Fibrillins: From Biogenesis of Microfibrils to Signaling Functions

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Fibrillins: From Biogenesis of Microfibrils to Signaling Functions Dirk Hubmacher,* Kerstin Tiedemann,* and Dieter P. Reinhardt *,{ *Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University Montreal, Quebec, H3A 2B2, Canada { Division of Biomedical Sciences, Faculty of Dentistry, McGill University Montreal, Quebec, H3A 2B2, Canada

I. Structure of Fibrillins II. Fibrillinopathies III. Fibrillin‐Containing Microfibrils A. Properties of Microfibrils B. Biogenesis of Microfibrils IV. Developmental Expression of Fibrillins A. Fibrillins in Early Avian Development B. Fibrillins in Mammalian Development V. Fibrillins and Growth Factors VI. Mouse Models VII. Conclusions Acknowledgments References

Fibrillins are large proteins that form extracellular microfibril suprastructures ubiquitously found in elastic and nonelastic tissues. Mutations in fibrillin‐1 and ‐2 lead to a number of heritable connective tissue disorders generally termed fibrillinopathies. Clinical symptoms in fibrillinopathies manifest in the skeletal, ocular, and cardiovascular systems and highlight the importance of fibrillins in development and homeostasis of tissues and organs, including blood vessels, bone, and eye. Microfibrils appear to have dual roles in (1) conferring mechanical stability and limited elasticity to tissues, and (2) modulating the activity of growth factors of the transforming growth factor beta (TGF‐ ) superfamily. This chapter’s focus is on the biogenesis of microfibrils, developmental expression patterns of fibrillins, signaling functions of microfibrils, and mouse models deficient in fibrillins. ß 2006, Elsevier Inc.

Current Topics in Developmental Biology, Vol. 75 Copyright 2006, Elsevier Inc. All rights reserved.

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0070-2153/06 $35.00 DOI: 10.1016/S0070-2153(06)75004-9

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I. Structure of Fibrillins The fibrillins constitute a family of large approximately 350‐kDa extracellular cysteine‐rich glycoproteins. Fibrillins are phylogenetically conserved proteins present in species from jellyfish to man (Corson et al., 1993; Lee et al., 1991; Maslen et al., 1991; Nagase et al., 2001; Reber‐Mu¨ller et al., 1995; Zhang et al., 1994). The three members of this family, fibrillin‐1, ‐2, and ‐3, are characterized by a highly conserved modular domain organization, while the homology on the amino acid level is about 61–69% (Fig. 1). The most prominent domain in fibrillins is the epidermal growth factor (EGF)‐like domain present 46–47 times. It contains six highly conserved cysteine residues stabilizing the structure by three disulfide bonds in a 1–3, 2–4, 5–6 arrangement (Campbell and Bork, 1993; Downing et al., 1996). The majority of the EGF domains in fibrillins (42–43) have a (D/N)X(D/N)(E/Q) Xm(D/N*)Xn(Y/F) consensus sequence for calcium binding (cb) in the N‐terminal pocket of the domain, where m and n are variable numbers of amino acid residues and the asterisk indicates a potential ‐hydroxylation site (Handford et al., 1991). Depending on the adjacent domains, the cbEGF domains bind calcium with diVerent aYnities ranging from the low nanomolar to the low micromolar range (Handford, 2000; Jensen et al., 2005). Homologous cbEGF domains are widely distributed in numerous extracellular matrix (ECM) and serum proteins. In contrast, the transforming growth factor (TGF)‐binding protein domain (TB or 8‐Cys) is only present in fibrillins and latent TGF‐ –binding proteins (LTBPs), which led to the concept of the fibrillin/LTBP superfamily (Fig. 1). The TB/8‐Cys domain is characterized by the presence of eight cysteine residues, three of which are arranged in tandem as an unusual Cys–Cys–Cys motif. All of the eight cysteine residues are involved in intradomain disulfide bonds organized in a 1–3, 2–6, 4–7, 5–8 pattern (Lack et al., 2003; Lee et al., 2004; Yuan et al., 1997). The TB/8‐Cys domain is found seven times in fibrillins and three times in LTBPs typically interrupting arrays of cbEGF domains (Fig. 1). In addition, fibrillins and LTBPs contain two and one hybrid domain respectively, which may have phylogenetically evolved by fusion of EGF and TB/8‐Cys domains (Corson et al., 1993). The structure of the hybrid domain is still unknown, but it is predicted that its cysteine residues also form intradomain disulfide bonds. However, the first hybrid domain in fibrillins contains nine cysteine residues and, consequently, at least one is not involved in intradomain disulfide bonds (Corson et al., 1993; Reinhardt et al., 2000). The N‐terminal domain contains four conserved cysteine residues and shares minor homology with some of the four‐cysteine domains in LTBPs. The C‐terminal domain is characterized by two conserved cysteine residues and shares some moderate

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Figure 1 Modular structure of human fibrillin/LTBP family members. Numbers above fibrillin‐1 indicate the relative numbers of cbEGF domains in the molecule. For simplicity, only the longest splice variant of each LTBP is indicated and suYxes correlating to the splice variant are omitted in the names. For a detailed overview about LTBP splice variants see Hyytia¨inen et al., 2004. The red bar indicates a region in fibrillin‐1 where mutations often lead to the severe neonatal Marfan syndrome. Binding sites for SL‐TGF‐ in LTBPs are indicated by asterisks.

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homology with C‐terminal domains of members of the fibulin family (Giltay et al., 1999). The three fibrillin isoforms diVer significantly in a few important features (Fig. 1). A small domain located in the first quarter of the molecule is rich in proline residues in fibrillin‐1, rich in glycine residues in fibrillin‐2, and rich in both proline and glycine residues in fibrillin‐3. The homologies of these domains on the amino acid level are relatively low, indicating a specific function for these domains in each fibrillin isoform. Other structural diVerences include the number and position of integrin‐binding sequences Arg‐Gly‐Asp (RGD) and N‐glycosylation sites (Asn‐Xaa‐Ser/Thr).

II. Fibrillinopathies Mutations in fibrillins give rise to a number of heritable connective tissue disorders summarized as fibrillinopathies. Mutations in fibrillin‐1 have been found to cause various forms of Marfan syndrome, familial ectopia lentis, MASS syndrome, familial aortic aneurysm/dissection, Shprintzen–Goldberg syndrome, systemic sclerosis, and dominant Weill–Marchesani syndrome (for reviews see Charbonneau et al., 2004; Pyeritz, 2000; Robinson et al., 2002). Mutations in fibrillin‐2 are known to cause congenital contractural arachnodactyly (CCA) or Beals syndrome (Gupta et al., 2002; Park et al., 1998), and fibrillin‐3 may be involved in recessive Weill–Marchesani syndrome (Corson et al., 2004; Faivre et al., 2002). The autosomal dominant Marfan syndrome is the most common disorder associated with mutations in fibrillin‐1. Clinical symptoms develop primarily in the cardiovascular, skeletal, and ocular systems including mitral valve disease, progressive dilation of the aortic root, dolichostenomelia, arachnodactyly, scoliosis, and ectopia lentis. Dissection and rupture of the aortic wall is the major life‐threatening clinical complication. With the exception of a small number of recurrent mutations, the vast majority of the approximately 600 mutations in fibrillin‐1 known today are unique to families and include missense and nonsense mutations, as well as deletions, insertions, and splice site mutations (Collod‐Beroud et al., 2003). Mutations in the center of fibrillin‐1 (exons 24–32) frequently, but not always, result in the very severe neonatal Marfan syndrome (Gupta et al., 2002; Park et al., 1998) (Fig. 1). Inter‐ and intrafamilial variability is a common feature of Marfan syndrome, suggesting that other gene products play a modifying role in the pathogenesis of the disease. An in‐frame deletion in exon 41 of the fibrillin‐1 gene (FBN1) was identified in a family with autosomal dominant Weill–Marchesani syndrome (Faivre et al., 2003b). Weill–Marchesani syndrome is a connective tissue disorder characterized by short stature, brachydactyly, joint stiVness, and

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eye symptoms including ectopia lentis and microspherophakia (Faivre et al., 2003a). It will be of particular interest in the future to unravel the molecular pathogenetic mechanisms that lead to the very diVerent clinical symptoms observed in Marfan syndrome as opposed to Weill–Marchesani syndrome. Clinical symptoms in CCA overlap with some skeletal features found in Marfan syndrome. In contrast to Marfan syndrome, individuals with CCA are characterized by joint contractures and abnormally shaped (crumpled) ears. Cardiovascular and ocular complications are usually absent (Viljoen, 1994). Similar to the mutations in FBN1 causing neonatal Marfan syndrome, mutations in FBN2 resulting in CCA are clustered in the central region of fibrillin‐2 suggesting that this region has important properties presumably in all fibrillin isoforms.

III. Fibrillin‐Containing Microfibrils A. Properties of Microfibrils Fibrillins comprise the major part of multicomponent aggregates called microfibrils, which are ubiquitously distributed in most tissues (Low, 1962). In elastic tissues, such as blood vessels, lung, and skin, microfibrils are thought to play a crucial—but yet unknown—role in the formation of elastic fibers by providing a scaVold for the developmentally regulated deposition of tropoelastin, the precursor of mature elastin (Mecham and Davis, 1994). In mature elastic fibers, microfibrils provide the outer fibrous layer. However, microfibrils are also found in the absence of elastin in many tissues, such as kidney or the ciliary zonules of the eye, either as individual entities or intersecting with basement membranes (Kriz et al., 1990; Raviola, 1971). Microfibrils without elastin appear to function as stress‐bearing entities. Ultrastructural analyses of microfibrils in tissues have revealed relatively uniform and threadlike structures with 10–12 nm in diameter (Fahrenbach et al., 1966; Greenlee et al., 1966; Low, 1962). Isolated microfibrils extracted from tissues using enzymatic digestion or tissue homogenization, however, display a typical beads‐on‐a‐string ultrastructure with periodicities of 50–55 nm in the relaxed state (Keene et al., 1991; Ren et al., 1991; Wallace et al., 1991; Wright and Mayne, 1988) (Fig. 2). Microfibrils not treated with enzymes or mechanical disruption do not display the typical interbead domains, suggesting that components are lost from the interbead region during conventional extraction procedures (Davis et al., 2002). It has been shown that the periodicities of a beads‐on‐a‐string microfibril can be stretched to more than 100 nm (Keene et al., 1991; Reinhardt et al., 1996). Bundles of microfibrils as well as individual microfibrils can be reversibly extended up to about 100 nm of periodicity, but irreversible deformation

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Figure 2 Sequence of fibrillin assembly steps into microfibrils. Mesenchymal cells synthesize and secrete fibrillins. Processing of the terminal ends by proprotein convertases occurs during or directly after secretion. Processed molecules possibly interact with cell surface proteoglycans (yellow) or integrins (red) for proper alignment and concentration. N‐ to C‐terminal self‐ assembly is mediated through regions at the molecular ends followed by reducible (disulfide bonds) and nonreducible transglutaminase‐mediated cross‐link formation. Elongation is likely mediated through central parts of the fibrillin molecules leading to a detectable fibrillin network by indirect immunofluorescence. Other microfibril components interact with this network. Further maturation events lead to the ‘‘beads‐on‐a‐string’’ microfibrils and ultimately to tissue microfibrils.

typically occurs at higher periodicities (Baldock et al., 2001; Eriksen et al., 2001; Haston et al., 2003). Bundles of parallel microfibrils are regularly aligned and spaced by an axial 1/3 stagger (Wess et al., 1998). Besides the organization in microfibrils, fibrillins may be organized diVerently in association with basement membranes. For example, specific antibodies against fibrillin‐1 label some zones in the epidermal–dermal basement membrane in the absence of any microfibrillar structures (Dzamba et al., 2001). The structural basis for this type of organization, however, remains to be clarified. The molecular organization of fibrillin monomers in microfibrils has been analyzed by various groups. Labeling of extracted microfibrils with specific antibodies, high‐resolution structure of cbEGF and TB/8‐Cys domains, analysis of intramolecular transglutaminase cross‐links, scanning

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transmission electron microscopy, atomic force microscopy, and automated electron tomography resulted in various models for the static alignment of fibrillins in microfibrils (Baldock et al., 2001; Downing et al., 1996; Lee et al., 2004; Qian and Glanville, 1997; Reinhardt et al., 1996; Sakai et al., 1991). A recent review highlighted these aspects and discussed the controversial models (Kielty et al., 2005). Despite the diVerences of these models in terms of stagger and molecular condensation of individual molecules, common to all models is a head‐to‐tail orientation of fibrillin‐1 molecules in the microfibril as originally proposed by Sakai and coworkers in 1991. Another commonly accepted property of microfibrils is the involvement of 6–8 fibrillin molecules per cross section of the interbead region (Baldock et al., 2001; Wallace et al., 1991; Wright and Mayne, 1988).

B. Biogenesis of Microfibrils The individual steps of the biogenesis of fibrillin‐containing microfibrils described in the following paragraphs are depicted graphically in Fig. 2. Fibrillins are secreted from the cells as proproteins of approximately 350 kDa, which are processed to a mature approximately 320‐kDa form (Milewicz et al., 1992, 1995). A number of studies identified members of the proprotein convertase family to be responsible for processing of fibrillin‐1 (Lo¨nnqvist et al., 1998; Milewicz et al., 1995; Raghunath et al., 1999; Ritty et al., 1999; Wallis et al., 2003). This endoprotease family includes furin and various related enzymes processing numerous proproteins after the tribasic consensus motif Arg‐Xaa‐(Lys/Arg)Arg (Molloy et al., 1992; Taylor et al., 2003). In fibrillins, matching sequences are located within both the N‐ and the C‐terminal domains. These sequence motifs are conserved between all fibrillin isoforms of all species analyzed so far. Evidence for utilization of this consensus sequences in fibrillin‐1 comes from site‐directed mutagenesis of the C‐terminal recognition sequence at various positions and from direct sequencing of authentic fibrillin‐1 isolated from cell culture and of recombinant N‐ and C‐terminal fibrillin‐1 fragments (Lo¨nnqvist et al., 1998; Raghunath et al., 1999; Reinhardt et al., 1996, 2000; Ritty et al., 1999). Although the majority of studies focused on processing of fibrillin‐1, by analogy it is predicted that other fibrillin isoforms are processed in an identical manner. Proprotein processing of fibrillin isoforms is predicted to result in the release of a small propeptide (16–48 amino acid residues) from the N‐termini and a larger fragment (120–140 amino acid residues) from the C‐termini. The precise location for fibrillin processing is discussed controversially, but evidence accumulates that processing occurs as fibrillin is secreted from the cells into the extracellular compartment or shortly thereafter as opposed to intracellular processing early in the secretory pathway

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(Milewicz et al., 1992, 1995; Ritty et al., 1999; Wallis et al., 2003). How fibrillins escape processing in the trans‐Golgi network in which proprotein convertases are active remains to be established. The fact that only the processed form of fibrillin‐1 becomes incorporated into the ECM suggested that profibrillin‐1 conversion to mature fibrillin‐1 plays a regulatory role in fibrillin‐1 assembly into microfibrils (Milewicz et al., 1992). Presently, the mechanism how propeptides can prevent assembly of fibrillin‐1 and other fibrillin isoforms is not known. Perhaps they mask important epitopes at the N‐ and C‐termini of fibrillin monomers important for self‐assembly. The C‐terminal propeptide was detected in proteomic analyses of mature isolated microfibrils, suggesting an additional role of this propeptide in microfibrils after it is cleaved during proprotein processing (Cain et al., 2006). After processing of the propeptides, fibrillin assembly into multimeric structures proceeds further likely on or close to the cell surface. An RGD sequence motif in TB/8‐Cys4 of fibrillin‐1 mediates cell binding via integrin receptors 5 3 (Lee et al., 2004; PfaV et al., 1996; Sakamoto et al., 1996) and 5 1 (Bax et al., 2003). In addition, it has been demonstrated that integrin 8 1 on cardiac fibroblasts can interact with fibrillin‐1 (Bouzeghrane et al., 2005). It will be important to define the potential role of integrin receptors in the fibrillin assembly process. It is possible that the fibrillin–integrin interaction is similarly important for multimerization as it is for fibronectin assembly in which 5 1 integrin induces conformational activation necessary for fibril formation (Mao and Schwarzbauer, 2005). Regardless of the potential functions of integrins, initial steps in microfibril biogenesis involve fibrillin self‐assembly mechanisms. Full length recombinant fibrillin‐1 spontaneously forms multimers in solution and the N‐ and C‐terminal halves of recombinant fibrillin‐1 interact with each other with high aYnity (Lin et al., 2002). These results were further substantiated by analyses of smaller overlapping fibrillin‐1 fragments in various ligand interaction assays, positioning the interaction sites to the N‐terminal region encoded by exons 1–8 (N‐terminus to cbEGF2) and the C‐terminal region encoded by exons 57–65 (TB/8‐Cys7 to processed C‐terminus) (Marson et al., 2005). These data explain the exclusive head‐to‐tail arrangement of fibrillin molecules in microfibrils. In addition to linear head‐to‐tail interactions, lateral homotypic interactions in diVerent regions of the fibrillin‐1 molecule may play a role in stabilizing initial multimers or lateral associations of individual microfibrils (Ashworth et al., 1999; Marson et al., 2005; Trask et al., 1999). Mature microfibrils can contain both fibrillin‐1 and fibrillin‐2 in the same microfibril and both molecules can heterotypically interact in an N‐to‐C‐terminal fashion (Charbonneau et al., 2003; Lin et al., 2002). Fibrillin‐3 is also present in microfibrils, but it is currently not known whether it can interact with the other fibrillin isoforms to form heterotrimeric fibrillin aggregates (Corson et al., 2004).

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Self‐assembly mechanisms are essential for initial steps in microfibril biogenesis, but they are not suYcient for further maturation of microfibrils. Intermolecular disulfide bonds between fibrillins or between fibrillin and other molecules form within a few hours in organ cultures of chick aorta (Reinhardt et al., 2000). One cysteine residue in the first hybrid domain of fibrillin‐1 (Cys204) and fibrillin‐2 (Cys233) has been identified as a free thiol and may, thus, be able to contribute to such cross‐links (Reinhardt et al., 2000). It is presently not clear whether intermolecular disulfide bond formation requires the presence of specific enzymes in the extracellular space, or whether they origin from spontaneous oxidation of exposed and properly aligned cysteine residues. Another type of cross‐links involved in biogenesis of microfibrils are the nonreducible e( ‐glutamyl)lysine cross‐links catalyzed by transglutaminases. It has been reported that mature microfibrils contain a significant number of transglutaminase cross‐links (Bowness and Tarr, 1997; Qian and Glanville, 1997; Thurmond et al., 1997). However, it is not clear at what stage of microfibril formation these cross‐links form. Covalent cross‐links between individual fibrillin monomers or between fibrillins and other components may provide mechanical stability (Thurmond and Trotter, 1996). Potentially, transglutaminase as well as disulfide cross‐links are critical for correct lateral alignment of fibrillin molecules to facilitate downstream assembly events. In addition to self‐assembly and cross‐linking mechanisms, other molecules may have essential roles in microfibril biogenesis. At least 17 components have been reported to be associated with microfibrils either as integral or peripherally associated constituents (for review see Kielty et al., 2002). For most of these microfibril‐associated ligands it is currently not known whether they have a role in microfibril biogenesis, stability, or homeostasis. A number of articles reported the presence of heparin/heparan sulfate‐ binding sites in the N‐terminal, the central, and the C‐terminal region of fibrillin‐1 (Cain et al., 2005; Ritty et al., 2003; Tiedemann et al., 2001). Addition of heparin or heparan sulfate to fibroblasts inhibited the formation of a fibrillin‐1 network, which is the precursor of mature microfibrils in cell culture systems (Ritty et al., 2003; Tiedemann et al., 2001). This observation led to the hypothesis that heparan sulfate, which is a component of various proteoglycans, plays a role in nucleating or modifying the assembly process of fibrillin‐1 (Tiedemann et al., 2001). This view is further substantiated by the fact that inhibition of sulfation, a critical process in maturation of heparan sulfate, or inhibition of heparan sulfate biosynthesis also compromises fibrillin network formation in cell culture (Tiedemann et al., 2001; Trask et al., 2000). Since fibrillin assembly is believed to take place on the cell surface or in the pericellular space, it is possible that proteoglycans present in these locations may play a critical role in fibrillin assembly. In this regard, Tiedemann et al. (2005) demonstrated that perlecan interacts

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with fibrillin‐1 and that the heparan sulfate component of perlecan is involved in this interaction. This study demonstrated a reduced number of microfibrils at basement membrane–microfibril interfaces in perlecan deficient mice, suggesting a potential role for perlecan in the biogenesis of microfibrils.

IV. Developmental Expression of Fibrillins A. Fibrillins in Early Avian Development The development of specific monoclonal antibodies for fibrillin‐1 (Sakai et al., 1986) and fibrillin‐2 (Wunsch et al., 1994), both reactive against the respective avian fibrillin isoforms, allowed detailed analyses of protein expression in developing chicken and quail embryos. In general, both fibrillin isoforms are expressed early in development starting in gastrulation stage avian embryos. Fibrillin‐1 is deposited at multiple sites in the early chicken embryo primarily at regions where cellular rearrangements occur along the primary axis, including Hensen’s node followed by an association with the mesocardium, the notochord, and the margins of the somitic field (Gallagher et al., 1993). In subsequent stages, fibrillin‐1 shows a broad distribution in all tissues (Burke et al., 2000). Similarly, fibrillin‐2 immunolocalizes to early midline structures, including Hensen’s node, the primitive streak, notochord, and mesodermal structures flanking the midline (Sugi and Markwald, 1996; Wunsch et al., 1994). Subsequent spatiotemporal distribution of fibrillin‐2 in the developing heart suggested a relationship to the earliest events in cardiac development, including definition of the heart‐forming fields, formation of the primary heart tube, and segmental transformation of a subpopulation of endothelial cells into cushion mesenchyme (Wunsch et al., 1994). Fibrillin‐2 is expressed asymmetrically between the left and right heart‐forming fields of the presomitic stage chicken embryo suggesting that interactions between cardiocytes and fibrillin‐2 may contribute to heart laterality determination and looping (Smith et al., 1997). Rongish and colleagues (Rongish et al., 1998) have demonstrated that fibrillin‐2 incorporates into microfibrils that surround the newly formed somites and the lateral splanchnic mesoderm. Whether these microfibrils are homotypic, containing only the fibrillin‐2 isoform, or whether they are heterotypic in nature containing other fibrillin isoforms remains to be established. The association of fibrillin‐2 with early blood vessels appears to be variable. While fibrillin‐2 is expressed in the intima and the adventitia of coronary arteries and the aortic media in later stage avian embryos, as well as in small peripheral vessels of the avian body wall (Bouchey et al., 1996; Hungerford et al., 1996), it is absent from the

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dorsal aorta and lateral vascular networks in stage 7 embryos (Rongish et al., 1998). Only very limited information for fibrillin‐3 expression in chicken is available today. It was shown that the fibrillin‐3 protein is expressed in perichondrial layers surrounding the developing limbs (7 days) (Corson et al., 2004). Collectively, the data suggest that fibrillin‐1 and ‐2 are early markers for the morphogenesis of a number of rudiments derived from the mesoderm. Fibrillins are associated with primordial structures responsible for generation of cranial‐to‐caudal morphogenesis, including regression of Henson’s node, extension of the notochord, somite formation, and regression of the anterior intestinal portal.

B. Fibrillins in Mammalian Development In most tissues of the developing mouse including lung, blood vessels, bone, and cartilage, the fibrillin‐1 and ‐2 genes (Fbn1 and Fbn2) exhibit a diphasic expression pattern in which the onset of Fbn2 transcription occurs typically earlier than Fbn1 expression. In the cardiovascular system, however, Fbn1 transcription can be detected very early (E8.5–9) and is always higher than the Fbn2 gene activity (Yin et al., 1995; Zhang et al., 1995). It was concluded that fibrillin‐2 expression coincides with early morphogenesis, while fibrillin‐1 expression correlates with late morphogenesis and the development of well‐ defined organ structures (Zhang et al., 1995). The expression patterns originally supported the hypothesis that fibrillin‐1 provides structural support, whereas fibrillin‐2 regulates early processes of elastic fiber assembly. However, this hypothesis was not confirmed by subsequent studies since elastic fiber formation was apparently normal in fibrillin‐2‐deficient mice (Arteaga‐Solis et al., 2001; Carta et al., 2006). On the other hand, mice completely lacking fibrillin‐1 in an Fbn2þ/þ background demonstrated disorganized elastic fibers in the aortic wall of postnatal animals, whereas loss of both Fbn1 alleles in an Fbn2‐null background causes embryonic death after E14.5 (Carta et al., 2006). These findings emphasize a critical role for fibrillin‐1 in the maturation of the aortic wall and suggest a partial functional overlap of both fibrillins. In addition, identification of fibrillin‐1‐containing microfibrils anchoring endothelial cells to the subendothelial matrix in the developing mouse (E15) suggested a role for fibrillin‐1 for the integrity of the endothelial cell layer during early development of the vessel wall (Davis, 1994). The fibrillin‐3 gene is inactivated in the rodent genome possibly due to chromosome rearrangement events during mouse evolution, while the gene appears to be active in man, cow, and chicken (Corson et al., 2004).

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Fibrillins appear to have a morphogenetic role in normal lung and kidney development. In a rat model, fibrillin‐2 mRNA and protein have been detected during lung branching morphogenesis and it has been shown that treatment with fibrillin‐2 antisense oligonucleotides perturbs normal morphogenesis (Yang et al., 1999). Mice deficient in fibrillin‐1 show airspace enlargement of the lung resulting from developmental failure of distal alveolar septation. These perturbations predispose the mice to late onset destructive emphysema and were correlated with dysregulation of TGF‐ signaling and activation (Neptune et al., 2003). In rat kidney at E15, fibrillin‐1 is expressed in the metanephric mesenchyme, while at E18 expression was confined to blood vessels and glomeruli. Treatment with fibrillin‐1 antisense oligonucleotides induced marked dysmorphogenesis of the embryonic metanephroi (Kanwar et al., 1998). Studies in early human development from the fifth gestational week onward established that fibrillin‐1 and fibrillin‐2 followed a similar temporospatial distribution pattern in most embryonic and early fetal organs (Quondamatteo et al., 2002; Zhang et al., 1994). DiVerential expression of both fibrillins was observed in organs such as kidney, liver, rib anlagen, and notochord. Similar to the temporal expression pattern of fibrillin‐2, fibrillin‐3 mRNA and protein is found most abundantly in human fetal tissues, suggesting that fibrillin‐3 expression is also largely limited to early development (Charbonneau et al., 2003; Corson et al., 2004). The spatial expression patterns of fibrillin‐3 overlap with those of the other fibrillin isoforms in some tissues including skeletal elements and skin but diVer in other tissues such as kidney, lung, blood vessels, and brain (Corson et al., 2004; Nagase et al., 2001). Other studies focused on human skeletal development from the ninth gestational week onward (Keene et al., 1997; Zhang et al., 1994). In human fetal limbs (10–11 weeks of gestation), fibrillin‐1 is expressed in loose connective tissue around skeletal muscles and tendons and is widely expressed in developing limbs and digits at 16 weeks of gestation, except for the cartilage matrix. At this time and continuing through adulthood, the perichondrium contains abundant fibrillin‐1 microfibrils. By 20 weeks of gestation, a loose meshwork of immunofluorescent fibrillin‐1 fibers is also detected in the cartilage matrix. In postnatal (3‐day) long bones, fibrillin‐1 is found in fibrils that colocalize with LTBP‐1 in the outer periosteum, and in the cartilage fibrillin‐1 localizes to the perichondrium (Dallas et al., 2000). From the clinical phenotypes seen in Marfan syndrome, it is clear that fibrillin‐1 plays a pivotal role in the regulation of bone growth. The underlying molecular mechanism, however, is still obscure. Fibrillin‐containing microfibrils may limit bone growth by exerting tension in the periosteum or perichondrium. Alternatively, fibrillin‐1 may play a regulatory role in the growth plate during bone deposition.

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In summary, fibrillins exhibit a broad overlapping but distinct temporal‐ and tissue‐specific regulation during mammalian development. Fibrillin‐2 and ‐3 are preferentially expressed in many tissues during the developmental period, while fibrillin‐1 expression persists throughout life.

V. Fibrillins and Growth Factors A number of publications demonstrated that fibrillins and fibrillin‐containing microfibrils are involved in matrix deposition, storage, and activation of growth factors of the TGF‐ superfamily. These mediators regulate a broad array of developmental and homeostatic processes. This chapter’s focus is on the structural and functional relationship of TGF‐ s and bone morphogenetic protein (BMP)‐7 with the fibrillin/LTBP superfamily. The mammalian TGF‐ ‐1, ‐2, and ‐3 are synthesized as proproteins, containing the latency‐associated protein (LAP) and mature TGF‐ . Two polypeptide chains associate to form a disulfide‐bonded homodimer, which is proteolytically processed resulting in a noncovalent complex between LAP and mature TGF‐ (Gentry et al., 1988; Lawrence et al., 1984). This latent complex is referred to as small latent TGF‐ complex (SL‐TGF‐ ). LAP controls the activity of TGF‐ by maintaining its latency (Gentry et al., 1988). In most studied cell lines TGF‐ s are secreted as large latent TGF‐ complexes (LL‐TGF‐ ) consisting of SL‐TGF‐ covalently bound to a member of the LTBPs (reviewed in Hyytia¨inen et al., 2004; Koli et al., 2001; Rifkin, 2005; Saharinen et al., 1999). However, the major fraction of secreted LTBPs does not contain TGF‐ , suggesting a dual role for LTBPs as TGF‐ targeting molecules, and as structural components in the ECM (Miyazono et al., 1991; Taipale et al., 1994). LTBP‐1, ‐3, and ‐4, but not LTBP‐2, can interact with SL‐TGF‐ by direct disulfide bond formation between Cys33 in each LAP monomer and two cysteine residues in the penultimate TB/8‐Cys domain of the LTBP proteins (Gleizes et al., 1996; Saharinen and Keski‐Oja, 2000; Saharinen et al., 1996). LTBP‐1 and ‐3 can associate with the LAP propeptide of all three TGF‐ isoforms, while LTBP‐4 only associates with that of TGF‐ 1 (Chen et al., 2005; Saharinen and Keski‐Oja, 2000). A two amino acid insertion between cysteine residues 6 and 7 of the penultimate TB/8‐Cys domain of LTBPs is critical for the interaction with LAP (Saharinen and Keski‐Oja, 2000). Based on the solution structure, cysteine residues 2 and 6 of this domain have been suggested to participate in the intermolecular covalent interaction with the LAP dimer (Lack et al., 2003). The initial molecular contact between LAP and the TB/8‐Cys domain is mediated by electrostatic interactions (Chen et al., 2005; Lack et al., 2003). Since TB/8‐Cys domains are present in LTBPs and fibrillins, but not in other proteins, it was hypothesized that one or more

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of these domains in fibrillins may also mediate interaction with SL‐TGF‐ . However, TB/8‐Cys domains in fibrillins as well as in LTBP‐2 are missing the critical residues necessary for interaction with the LAP protein and are, thus, not able to interact directly with SL‐TGF‐ (Lack et al., 2003; Saharinen and Keski‐Oja, 2000). However, it has been demonstrated that fibrillins and fibrillin‐containing microfibrils can indirectly interact with TGF‐ through their interactions with LTBPs. In cell culture studies, LTBP‐1 colocalizes with fibrillin‐1 and fibronectin (Dallas et al., 2000, 2005; Taipale et al., 1996). In tissues, LTBP‐1 and latent TGF‐ 1 localization to fibrillin‐containing microfibrils was described in the following studies. In skin, LTBP‐1 and latent TGF‐ 1 are both detectable during the earliest stages of microfibril formation (Raghunath et al., 1998). In the developing long bone, LTBP‐1 colocalizes with fibrillin‐1 immunoreactive fibrils in the outer periosteum (Dallas et al., 2000). In the developing heart and in the cardiovascular system, LTBP‐1 is present on microfibrils in the endocardial cushion tissue and the aorta, and was found prominently colocalized with fibrillin‐1 in the neointima in an arterial injury model (Isogai et al., 2003; Nakajima et al., 1997; Sinha et al., 2002). LTBP‐2 has also been immunolocalized to microfibrils located on the surface of elastic fibers in fetal aorta and to fibrillin‐1‐labeled structures in arteries (Gibson et al., 1995; Sinha et al., 2002). Although LTBP‐2 cannot interact with LAP, it has been speculated that this isoform may target other growth factors to the microfibril system (Chen et al., 2005). However, experimental evidence to support this hypothesis is lacking. The TGF‐ ‐binding isoforms LTBP‐1 and ‐4, but not LTBP‐3, appear to interact with their C‐termini with fibrillin network structures produced by fibroblasts (Koli et al., 2005; Unso¨ld et al., 2001). This observation is consistent with in vitro studies that showed interaction of fibrillin‐1 with the C‐terminal region of LTBP‐1 and ‐4 but not of LTBP‐3 (Isogai et al., 2003). While the major interaction sites of LTBPs with ECM components are located at their N‐terminal regions and are stabilized by transglutaminase cross‐links (Nunes et al., 1997; Olofsson et al., 1995; Saharinen et al., 1996), the C‐terminal interactions with fibrillin appear to be of lower aYnity (Koli et al., 2005; Unso¨ld et al., 2001). It is possible that this particular property may be important for the physiological role of fibrillin and microfibrils in activation of TGF‐ . The molecular interaction between LTBP‐1 and microfibrils are mediated by noncovalent forces (Isogai et al., 2003). In fibrillin‐1, the interaction site with LTBP‐1 has been mapped to a multifunctional N‐terminal region spanning EGF2‐cbEGF1 (Charbonneau et al., 2004; Isogai et al., 2003). Sakai and coworkers suggested a model in which the C‐terminal association of LTBPs with microfibrils in addition to N‐terminal interactions with other matrix components is necessary to stabilize the LL‐TGF‐ in ECM, and it was speculated that loss of fibrillin‐1 may

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lead to destabilization of LL‐TGF‐ and activation of TGF‐ (Isogai et al., 2003). This hypothesis was further supported by the analysis of fibrillin‐ 1‐deficient mice demonstrating a developmental failure in distal alveolar septation in the lung, which was attributed to abnormal TGF‐ activation (Neptune et al., 2003). The molecular mechanism, however, how the LL‐ TGF‐ microfibril complex becomes destabilized remains to be established. Additional evidence for a role of microfibrils in TGF‐ regulation comes from a genetic duplication of the Fbn1 gene in the tight skin (Tsk) mouse (see Chapter 6 for a detailed description). The Tsk mouse is a model for human scleroderma and this disorder is associated with abnormal TGF‐ signaling (Denton and Abraham, 2001). Despite the controversial reports about the precise composition of microfibrils isolated from Tsk mice and fibroblasts, it seems clear that these microfibrils contain the abnormal Tsk fibrillin‐1 molecules (Gayraud et al., 2000; Kielty et al., 1998). Abnormal microfibrils in these mice may compromise the stabilization of LL‐TGF‐ in the ECM. This concept is further strengthened by the fact that autoantibodies against fibrillin‐1, which have been detected in human scleroderma, have been suggested to play a role in a competitive release mechanism in which the autoantibodies displace LL‐TGF‐ from the fibrillin molecule (Tan et al., 1999; Zhou et al., 2005). Normal activation of TGF‐ in LL‐TGF‐ can occur through diVerent mechanisms, including binding to various cell surface integrin receptors, interaction with thrombospondin‐1, and by proteolytic events in LL‐TGF‐ mediated, for example, by plasmin and matrix metalloproteinases (reviewed in Annes et al., 2003; Hyytia¨inen et al., 2004; Koli et al., 2001). In this regard, destabilization of the LL‐TGF‐ by structurally compromised fibrillin and microfibrils may trigger activation through one of these mechanisms. It is tempting to speculate that TB/8‐Cys domains in fibrillins as well as in LTBPs, which are not able to interact directly with SL‐TGF‐ , may be able to interact with proregions of other members of the TGF‐ superfamily including the BMPs. Recently, BMP‐7 was immunolocalized to fibrillin networks in skin and kidney capsules (Gregory et al., 2005). In this study, it was found that the prodomain of BMP‐7 directly interacts with an N‐terminal region of fibrillin‐1. BMP‐7 does not contain cysteine residues in its prodomains and, thus, it is predicted that the interaction mechanism with fibrillin‐1 and presumably with fibrillin‐2 and ‐3 is diVerent to what is known about the interaction of SL‐TGF‐ with LTBPs. The molecular interactions patterns of the BMP‐7 prodomain with overlapping recombinant fibrillin‐1 polypeptides suggested that the interaction cannot be attributed to a single TB/8‐Cys domain in fibrillin‐1 (Gregory et al., 2005). Fibrillin‐2 and BMP‐7 have been linked to the same genetic pathway by gene‐targeting experiments in mice (Arteaga‐Solis et al., 2001). In this study, homozygous mice deficient for fibrillin‐2 (Fbn2
/
) were born with

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temporary joint contractures and a limb‐patterning defect in form of bilateral syndactyly of soft and hard tissue. HaploinsuYcient mice (Fbn2þ/
) displayed no obvious phenotype. BMP‐7‐null mice are characterized by several developmental abnormalities including polydactyly, whereas mice heterozygous for BMP‐7 are phenotypically silent (Dudley et al., 1995; Luo et al., 1995). Analysis of combined heterozygosity for fibrillin‐2 and BMP‐7 revealed a limb phenotype that combines the patterning defects (polydactyly and syndactyly) of each homozygous mouse, suggesting that fibrillin‐2 and BMP‐7 interact with each other in some stage of the autopod development (Arteaga‐Solis et al., 2001). Complete absence of fibrillin‐2 may abolish targeting and/or deposition of BMP‐7 to microfibrils. In summary, new concepts have emerged over the last few years for a role of fibrillin‐containing microfibrils in extracellular storage and potential activation of growth factors of the TGF‐ superfamily either mediated through LTBPs or through direct interactions. This type of storage provides tissues rich in microfibrils with rapidly inducible and highly localized growth factor signals. It will be important in the future to identify all growth factors involved in this pathway and to understand the significance of microfibrils in the molecular physiology of these growth factors.

VI. Mouse Models The creation of a number of mouse models revealed important insights in functional roles of fibrillin‐1 and fibrillin‐2 in the development and homeostasis of microfibril‐rich tissues and contributed significantly to the understanding of pathogenetic mechanisms in fibrillinopathies (Table I). In mice, the fibrillin‐3 gene is inactive and is thus not accessible to gene targeting experiments (Corson et al., 2004). The mgD mice express a fibrillin‐1 protein with a central deletion at about 10% of the wild‐type level (Pereira et al., 1997). The deletion spans exons 19–24 of Fbn1 coding for domains cbEGF8‐TB/8‐Cys3 of fibrillin‐1. While heterozygous mgD/þ mice are indistinguishable from wild‐type littermates, the homozygous mgD/mgD mice die approximately 3 weeks after birth of cardiovascular complications including aneurysmal dilatation and dissection of the aortic wall. These data suggested that failure of microfibril structures to sustain hemodynamic stress in the adventitia is the primary reason for aortic dilatation. No skeletal abnormalities were observed in homozygous mutant mice. The fibrillin‐1 network produced by homozygous mgD/mgD fibroblasts was strongly reduced and resembled the network seen in fibroblasts derived from individuals with Marfan syndrome. However, microfibrils containing the mutant fibrillin‐1 were still assembled and elastic fibers developed normally, although focal fragmentation of elastic fibers was observed.

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Table I Overview of Fibrillin Mouse Modelsa

Model mgD

mgR

Tissue Phenotypeb

Deletion of exons 19–24 (cbEGF8‐ TB/8‐Cys3) in fibrillin‐1; mutant fibrillin‐1 is expressed at 10% of wild‐type level Normal fibrillin‐1 is expressed at 20–25% of the wild‐type level

Mice die at 3 weeks after birth due to cardiovascular complications (aortic dilatation and dissection); no skeletal phenotype

Fibrillin‐1 network from fibroblasts is reduced; mutant fibrillin‐1 assembles into microfibrils; focal fragmentation of elastic fibers

Mice die after 3–4 months of pulmonary and vascular insuYciency; kyphosis and overgrowth of ribs, but other long bones not aVected Mice die within 2 weeks after birth of vascular and pulmonary failure; elongated ribs but no additional bone phenotype; thinner skin; detached endothelial lining No phenotype

Reduced fibrillin‐1 deposition; 6 weeks after birth onset of focal calcification of aortic elastic lamellae

mgN

Fibrillin‐1 null

C1663R

Transgenic overexpression of human fibrillin‐1– containing mutation C1663R in a normal mouse background Mouse fibrillin‐1 with missense mutation C1039G

C1039G

Microfibril/Elastic Fiber Phenotypeb

Fibrillin AVected

Heterozygous mice live normal life span; aortic wall deterioration (2 months after birth); no death due to aortic dissection. Postnatal development of kyphosis and rib overgrowth Homozygous mice die perinatally due to vascular failure

Thin, wavy, and fragmented elastic fibers in whole aorta

No phenotype

Reduced microfibril deposition from hetero‐ and homozygous fibroblasts; late onset of elastic fiber fragmentation

(Continued)

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Table I Continued

Model

Fibrillin AVected

Tsk

In‐frame duplication of exon 17–40 in fibrillin‐1

Fbn2

Fibrillin‐2 null

sy

Multigene deletion including Fbn2 locus on chromosome 18 Frameshift mutation in Fbn2 (5051del A) generates a premature stop codon Exon skipping mutation leading to loss of exon 38 in Fbn2 coding for the second half of the fourth TB/8‐Cys domain in fibrillin‐2 Fibrillin‐1 and fibrillin‐2 double null

syfp

syfp‐2J

Fbn1
/
; Fbn2
/


Tissue Phenotypeb Heterozygous mice have thickened skin with decreased elasticity; myocardial fibrosis; emphysemalike condition; increased growth of bone and cartilage; normal life span Homozygous mice die at embryonic day 7–8 Mice are viable and fertile; bilateral syndactyly; temporary joint contractures; absence of vascular phenotype Variable fore‐ and hindlimb syndactyly; deafness; abnormal behavior Variable fore‐ and hindlimb syndactyly

Microfibril/Elastic Fiber Phenotypeb Tsk fibrillin‐1 incorporates into abnormal microfibrils

Disorganized microfibrillar patterns in interdigital tissues

Intact microfibrils

Intact microfibrils

Variable fore‐ and hindlimb syndactyly

Intact microfibrils

Embryonic lethality around E14.5

Delayed elastic fiber formation in aortic media

a

References are indicated in the text. Except where specifically mentioned, heterozygous animals or fibroblasts do not show any phenotype and the description is limited to homozygosity. b

In the mgR model, mice express a normal full‐length fibrillin‐1 protein at about 20–25% of the wild‐type level (Pereira et al., 1999). Homozygous mgR/mgR mice live significantly longer than the mgD/mgD mice and die at

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the age of a few months from pulmonary and vascular insuYciency. The mice display phenotypic features in the skeleton, including significant kyphosis and overgrowth of the ribs. However, other long bones showed only very little overgrowth, which is in contrast to clinical findings in Marfan syndrome. In the vascular system, medial calcification of elastic lamellae is the first pathological sign seen in these animals indicating that fibrillin‐1 or associated components are involved in the protection of elastic fibers against calcification. Intimal hyperplasia was typically evident by 9 weeks of age. A later stage of the vascular disease in mgR/mgR mice is characterized by an inflammatory‐fibroproliferative response, and inflammation‐mediated elastolysis may participate in the mechanical collapse of the aortic wall. Loss of the microfibrillar connections between smooth muscle cells and elastic laminae are believed to initiate this destructive process, which involves overproduction of structural matrix components and proteolytic enzymes such as matrix metalloproteinase‐9 (Bunton et al., 2001; Davis, 1993). A threshold theory was suggested for the development of aortic aneurysms and dissection depending on the total amount of functional microfibrils present in the tissue (Pereira et al., 1999). Ruptured aortic aneurysms in the mgD and the mgR mice are associated with morphologically normal elastic fibers between focal lesions and in unaVected tissues. Thus, it was originally concluded that fibrillin‐1 plays a role in tissue homeostasis rather than in the development of elastic fibers and tissues. In this scenario, disruption of the elastic network of the media may be a secondary event. Since fibrillin‐2 is generally expressed earlier than fibrillin‐1 and primarily during development, it was further hypothesized that fibrillin‐2 is predominantly involved in organizing microfibrils. However, generation of complete knockout mouse models of either fibrillin‐1 or fibrillin‐2 corrected this view. Mice without fibrillin‐1 (mgN/mgN) die within the first two weeks of postnatal life from ruptured aortic aneurysms and impaired pulmonary function, while heterozygous mgN/þ mice were viable and fertile (Carta et al., 2006). Similar to the mgR/mgR mice, homozygous null mice show malformed and elongated ribs, while overgrowth of other bones was not reported. Unlike the mgD and the mgR mutant mice and unlike individuals with Marfan syndrome, the aneurysms in the Fbn1‐null mice involved the ascending aorta rather than the aortic root. The elastic lamellar units in the medial layer were disorganized not only in lesions as found in the mgD and the mgR mutant mice but in the whole aorta. Thinner and disorganized elastic lamellae in the mgN/mgN mutant mice now suggest a key role for fibrillin‐1 in development and maturation of the elastic lamellae and the aortic wall especially during early postnatal life. Another unique finding in mgN/mgN mice was a detachment of the endothelial lining associated with loss of structural connections between the intima and the medial layers.

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These observations demonstrate that fibrillin‐1 is crucial for stabilization of microfibrils, which support this type of anchorage (Davis, 1994). Microfibrils isolated from mgN/mgN mice, consisting only of fibrillin‐2, showed an asymmetric ultrastructural appearance, which diVers from the appearance of other microfibrils. This suggests diVerences in the composition and/or assembly process of fibrillin‐2 into microfibrils compared to fibrillin‐1 microfibrils. Since fibrillin‐2 is expressed during development and typically earlier than fibrillin‐1, it was expected that fibrillin‐2 deficiency would result in a severe phenotype. However, complete ablation of fibrillin‐2 (Fbn2
/
) generated a relatively mild phenotype (Arteaga‐Solis et al., 2001). Homozygous mice are born with temporary joint contractures mimicking the clinical symptoms observed in CCA. The Fbn2
/
mice also showed bilateral syndactyly of the central digits in forelimbs and hindlimbs associated with reduced apoptosis of interdigital tissue during autopod development. Analysis of compound heterozygosity for fibrillin‐2 and BMP‐7 (Fbn2þ/
; Bmp7þ/
) revealed both syn‐ and polydactyly similar to the defects of each homozygous mouse, suggesting that fibrillin‐2 and BMP‐7 are in the same genetic program (Chapter 5). Carta and coworkers (Carta et al., 2006) proposed that fibrillin‐1 may compensate for the loss of fibrillin‐2 in Fbn2
/
mice based on the following observations. Complete loss of both fibrillin‐1 and ‐2 is incompatible with embryonic viability. Homozygous double mutants (Fbn1
/
; Fbn2
/
) die around E14.5 due to impaired or delayed elastogenesis in the medial layers of the aorta. About half of the Fbn1þ/
; Fbn2
/
mutant mice fail to complete fetal development. These results suggested that fibrillin (either fibrillin‐1 or ‐2) is absolutely required for the initial assembly of elastic fibers, although fibrillin‐2 is dispensable during later phases of the elastic fiber development. The connective tissue phenotype of the Fbn2
/
mutant correlate very well with that of the radiation‐induced classical mouse mutant shaker‐with‐ syndactylism (sy) caused by a multigene deletion, including the locus for Fbn2 on chromosome 18 (Arteaga‐Solis et al., 2001; Chaudhry et al., 2001; Johnson et al., 1998). The phenotypic consequences of the sy mutation include, besides auditory/vestibular defects and early lethality, variable fusion of the digits (syndactyly) (Deol, 1963; Gru¨neberg, 1962; Hertwig, 1942). Two additional alleles of sy, which mutated spontaneously, are also characterized by syndactyly. The syfp homozygous animals show variable fusion of the three central digits of the hindfeet and forefeet, which has been attributed to the frameshift mutation 5051del A in exon 39 of Fbn2 introducing a premature termination codon (Chaudhry et al., 2001; Hummel and Chapman, 1971; Lane and Hummel, 1973). The syfp‐2J homozygous mice are also characterized by syndactyly, although they are less severely aVected as compared to the sy and the syfp mutants (Sweet, 1996). The underlying mutation in the syfp‐2J

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mutant is an exon‐skipping mutation leading to the loss of exon 38 in Fbn2, which encodes the second half of the fourth TB/8‐Cys domain in fibrillin‐2 (Chaudhry et al., 2001). No abnormalities were observed in any of the heterozygous mice. Intact microfibrils could be extracted from all three mutant mice (Chaudhry et al., 2001). To address the question, whether Marfan syndrome is caused by a dominant negative mechanism or by haploinsuYciency, Judge and coworkers (Judge et al., 2004) generated transgenic mice overexpressing human fibrillin‐1 with the mutation C1663R in cbEGF24 (leading to classical Marfan syndrome) in a normal mouse background. Although it was demonstrated that the human fibrillin‐1 was expressed in relevant tissues during pertinent developmental stages and the human fibrillin interacted with the normal mouse fibrillin‐1, these mice did not show any abnormalities. On the other hand, mice heterozygous for the C1039G mutation in cbEGF11 of mouse fibrillin‐1 showed skeletal deformity, progressive deterioration of the aortic wall including elastic fiber fragmentation and excessive deposition of collagen and proteoglycans. The corresponding cysteine mutation in humans (C1039Y) leads to the classical form of Marfan syndrome (Schrijver et al., 1999). In addition, deposition of microfibrils by fibroblasts isolated from these mice showed a diminished fibrillin‐1 network. Introduction of a wild‐type human FBN1 transgene in the heterozygous C1039G mouse background rescued the aortic phenotype. These observations suggest that haploinsuYciency rather than the production of a dominant negative mutant fibrillin‐1 may be a critical determinant for the pathogenesis of Marfan syndrome. Tsk is an autosomal dominant mutation that occurred spontaneously (Green et al., 1976). Mice homozygous for the Tsk mutation (Tsk/Tsk) are not viable and die in utero at 7–8 days of gestation. Heterozygous Tsk/þ mice are characterized by tight skins with hyperplasia of subcutaneous loose connective tissues, increased growth of cartilage and bone, and small tendons with hyperplasia of the tendon sheaths. Large accumulations of microfibrils are found in the loose connective tissue (Green et al., 1976). The Tsk mutation is a tandem genomic in frame duplication of exons 17–40 inserted between exon 40 and 41 of the Fbn1 gene, resulting in a larger approximately 420‐kDa fibrillin‐1 protein, compared to the approximately 350‐kDa wild‐ type protein (Saito et al., 1999; Siracusa et al., 1996). The mutation roughly duplicates the neonatal region in Fbn1 including adjacent regions. Tsk/þ mice produce both the normal and the large abnormal fibrillin‐1 protein (Saito et al., 1999). Conflicting evidence have been reported in terms of whether and how the mutant fibrillin‐1 becomes incorporated into microfibrils. Kielty and coworkers (Kielty et al., 1998) analyzed microfibrils isolated from Tsk/þ mice and found two mutually exclusive populations

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whereas one contained normal fibrillin‐1 and the other one contained the abnormally long Tsk fibrillin‐1. This suggested for the fibrillin‐1 assembly a molecular selection based on the length of the molecules. However, Gayraud and coworkers (Gayraud et al., 2000) found that the longer Tsk fibrillin‐1 is able to copolymerize together with the normal wild‐type fibrillin‐1 into abnormal beaded microfibrils. Despite this controversy about the homo‐ and heterotypic assembly of these microfibrils, it seems clear that the mutant Tsk fibrillin‐1 is able to incorporate into microfibrils. This is in line with the current view of fibrillin‐1 self‐assembly, indicating that the process is guided by regions located in the N‐ and C‐terminus of the monomeric proteins (Lin et al., 2002; Marson et al., 2005). These regions are present in the mutated Tsk fibrillin‐1 and, therefore, should allow self‐assembly or heterotypic assembly with wild‐type fibrillin‐1. How the altered structure of Tsk fibrillin‐1 and microfibrils ultimately result in the Tsk phenotype is presently not clear. Enhanced proteolytic susceptibility of the Tsk fibrillin‐1 may lead to decreased numbers of fully functional microfibrils in tissue, which in turn may destabilize LL‐TGF‐ leading to activation of TGF‐ (Chapter 5) (Gayraud et al., 2000). Additionally, abnormal interactions between fibrillin‐1 and microfibril‐associated glycoprotein‐2 may play a role in this process (Lemaire et al., 2004).

VII. Conclusions Over the years, our understanding about the roles of fibrillins in development and homeostasis of tissues and organs has increased significantly. Important mechanisms have been elucidated for the biogenesis of microfibrils. It is becoming clear that fibrillins and microfibrils are not only structural entities but also function as important ECM regulators in developmental and signaling processes. Mouse models provided new concepts for pathogenetic mechanisms in fibrillinopathies and oVer the possibility to test therapeutic strategies for these disorders. Future research should aim at integrating specific functions of fibrillins and microfibrils in the cellular and organismal context.

Acknowledgments This work was supported by the Canadian Institutes of Health Research (MOP‐68836 to DPR) and the German Academic Exchange Service DAAD (postdoctoral fellowship to DH).

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References Annes, J. P., Munger, J. S., and Rifkin, D. B. (2003). Making sense of latent TGFbeta activation. J. Cell Sci. 116, 217–224. Arteaga‐Solis, E., Gayraud, B., Lee, S. Y., Shum, L., Sakai, L. Y., and Ramirez, F. (2001). Regulation of limb patterning by extracellular microfibrils. J. Cell Biol. 154, 275–281. Ashworth, J. L., Kelly, V., Wilson, R., Shuttleworth, C. A., and Kielty, C. M. (1999). Fibrillin assembly: Dimer formation mediated by amino‐terminal sequences. J. Cell Sci. 112, 3549–3558. Baldock, C., Koster, A. J., Ziese, U., Rock, M. J., Sherratt, M. J., Kadler, K. E., Shuttleworth, C. A., and Kielty, C. M. (2001). The supramolecular organization of fibrillin‐rich microfibrils. J. Cell Biol. 152, 1045–1056. Bax, D. V., Bernard, S. E., Lomas, A., Morgan, A., Humphries, J., Shuttleworth, A., Humphries, M. J., and Kielty, C. M. (2003). Cell adhesion to fibrillin‐1 molecules and microfibrils is mediated by alpha5 beta1 and alphav beta3 integrins. J. Biol. Chem. 278, 34605–34616. Bouchey, D., Drake, C. J., Wunsch, A. M., and Little, C. D. (1996). Distribution of connective tissue proteins during development and neovascularization of the epicardium. Cardiovasc. Res. 31, E104–E115. Bouzeghrane, F., Reinhardt, D. P., Reudelhuber, T., and Thibault, G. (2005). Enhanced expression of fibrillin‐1, a constituent of the myocardial extracellular matrix, in fibrosis. Am. J. Physiol. Heart Circ. Physiol. 289, H982–H991. Bowness, J. M., and Tarr, A. H. (1997). Epsilon(gamma‐Glutamyl)lysine crosslinks are concentrated in a non‐collagenous microfibrillar fraction of cartilage. Biochem. Cell Biol. 75, 89–91. Bunton, T. E., Biery, N. J., Myers, L., Gayraud, B., Ramirez, F., and Dietz, H. C. (2001). Phenotypic alteration of vascular smooth muscle cells precedes elastolysis in a mouse model of Marfan syndrome. Circ. Res. 88, 37–43. Burke, R. D., Wang, D., Mark, S., and Martens, G. (2000). Distribution of fibrillin I in extracellular matrix and epithelia during early development of avian embryos. Anat. Embryol. (Berl.) 201, 317–326. Cain, S. A., Baldock, C., Gallagher, J., Morgan, A., Bax, D. V., Weiss, A. S., Shuttleworth, C. A., and Kielty, C. M. (2005). Fibrillin‐1 interactions with heparin: Implications for microfibril and elastic fibre assembly. J. Biol. Chem. 280, 30526–30537. Cain, S. A., Morgan, A., Sherratt, M. J., Ball, S. G., Shuttleworth, C. A., and Kielty, C. M. (2006). Proteomic analysis of fibrillin‐rich microfibrils. Proteomics 6, 111–122. Campbell, I. D., and Bork, P. (1993). Epidermal growth factor‐like modules. Curr. Opin. Struct. Biol. 3, 385–392. Carta, L., Pereira, L., Arteaga‐Solis, E., Lee‐Arteaga, S. Y., Lenart, B., Starcher, B., Merkel, C. A., Sukoyan, M., Kerkis, A., Hazeki, N., Keene, D. R., Sakai, L. Y., et al. (2006). Fibrillins 1 and 2 perform partially overlapping functions during aortic development. J. Biol. Chem. 281, 8016–8023. Charbonneau, N. L., Dzamba, B. J., Ono, R. N., Keene, D. R., Corson, G. M., Reinhardt, D. P., and Sakai, L. Y. (2003). Fibrillins can co‐assemble in fibrils, but fibrillin fibril composition displays cell‐specific diVerences. J. Biol. Chem. 278, 2740–2749. Charbonneau, N. L., Ono, R. N., Corson, G. M., Keene, D. R., and Sakai, L. Y. (2004). Fine tuning of growth factor signals depends on fibrillin microfibril networks. Birth Defects Res. C. Embryo Today 72, 37–50.

116

Hubmacher et al.

Chaudhry, S. S., Gazzard, J., Baldock, C., Dixon, J., Rock, M. J., Skinner, G. C., Steel, K. P., Kielty, C. M., and Dixon, M. J. (2001). Mutation of the gene encoding fibrillin‐2 results in syndactyly in mice. Hum. Mol. Genet. 10, 835–843. Chen, Y., Ali, T., Todorovic, V., O’Leary, J. M., Kristina, D. A., and Rifkin, D. B. (2005). Amino acid requirements for formation of the TGF‐beta‐latent TGF‐beta binding protein complexes. J. Mol. Biol. 345, 175–186. Collod‐Beroud, G., Le Bourdelles, S., Ades, L., Ala‐Kokko, L., Booms, P., Boxer, M., Child, A., Comeglio, P., De Paepe, A., Hyland, J. C., Holman, K., Kaitila, I., et al. (2003). Update of the UMD‐FBN1 mutation database and creation of an FBN1 polymorphism database. Hum. Mutat. 22, 199–208. Corson, G. M., Chalberg, S. C., Dietz, H. C., Charbonneau, N. L., and Sakai, L. Y. (1993). Fibrillin binds calcium and is coded by cDNAs that reveal a multidomain structure and alternatively spliced exons at the 50 end. Genomics 17, 476–484. Corson, G. M., Charbonneau, N. L., Keene, D. R., and Sakai, L. Y. (2004). DiVerential expression of fibrillin‐3 adds to microfibril variety in human and avian, but not rodent, connective tissues. Genomics 83, 461–472. Dallas, S. L., Keene, D. R., Bruder, S. P., Saharinen, J., Sakai, L. Y., Mundy, G. R., and Bonewald, L. F. (2000). Role of the latent transforming growth factor beta binding protein 1 in fibrillin‐containing microfibrils in bone cells in vitro and in vivo. J. Bone Miner. Res. 15, 68–81. Dallas, S. L., Sivakumar, P., Jones, C. J., Chen, Q., Peters, D. M., Mosher, D. F., Humphries, M. J., and Kielty, C. M. (2005). Fibronectin regulates latent transforming growth factor‐beta (TGF beta) by controlling matrix assembly of latent TGF beta‐binding protein‐1. J. Biol. Chem. 280, 18871–18880. Davis, E. C. (1993). Smooth muscle cell to elastic lamina connections in developing mouse aorta. Lab. Invest. 68, 89–99. Davis, E. C. (1994). Immunolocalization of microfibril and microfibril‐associated proteins in the subendothelial matrix of the developing mouse aorta. J. Cell Sci. 107, 727–736. Davis, E. C., Roth, R. A., Heuser, J. E., and Mecham, R. P. (2002). Ultrastructural properties of ciliary zonule microfibrils. J. Struct. Biol. 139, 65–75. Denton, C. P., and Abraham, D. J. (2001). Transforming growth factor‐beta and connective tissue growth factor: Key cytokines in scleroderma pathogenesis. Curr. Opin. Rheumatol. 13, 505–511. Deol, M. S. (1963). The development of the inner ear in mice homozygous for shaker‐with‐ syndactylism. J. Embryol. Exp. Morphol. 11, 493–512. Downing, A. K., Knott, V., Werner, J. M., Cardy, C. M., Campbell, I. D., and Handford, P. A. (1996). Solution structure of a pair of calcium‐binding epidermal growth factor‐like domains: Implications for the Marfan syndrome and other genetic disorders. Cell 85, 597–605. Dudley, A. T., Lyons, K. M., and Robertson, E. J. (1995). A requirement for bone morphogenetic protein‐7 during development of the mammalian kidney and eye. Genes Dev. 9, 2795–2807. Dzamba, B. J., Keene, D. R., Isogai, Z., Charbonneau, N. L., Karaman‐Jurukovska, N., Simon, M., and Sakai, L. Y. (2001). Assembly of epithelial cell fibrillins. J. Invest. Dermatol. 117, 1612–1620. Eriksen, T. A., Wright, D. M., Purslow, P. P., and Duance, V. C. (2001). Role of Ca(2þ) for the mechanical properties of fibrillin. Proteins 45, 90–95. Fahrenbach, W. H., Sandberg, L. B., and Cleary, E. G. (1966). Ultrastructural studies on early elastogenesis. Anat. Rec. 155, 563–576. Faivre, L., Megarbane, A., Alswaid, A., Zylberberg, L., Aldohayan, N., Campos‐Xavier, B., Bacq, D., Legeai‐Mallet, L., Bonaventure, J., Munnich, A., and Cormier‐Daire, V. (2002).

4. Structure and Function of Fibrillins in the Extracellular Matrix

117

Homozygosity mapping of a Weill‐Marchesani syndrome locus to chromosome 19p13.3‐ p13.2. Hum. Genet. 110, 366–370. Faivre, L., Dollfus, H., Lyonnet, S., Alembik, Y., Megarbane, A., Samples, J., Gorlin, R. J., Alswaid, A., Feingold, J., Le, M. M., Munnich, A., and Cormier‐Daire, V. (2003a). Clinical homogeneity and genetic heterogeneity in Weill‐Marchesani syndrome. Am. J. Med. Genet. A 123, 204–207. Faivre, L., Gorlin, R. J., Wirtz, M. K., Godfrey, M., Dagoneau, N., Samples, J. R., Le, M. M., Collod‐Beroud, G., Boileau, C., Munnich, A., and Cormier‐Daire, V. (2003b). In frame fibrillin‐1 gene deletion in autosomal dominant Weill‐Marchesani syndrome. J. Med. Genet. 40, 34–36. Gallagher, B. C., Sakai, L. Y., and Little, C. D. (1993). Fibrillin delineates the primary axis of the early avian embryo. Dev. Dyn. 196, 70–78. Gayraud, B., Keene, D. R., Sakai, L. Y., and Ramirez, F. (2000). New insights into the assembly of extracellular microfibrils from the analysis of the fibrillin 1 mutation in the tight skin mouse. J. Cell Biol. 150, 667–680. Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988). Molecular events in the processing of recombinant type 1 pre‐pro‐transforming growth factor beta to the mature polypeptide. Mol. Cell Biol. 8, 4162–4168. Gibson, M. A., Hatzinikolas, G., Davis, E. C., Baker, E., Sutherland, G. R., and Mecham, R. P. (1995). Bovine latent transforming growth factor beta 1‐binding protein 2: Molecular cloning, identification of tissue isoforms, and immunolocalization to elastin‐associated microfibrils. Mol. Cell. Biol. 15, 6932–6942. Giltay, R., Timpl, R., and Kostka, G. (1999). Sequence, recombinant expression and tissue localization of two novel extracellular matrix proteins, fibulin‐3 and fibulin‐4. Matrix Biol. 18, 469–480. Gleizes, P. E., Beavis, R. C., Mazzieri, R., Shen, B., and Rifkin, D. B. (1996). Identification and characterization of an eight‐cysteine repeat of the latent transforming growth factor‐beta binding protein‐1 that mediates bonding to the latent transforming growth factor‐beta1. J. Biol. Chem. 271, 29891–29896. Green, M. C., Sweet, H. O., and Bunker, L. E. (1976). Tight‐skin, a new mutation of the mouse causing excessive growth of connective tissue and skeleton. Am. J. Pathol. 82, 493–512. Greenlee, T. K., Ross, R., and Hartman, J. L. (1966). The fine structure of elastic fibers. J. Cell Biol. 30, 59–71. Gregory, K. E., Ono, R. N., Charbonneau, N. L., Kuo, C. L., Keene, D. R., Ba¨chinger, H. P., and Sakai, L. Y. (2005). The prodomain of BMP‐7 targets the BMP‐7 complex to the extracellular matrix. J. Biol. Chem. 280, 27970–27980. Gru¨neberg, H. (1962). Genetical studies on the skeleton of the mouse. XXXII. The development of shaker with syndactylism. Genet. Res. 3, 157–166. Gupta, P. A., Putnam, E. A., Carmical, S. G., Kaitila, I., Steinmann, B., Child, A., Danesino, C., Metcalfe, K., Berry, S. A., Chen, E., Delorme, C. V., Thong, M. K., et al. (2002). Ten novel FBN2 mutations in congenital contractural arachnodactyly: Delineation of the molecular pathogenesis and clinical phenotype. Hum. Mutat. 19, 39–48. Handford, P. A. (2000). Fibrillin‐1, a calcium binding protein of extracellular matrix. Biochim. Biophys. Acta 1498, 84–90. Handford, P. A., Mayhew, M., Baron, M., Winship, P. R., Campbell, I. D., and Brownlee, G. G. (1991). Key residues involved in calcium‐binding motifs in EGF‐like domains. Nature 351, 164–167. Haston, J. L., Engelsen, S. B., Roessle, M., Clarkson, J., Blanch, E. W., Baldock, C., Kielty, C. M., and Wess, T. J. (2003). Raman microscopy and X‐ray diVraction, a combined study of fibrillin‐rich microfibrillar elasticity. J. Biol. Chem. 278, 41189–41197. Hertwig, P. (1942). Neue Mutationen und Koppelungsgruppen bei der Hausmaus. Z. Indukt. Abstamm. Vererbungsl. 80, 220–246.

118

Hubmacher et al.

Hummel, K. P., and Chapman, D. B. (1971). Fused phalanges. Mouse News Lett. 45, 28. Hungerford, J. E., Owens, G. K., Argraves, W. S., and Little, C. D. (1996). Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers. Dev. Biol. 178, 375–392. Hyytia¨inen, M., Penttinen, C., and Keski‐Oja, J. (2004). Latent TGF‐beta binding proteins: Extracellular matrix association and roles in TGF‐beta activation. Crit Rev. Clin. Lab Sci. 41, 233–264. Isogai, Z., Ono, R. N., Ushiro, S., Keene, D. R., Chen, Y., Mazzieri, R., Charbonneau, N. L., Reinhardt, D. P., Rifkin, D. B., and Sakai, L. Y. (2003). Latent transforming growth factor beta‐binding protein 1 interacts with fibrillin and is a microfibril‐associated protein. J. Biol. Chem. 278, 2750–2757. Jensen, S. A., Corbett, A. R., Knott, V., Redfield, C., and Handford, P. A. (2005). Ca2þ‐ dependent interface formation in fibrillin‐1. J. Biol. Chem. 280, 14076–14084. Johnson, K. R., Cook, S. A., and Zheng, Q. Y. (1998). The original shaker‐with‐syndactylism mutation (sy) is a contiguous gene deletion syndrome. Mamm. Genome 9, 889–892. Judge, D. P., Biery, N. J., Keene, D. R., Geubtner, J., Myers, L., Huso, D. L., Sakai, L. Y., and Dietz, H. C. (2004). Evidence for a critical contribution of haploinsuYciency in the complex pathogenesis of Marfan syndrome. J. Clin. Invest 114, 172–181. Kanwar, Y. S., Ota, K., Yang, Q., Kumar, A., Wada, J., Kashihara, N., and Peterson, D. R. (1998). Isolation of rat fibrillin‐1 cDNA and its relevance in metanephric development. Am. J. Physiol. 275, F710–F723. Keene, D. R., Maddox, B. K., Kuo, H. J., Sakai, L. Y., and Glanville, R. W. (1991). Extraction of extendable beaded structures and their identification as fibrillin‐containing extracellular matrix microfibrils. J. Histochem. Cytochem. 39, 441–449. Keene, D. R., Jordan, C. D., Reinhardt, D. P., Ridgway, C. C., Ono, R. N., Corson, G. M., Fairhurst, M., Sussman, M. D., Memoli, V. A., and Sakai, L. Y. (1997). Fibrillin‐1 in human cartilage: Developmental expression and formation of special banded fibers. J. Histochem. Cytochem. 45, 1069–1082. Kielty, C. M., Raghunath, M., Siracusa, L. D., Sherratt, M. J., Peters, R., Shuttleworth, C. A., and Jimenez, S. A. (1998). The tight skin mouse: Demonstration of mutant fibrillin‐1 production and assembly into abnormal microfibrils. J. Cell Biol. 140, 1159–1166. Kielty, C. M., Wess, T. J., Haston, L., Ashworth, J. L., Sherratt, M. J., and Shuttleworth, C. A. (2002). Fibrillin‐rich microfibrils: Elastic biopolymers of the extracellular matrix. J. Muscle Res. Cell Motil. 23, 581–596. Kielty, C. M., Sherratt, M. J., Marson, A., and Baldock, C. (2005). Fibrillin microfibrils. Adv. Protein Chem. 70, 405–436. Koli, K., Saharinen, J., Hyytia¨inen, M., Penttinen, C., and Keski‐Oja, J. (2001). Latency, activation, and binding proteins of TGF‐beta. Microsc. Res. Tech. 52, 354–362. Koli, K., Hyytia¨inen, M., Ryynanen, M. J., and Keski‐Oja, J. (2005). Sequential deposition of latent TGF‐beta binding proteins (LTBPs) during formation of the extracellular matrix in human lung fibroblasts. Exp. Cell Res. 310, 370–382. Kriz, W., Elger, M., Lemley, K., and Sakai, T. (1990). Structure of the glomerular mesangium: A biomechanical interpretation. Kidney Int. Suppl. 30, S2–S9. Lack, J., O’Leary, J. M., Knott, V., Yuan, X., Rifkin, D. B., Handford, P. A., and Downing, A. K. (2003). Solution structure of the third TB domain from LTBP1 provides insight into assembly of the large latent complex that sequesters latent TGF‐beta. J. Mol. Biol. 334, 281–291. Lane, P. W., and Hummel, K. P. (1973). Fused phalanges allelic with sy. Mouse News Lett. 49, 32. Lawrence, D. A., Pircher, R., Kryceve‐Martinerie, C., and Jullien, P. (1984). Normal embryo fibroblasts release transforming growth factors in a latent form. J. Cell Physiol 121, 184–188.

4. Structure and Function of Fibrillins in the Extracellular Matrix

119

Lee, B., Godfrey, M., Vitale, E., Hori, H., Mattei, M. G., Sarfarazi, M., Tsipouras, P., Ramirez, F., and Hollister, D. W. (1991). Linkage of Marfan syndrome and a phenotypically related disorder to two diVerent fibrillin genes. Nature 352, 330–334. Lee, S. S., Knott, V., Jovanovic, J., Harlos, K., Grimes, J. M., Choulier, L., Mardon, H. J., Stuart, D. I., and Handford, P. A. (2004). Structure of the integrin binding fragment from fibrillin‐1 gives new insights into microfibril organization. Structure (Camb.) 12, 717–729. Lemaire, R., Farina, G., Kissin, E., Shipley, J. M., Bona, C., Korn, J. H., and Lafyatis, R. (2004). Mutant fibrillin 1 from tight skin mice increases extracellular matrix incorporation of microfibril‐associated glycoprotein 2 and type I collagen. Arthritis Rheum. 50, 915–926. Lin, G., Tiedemann, K., Vollbrandt, T., Peters, H., Ba¨tge, B., Brinckmann, J., and Reinhardt, D. P. (2002). Homo‐ and heterotypic fibrillin‐1 and ‐2 interactions constitute the basis for the assembly of microfibrils. J. Biol. Chem. 277, 50795–50804. Lo¨nnqvist, L., Reinhardt, D. P., Sakai, L. Y., and Peltonen, L. (1998). Evidence for furin‐type activity‐mediated C‐terminal processing of profibrillin‐1 and interference in the processing by certain mutations. Hum. Mol. Genet. 7, 2039–2044. Low, F. N. (1962). Microfibrils: Fine filamentous components of the tissue space. Anat. Rec. 142, 131–137. Luo, G., Hofmann, C., Bronckers, A. L., Sohocki, M., Bradley, A., and Karsenty, G. (1995). BMP‐7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. Genes Dev. 9, 2808–2820. Mao, Y., and Schwarzbauer, J. E. (2005). Fibronectin fibrillogenesis, a cell‐mediated matrix assembly process. Matrix Biol. 24, 389–399. Marson, A., Rock, M. J., Cain, S. A., Freeman, L. J., Morgan, A., Mellody, K., Shuttleworth, C. A., Baldock, C., and Kielty, C. M. (2005). Homotypic fibrillin‐1 interactions in microfibril assembly. J. Biol. Chem. 280, 5013–5021. Maslen, C. L., Corson, G. M., Maddox, B. K., Glanville, R. W., and Sakai, L. Y. (1991). Partial sequence of a candidate gene for the Marfan syndrome. Nature 352, 334–337. Mecham, R. P., and Davis, E. (1994). Elastic fiber structure and assembly. In ‘‘Extracellular Matrix Assembly and Structure’’ (P. D. Yurchenco, Ed.), pp. 281–314. Academic Press, New York. Milewicz, D., Pyeritz, R. E., Crawford, E. S., and Byers, P. H. (1992). Marfan syndrome: Defective synthesis, secretion, and extracellular matrix formation of fibrillin by cultured dermal fibroblasts. J. Clin. Invest. 89, 79–86. Milewicz, D. M., Grossfield, J., Cao, S. N., Kielty, C., Covitz, W., and Jewett, T. (1995). A mutation in FBN1 disrupts profibrillin processing and results in isolated skeletal features of the Marfan syndrome. J. Clin. Invest. 95, 2373–2378. Miyazono, K., Olofsson, A., Colosetti, P., and Heldin, C. H. (1991). A role of the latent TGF‐ 1‐binding protein in the assembly and secretion of TGF‐ 1. EMBO J. 10, 1091–1101. Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., and Thomas, G. (1992). Human furin is a calcium‐dependent serine endoprotease that recognizes the sequence Arg‐X‐X‐Arg and eYciently cleaves anthrax toxin protective antigen. J. Biol. Chem. 267, 16396–16402. Nagase, T., Nakayama, M., Nakajima, D., Kikuno, R., and Ohara, O. (2001). Prediction of the coding sequences of unidentified human genes. XXII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA. Res. 8, 85–95. Nakajima, Y., Miyazono, K., Kato, M., Takase, M., Yamagishi, T., and Nakamura, H. (1997). Extracellular fibrillar structure of latent TGF beta binding protein‐1: Role in TGF beta‐ dependent endothelial‐mesenchymal transformation during endocardial cushion tissue formation in mouse embryonic heart. J. Cell Biol. 136, 193–204. Neptune, E. R., Frischmeyer, P. A., Arking, D. E., Myers, L., Bunton, T. E., Gayraud, B., Ramirez, F., Sakai, L. Y., and Dietz, H. C. (2003). Dysregulation of TGF‐beta activation contributes to pathogenesis in Marfan syndrome. Nat. Genet. 33, 407–411.

120

Hubmacher et al.

Nunes, I., Gleizes, P. E., Metz, C. N., and Rifkin, D. B. (1997). Latent transforming growth factor‐ binding protein domains involved in activation and transglutaminase‐dependent cross‐linking of latent transforming growth factor‐ . J. Cell Biol. 136, 1151–1163. Olofsson, A., Ichijo, H., More´n, A., Ten Dijke, P., Miyazono, K., and Heldin, C. H. (1995). EYcient association of an amino‐terminally extended form of human latent transforming growth factor‐beta binding protein with the extracellular matrix. J. Biol. Chem. 270, 31294–31297. Park, E. S., Putnam, E. A., Chitayat, D., Child, A., and Milewicz, D. M. (1998). Clustering of FBN2 mutations in patients with congenital contractural arachnodactyly indicates an important role of the domains encoded by exons 24 through 34 during human development. Am. J. Med. Genet. 78, 350–355. Pereira, L., Andrikopoulos, K., Tian, J., Lee, S. Y., Keene, D. R., Ono, R. N., Reinhardt, D. P., Sakai, L. Y., Jensen‐Biery, N., Bunton, T., Dietz, H. C., and Ramirez, F. (1997). Targeting of fibrillin‐1 recapitulates the vascular phenotype of Marfan syndrome in the mouse. Nat. Genet. 17, 218–222. Pereira, L., Lee, S. Y., Gayraud, B., Andrikopoulos, K., Shapiro, S. D., Bunton, T., Biery, N. J., Dietz, H. C., Sakai, L. Y., and Ramirez, F. (1999). Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin‐1. Proc. Natl. Acad. Sci. USA 96, 3819–3823. PfaV, M., Reinhardt, D. P., Sakai, L. Y., and Timpl, R. (1996). Cell adhesion and integrin binding to recombinant human fibrillin‐1. FEBS Lett. 384, 247–250. Pyeritz, R. E. (2000). The Marfan syndrome. Annu. Rev. Med. 51, 481–510. Qian, R. Q., and Glanville, R. W. (1997). Alignment of fibrillin molecules in elastic microfibrils is defined by transglutaminase‐derived cross‐links. Biochemistry 36, 15841–15847. Quondamatteo, F., Reinhardt, D. P., Charbonneau, N. L., Pophal, G., Sakai, L. Y., and Herken, R. (2002). Fibrillin‐1 and fibrillin‐2 in human embryonic and early fetal development. Matrix Biol. 21, 637–646. Raghunath, M., Unso¨ld, C., Kubitscheck, U., Bruckner‐Tuderman, L., Peters, R., and Meuli, M. (1998). The cutaneous microfibrillar apparatus contains latent transforming growth factor‐beta binding protein‐1 (LTBP‐1) and is a repository for latent TGF‐beta1. J. Invest. Dermatol. 111, 559–564. Raghunath, M., Putnam, E. A., Ritty, T., Hamstra, D., Park, E. S., Tscho¨drich‐Rotter, M., Peters, R., Rehemtulla, A., and Milewicz, D. M. (1999). Carboxy‐terminal conversion of profibrillin to fibrillin at a basic site by PACE/furin‐like activity required for incorporation in the matrix. J. Cell Sci. 112, 1093–1100. Raviola, G. (1971). The fine structure of the ciliary zonule and ciliary epithelium. Invest. Ophthalmol. 10, 851–869. Reber‐Mu¨ller, S., Spissinger, T., Schuchert, P., Spring, J., and Schmid, V. (1995). An extracellular matrix protein of jellyfish homologous to mammalian fibrillins forms diVerent fibrils depending on the life stage of the animal. Dev. Biol. 169, 662–672. Reinhardt, D. P., Keene, D. R., Corson, G. M., Po¨schl, E., Ba¨chinger, H. P., Gambee, J. E., and Sakai, L. Y. (1996). Fibrillin 1: Organization in microfibrils and structural properties. J. Mol. Biol. 258, 104–116. Reinhardt, D. P., Gambee, J. E., Ono, R. N., Ba¨chinger, H. P., and Sakai, L. Y. (2000). Initial steps in assembly of microfibrils. Formation of disulfide‐cross‐linked multimers containing fibrillin‐1. J. Biol. Chem. 275, 2205–2210. Ren, Z. X., Brewton, R. G., and Mayne, R. (1991). An analysis by rotary shadowing of the structure of the mammalian vitreous humor and zonular apparatus. J. Struct. Biol. 106, 57–63. Rifkin, D. B. (2005). Latent transforming growth factor‐beta (TGF‐beta) binding proteins: Orchestrators of TGF‐beta availability. J. Biol. Chem. 280, 7409–7412. Ritty, T. M., Broekelmann, T., Tisdale, C., Milewicz, D. M., and Mecham, R. P. (1999). Processing of the fibrillin‐1 carboxyl‐terminal domain. J. Biol. Chem. 274, 8933–8940.

4. Structure and Function of Fibrillins in the Extracellular Matrix

121

Ritty, T. M., Broekelmann, T. J., Werneck, C. C., and Mecham, R. P. (2003). Fibrillin‐1 and ‐2 contain heparin‐binding sites important for matrix deposition and that support cell attachment. Biochem. J. 375, 425–432. Robinson, P. N., Booms, P., Katzke, S., Ladewig, M., Neumann, L., Palz, M., Pregla, R., Tiecke, F., and Rosenberg, T. (2002). Mutations of FBN1 and genotype‐phenotype correlations in Marfan syndrome and related fibrillinopathies. Hum. Mutat. 20, 153–161. Rongish, B. J., Drake, C. J., Argraves, W. S., and Little, C. D. (1998). Identification of the developmental marker, JB3‐antigen, as fibrillin‐2 and its de novo organization into embryonic microfibrous arrays. Dev. Dyn. 212, 461–471. Saharinen, J., and Keski‐Oja, J. (2000). Specific sequence motif of 8‐Cys repeats of TGF‐beta binding proteins, LTBPs, creates a hydrophobic interaction surface for binding of small latent TGF‐beta. Mol. Biol. Cell 11, 2691–2704. Saharinen, J., Taipale, J., and Keski‐Oja, J. (1996). Association of the small latent transforming growth factor‐ with an eight cysteine repeat of its binding protein (LTBP‐1). EMBO J. 15, 245–253. Saharinen, J., Hyytia¨inen, M., Taipale, J., and Keski‐Oja, J. (1999). Latent transforming growth factor‐binding proteins (LTBPs)‐structural extracellular matrix proteins for targeting TGF‐action. Cytokine Growth Factor Rev. 10, 99–117. Saito, S., Nishimura, H., Brumeanu, T. D., Casares, S., Stan, A. C., Honjo, T., and Bona, C. A. (1999). Characterization of mutated protein encoded by partially duplicated fibrillin‐1 gene in tight skin (TSK) mice. Mol. Immunol. 36, 169–176. Sakai, L. Y., Keene, D. R., and Engvall, E. (1986). Fibrillin, a new 350‐kD glycoprotein, is a component of extracellular microfibrils. J. Cell Biol. 103, 2499–2509. Sakai, L. Y., Keene, D. R., Glanville, R. W., and Ba¨chinger, H. P. (1991). Purification and partial characterization of fibrillin, a cysteine‐rich structural component of connective tissue microfibrils. J. Biol. Chem. 266, 14763–14770. Sakamoto, H., Broekelmann, T., Cheresh, D. A., Ramirez, F., Rosenbloom, J., and Mecham, R. P. (1996). Cell‐type specific recognition of RGD‐ and non‐RGD‐containing cell binding domains in fibrillin‐1. J. Biol. Chem. 271, 4916–4922. Schrijver, I., Liu, W., Brenn, T., Furthmayr, H., and Francke, U. (1999). Cysteine substitutions in epidermal growth factor‐like domains of fibrillin‐1: Distinct eVects on biochemical and clinical phenotypes. Am. J. Hum. Genet. 65, 1007–1020. Sinha, S., Heagerty, A. M., Shuttleworth, C. A., and Kielty, C. M. (2002). Expression of latent TGF‐beta binding proteins and association with TGF‐beta 1 and fibrillin‐1 following arterial injury. Cardiovasc. Res. 53, 971–983. Siracusa, L. D., McGrath, R., Ma, Q., Moskow, J. J., Manne, J., Christner, P. J., Buchberg, A. M., and Jimenez, S. A. (1996). A tandem duplication within the fibrillin 1 gene is associated with the mouse tight skin mutation. Genome Res. 6, 300–313. Smith, S. M., Dickman, E. D., Thompson, R. P., Sinning, A. R., Wunsch, A. M., and Markwald, R. R. (1997). Retinoic acid directs cardiac laterality and the expression of early markers of precardiac asymmetry. Dev. Biol. 182, 162–171. Sugi, Y., and Markwald, R. R. (1996). Formation and early morphogenesis of endocardial endothelial precursor cells and the role of endoderm. Dev. Biol. 175, 66–83. Sweet, H. O. (1996). Remutations at the Jackson Laboratory. Mouse Genome 94, 487. Taipale, J., Miyazono, K., Heldin, C. H., and Keski‐Oja, J. (1994). Latent transforming growth factor‐ 1 associates to fibroblast extracellular matrix via latent TGF‐ binding protein. J. Cell Biol. 124, 171–181. Taipale, J., Saharinen, J., Hedman, K., and Keski‐Oja, J. (1996). Latent transforming growth factor‐beta 1 and its binding protein are components of extracellular matrix microfibrils. J. Histochem. Cytochem. 44, 875–889.

122

Hubmacher et al.

Tan, F. K., Arnett, F. C., Antohi, S., Saito, S., Mirarchi, A., Spiera, H., Sasaki, T., Shoichi, O., Takeuchi, K., Pandy, J. P., Silver, R. M., LeRoy, C., et al. (1999). Autoantibodies to the extracellular matrix microfibrillar protein, fibrillin‐1, in patients with scleroderma and other connective tissue diseases. J. Immunol. 163, 1066–1072. Taylor, N. A., Van, D. V., and Creemers, J. W. (2003). Curbing activation: Proprotein convertases in homeostasis and pathology. FASEB J. 17, 1215–1227. Thurmond, F. A., and Trotter, J. A. (1996). Morphology and biomechanics of the microfibrillar network of sea cucumber dermis. J. Exp. Biol. 199, 1817–1828. Thurmond, F. A., Koob, T. J., Bowness, J. M., and Trotter, J. A. (1997). Partial biochemical and immunologic characterization of fibrillin microfibrils from sea cucumber dermis. Connect. Tissue Res. 36, 211–222. Tiedemann, K., Ba¨tge, B., Mu¨ller, P. K., and Reinhardt, D. P. (2001). Interactions of fibrillin‐1 with heparin/heparan sulfate: Implications for microfibrillar assembly. J. Biol. Chem. 276, 36035–36042. Tiedemann, K., Sasaki, T., Gustafsson, E., Go¨hring, W., Ba¨tge, B., Notbohm, H., Timpl, R., Wedel, T., Schlo¨tzer‐Schrehardt, U., and Reinhardt, D. P. (2005). Microfibrils at basement membrane zones interact with perlecan via fibrillin‐1. J. Biol. Chem. 280, 11404–11412. Trask, B. C., Trask, T. M., Broekelmann, T., and Mecham, R. P. (2000). The microfibrillar proteins MAGP‐1 and fibrillin‐1 form a ternary complex with the chondroitin sulfate proteoglycan decorin. Mol. Biol. Cell 11, 1499–1507. Trask, T. M., Ritty, T. M., Broekelmann, T., Tisdale, C., and Mecham, R. P. (1999). N‐terminal domains of fibrillin 1 and fibrillin 2 direct the formation of homodimers: A possible first step in microfibril assembly. Biochem. J. 340, 693–701. Unso¨ld, C., Hyytia¨inen, M., Bruckner‐Tuderman, L., and Keski‐Oja, J. (2001). Latent TGF‐ beta binding protein LTBP‐1 contains three potential extracellular matrix interacting domains. J. Cell Sci. 114, 187–197. Viljoen, D. (1994). Congenital contractural arachnodactyly. J. Med. Genet. 31, 640–643. Wallace, R. N., Streeten, B. W., and Hanna, R. B. (1991). Rotary shadowing of elastic system microfibrils in the ocular zonule, vitreous, and ligament nuchae. Curr. Eye Res. 10, 99–109. Wallis, D. D., Putnam, E. A., Cretoiu, J. S., Carmical, S. G., Cao, S. N., Thomas, G., and Milewicz, D. M. (2003). Profibrillin‐1 maturation by human dermal fibroblasts: Proteolytic processing and molecular chaperones. J. Cell Biochem. 90, 641–652. Wess, T. J., Purslow, P. P., Sherratt, M. J., Ashworth, J., Shuttleworth, C. A., and Kielty, C. M. (1998). Calcium determines the supramolecular organization of fibrillin‐rich microfibrils. J. Cell Biol. 141, 829–837. Wright, D. W., and Mayne, R. (1988). Vitreous humor of chicken contains two fibrillar systems: An analysis of their structure. J. Ultrastruct. Mol. Struct. R. 100, 224–234. Wunsch, A. M., Little, C. D., and Markwald, R. R. (1994). Cardiac endothelial heterogeneity defines valvular development as demonstrated by the diverse expression of JB3, an antigen of the endocardial cushion tissue. Dev. Biol. 165, 585–601. Yang, Q., Ota, K., Tian, Y., Kumar, A., Wada, J., Kashihara, N., Wallner, E., and Kanwar, Y. S. (1999). Cloning of rat fibrillin‐2 cDNA and its role in branching morphogenesis of embryonic lung. Dev. Biol. 212, 229–242. Yin, W., Smiley, E., Germiller, J., Sanguineti, C., Lawton, T., Pereira, L., Ramirez, F., and Bonadio, J. (1995). Primary structure and developmental expression of Fbn‐1, the mouse fibrillin gene. J. Biol. Chem. 270, 1798–1806. Yuan, X., Downing, A. K., Knott, V., and Handford, P. A. (1997). Solution structure of the transforming growth factor ‐binding protein‐like module, a domain associated with matrix fibrils. EMBO J. 16, 6659–6666.

4. Structure and Function of Fibrillins in the Extracellular Matrix

123

Zhang, H., Apfelroth, S. D., Hu, W., Davis, E. C., Sanguineti, C., Bonadio, J., Mecham, R. P., and Ramirez, F. (1994). Structure and expression of fibrillin‐2, a novel microfibrillar component preferentially located in elastic matrices. J. Cell Biol. 124, 855–863. Zhang, H., Hu, W., and Ramirez, F. (1995). Developmental expression of fibrillin genes suggests heterogeneity of extracellular microfibrils. J. Cell Biol. 129, 1165–1176. Zhou, X., Tan, F. K., Milewicz, D. M., Guo, X., Bona, C. A., and Arnett, F. C. (2005). Autoantibodies to fibrillin‐1 activate normal human fibroblasts in culture through the TGF‐ beta pathway to recapitulate the ‘‘scleroderma phenotype.’’ J. Immunol. 175, 4555–4560.