Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo

Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat-containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5-deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171-175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168-171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.     

Analysis of dermal elastic fibers in the absence of fibulin-5 reveals potential roles for fibulin-5 in elastic fiber assembly

Fibulin-5 is a 66 kDa modular, extracellular matrix protein that localizes to elastic fibers. Although in vitro protein–protein binding studies have shown that fibulin-5 binds many proteins involved in elastic fiber formation, the specific role of fibulin-5 in elastogenesis remains unclear. To provide a more detailed analysis of elastic fiber assembly in the absence of fibulin-5, the dermis of wild-type and fibulin-5 gene knockout (Fbln5^?/?) mice was examined with electron microscopy (EM). Although light microscopy showed apparently normal elastic fibers near the hair follicles and the absence of elastic fibers in the intervening dermis of the Fbln5^?/? mouse, EM revealed the presence of aberrantly assembled elastic fibers in both locales. Instead of the elastin being incorporated into the microfibrillar scaffold, the elastin appeared as globules juxtaposed to the microfibrils. Desmosine analysis showed significantly lower levels of mature cross-linked elastin in the Fbln5^?/? dermis, however, gene expression levels for tropoelastin and fibrillin-1, the major elastic fiber components, were unaffected. Based on these results, the nature of tropoelastin cross-linking was investigated using domain specific antibodies to lysyl oxidase like-1 (LOXL-1). Immunolocalization with an antibody to the N-terminal pro-peptide, which is cleaved to generate the active enzyme, revealed abundant staining in the Fbln5^?/? dermis and no staining in the wild-type dermis. Overall, these results suggest two previously unrecognized functions for fibulin-5 in elastogenesis; first, to limit the extent of aggregation of tropoelastin monomers and/or coacervates and aid in the incorporation of elastin into the microfibril bundles, and second, to potentially assist in the activation of LOXL-1.     

Targeted disruption of fibulin-4 abolishes elastogenesis and causes perinatal lethality in mice

Elastic fibers provide tissues with elasticity which is critical to the function of arteries, lungs, skin, and other dynamic organs. Loss of elasticity is a major contributing factor in aging and diseases. However, the mechanism of elastic fiber development and assembly is poorly understood. Here, we show that lack of fibulin-4, an extracellular matrix molecule, abolishes elastogenesis. fibulin-4-/- mice generated by gene targeting exhibited severe lung and vascular defects including emphysema, artery tortuosity, irregularity, aneurysm, rupture, and resulting hemorrhages. All the homozygous mice died perinatally. The earliest abnormality noted was a uniformly narrowing of the descending aorta in fibulin-4-/- embryos at embryonic day 12.5 (E12.5). Aorta tortuosity and irregularity became noticeable at E15.5. Histological analysis demonstrated that fibulin-4-/- mice do not develop intact elastic fibers but contain irregular elastin aggregates. Electron microscopy revealed that the elastin aggregates are highly unusual in that they contain evenly distributed rod-like filaments, in contrast to the amorphous appearance of normal elastic fibers. Desmosine analysis indicated that elastin cross-links in fibulin-4-/- tissues were largely diminished. However, expression of tropoelastin or lysyl oxidase mRNA was unaffected in fibulin-4-/- mice. In addition, fibulin-4 strongly interacts with tropoelastin and colocalizes with elastic fibers in culture. These results demonstrate that fibulin-4 plays an irreplaceable role in elastogenesis.     

Significant decrease in tropoelastin gene expression in fibroblasts from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation

Costello syndrome is a connective tissue disorder associated with sparse, thin, and fragmented elastic fibers in tissues. In this study we demonstrated a significant decrease in the expression of tropoelastin mRNA in fibroblasts derived from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation. In contrast, there were no changes in expression of the Harvey ras (HRAS), fibrillin-1, fibulin-5, microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), or 67-kDa non-integrin elastin-binding protein (EBP) gene. The proliferative activity of the Costello fibroblasts was about 4-fold higher than that of the normal and pathological control ones. However, no mutations were detected in the coding region of HRAS mRNA. Transduction of the bovine tropoelastin (bTE) gene with the lentiviral vector restored the elastic fiber formation and decreased the growth rate in the Costello fibroblasts. These results strongly suggest that the defect of human tropoelastin (hTE) gene expression should induce the impaired elastogenesis and enhanced proliferation of Costello fibroblasts, and that a primary cause other than the HRAS gene mutation should contribute to the pathogenesis in the present Costello case.     

Microfibril-associated MAGP-2 stimulates elastic fiber assembly

Elastic fibers are complex structures composed of a tropoelastin inner core and microfibril outer mantle guiding tropoelastin deposition. Microfibrillar proteins mainly include fibrillins and microfibril-associated glycoproteins (MAGPs). MAGP-2 exhibits developmental expression peaking at elastic fiber onset, suggesting that MAGP-2 mediates elastic fiber assembly. To determine whether MAGP-2 regulates elastic fiber assembly, we used an in vitro model featuring doxycycline-regulated cells conditionally overexpressing exogenous MAGP-2 and constitutively expressing enhanced green fluorescent protein-tagged tropoelastin. Analysis by immunofluorescent staining showed that MAGP-2 overexpression dramatically increased elastic fibers levels, independently of extracellular levels of soluble tropoelastin, indicating that MAGP-2 stimulates elastic fiber assembly. This was associated with increased levels of matrix-associated MAGP-2. Electron microscopy showed that MAGP-2 specifically associates with microfibrils and that elastin globules primarily colocalize with MAGP-2-associated microfibrils, suggesting that microfibril-associated MAGP-2 facilitates elastic fiber assembly. MAGP-2 overexpression did not change levels of matrix-associated fibrillin-1, MAGP-1, fibulin-2, fibulin-5, or emilin-1, suggesting that microfibrils and other elastic fiber-associated proteins known to regulate elastogenesis do not mediate MAGP-2-induced elastic fiber assembly. Moreover, mutation analysis showed that MAGP-2 does not stimulate elastic fiber assembly through its RGD motif, suggesting that integrin receptor binding does not mediate MAGP-2-induced elastic fiber assembly. Because MAGP-2 interacts with Jagged-1 that controls cell-matrix interaction and cell motility, two key factors in elastic fiber macroassembly, microfibril-associated MAGP-2 may stimulate elastic fiber macroassembly by targeting the release of elastin globules from the cell membrane onto developing elastic fibers.     

Fibrillin-1 interactions with fibulins depend on the first hybrid domain and provide an adaptor function to tropoelastin

Fibrillin-containing microfibrils in elastic and nonelastic extracellular matrices play important structural and functional roles in various tissues, including blood vessels, lung, skin, and bone. Microfibrils are supramolecular aggregates of several protein and nonprotein components. Recently, a large region in the N-terminal portion of fibrillin-1 was characterized as a multifunctional protein interaction site, including binding sites for fibulin-2 and -5 among others. Using a panel of recombinant fibrillin-1 swapped domain and deletion fragments, we demonstrate here that the conserved first hybrid domain in fibrillin-1 is essential for binding to fibulin-2, -4, and -5. Fibulin-3 and various isoforms of fibulin-1 did not interact with fibrillin-1. Although the first hybrid domain in fibrillin-1 is located in close vicinity to the self-assembly epitope, binding of fibulin-2, -4, and -5 did not interfere with self-assembly. However, these fibulins can associate with microfibrils at various levels of maturity. Formation of ternary complexes between fibrillin-1, fibulins, and tropoelastin demonstrated that fibulin-2 and -5 but much less fibulin-4, are able to act as molecular adaptors between fibrillin-1 and tropoelastin.     

Oxidative and Nitrosative Modifications of Tropoelastin Prevent Elastic Fiber Assembly in Vitro

Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. Tropoelastin (TE), the precursor of elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO⁻) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO⁻ and HOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.     

Coacervation is promoted by molecular interactions between the PF2 segment of fibrillin-1 and the domain 4 region of tropoelastin

In forming elastic fibers, microfibrils act as the scaffold sites for depositing the elastin precursor tropoelastin. We examined key binding interactions that promote massive tropoelastin association through coacervation. Using a segment of the microfibril protein fibrillin-1, PF2, known to bind full-length tropoelastin, we mapped its interaction site to the N-terminal region of tropoelastin bounded by domains 2 and 18. Precise contact residues between domain 4 of tropoelastin and domain 16 of fibrillin-1 were discovered through a novel combination of transglutaminase cross-linking and mass spectroscopy, with contact sites at residues K38 of tropoelastin and Q669 of fibrillin-1. This is the first report of a role for this region of tropoelastin in microfibril interactions. The addition of PF2 thermodynamically facilitated the coacervation of tropoelastin, resulting in smaller changes in entropy and enthalpy values for the coacervating system. A novel multicomponent in vitro tropoelastin assembly reaction system demonstrated that amassed tropoelastin was spatially and preferentially directed to surfaces coated with PF2 as expected for organized three-dimensional distribution during tissue elastogenesis. This study underscores the role of this part of fibrillin-1 as an anchor point for tropoelastin at the microfibril-elastin junction during the initial stages of elastic fiber assembly.     

Fibulin-2 is dispensable for mouse development and elastic fiber formation

Fibulin-2 is an extracellular matrix protein belonging to the five-member fibulin family, of which two members have been shown to play essential roles in elastic fiber formation during development. Fibulin-2 interacts with two major constituents of elastic fibers, tropoelastin and fibrillin-1, in vitro and localizes to elastic fibers in many tissues in vivo. The protein is prominently expressed during morphogenesis of the heart and aortic arch vessels and at early stages of cartilage development. To examine its role in vivo, we generated mice that do not express the fibulin-2 gene (Fbln2) through homologous recombination of embryonic stem cells. Unexpectedly, the fibulin-2-null mice were viable and fertile and did not display gross and anatomical abnormalities. Histological and ultrastructural analyses revealed that elastic fibers assembled normally in the absence of fibulin-2. No compensatory up-regulation of mRNAs for other fibulin members was detected in the aorta and skin tissue. However, in the fibulin-2 null aortae, fibulin-1 immunostaining was increased in the inner elastic lamina, where fibulin-2 preferentially localizes. The results demonstrate that fibulin-2 is not required for mouse development and elastic fiber formation and suggest possible functional redundancy between fibulin-1 and fibulin-2.     

Induction of fibulin-5 gene is regulated by tropoelastin gene, and correlated with tropoelastin accumulation in vitro

Fibulin-5 (also known as DANCE) is an elastin-binding protein that is thought to play a role in elastogenesis. We examined the relationship between the gene expression of fibulin-5 and the gene expression and accumulation of tropoelastin by comparing elastin-producing cells (human gingival fibroblasts) with non-elastin-producing cells (human periodontal ligament fibroblasts) by Northern blot analysis. Fibulin-5 gene induction was found only in elastin-producing cells. Induction of the fibulin-5 gene in elastin-producing cells occurred after induction of the tropoelastin gene, and the fibulin-5 level was reduced upon RNA interference-mediated down-regulation of tropoelastin. Fibulin-5 gene induction was also correlated with a rapid increase of tropoelastin accumulation within the cell layer. These results may suggest that the fibulin-5 gene induction is directly or indirectly regulated by tropoelastin gene expression and plays a role in the accumulation of elastic fibers within matrices.     

Tropoelastin binding to fibulins, nidogen-2 and other extracellular matrix proteins.

Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K(d) about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.     

Latent TGF-beta-binding protein 2 binds to DANCE/fibulin-5 and regulates elastic fiber assembly

Elastic fibers play the principal roles in providing elasticity and integrity to various types of human organs, such as the arteries, lung, and skin. However, the molecular mechanism of elastic fiber assembly that leads to deposition and crosslinking of elastin along microfibrils remains largely unknown. We have previously shown that developing arteries and neural crest EGF-like protein (DANCE) (also designated fibulin-5) is essential for elastogenesis by studying DANCE-deficient mice. Here, we report the identification of latent transforming growth factor-beta-binding protein 2 (LTBP-2), an elastic fiber-associating protein whose function in elastogenesis is not clear, as a DANCE-binding protein. Elastogenesis assays using human skin fibroblasts reveal that fibrillar deposition of DANCE and elastin is largely dependent on fibrillin-1 microfibrils. However, downregulation of LTBP-2 induces fibrillin-1-independent fibrillar deposition of DANCE and elastin. Moreover, recombinant LTBP-2 promotes deposition of DANCE onto fibrillin-1 microfibrils. These results suggest a novel regulatory mechanism of elastic fiber assembly in which LTBP-2 regulates targeting of DANCE on suitable microfibrils to form elastic fibers.     

Fibulin-4 and fibulin-5 in elastogenesis and beyond: Insights from mouse and human studies

The fibulin family of extracellular matrix/matricellular proteins is composed of long fibulins (fibulin-1, -2, -6) and short fibulins (fibulin-3, -4, -5, -7) and is involved in protein–protein interaction with the components of basement membrane and extracellular matrix proteins. Fibulin-1, -2, -3, -4, and -5 bind the monomeric form of elastin (tropoelastin) in vitro and fibulin-2, -3, -4, and -5 are shown to be involved in various aspects of elastic fiber development in vivo. In particular, fibulin-4 and -5 are critical molecules for elastic fiber assembly and play a non-redundant role during elastic fiber formation. Despite manifestation of systemic elastic fiber defects in all elastogenic tissues, fibulin-5 null (Fbln5^−/−) mice have a normal lifespan. In contrast, fibulin-4 null (Fbln4^−/−) mice die during the perinatal period due to rupture of aortic aneurysms, indicating differential functions of fibulin-4 and fibulin-5 in normal development. In this review, we will update biochemical characterization of fibulin-4 and fibulin-5 and discuss their roles in elastogenesis and outside of elastogenesis based on knowledge obtained from loss-of-function studies in mouse and in human patients with FBLN4 or FBLN5 mutations. Finally, we will evaluate therapeutic options for matrix-related diseases.     

Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils.Fibulins are a family of extracellular glycoproteins containing contiguous calcium-binding epidermal growth factor-like domains (cbEGFs)³ and a characteristic C-terminal fibulin (FC) domain (1–3). Recent studies have revealed that fibulin-4 and -5 are both essential for elastic fiber formation (4–7). Fibulin-4 is widely expressed from early embryogenesis and is necessary for normal vascular, lung, and skin development, since mice that lack fibulin-4 do not form elastic fibers and die perinatally (5). Furthermore, mice with reduced fibulin-4 expression develop aneurysms (8). Fibulin-5 is abundant in the aorta and large arteries during embryogenesis and following vascular injury (9, 10). Lack of fibulin-5 causes a less severe phenotype, with viable homozygous mice, but the elastic fibers in skin, lungs, and aorta are irregular and fragmented (6, 7), and there is altered vascular remodeling (11). These mice models also highlight that fibulin-4 and -5 have non-compensatory roles in elastic fiber formation. Mutations in both molecules can cause cutis laxa, a heritable disorder associated with elastic fiber degeneration leading to sagging skin, vascular tortuosity, and emphysematous lungs (12–15). A third isoform, fibulin-3, may play a minor role in elastic fiber formation, since its deficiency disrupts elastic fibers in Bruch''s membrane of the eye (16) and vaginal tissues (17).Elastic fiber formation is a complex multistep process (18–20). Initial pericellular microassembly of tropoelastin, which may involve the 67-kDa elastin-binding protein receptor, generates elastin globules that are stabilized by desmosine cross-links catalyzed mainly by lysyl oxidase (LOX) but also by LOXL1 (LOX-like 1). These globules are deposited on a fibrillin microfibril template, where they coalesce and undergo further cross-linking to form the elastin core of mature fibers. The ability of fibulin-4 and -5 to bind tropoelastin and fibrillin-1, the major structural component of microfibrils, supports a model in which these fibulins direct elastin deposition on microfibrils (4–7, 21–25). This model does not delineate the unique molecular contributions of fibulin-4 and -5 to elastic fiber formation, but some molecular differences have emerged. Tropoelastin was bound more strongly by fibulin-5 than by fibulin-4, whereas fibulin-5 was at the microfibril-elastin interface, but perichondrial fibulin-4 localized mainly to microfibrils (4).Fibulin-4 null mice offer tantalizing clues to how fibulin-4 contributes to elastic fiber formation (5). They had dramatically reduced (94%) desmosine cross-links despite no change in elastin or LOX expression levels, and electron-dense rodlike structures were prominent within elastin aggregates. Morphologically similar structures seen after chemically inhibiting LOX were previously identified as glycosaminoglycans, which can bind charged free ϵ-amino groups on lysines in tropoelastin (26). However, fibulin-4^+/− mice showed ∼20% increase in desmosine (5). LOX-null mice have phenotypic features similar to those of fibulin-4 null mice, dying perinatally with 60% reduced desmosine cross-links and major abnormalities in vascular and other elastic tissues (27, 28). In contrast, LOXL1-null mice are viable but have reduced desmosine (29), whereas fibulin-5 null mice have a 16% reduction in desmosine cross-links and survive well into adulthood (7). Detection of the LOXL1 pro-domain in fibulin-5 null mice skin but not wild-type skin implicates fibulin-5 in activation of LOXL1 (30).We and others have shown that fibrillin-1 and the microfibrillar protein MAGP-1 can both directly bind tropoelastin (31–34). However, the fibulin-null mice show that the fibrillin-1 interaction with tropoelastin is insufficient to support elastic fiber formation in vivo. Fibulin-5 has been reported to facilitate tropoelastin binding to the N-terminal half of fibrillin-1 (21). A study of elastin polypeptide self-assembly through coacervation and maturation phases showed that, although the N-terminal half of fibrillin-1 increased maturation velocity and droplet clustering, fibulin-4 and -5 both slowed maturation and limited globule growth (35). These studies imply that fibulins and fibrillin-1 act together to regulate elastin accretion on microfibrils.To gain further insights into the contributions of fibulin-4 and -5 to elastic fiber formation, we have delineated how they interact with tropoelastin, LOX, and fibrillin-1. Novel findings are that fibulin-4 directly binds LOX, and this interaction enhances fibulin-4 binding to tropoelastin, thus forming a ternary complex that may be critical for elastin cross-linking. Fibulin-5 can concurrently bind fibulin-4 and tropoelastin, but the interaction of both fibulins with fibrillin-1 strongly inhibits their binding to tropoelastin. These interactions indicate the molecular basis of how fibulins act as chaperones for deposition of elastin onto microfibrils. Our study thus provides a molecular account of the differential roles of fibulins-4 and -5 in elastic fiber formation.     

Interaction of tropoelastin with the amino-terminal domains of fibrillin-1 and fibrillin-2 suggests a role for the fibrillins in elastic fiber assembly

Alignment of tropoelastin molecules during the process of elastogenesis is thought to require fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, fibrillin-1 and fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of fibrillin-2 and fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.     

LTBP-2 competes with tropoelastin for binding to fibulin-5 and heparin,and is a negative modulator of elastinogenesis

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of ill-defined function associated with elastic fibers during elastinogenesis. Although LTBP-2 binds fibrillin-1, fibulin-5, and heparin/heparan sulfate, molecules critical for normal elastic fiber assembly, it does not interact directly with elastin or its precursor, tropoelastin. We investigated the modulating effect of LTBP-2 on two key interactions of tropoelastin during elastinogenesis a) with fibulin-5 and b) with heparan sulfate (using heparin). Firstly, using solid phase assays we showed that LTBP-2 bound fibulin-5 (Kd = 26.47 ± 5.68 nM) with an affinity similar to that of the tropoelastin-fibulin-5 interaction (Kd = 24.66 ± 5.64 nM). Then using a competitive binding assay we showed that LTBP-2 inhibited the tropoelastin-fibulin-5 interaction in a dose dependent manner with almost complete inhibition obtained with 5-fold molar excess of LTBP-2. Interestingly, a fragment of LTBP-2 containing the fibulin-5 binding sequence only partially inhibited the tropoelasin-fibulin-5 interaction suggesting that LTBP-2 was directly blocking only the C-terminal tropoelastin binding site on fibulin-5 and indirectly blocking tropoelastin binding to the N-terminal region. In parallel experiments heparin was shown to have minor inhibitory effects on fibulin-5 interactions with tropoelastin and LTBP-2. However, LTBP-2 was shown to significantly inhibit the binding of heparin to tropoelastin with 50% inhibition achieved with 10 fold molar excess of LTBP-2. Confocal microscopy of fibroblast matrix showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial co-distribution with heparan sulfate proteoglycans, perlecan and syndecan-4. Also addition of exogenous LTBP-2 to ear cartilage chondrocyte cultures blocked elastinogenesis in a concentration-dependent manner. Overall the results indicate that LTBP-2 may have a negative regulatory role during elastic fiber assembly, perhaps in displacing elastin microassemblies from complexes with fibulin-5 and/or cell surface heparan sulfate proteoglycans.     

Ultrastructural distribution of 36-kD microfibril-associated glycoprotein (MAGP-36) in human and bovine tissues.

We observed the ultrastructural distribution of MAGP-36 by immunoelectron microscopy in human and bovine tissues. MAGP-36 was present in microfibrils associated with tropoelastin in skin, aorta, and spleen. It was not detected in microfibrils from the ocular zonule and kidney mesangium that were not associated with tropoelastin. In skin, MAGP-36 was present in both early immature elastic fibers and mature elastic fibers. In mature elastic fibers, MAGP-36 was localized around amorphous elastic cores at the elastin-microfibril interface and in electron-dense bundles. Localization of MAGP-36 in elastic fibers coincided with the distribution of lysyl oxidase, an enzyme that plays a pivotal role in the deposition of tropoelastin. These findings suggest that MAGP-36 may be involved in elastogenesis.     

Neuraminidase-1 is required for the normal assembly of elastic fibers

The assembly of elastic fibers in tissues that undergo repeated cycles of extension and recoil, such as the lungs and blood vessels, is dependent on the proper interaction and alignment of tropoelastin with a microfibrillar scaffold. Here, we describe in vivo histopathological effects of neuraminidase-1 (Neu1) deficiency on elastin assembly in the lungs and aorta of mice. These mice exhibited a tight-skin phenotype very similar to the Tsk mouse. Normal septation of Neu1-null mice did not occur in neonatal mice, resulting in enlarged alveoli that were maintained in adults. The abnormal development of elastic fibers was remarkable under electron microscopy and confirmed by the overlapping distribution of elastin, fibrillin-1, fibrillin-2, and fibulin-5 (Fib-5) by the light microscopy immunostainings. Fib-5 fibers appeared diffuse and unorganized around the alveolar walls and the apex of developing secondary septal crests. Fibrillin-2 deposition was also abnormal in neonatal and adult lungs. Dispersion of myofibroblasts appeared abnormal in developing lungs of Neu1-null mice, with a random distribution of myofibroblast around the alveolar walls, rather than concentrating at sites of elastin synthesis. The elastic lamellae in the aorta of the Neu1-null mice were thinner and separated by hypertrophic smooth muscle cells that were surrounded by an excess of the sialic acid-containing moieties. The concentration of elastin, as measure by desmosine levels, was significantly reduced in the aorta of Neu1-null mice. Message levels for tropoelastin and Fib-5 were normal, suggesting the elastic fiber defects in Neu1-null mice result from impaired extracellular assembly.     

Elastic fiber assembly in the adult mouse pubic symphysis during pregnancy and postpartum

Impairment of pelvic organ support has been described in mice with genetic modifications of the proteins involved in elastogenesis, such as lysyl oxidase-like 1 (LOXL1) and fibulin 5. During pregnancy, elastic fiber-enriched pelvic tissues are modified to allow safe delivery. In addition, the mouse pubic symphysis is remodeled in a hormone-controlled process that entails the modification of the fibrocartilage into an interpubic ligament (IpL) and the relaxation of this ligament. After first parturition, recovery occurs to ensure pelvic tissue homeostasis. Because ligaments are the main supports of the pelvic organs, this study aimed to evaluate elastogenesis in the IpL during mouse pregnancy and postpartum. Accordingly, virgin, pregnant, and postpartum C57BL/6 mice were studied using light, confocal, and transmission electron microscopy as well as Western blots and real-time PCR. Female mice exhibited the separation of the pubic bones and the formation, relaxation, and postpartum recovery of the IpL. By the time the IpL was formed, the elastic fibers had increased in profile length and diameter, and they consisted of small conglomerates of amorphous material distributed among the bundles of microfibrils. Our analyses also indicated that elastin/tropoelastin, fibrillin 1, LOXL1/Loxl1, and fibulin 5 were spatially and temporally regulated, suggesting that these molecules may contribute to the synthesis of new elastic fibers during IpL development. Overall, this work revealed that adult elastogenesis may be important to assure the elasticity of the pelvic girdle during preparation for parturition and postpartum recovery. This finding may contribute to our understanding of pathological processes involving elastogenesis in the reproductive tract.     

JNK suppresses pulmonary fibroblast elastogenesis during alveolar development

Background

The formation of discrete elastin bands at the tips of secondary alveolar septa is important for normal alveolar development, but the mechanisms regulating the lung elastogenic program are incompletely understood. JNK suppress elastin synthesis in the aorta and is important in a host of developmental processes. We sought to determine whether JNK suppresses pulmonary fibroblast elastogenesis during lung development.

Methods

Alveolar size, elastin content, and mRNA of elastin-associated genes were quantitated in wild type and JNK-deficient mouse lungs, and expression profiles were validated in primary lung fibroblasts. Tropoelastin protein was quantitated by Western blot. Changes in lung JNK activity throughout development were quantitated, and pJNK was localized by confocal imaging and lineage tracing.

Results

By morphometry, alveolar diameters were increased by 7% and lung elastin content increased 2-fold in JNK-deficient mouse lungs compared to wild type. By Western blot, tropoelastin protein was increased 5-fold in JNK-deficient lungs. Postnatal day 14 (PND14) lung JNK activity was 11-fold higher and pJNK:JNK ratio 6-fold higher compared to PN 8 week lung. Lung tropoelastin, emilin-1, fibrillin-1, fibulin-5, and lysyl oxidase mRNAs inversely correlated with lung JNK activity during alveolar development. Phosphorylated JNK localized to pulmonary lipofibroblasts. PND14 JNK-deficient mouse lungs contained 7-fold more tropoelastin, 2,000-fold more emilin-1, 800-fold more fibrillin-1, and 60-fold more fibulin-5 than PND14 wild type lungs. Primarily lung fibroblasts from wild type and JNK-deficient mice showed similar differences in elastogenic mRNAs.

Conclusions

JNK suppresses fibroblast elastogenesis during the alveolar stage of lung development.