Analyses of Integrin Signaling

Sara A. Wickstro¨m1, Korana Radovanac2, and Reinhard Fa¨ssler2

1Paul Gerson Una Group, Skin Homeostasis and Ageing, Max Planck Institute for Biology of Ageing, 50937 Cologne, Germany 2Department of Molecular Medicine, Max Planck Institute for Biochemistry, 82152 Martinsried, Germany Correspondence: [email protected]; [email protected]

The development of multicellular organisms, as well as maintenance of organ architecture and function, requires robust regulation of cell fates. This is in part achieved by conserved signaling pathways through which cells process extracellular information and translate this information into changes in proliferation, differentiation, migration, and cell shape. Gene deletion studies in higher eukaryotes have assigned critical roles for components of the extracellular matrix (ECM) and their cellular receptors in a vast number of developmental processes, indicating that a large proportion of this signaling is regulated by cell-ECM inter- actions. In addition, genetic alterations in components of this signaling axis play causative roles in several human diseases. This review will discuss what genetic analyses in mice and lower organisms have taught us about adhesion signaling in development and disease.

lmost all cells in multicellular organisms are the ECM is recognized by multiple cell surface Asurrounded by a three-dimensional organ- receptors that transmit information from the ized meshwork of macromolecules that consti- extracellular environment by propagating intra- tute the extracellular matrix (ECM). The ECM cellular signals (for a review, see Hynes 2009). is a dynamic structure that is generated and The major cell surface receptors that recog- constantly remodeled by cells that secrete and nize and assemble the ECM are integrins. Integ- manipulate its components into a precise con- rins are heterodimeric transmembrane proteins figuration. It functions as a structural frame- composed of a and b subunits. Eighteen a sub- work that provides cells with positional and units and eight b subunits can assemble in 24 environmental information, but also forms different combinations with overlapping sub- specialized structures such as cartilage, tendons, strate specificity and cell-type-specific expres- basement membranes (BM), bone, and teeth. sion patterns (Hynes 2002; Humphries et al. In addition to its structural properties, the 2006). This, together with the ability of different ECM acts as a signaling platform that regulates heterodimers to assemble specific intracellular a large number of cellular functions. It is capa- signaling complexes, provides multiple layers ble of binding growth factors, chemokines, of signaling specificity to these receptors. Con- and cytokines thereby modulating their bio- versely, the integrin expression profile of a given availability and activity. On the other hand, cell type determines which ECM components it

Editors: Richard Hynes and Kenneth Yamada Additional Perspectives on Extracellular Matrix Biology available at www.cshperspectives.org Copyright # 2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a005116 Cite this article as Cold Spring Harb Perspect Biol 2011;3:a005116

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S.A. Wickstro¨m et al.

can bind. Signals arising from integrins regulate manner, as well as temporally during develop- virtually all aspects of cell behavior, including ment (Miner 2008; Yurchenco 2010). The cen- cell migration, survival, cell cycle progression, tral role of laminins in BM assembly is and differentiation. illustrated by gene deletion studies in mice. Genetics has proven to be a powerful tool to Laminin-111 (containing a1, b1, and g1 dissect the functions of ECM–cell interactions chains) and laminin-511 (containing a5, b1, in complex organisms. To date, all of the integ- and g1 chains) are the central constituents of rin subunits and their major ligands have been peri-implantation stage BMs. Mice deficient in deleted in mice. Given the large variety of cellu- g1orb1 subunits are unable to generate lami- lar processes regulated by adhesion signaling, nin trimers, and therefore lack BMs (Smyth it is not surprising that a significant subset of et al. 1999; Miner et al. 2004). these proteins has proven to be essential for Laminin-332 (containing a3, b3, and g2 embryonic development and/or tissue mainte- chains; previously termed laminin-5) is a com- nance. However, in addition to underlining the ponent of epithelial BMs and thus present in importance of cell-ECM interactions in devel- skin, stratified squamous mucosa, amnion, and opment, genetic studies also revealed critical cornea. Its main task is to maintain epithelial roles for tissue- and cell-type-specific modes integrity and epithelial-mesenchymal cohesion of adhesion signaling and provided important in tissues exposed to high mechanical forces. insights into human disease. This function is facilitated by the unique ability of laminin-322 to interact with two dis- tinct integrin heterodimers. Its interaction with THE INTEGRIN-INTERMEDIATE a3b1 integrin results in the assembly of canonical FILAMENT AXIS focal adhesions (FA), whereas the interaction Basement membranes (BMs) are dense sheets of with a6b4 integrin results in the formation of extracellular matrix that function as structural specialized adhesion complexes termed hemides- barriers that separate epithelial and endothelial mosomes (Tsuruta et al. 2008). cells as well as peripheral nerve axons, fat cells, and muscle cells from the underlying tissue Hemidesmosomes—Structure and Assembly stroma. BMs provide structural support,separate tissues into compartments, and regulate cell Hemidesmosomes were first characterized at the behavior. All cell types are known to produce ultrastructural level as electron-dense clusters at components of BMs, which include type IV col- the plasma membrane-ECM interphase (Weiss lagen, laminin, fibronectin, heparan sulfate and Ferris 1954). Further studies identified proteoglycans, and nidogen/entactin as well as them as multiprotein adhesion complexes pre- severalminorcomponents(EricksonandCouch- sent in stratified and simple epithelia. Twotypes man 2000; Yurchenco 2010). The molecularcom- of hemidesmosomes can be distinguished based position ofthe BMvariesamong different tissues, on their molecular composition. Type I or clas- conferring signaling specificity important for sical hemidesmosomes are found in stratified defining the specialized functions of epithelial epithelium such as the skin and contain three and endothelial cells in different organs. transmembrane proteins: a6b4 integrin, tetra- All BMs contain laminins, a family of 16 spanin CD151, and type XVII collagen (also heterotrimeric glycoproteins generated by the called bullous pemphigoid [BP] antigen 180) combination of 5a,4b, and 3g chains. When (Fig. 1). Type II hemidesmosomes are found present in sufficient concentrations, laminin in simple epithelia such as intestine, and contain networks can self-assemble into polymers that only integrin a6b4 (Uematsu et al. 1994; interact with other ECM components, as well reviewed in Litjens et al. 2006). as cell surface receptors such as integrins and The unique feature of hemidesmosomes dystroglycan. The laminin isoforms present is that they connect the ECM to the intermedi- in BMs are regulated in a tissue-specific ate filament (IF) network. This interaction is

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Genetic Analyses of Integrin Signaling

IF Stable adhesion

Plectin

BP230 Basal keratinocyte P - S1364 P - S1360 Hemidesmosome Cell migration P - S1356 disassembly signaling

Collagen XVII

α6 Integrin β4 Integrin

CD151 Lamina lucida BM LM 322 Lamina densa

Figure 1. Molecular architecture of type I hemidesmosomes. Schematic depiction of a type I hemidesmosome found in stratified epithelia such as in basal skin keratinocytes. The core component is a6b4 integrin, which binds the basement membrane (BM) component laminin (LM)-322. a6b4 integrin recruits the plakin protein plectin through multiple interactions with the b4 integrin cytoplasmic tail, which initiates the formation of hemidesmosomes. This is followed by the recruitment of collagen XVII, which interacts both with b4 integrin and plectin as well as with LM 322. Collagen XVII in turn mediates the recruitment of another plakin, bullous pemphigoid antigen 230 (BP 230), which together with plectin provides the connection to intermediate fila- ments (IF). This linkage is essential to stabilize the hemidesmosome and to provide stable adhesion of the basal keratinocyte to the BM. Also, the transmembrane protein tetraspanin CD151 that interacts with a6 integrin is found in type I hemidesmosomes. Phosphorylation of serines (S) 1356, 1360, and 1364 on the cytoplasmic tail of b4 integrin by growth factors induces disassembly of hemidesmosomes, which promotes cell migration and sig- naling. The molecules are not drawn to scale.

established by two plakin proteins, plectins and explains the absence of actin in hemides- BP230 (also called BPAG1), of which plectin is mosomes (Rezniczek et al. 1998; Geerts et al. present both in type I and II hemidesmosomes. 1999; Litjens et al. 2003). The interaction of Plectins are large cytoplasmic proteins, which plectin with the cytoplasmic tail of b4 integrin at their carboxyl terminus contain six plakin is considered as the initial step of hemidesmo- repeats. A stretch of basic residues linking the some assembly (Geerts et al. 1999; Koster et al. fourth and fifth plakin repeat mediates the 2004). The cytoplasmic tail of b4 integrin is interaction of plectin with IFs. The amino ter- unusually large and shares no homology with minus contains two calponin-homology (CH) other b integrin subunits. Its interaction with domains that constitute an actin-binding do- plectin has been shown to induce a conforma- main. Through the actin-binding domain, tional change in the integrin tail (de Pereda plectins can associate with either the cytoplas- et al. 2009). Because the recruitment of type mic domain of the b4 integrin subunit or actin XVII collagen and the plakin BP230 to hemides- filaments in a mutually exclusive manner, which mosomes requires prior interaction of plectin

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S.A. Wickstro¨m et al.

with b4, this conformational change might epidermis of the zebrafish, the basolateral facilitate the interactions of both proteins with identity protein Lgl2 is required for hemides- the integrin (Koster et al. 2004). mosome formation through recruitment of b4 Deletion of a6b4 leads to the loss of hemi- integrin to the basal plasma membrane (Sona- desmosomes and epithelial detachment in mice wane et al. 2005, 2009). Taken together, the (Dowling et al. 1996; van der Neut et al. 1996), formation of hemidesmosomes requires the indicating that BP180, despite its ability to bind establishment of a basolateral membrane iden- laminin-332 and plectin in vitro, is not suffi- tity through polarity proteins and possibly also cient to maintain adhesion of cells to laminin- other integrin-based adhesion structures to re- 332 in the absence of a6b4 (Tasanen et al. cruit a6b4 integrin to these sites. a6b4 integrin 2004) and identifying a6b4 as the core compo- then interacts with BM laminin and together nent of hemidesmosomes. Another critical with plectin nucleates stable, IF-bound hemi- event in hemidesmosome assembly is the inter- desmosomes. Interestingly, however, keratino- action between b4 integrin tails and plectin. cytes expressing a b4 integrin subunit unable Blocking this interaction compromises hemi- to bind laminin can still form structures that desmosome assembly in vitro (Geerts et al. contain all components of type I hemides- 1999; Koster et al. 2001). In plectin-deficient mosomes, which implies that ligand binding mice hemidesmosomes appear ultrastructurally is not necessary for their formation (Nievers normal, but their number and mechanical sta- et al. 1998, 2000). These complexes, however, dif- bility are reduced (Andra et al. 1997). The fact fer in their morphology, density, and dynamics that type II hemidesmosomes lack both type from normal hemidesmosomes, suggesting that XVII collagen and BP230 further shows that the integrin-laminin interaction plays a role in a6b4 and plectin alone are sufficient to initiate the stability and structural organization of hemi- the formation of these structures and to main- desmosomes (Geuijen and Sonnenberg 2002). tain their integrity. Although a6b4 is the only integrin found Hemidesmosomes—Guardians of in hemidesmosomes, other integrins can indi- Epidermal Integrity rectly contribute to their assembly. Integrin a3b1 has been shown to cluster in “pre-hemi- A central function of the skin is to act as a bidi- desmosomal” structures on the basal cell surface rectional barrier to prevent dehydration and to of human keratinocytes together with CD151. protect against injury and pathogens. In addi- However, as hemidesmosomes mature, a3b1 tion, it is exposed to constant mechanical stress. integrins become recruited to cell-cell or focal On the other hand, this tissue is continuously contacts, whereas CD151 remains in the hemi- renewed through a terminal differentiation pro- desmosomes (Sterk et al. 2000). The initial gram, in which basal keratinocytes detach from a3b1-containing adhesions have been pro- the BM and move to the upper layers of the epi- posed to contribute to the recruitment of a6b4 thelium where they slough off. Finally, in case to the plasma membrane, and thereby increase of injury, keratinocytes need to rapidly acquire the efficacy with which hemidesmosomes are a motile phenotype and migrate to close the formed. This is supported by the observation wound. These functions require mechanically that b1-deficient mice have reduced numbers stable but dynamic modes of cell adhesion. of hemidesmosomes in the skin, although it Hemidesmosomes are found in the basal plasma should be noted that the expression level of membrane of basal keratinocytes, where they a6b4 is also reduced in these mice, suggesting function to attach these cells firmly to the BM that the mechanism could also be more indirect separating the epidermis from the dermis (Fig. (Brakebusch et al. 2000). Polarity proteins 1). Interestingly, a6b4 integrins and thus also have also been shown to play a role in hemides- hemidesmosomes are not required for skin mosome assembly through the regulation of morphogenesis or developmental homeostasis polarized targeting of integrins. In the larval (DiPersio et al. 2000). The importance of

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Genetic Analyses of Integrin Signaling

hemidesmosomes in maintaining epidermal mouse lung carcinoma and melanoma cell lines integrity is, however, shown by multiple lines (Kennel et al. 1981), and has later been shown to of genetic evidence. Ablation of genes encoding be up-regulated in several human cancers, sug- a6 integrin, b4 integrin, or plectin in mice results gesting that the expression of b4 integrin is ben- in severe blistering of the skin causing neonatal eficial for tumor cells (Giancotti 2007). The role death because of a severe epithelial barrier defect of a6b4 integrin signaling in tumorigenesis (Dowling et al. 1996; Georges-Labouesse et al. has been studied in mice carrying a truncated 1996a; van der Neut et al. 1996; Andra et al. cytoplasmic tail of b4 lacking the tyrosine 1997). In linewith their dispensable role in hemi- phosphorylation sites as well as other potential desmosome assembly, mice lacking type XVII interaction motifs. In contrast to mice lacking collagen or BP230 display only mild forms of the entire b4 cytoplasmic domain, which skin blistering (Guo et al. 1995; Nishie et al. display extensive skin blistering because of the 2007), whereas deletion of CD151 is dispensable absence of hemidesmosomes (Murgia et al. for both hemidesmosome stability and the integ- 1998), mice with a more restricted truncation rity of skin (Wright et al. 2004). do not show these defects. However, they show No experimental data exist on the dynamic delayed wound healing and defective neoangio- behavior of hemidesmosomes in vivo, but it has genesis in tumor xenografts (Nikolopoulos et al. been analyzed in keratinocyte culture, in which 2004, 2005). In addition, when crossed to the hemidesmosomes have been shown to display MMTV-Neu mice that carry an activated form a certain dynamics also in nonmigratory cells of epidermal growth factor receptor-2 (ErbB2) (Tsuruta et al. 2003). When cells are stimu- driven by a mouse mammary tumor virus lated to migrate, a6b4 integrin is mobilized (MMTV) promoter leading to mammary tu- from hemidesmosomes to actin-based struc- mors, the b4 mutant mice display delayed tures such as lamellipodia and filopodia to pro- tumor onset, impaired tumor growth and de- mote cell motility, resulting in hemidesmosome creased metastatic potential (Guo et al. 2006). disassembly (Geuijen and Sonnenberg 2002; Whether this applies to other tumor types as Tsuruta et al. 2003). This type of relocalization well remains to be shown. has also been visualized in human wound edges, The tumor-promoting properties of a6b4 where b4 integrins can be seen in trailing-edge integrin seem to result from both promigratory hemidesmosomes as well as in lamellipodia of and signaling functions. b4 integrin has been the leading edge (Underwood et al. 2009). In shown to cross-talk with several receptor tyro- vitro analyses have shown that the mobilization sine kinases (RTK), including ErbB2, epidermal of a6b4 integrin from hemidesmosomes is growth factor receptor (EGF-R), Met and Ron facilitated by growth factor-mediated phos- (Mariotti et al. 2001; Trusolino et al. 2001; phorylation of multiple residues in the cyto- Santoro et al. 2003; Guo et al. 2006). Following plasmic tail of b4. Both serines and tyrosines stimulation, these RTKs induce activation of can be phosphorylated, but the exact sites and phosphatidylinositol 3-kinase (PI3-K) or Src their relevance remain controversial. The cur- family kinases (SFK), leading to phosphoryla- rent data suggest that serine phosphorylation tion of the b4 integrin tail, disassembly of hemi- might be more relevant under physiological desmosomes, and induction of cell motility conditions, leading to hemidesmosome disas- (Mariotti et al. 2001; Santoro et al. 2003). On sembly through the release of plectin from the the other hand, b4 integrin has been shown to integrin (Margadant et al. 2008) (Fig. 1) promote SFK-dependent phosphorylation of the catalytic sites of RTKs and their substrates (Bertotti et al. 2006; Guo et al. 2006), thereby a6b4 Integrin and Cancer amplifying their signaling ability. Taken b4 integrin was originally identified as a “tu- together, a6b4 integrin seems to have a proin- mor-specific” protein (Tumor Surface Protein vasive and proangiogenic activity in tumors, 180) up-regulated in metastatic variants of but additional genetic studies are needed to

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S.A. Wickstro¨m et al.

determine whether this role is limited to malig- FN is secreted as a disulfide-bonded dimer, nancies involving hyperactivation of specific and its deposition into a fibrillar matrix is a RTKs. In addition, several groups have failed cell-driven process that critically depends on to detect significant tyrosine phosphorylation integrins. So far a5b1, avb3, a4b1, and aIIbb3 of b4 integrin in keratinocytes or transformed integrins have been shown to induce fibri- cell lines in response to stimulation with EGF llogenesis in vitro. These integrins bind FN and hemidesmosome disassembly, making the that is secreted as a compact globular structure relevance of tyrosine phosphorylation events where binding sites for other FN molecules are controversial (Rabinovitz et al. 1999, 2004; Alt buried within the protein (see Schwarzbauer et al. 2001; Wilhelmsen et al. 2007). Knockin and DeSimone 2011). Binding is followed by mice carrying mutations in specific phosphory- the activation of integrin signaling, leading to lation sites of the b4 integrin subunit would the recruitment of cytoplasmic proteins to provide more conclusive information on the form focal complexes (FCs). Several FC com- in vivo significance of the various phosphoryla- ponents are actin-binding and modulatory tion events in a6b4 integrin signaling, hemi- proteins, allowing the recruitment and reorgan- desmosome turnover, and invasion. ization of the actin cytoskeleton at these sites and their maturation into FAs (Geiger and Yamada 2011). The major FN-fibril-forming THE INTEGRIN-ACTIN AXIS integrin a5b1 leaves FAs and moves along F- In contrast to a6b4, most integrins engage the actin to the cell center to form fibrillar adhe- actin cytoskeleton following ligand binding. A sions and to facilitate the generation of mechan- central ligand of actin-associated integrins is ical tension via the actin cytoskeleton, leading to FN, which is recognized by 11 integrin hetero- stretching of the bound FN molecule, unravel- dimers in mice and humans, and it will be used ing of the cryptic self-association sites, and in this section as an example to discuss the prin- finally the binding to other FN molecules ciples of ECM-integrin interactions leading to resulting in fibril formation (Mao and Schwarz- engagement and remodeling of the actin cytoske- bauer 2005; Leiss et al. 2008; Schwarzbauer and leton. FN is a large, modular glycoprotein that DeSimone 2011). exists in two forms; cellular FN which is present in tissues where it is assembled into a fibrillar Interactions via the RGD Motif matrix, and plasma FN, which is produced by hepatocytes and is secreted into the blood where Cell adhesion to FN critically depends on the it remains in a nonfibrillar, soluble form, or fol- Arg-Gly-Asp (RGD) motif of FN, which is rec- lowing entry into tissues becomes incorporated ognized by a5b1, the av family, a8b1, a9b1, into the ECM (Leiss et al. 2008). FN is found and the platelet-specific aIIbb3 integrins only invertebrates, and it has co-evolved together (Fig. 2). a5b1 is considered the major integrin with the cardiovascular system. Consistently, FN responsible for FN assembly, and its interaction is critical for the development of the vasculature, with the RGD motif is required for this func- where it localizes between the endothelium and tion. However, although deletion of a5 integrin perivascular cells. Deletion of FN in mice results in mice also leads to embryonic lethality and in embryonic lethal cardiovasculardefects, which vascular defects, these mice develop signifi- vary depending on the genetic background. cantly further than the FN-null mice (Yang FN-null embryos from 129S4 mice are unable et al. 1993). This is attributable to the ability to form the dorsal aorta, indicative of an early of cells to assemble FN using other integrins, defect already in vasculogenesis, whereas this mainly of the av subfamily. The deletion of all structure is present in C57BL/6 derived embryos five av heterodimers also leads to late embry- that display defects in vascular lumen formation onic defects in vascular development mainly (George et al. 1993, 1997; Georges-Labouesse in the placenta. A subset of animals can even et al. 1996b). proceed through embryonic development and

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Genetic Analyses of Integrin Signaling

LDV NGR RGD EDGIHEL REDV Motif SS

Domain NH2 COOH

αvβ1 α5β1 α8β1 α9β1 αvβ3 α5β1 αIIβ3 α4β1 α4β1 Integrin αvβ3 αvβ5 α9β1 α4β7 αvβ6

Lymphatic Reported Somitogenesis Lung Lymphangio- Placental Platelet valve Vascular remodelling developmental Vasculogenesis ? morphogenesis genesis angiogenesis aggregation morphogenesis Pericyte recruitment Angiogenesis function (α9β1)

Fn1 Fn2 Fn3 Extra domain Variable region

Figure 2. Developmental functions of fibronectin-integrin interactions. Fibronectin (FN) is a dimeric glycyo- protein consisting of type I, type II, and type III modular repeats. Dimerization is achieved by disulfide bonds mediated by the two cysteines (S) in the COOH-terminus of the protein. The binding domains and motifs for integrins, as well as mouse developmental functions reported to depend on the particular interaction are indi- cated. Integrins that have been shown to mediate FN fibrillogenesis in vitro are marked by gray boxes.

die after birth because of hemorrhages, indicat- FN signaling in embryogenesis occurs through ing that, unlike a5, av integrins are dispensable a5b1 and that av and a5b1 integrins relay for vasculogenesis and partly also for angiogen- distinct signals on FN binding. The severity of esis (Bader et al. 1998). Only a double knockout the FN-null phenotype in contrast to the integ- of the av and a5 integrin genes results in a loss rin deletions also suggests that the functions of FN fibrillogenesis (Yanget al. 1999). Interest- of FN during development extend beyond ingly, although the RGD motif is central for the the FN-integrin signaling axis. These functions interaction of FN with a5b1 and avb3, the could be related to its mechanical role in the inactivation of this motif by a RGD to RGE ECM, or its ability to induce integrin-indepen- point mutation also allows FN fibrillogenesis dent signaling, for example, through sequester- in vivo. The knockin mice carrying this muta- ing growth factors such as vascular endothelial tion display a phenotype closely resembling growth factor (VEGF) or transforming growth that of the a5-null mice (Takahashi et al. factor-b (TGF-b). 2007). This highlights the importance of this motif in FN signaling through a5b1, but also Non-RGD Binding Integrins shows the ability of other integrin interaction sites on FN to take over the role of RGD in FN The FN gene can be alternatively spliced allow- fibrillogenesis. Whether these sites play a role ing the expression of up to 20 possible mo- in FN assembly when the RGD sequence is nomeric isoforms in humans and up to 12 in intact, remains open. Together, these results mouse, potentially giving rise to a larger variety indicate that FN fibrillogenesis is not sufficient of FN dimers (Pankov and Yamada2002). Some for it to carry out its functions during develop- of these splice variants can generate additional ment. They further illustrate that the majority of integrin interaction sites on FN. One of them

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S.A. Wickstro¨m et al.

is the variable region (v-region) that can inter- of tumors, whereas other integrin subunits act with two non-RGD-binding integrins, a4b1 seem not to be up-regulated (Garmy-Susini and a4b7 (Wayner et al. 1989; Guan and Hynes et al. 2010). Whether this function really 1990; Fig. 2). These integrins are mainly ex- depends on the EDA domain was, however, pressed by hematopoietic cells, although a4b1 not analyzed. In addition, it is not clear whether is also found in several other cell types, such a4b1 integrin is also regulating developmental as neural crest cells and cellular components lymphangiogenesis, as the constitutive knock- of the cardiovascular and peripheral nervous out mice die before the initiation of this process. systems. The two a4 integrins have many im- The EDA domain itself, however, plays a role portant roles during development. Deletion of in developmental lymphangiogenesis through the a4 integrin gene results in embryonic le- integrin a9b1. Interestingly, this integrin is thality because of early placental and cardiovas- highly expressed in lymphatic but not blood cular defects (Yang et al. 1995). However, these endothelial cells (Huang et al. 2000). Deletion defects are probably not attributed to the FN- of a9 integrin gene leads to a severe chylothorax a4b1/a4b7 integrin interaction, as these two caused by defects in lymphatic valve morpho- integrins also bind cell counter receptors such genesis and subsequent respiratory failure as vascular-cell adhesion molecule-1 (VCAM-1) (Huang et al. 2000; Bazigou et al. 2009). This and MadCAM, respectively. The developmen- phenotype was partially recapitulated by dele- tal defects in the a4 integrin-null mice are tion of EDA. Furthermore, both a9b1 integrin very similar to those found in mice lacking as well as the EDA domain were shown to be VCAM-1, indicating that the observed defects critical for FN assembly in lymphatic endothe- are caused by an abrogated interaction between lial cells in vitro (Bazigou et al. 2009), in con- a4b1 integrin and VCAM-1 (Kwee et al. 1995; trast with previous studies showing that EDA Yang et al. 1995). From a clinical point of view, is not required for FN assembly or mouse devel- it is particularly interesting that the interaction opment because of compensation by EDB (Tan between a4b1 and VCAM-1 is important for et al. 2004; Astrof et al. 2007). This study sug- the homing of autoreactive T cells to the central gests that matrix assembly might be regulated nervous system during the pathogenesis of an by integrins that are expressed in a tissue-spe- autoimmune disease called multiple sclerosis cific manner to allow more stringent control (Yednocket al. 1992; Vajkoczy et al. 2001; Bauer of distinct anatomical structures. et al. 2009). This disease is characterized by axon demyelination and damage, leading to Linkage to the Actin Cytoskeleton numbness, weakness, paresis as well as cogni- tive problems. Blocking antibodies against a4 In addition to catalyzing matrix assembly, the integrin have proven to be an effective treatment binding of integrins to FN or other ligands leads of multiple sclerosis (Steinman 2009), and to the induction of multiple modes of intracel- genetic analysis in mice showed that a4b1is lular signaling. These include activation of clas- required for the arrest of T cells on the brain sical signaling pathways through tyrosine and endothelial surface (Bauer et al. 2009). serine phosphorylation of specific substrates, a4b1 integrins can also interact with the resulting ultimately in the regulation of cell sur- alternative spliced extra domain A (EDA) of FN vival, growth and differentiation. This signal- (Fig. 2), which is highly expressed during de- ing operates in close collaboration with growth velopment and during pathological conditions factor signaling, making it difficult to dissect such as tumorigenesis. This “reactivation” of which signals specifically emanate from ligated the embryonic splice pattern is interesting, as integrins (Legate et al. 2009; Ivaska and Heino a4b1 has recently been shown to play a criti- 2010). The other central signaling mode is the cal role in tumor lymphangiogenesis in a FN- recruitment of filamentous (F-) actin to integ- dependent manner. Both a4b1 and FN are rin adhesion sites, a process tightly coupled to highly expressed in lymphatic endothelial cells the active remodeling of the actin cytoskeleton.

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Genetic Analyses of Integrin Signaling

The integrin-actin linkage is central to integrin of FA proteins by their ability to regulate both function in several respects. First, active actin integrin activation (inside-out signaling) and polymerization, crosslinking, and subsequent intracellular signaling downstream of ligand force generation are critical for the maturation binding (outside-in signaling). Talin is present of nascent and short-lived integrin adhesions in all multicellular eukaryotes; lower eukary- into signaling-competent and stable FAs. Sec- otes encode only a single talin isoform, whereas ond, it is required for the precise spatiotemporal vertebrates have two talin isoforms, talin-1 control of cell protrusion and retraction during and talin-2. Talin consists of a large carboxy- cell migration. Third, it allows cells to adopt or terminal rod and an amino-terminal head change their characteristic shape, for example domain composed of a FERM domain with to polarize. Finally, it is critical for FN matrix four subdomains: F0, F1, F2, and F3. The F3 assembly. Integrins do not bind actin directly, subdomain contains a phosphotyrosine-bind- but regulate this linkage by recruiting a large ing motif, which harbors a high affinity binding number of actin-binding and regulatory pro- site for b-integrin tails. The rod domain con- teins. A recent study based on database mining tains multiple binding sites for the actin–bind- identified 156 signaling, structural and adaptor ing protein vinculin as well as for actin itself. In molecules that can be found in integrin adhe- addition it contains a second integrin-binding sions (Zaidel-Bar et al. 2007). Only a small sub- site (Critchley and Gingras 2008). The talin set of these proteins binds directly to integrins, head has been shown to be sufficient for integ- and the large majority is recruited through rin binding and activation, whereas the rod is protein-protein interactions between the vari- required for the scaffolding function of talin ous scaffold proteins (for reviews, see Legate in outside-in signaling (Garcia-Alvarez et al. and Fa¨ssler 2009; Geiger and Yamada 2011). A 2003; Tanentzapf and Brown 2006; Tanentzapf large body of structural, biochemical, and et al. 2006). genetic evidence points to talin, kindlin, and Kindlins are an evolutionarily conserved the integrin-linked kinase (ILK)-pinch-parvin familyof multidomain proteins, which in mam- (IPP) complex as some of the central FA com- mals consists of three members: kindlin-1 (also ponents regulating the integrin-actin linkage. known as FERMT1), kindlin-2 (also known All of these proteins are ubiquitously expressed as FERMT2 or MIG-2), and kindlin-3 (also and regulate a wide range of integrin hetero- known as FERMT3 or URP2). Although en- dimers. Deletion of any of these proteins leads coded by separate genes, they show identical to early embryonic lethality, which results domain architecture and high sequence simi- from compromised integrin function and larity (Ussar et al. 2006). Like talin, kindlins defects in their connection to the cytoskeleton. contain a FERM domain through which they On the cell-biological level these defects span interact with b1, b2, and b3 integrins, but the entire range of functions attributed to the this FERM domain is unique in that it is inter- integrin-actin linkage. Hence, they can be rupted by a pleckstrin homology (PH) domain, viewed as global regulators of integrin function. which provides a putative binding motif for The precise functions of these proteins will be membrane lipids. discussed in the next section. Consequences of Loss of Integrin IN VIVO MECHANISMS OF INTEGRIN Inside-Out Signaling SIGNALING THROUGH INTRACELLULAR Integrins are present on the plasma membrane EFFECTORS AND SCAFFOLDS in low-, intermediate-, and high-affinity states. Talin and kindlin are two FA proteins that Structural studies suggest that a low-affinity state directly bind integrins and thereby are involved is characterized by a bent, “closed” conformation in the very early events of integrin signaling. of the extracellular domains and a high-affinity They are distinguished from the large group state by an extended, “open” conformation

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(Campbell and Humphries 2011). The extended Moser et al. 2008, 2009a). The molecular details conformation is thought to be achieved by of this cooperation are not clear. separation of the a and b cytoplasmic tails Regulation of the ligand binding affinity is through binding of talin to the b subunit fundamental for various cellular functions. (Moser et al. 2009b; Shattil et al. 2010). Studies For example, the platelet integrin aIIbb3 needs in cultured cells have shown that the binding to be maintained inactive to prevent it from of talin-1 to the cytoplasmic domain of the binding fibrinogen present in the blood stream b-integrin subunit is a common step in b1 thus prohibiting platelet aggregation and and b3 integrin activation in vitro (Tadokoro thrombus formation. On the other hand, this et al. 2003). In contrast, kindlin alone is not integrin needs to be rapidly activated to mediate sufficient to activate these integrins, but its platelet adhesion on vascular injury to prevent co-operation with talin is required to generate bleeding (Fig. 3). Similarly, leukocytes require fully active integrins (Montanez et al. 2008; integrin activation to adhere to and migrate

1

5 2 3 4

GPI

αβ Integrin

Talin

Kindlin

Adaptor proteins F-Actin

1 2 3 4 5

Resting platelet Inside-out signaling Outside-in signaling Platelet spreading Platelet aggregation

Inactive integrins Talin and kindlin Ligation of active integrins Cytoskeletal rearrangement Binding to soluble fibrinogen recruitment Recruitment of adaptors Adhesion strengthening Thrombus formation Activation of integrin

Figure 3. Talin and kindlin regulate bidirectional integrin signaling. Bidirectional integrin signaling is essential for platelets (tan) to seal the injured blood vessel endothelium (red) and stop bleeding. Integrins in circulating, resting platelets exist in a low affinity state indicated by a bent confirmation (1). Following vessel injury, von Wil- lebrand factor (vWF) and collagen are exposed to bind their receptors GPIb and GPVI that are expressed on the surface of platelets. Together with locally produced thrombin these receptors trigger the activation of aIIbb3 integrin. This is achieved by promoting the association of talin-1 and kindlin-3 with the cytoplasmic tail of b3 integrin, facilitating a conformational change in the integrin (inside-out signaling) (2). The conformational change allows integrins to bind fibrinogen, vWF,and fibronectin with high affinity. As a result, platelets adhere to the vessel wall. Integrin ligation subsequently initiates signaling through kindlin and talin (outside-in signaling) (3) resulting in the recruitment of adaptor proteins and rearrangement of the cytoskeleton to promote cell spreading (4). This, together with integrin-mediated binding of soluble fibrinogen, results in platelet aggrega- tion and formation of a stable clot (5). The molecules are not drawn to scale.

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Genetic Analyses of Integrin Signaling

across the endothelium to combat pathogens. indicating that talin providesan essential linkage The importance of this process is illustrated by to the cytoskeleton independent of its ability to the phenotypes of talin- and kindlin-deficient activate integrins. Kindlins do not bind actin mice. Deletion of talin-1 or kindlin-3 in plate- directly, but connect to the actin cytoskele- lets blocks activation of b1 and b3 integrins, ton via a migfilin-filamin interaction as well leading to the inability of platelets to bind fibri- as through the IPP complex (Montanez et al. nogen and to form clots. As a result, these mice 2008). Platelets lacking kindlin-3 expression suffer from severe bleeding (Nieswandt et al. are unable to organize their cytoskeleton or 2007; Petrich et al. 2007; Moser et al. 2008). to establish stable lamellipodia (Moser et al. Similarly, neutrophils lacking kindlin-3 are 2008), which probably contributes to their unable to activate b2 integrins, resulting in inability to spread and form a crosslinked clot loss of neutrophil adhesion to activated endo- (see Fig. 3). Loss of kindlin-2 impairs actin thelial cells (Moser et al. 2009a). Talin has also organization and FA formation in endoderm been shown to bind and activate neutrophil cells, which together with defective integrin integrin b2 in vitro (Simonson et al. 2006). activation leads to early embryonic lethality. The importance of integrin affinity regulation Interestingly, the rudimentary FAs in kindlin- in adherent cell types is not as clear. Although 2-deficient cells do not contain ILK, suggesting deletion of talin and kindlin in mesenchymal that kindlin acts as a nucleator of FA formation or epithelial cells leads to defects in integrin (Montanez et al. 2008). activation, these cells are still capable of adher- Despite the central role of talin and kindlin ing, albeit to a reduced extent (Ussar et al. in outside-in signaling, they are not sufficient to 2008; Zhang et al. 2008), suggesting that under establish a stable integrin-actin connection. An conditions of less stringent requirements for important scaffold also required for this func- rapid induction of adhesion, high concentra- tion is the IPP complex, which is a constituent tions of ligands, and the absence of high shear of at least b1 and b3 integrin-containing adhe- flow, avidity regulation might be the predomi- sion sites. The core component of this complex nant mechanism of integrin regulation. The is ILK, a pseudokinase that is also capable of fact that both talin and kindlin are also impor- binding these integrins directly, at least in vitro tant in regulation of outside-in signaling makes (Wickstro¨m et al. 2010). Whether the binding the in vivo dissection of these two processes is actually relevant for the function of this com- difficult. plex, or whether it occurs in vivo, has not been conclusively shown. The other two members of this complex are pinch and parvin. Pinch, Consequences of Loss of Outside-In which interacts with the amino-terminal Signaling ankyrin repeats of ILK, is a family of proteins A central piece of evidence that talin has impor- containing only LIM domains and consisting tant functions besides integrin activation comes of two members, pinch-1 and pinch-2 (Tu from studies performed with talin-null flies, et al. 1999, 2001; Chiswell et al. 2008). The par- which show that in the absence of talin, integ- vins, which interact with the carboxy-terminal rins are able to associate with the ECM, but kinase-homology domain of ILK, are a family are unable to connect to the cytoskeleton, lead- of CH-domain-containing proteins with three ing to muscle detachment at the integrin-actin members; the ubiquitously expressed a-parvin interphase (Brown et al. 2002). Consistent with (also known as actopaxin or CH-ILKBP), b- this finding, mouse fibroblasts lacking both parvin (also known as affixin), which is highly talin-1 and talin-2 are able to adhere, but not expressed in heart and skeletal muscle, and g- to link integrins to the cytoskeleton (Priddle parvin, which is restricted to the hematopoietic et al. 1998; Zhang et al. 2008). This function system (Nikolopoulos and Turner 2000; Olski requires the talin rod domain, which contains et al. 2001; Tu et al. 2001; Yamaji et al. 2001; binding sites for F-actin and vinculin Chu et al. 2006). The IPP complex is thought

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to be preassembled in the cytoplasm and is sub- recruitment of the cytoskeleton to adhesion sequently recruited to integrin adhesions in an sites, resulting in defects in FA maturation and ILK-dependent manner (Zhang et al. 2002). cytoskeletal remodeling. The defective mat- The stability of each individual component de- uration of ILK-deficient FAs into fibrillar pends on the assembly of the complex, as deple- adhesions subsequently leads to impaired tion of ILK or pinch leads to a decrease in the deposition of the FN matrix (Sakai et al. 2003; protein levels of the other two complex mem- Stanchi et al. 2009). The precise molecular bers (Fukuda et al. 2003; Li et al. 2005). mechanism by which ILK regulates the cyto- The central biological function of the IPP skeleton is not clear. Parvins are capable of complex is the regulation of the cytoskeleton interacting with F-actin through their two CH downstream of integrins, although a number domains, but as these domains also regulate of additional functions for this complex as the interaction of parvins with ILK and paxillin, well as its individual components have been they might not be available for actin binding. assigned by in vitro studies. The importance Therefore, it is likely that the IPP complex of these proteins in the integrin-actin connec- requires additional downstream partners for tion is highlighted by deletion studies in mice, the regulation of the actin cytoskeleton, and where deletion of ILK or pinch-1 results in peri- the identification of these proteins is a central implantation lethality caused by severe defects focus of future research. It is, however, clear in F-actin organization at adhesion sites, lead- that the presence of parvin in the IPP complex ing to a failure in epiblast polarization (Sakai is critical for the regulation of the actin cyto- et al. 2003; Li et al. 2005). The role of parvins skeleton. The absence of a-parvin in mice is more complex because of the presence of causes impaired migration of vascular smooth three structurally very similar isoforms. Mice muscle cells (vSMC) toward developing vessels lacking b-org-parvin show no obvious pheno- resulting in defective stabilization of the vascu- types, whereas a-parvin-null mice survive up lature and subsequent dilation of vessels, forma- to embryonic day 14.5 and die because of tion of microaneurysms and vessel rupture. The defects in cardiovascular development (Chu migration defect is caused by aberrant actomyo- et al. 2006; Montanez et al. 2009; I Thievessen sin contractility (Montanez et al. 2009), and can and R Fa¨ssler, unpubl.). The a/b-parvin double be phenocopied by vSMC-specific ablation of knock-out results in early embryonic lethality, ILK (Kogata et al. 2009). However, this function suggesting that parvins can compensate for seems to be restricted to certain cell types, sug- each other during early development (E. Mon- gesting that although the central role of the tanez and R. Fa¨ssler, unpubl.). Caenorhabditis IPP complex in integrin outside-in signaling is elegans expresses only single orthologs for pinch ubiquitous, the precise molecular mechanisms and parvin, and is thus a suitable model organ- might be cell type-specific to accommodate ism for a more straightforward analysis of the the specialized needs of various cell and tissue IPP complex. Studies in C. elegans have shown types. that ILK (PAT-4), pinch (UNC-97), and parvin (PAT-6) colocalize with b integrin (PAT-3) at GENETIC DISEASES OF CELL–MATRIX muscle attachment sites, where a robust con- INTERACTIONS nection of cells to the cytoskeleton is required during muscle contraction. Deletion of b in- Aberrant cell–matrix interactions are involved tegrin or any member of the IPP complex leads in a large number of pathological conditions to detachment of muscles from the body wall such as cancer and various inflammatory dis- and embryonic lethality (Mackinnon et al. eases. Altered function of this signaling axis is 2002; Lin et al. 2003; Norman et al. 2007). however rarely the actual cause of these disor- On the cellular level, ILK-deficiency leads ders. In contrast, recent studies have identified to compromised cell adhesion, spreading a panel of genetic diseases whose etiology can and migration. This is because of defective be pinpointed to a mutation in a component

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Genetic Analyses of Integrin Signaling

of the adhesive machinery. The high degree of expression (Mory et al. 2008; Kuijpers et al. conservation in the components of this pathway 2009; Malinin et al. 2009; Svensson et al. has allowed the generation of mouse models for 2009). Transfection of patient’s lymphocytes these diseases that can be analyzed to generate with wild-type kindlin-3 was shown to restore mechanistic insights and therapeutic modalities integrin activation (Malinin et al. 2009; Svens- for clinical applications. son et al. 2009). In addition, kindlin-3-defi- cient mice recapitulate all symptoms of LAD III, firmly identifying defective kindlin-3 as Integrin Activation Diseases the cause of LAD III (Moser et al. 2008; Moser As discussed earlier, leukocyte and platelet et al. 2009a). In this respect, reconstitution of adhesion are cellular events in which integrin kindlin-3 expression provides a possible thera- affinity regulation is of key importance. Inher- peutic modality for this disease. Interestingly, ited human diseases with defects in these proc- the naturally occurring mutations have also esses have been described more than 25 years provided information on the structure-func- ago, but only recently, after the discovery of tion aspects of kindlin-3. A homozygous stop the central role of talin and kindlin in integrin codon identified in three patients occurs distal activation, the causes for these diseases have to the PTB-containing integrin-binding subdo- been identified. main of kindlin-3, suggesting that the carboxyl There are currently three distinct syndromes terminus of kindlin is required for its function that together constitute the leukocyte adhesion in integrin activation (Mory et al. 2008). A point deficiency (LAD) family of diseases. They affect mutation leading to a truncation in the middle distinct phases of the leukocyte adhesion cas- of the PH-domain was further shown to impair cade and therefore cause symptoms of variable membrane localization of kindlin-3 and thereby severity. LAD I, a disease that affects several block both lymphocyte adhesion and migra- hundreds of patients worldwide, results from tion, whereas a point mutation in the F2 sub- impaired firm adhesion of leukocytes, leading domain inhibited only the migration of these to recurrent severe infections and impaired cells, demonstrating that the functions of kind- wound healing. This disease is caused by a range lin in inside-out and outside-in signaling can be of mutations in the b2 integrin (ITGB2) gene, uncoupled (McDowall et al. 2010). including deletions, truncations, substitutions, The bleeding tendency of LAD III patients frame shifts, and intronic mutations, resulting closely resembles that of Glanzmann’s throm- in loss of protein expression or expression of basthenia (GT), a rare, autosomal recessive a truncated protein. LAD III (also known as bleeding disorder caused by mutations leading LAD I/variant), which has been more recently to quantitative or qualitative defects in platelet described, is characterized by similar symptoms aIIbb3 integrin. The genetic background is as LAD I. Interestingly, these patients also suffer very heterogeneous: more than 100 different from a bleeding tendency, indicating that addi- mutations in either the aIIb (ITGA2B) or b3 tional b subunits are involved. Indeed, these integrin (ITGB3) genes have been reported so patients show defects in b1, b2, and b3 integrin far (Kannan and Saxena 2009). Genetic ablation activation (Kuijpers et al. 1997; Alon and of the b3 integrin in mice almost fully reca- Etzioni 2003). In contrast to LAD I, no muta- pitulates the human disease, including gastroin- tions in integrin genes have been identified, testinal and cutaneous hemorrhage, increased and the cause of this disease remained unknown bleeding time, reduced platelet aggregation, until the role of kindlin in integrin activation and clot retraction (Hodivala-Dilke et al. was discovered. Subsequently, genetic sequenc- 1999). However, the heterogeneity in the ing of LAD III patients has revealed mutations mutation spectrum of the integrins found in in the kindlin 3 (FRMT3) gene in all patients GT patients also applies to the symptoms; sib- tested so far, leading to expression of a trun- lings sharing the same mutation can display cated protein, or reduction or loss of protein symptoms of varying severity, suggesting that

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polymorphisms in other genes involved in skin-blistering diseases (Uitto 2009). EB is a hemostasis could influence the phenotype. In group of highly variable disorders characterized addition, several GT patients lacking a mutation by fragility of skin leading to blistering and ero- in the aIIbb3 integrin have been identified sions on mechanical trauma. Genetic studies on (Kannan and Saxena 2009). As the bleeding different variants of EB have so far shown muta- phenotype of the kindlin-3-deficient mice and tions in 12 distinct genes encoding structural the LADIII patients closely resemble GT, it is components of the BM and adhesion proteins possible that altered function of this protein (Table 2). Interestingly, some of the EB patients might play a role in this disease as well. How- also develop muscular dystrophy, underlining ever, no polymorphisms in the kindlin-3 gene the pathophysiological link between these two in GT patients have been reported so far. Taken diseases. As the histopathology of these diseases together, integrin activation plays a central role is very complex, the identification of the causa- in immune function and hemostasis. The tive mutation has become a central diagnostic prominent role of kindlin-3 in these processes tool of EB (Nagy and McGrath 2010). Genetic together with its restricted expression pattern mouse models for the different EB variants makes it a promising candidate for anti-inflam- are available, and they have been used to work matory and antithrombotic therapies. out therapeutic strategies for these diseases (Natsuga et al. 2010). As a result, gene delivery, ectopic protein replacement as well as cellular Adhesion Strengthening Diseases therapies have already been tested on EB Another “class” of diseases involving impaired patients (Uitto 2009). cell–matrix interactions can be found affecting tissues that are subjected to high levels of CONCLUDING REMARKS mechanical stress such as the skin and skeletal muscle. The common denominator of these Two decades of genetic studies on cell-ECM in- disorders, namely muscular dystrophies and teractions have illustrated the importance of skin-blistering diseases, is that the symptoms adhesion signaling in the development of mul- are caused by weakened interactions of cells ticellular organisms as well as in disease. It is with their extracellular environment, leading now clear that these interactions not only pro- to tissue dysfunction and disruption. Another vide structural support and positional cues to common feature is that causative mutations for guide morphogenesis, but also act as signaling a single disorder can be found in either compo- platforms to regulate cell fate in a highly tis- nents of the ECM, their integrin receptors, or sue-specific manner. It is also evident that the adhesion-strengthening scaffold proteins. In relationship between ECM proteins and their the case of congenital muscular dystrophies, integrin receptors is complex: Integrins trans- although the dystrophin-dystroglycan-laminin/ mit information from the ECM to the cell, but agrin axis is a common target (Yurchenco also regulate the deposition and remodeling of 2010), other mutations affect the laminin-211/ the matrix itself. In addition, the ECM func- a7b1 integrin/plectin axis, leading to defects tions as a reservoir for growth factors, whereas in organization and migration of myoblasts, integrins cross talk with growth factor signal- subsequent myofiber degeneration and progres- ing on various levels. Because of this complex- sive muscle weakness (for more detailed review ity, mechanistic interpretations of the various see Kanagawa and Toda 2006; Mendell et al. phenotypes caused by genetic ablation of indi- 2006). Clinical features, cellulardefects andtheir vidual components of the adhesion signaling corresponding causative mutations of muscular machinery have proven to be difficult. Thus, a dystrophies resulting from defective cell-ECM major challenge for the future is to understand interaction are summarized in Table 1. which types of intracellular signals directly ema- The same principles are true for epidermol- nate from integrin adhesions, and how these ysis bullosa (EB), the prototype of human signals are propagated. This requires generation

14 Cite this article as Cold Spring Harb Perspect Biol 2011;3:a005116 Downloaded from ieti ril as article this Cite Table 1. Selected mutations causing muscular dystrophy and their corresponding mouse models http://cshperspectives.cshlp.org/ Human disease Central clinical features Affected gene (protein) Cellular defect Mouse model Reference Limb-Girdle Early adulthood onset MYOT (myotilin) Sarcomeric organization N/A– muscular proximal muscle weakness, dystrophy nasal pattern of speech odSrn abPrpc Biol Perspect Harb Spring Cold Severe, early onset muscle SGCA (a sarcoglycan) Muscle membrane KO (Duclos et al. 1998) weakness SGCB (b sarcoglycan)integrity KO (Durbeej et al. 2000) SGCG (g sarcoglycan) KO (Sasaoka et al. 2003) SGCD (d sarcoglycan) KO (Coral-Vazquez et al. 1999) Late onset muscle weakness TTN (titin) Sarcomere contraction N/A–

and atrophy, /relaxation onOctober1,2021-PublishedbyColdSpringHarborLaboratoryPress cardiomyopathy Relatively mild muscle TCAP (telethonin) Sarcomere stability N/A– weakness, variable clinical features 2011;3:a005116 Congenital Early onset with variable ITGA7 (integrin a7) Impaired muscle KO (Mayer et al. 1997) muscular symptoms, hypotonia, cell–matrix interactions dystrophy torticollis Early onset hypotonia and COL6A1 Stability of extracellular KO a (Bonaldo et al. 1998) muscle weakness COL6A2 matrix COL6A3 (type VI collagen) Severe, early onset muscle LAMA2 (laminin 211) Attachment, migration Dy/Dy (naturally occurring (Sunada et al. 1994) Signaling Integrin of Analyses Genetic weakness, white matter and organization of mutant) hypodensity, mental myoblasts Dy2j/Dy2j (naturally (Xu et al. 1994) retardation occurring mutant) DyPas/DyPas (naturally (Besse et al. 2003) occurring null allele) Dy3K/Dy3K (KO) (Miyagoe et al. 1997) DyW/DyW (hypomorph) (Kuang et al. 1998) Duchenne Severe, early onset muscle DMD (dystrophin) Stabilization of membrane Mdx (naturally occurring null (Bulfield et al. 1984) muscular weakness with respiratory, during contraction allele) dystrophy orthopedic and cardiac Mdx 2Cv, Mdx 3Cv (Chapman et al. 1989) complications Mdx 4Cv, Mdx 5Cvb Mdx 52 (deletion of exon 52) (Araki et al. 1997) 15 aAblation of the col6a1 gene. bMutants recovered from ENU chemical mutagenesis screen. Abbreviations: N/A, not analyzed; KO, knockout mouse; Dy, Dystrophin; mdx, X chromosome-linked muscular dystrophy. Downloaded from ..Wcsr¨me al. Wickstro¨ et S.A. m 16 Table 2. Mutations causing epidermolysis bullosa (EB) and their corresponding mouse models http://cshperspectives.cshlp.org/ Site of tissue Human disease separation Affected gene (protein) Mouse model Mouse skin phenotype Reference Epidermolysis Intraepidermal PKP1 (plakophilin-1) N/A–– bullosa simplex DSP (desmoplakin) N/A–– (EBS) KRT5 (keratin-5) KOa Severe blistering, loss of keratin (Peters et al. 2001) filaments KRT14 (keratin-14) KO Blistering, decreased keratin (Lloyd et al. 1995) filaments K14-R131C KIa Severe blistering (Cao et al. 2001) K14-DCT TG Blistering (Vassar et al. 1991) onOctober1,2021-PublishedbyColdSpringHarborLaboratoryPress PLEC1 (plectin) KOa Blistering, reduced stability and (Andra et al. 1997) number of hemidesmosomes Conditional KO Blistering, skin fragility (Ackerl et al. 2007) Junctional Lamina lucida ITGA6 (integrin a6) KOa Severe blistering, loss of (Georges-Labouesse et al. 1996a) ieti ril as article this Cite epidermolysis hemidesmosomes bullosa (JBS) ITGB4 (integrin b4) KOa Severe blistering, loss of (Dowling et al. 1996; van der Neut hemidesmosomes et al. 1996) ITGB4-DCT KIa Severe blistering, loss of (Murgia et al. 1998) hemidesmosomes, hypoproliferation odSrn abPrpc Biol Perspect Harb Spring Cold Conditional KO Blistering, loss of hemidesmosomes (Raymond et al. 2005) LAMA3 (laminin-332) KOa Severe blistering, abnormal (Ryan et al. 1999) hemidesmosomes, reduced cell survival LAMB3 (laminin-332) Spontaneous null allele Blistering, abnormal (Kuster et al. 1997) hemidesmosomes LAMC2 (laminin-332) KOa Blistering, rudimentary (Meng et al. 2003) hemidesmosomes, apoptosis Spontaneous Blistering, ulcerations, (Bubier et al. 2010) hypomorphic allele hyperkeratosis, rudimentary 2011;3:a005116 hemidesmosomes Dystrophic Sub-lamina densa COL7A1 (type VII collagen) KOa Severe blistering, absence of (Heinonen et al. 1999) epidermolysis anchoring fibrils bullosa (DBS) COL7 hypomorph Severe blistering, decreased (Fritsch et al. 2008) anchoring fibrils Kindler syndrome Primarily lamina FERMT1 (kindlin-1) KOa Skin atrophy (Ussar et al. 2008) lucida aLeads to early postnatal lethality. Abbreviations used: N/A, not analyzed; KO, knock-out mouse; K14-R131C, Arginine-131 to Cysteine substitution in the Keratin-14 gene; KI, knock in mouse; K14-DCT, carboxy- terminal truncation of Keratin-14; ITGB4-DCT, deletion of the entire cytoplamsic domain of integrin b4; TG, transgenic mouse. Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

Genetic Analyses of Integrin Signaling

of more sophisticated mouse models, such as Astrof S, Crowley D, Hynes RO. 2007. Multiple cardiovascu- knock-in mice carrying point mutations that lar defects caused by the absence of alternatively spliced segments of fibronectin. Dev Biol 311: 11–24. only partially disrupt protein function together Bader BL, Rayburn H, Crowley D, Hynes RO. 1998. Exten- with reporter mice for specific signaling events, sive vasculogenesis, angiogenesis, and organogenesis transcriptional changes, or changes in protein- precede lethality in mice lacking all av integrins. Cell protein interactions or conformations. In addi- 95: 507–519. Bauer M, Brakebusch C, Coisne C, Sixt M, Wekerle H, tion, a large majority of the cell-biological anal- Engelhardt B, Fa¨ssler R. 2009. Beta1 integrins differen- yses on integrin signaling are still performed tially control extravasation of inflammatory cell subsets on rigid 2D-surfaces, which very poorly reca- into the CNS during autoimmunity. Proc Natl Acad Sci pitulate the more compliant and complex 3D 106: 1920–1925. Bazigou E, Xie S, Chen C, Weston A, Miura N, Sorokin L, environment of tissues. As our understanding Adams R, Muro AF, Sheppard D, Ma¨kinen T. 2009. of the biophysical properties of the ECM in tis- Integrin-a9 is required for fibronectin matrix assembly sues steadily increases, a major goal of future during lymphatic valve morphogenesis. Dev Cell 17: research is to translate this knowledge into gen- 175–186. Bertotti A, Comoglio PM, Trusolino L. 2006. b4 integrin eration of more relevant in vitro models for cell activates a Shp2-Src signaling pathway that sustains biological studies of integrin signaling. HGF-induced anchorage-independent growth. J Cell Biol 175: 993–1003. Besse S, Allamand V, Vilquin JT, Li Z, Poirier C, Vignier N, ACKNOWLEDGMENTS Hori H, Guenet JL, Guicheney P. 2003. Spontaneous muscular dystrophy caused by a retrotransposal insertion The authors apologize to all those whose work in the mouse laminin a2 chain gene. Neuromuscul Disord could not be cited because of space restrictions 13: 216–222. Bonaldo P,Braghetta P,Zanetti M, Piccolo S, VolpinD, Bres- and for not always citing all the primary litera- san GM. 1998. Collagen VI deficiency induces early onset ture. The authors would like to thank Max myopathy in the mouse: An animal model for Bethlem Iglesias for artwork. The work of the Wickstro¨m myopathy. Hum Mol Genet 7: 2135–2140. and Fa¨ssler laboratories are funded by the Max Brakebusch C, Grose R, Quondamatteo F, Ramirez A, Jor- cano JL, Pirro A, Svensson M, Herken R, Sasaki T, Timpl Planck Society. R, et al. 2000. Skin and hair follicle integrity is crucially dependent on b1 integrin expression on keratinocytes. EMBO J 19: 3990–4003. REFERENCES Brown NH, Gregory SL, Rickoll WL, Fessler LI, Prout M, White RA, Fristrom JW. 2002. 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Genetic Analyses of Integrin Signaling

Sara A. Wickström, Korana Radovanac and Reinhard Fässler

Cold Spring Harb Perspect Biol 2011; doi: 10.1101/cshperspect.a005116 originally published online December 30, 2010

Subject Collection Extracellular Matrix Biology

Extracellular Matrix in Development: Insights from Extracellular Matrix Degradation and Remodeling Mechanisms Conserved between Invertebrates in Development and Disease and Vertebrates Pengfei Lu, Ken Takai, Valerie M. Weaver, et al. Nicholas H. Brown Extracellular Matrix Proteins in Hemostasis and Overview of the Matrisome−−An Inventory of Thrombosis Extracellular Matrix Constituents and Functions Wolfgang Bergmeier and Richard O. Hynes Richard O. Hynes and Alexandra Naba The Thrombospondins Integrins in Cell Migration Josephine C. Adams and Jack Lawler Anna Huttenlocher and Alan Rick Horwitz Cross Talk among TGF-β Signaling Pathways, Fibronectins, Their Fibrillogenesis, and In Vivo Integrins, and the Extracellular Matrix Functions John S. Munger and Dean Sheppard Jean E. Schwarzbauer and Douglas W. DeSimone Heparan Sulfate Proteoglycans Extracellular Matrix: Functions in the Nervous Stephane Sarrazin, William C. Lamanna and System Jeffrey D. Esko Claudia S. Barros, Santos J. Franco and Ulrich Müller The Collagen Family Molecular Architecture and Function of Matrix Sylvie Ricard-Blum Adhesions Benjamin Geiger and Kenneth M. Yamada Tenascins and the Importance of Adhesion Cell-Extracellular Matrix Interactions in Normal Modulation and Diseased Skin Ruth Chiquet-Ehrismann and Richard P. Tucker Fiona M. Watt and Hironobu Fujiwara Integrin Structure, Activation, and Interactions Genetic Analyses of Integrin Signaling Iain D. Campbell and Martin J. Humphries Sara A. Wickström, Korana Radovanac and Reinhard Fässler

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