Fibrillin-containing Microfibrils Research Articles

Redox stress in Marfan syndrome: Dissecting the role of the NADPH oxidase NOX4 in aortic aneurysm.

Marfan syndrome (MFS) is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix fibrillin-containing microfibrils and dysfunction of TGF-β signaling. Here we identify the molecular targets of redox stress in aortic aneurysms from MFS patients, and investigate the role of NOX4, whose expression is strongly induced by TGF-β, in aneurysm formation and progression in a murine model of MFS. Working models included aortae and cultured vascular smooth muscle cells (VSMC) from MFS patients, and a NOX4-deficient Marfan mouse model (Fbn1C1039G/+-Nox4-/-). Increased tyrosine nitration and reactive oxygen species levels were found in the tunica media of human aortic aneurysms and in cultured VSMC. Proteomic analysis identified nitrated and carbonylated proteins, which included smooth muscle α-actin (αSMA) and annexin A2. NOX4 immunostaining increased in the tunica media of human Marfan aorta and was transcriptionally overexpressed in VSMC. Fbn1C1039G/+-Nox4-/- mice aortas showed a reduction of fragmented elastic fibers, which was accompanied by an amelioration in the Marfan-associated enlargement of the aortic root. Increase in the contractile phenotype marker calponin in the tunica media of MFS mice aortas was abrogated in Fbn1C1039G/+-Nox4-/- mice. Endothelial dysfunction evaluated by myography in the Marfan ascending aorta was prevented by the absence of Nox4 or catalase-induced H2O2 decomposition. We conclude that redox stress occurs in MFS, whose targets are actin-based cytoskeleton members and regulators of extracellular matrix homeostasis. Likewise, NOX4 have an impact in the progression of the aortic dilation in MFS and in the structural organization of the aortic tunica media, the VSMC phenotypic modulation, and endothelial function.

Fibrillin-containing microfibrils are key signal relay stations for cell function.

Fibrillins constitute the backbone of microfibrils in the extracellular matrix of elastic and non-elastic tissues. Mutations in fibrillins are associated with a wide range of connective tissue disorders, the most common is Marfan syndrome. Microfibrils are on one hand important for structural stability in some tissues. On the other hand, microfibrils are increasingly recognized as critical mediators and drivers of cellular signaling. This review focuses on the signaling mechanisms initiated by fibrillins and microfibrils, which are often dysregulated in fibrillin-associated disorders. Fibrillins regulate the storage and bioavailability of growth factors of the TGF-β superfamily. Cells sense microfibrils through integrins and other receptors. Fibrillins potently regulate pathways of the immune response, inflammation and tissue homeostasis. Emerging evidence show the involvement of microRNAs in disorders caused by fibrillin deficiency. A thorough understanding of fibrillin-mediated cell signaling pathways will provide important new leads for therapeutic approaches of the underlying disorders.

Enhanced SDF-1 and rantes attract the locally injected bone marrow stromal cells to remedy dental biomechanically induced temporomandibular joint osteoarthritic lesions

Enhanced SDF-1 and rantes attract the locally injected bone marrow stromal cells to remedy dental biomechanically induced temporomandibular joint osteoarthritic lesions

The function of elastic fibers in the arteries: Beyond elasticity

The main components of elastic fibers, elastin and fibrillin-containing microfibrils play a structural and mechanical role in the arteries and their essential function is to provide elasticity and resilience to the tissues. However, through control of the quiescent contractile phenotype of arterial smooth muscle cells, elastin also acts as an autocrine factor and, via the binding of ‘latent transforming growth factor (TGF)-β binding protein (LTBP) – latency-associated peptide (LAP) – TGF-β’ complexes, fibrillins regulate the activation and availability of TGF-βs. These recent discoveries are detailed in this review.

Bone and soft connective tissue alterations result from loss of fibrillin-2 expression

Fibrillins 1, 2 and 3 make up a family of genes that encode large, cysteine-rich extracellular matrix glycoproteins found in connective tissues, lung, blood vessels and other extensible tissues. Fibrillins 1 and 2 have both overlapping as well as separate distributions in human embryonic and adult tissues. Fibrillin-containing microfibrils are known to modulate morphogenetic events by proper targeting of growth factors to the extracellular matrix. Mutation of the fibrillin-2 gene causes a genetic disorder, congenital contractural arachnodactyly (CCA), that results in flexion contractures. Previously, we have shown a distinct fibrillin-2 distribution in the pericellular matrix of interior tenocytes and later demonstrated a unique fibrillin-2 containing structure that runs along the tendon cell arrays in the canine flexor tendon. We hypothesized that loss of these fibrillin-2 containing structures might affect normal tendon development. To test our hypothesis, connective tissues from mice null for fibrillin-2 gene expression were studied. Murine flexor digitorum longus tendons were evaluated for total collagen content, and the intermolecular collagen cross-links hydroxylysyl and lysyl pyridinoline. The results show decreased collagen cross-links in fibrillin-2 null mice, however total collagen content remained the same when compared to wild type. Bone morphology was studied using micro computed tomography (CT). Fibrillin-2 null mice display a focal area of decreased bone length in the extremities as compared to wild type mice. Together, these results demonstrate a role for fibrillin-2 in bone and soft connective tissue morphological and biochemical processes.

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.

Effects of Fibrillin-1 Degradation on Microfibril Ultrastructure

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.

LTBP-2 specifically interacts with the amino-terminal region of fibrillin-1 and competes with LTBP-1 for binding to this microfibrillar protein

LTBP-2 is a matrix protein of unknown function since, unlike other LTBPs, it does not form covalent complexes with latent TGF-β. We have previously shown that LTBP-2 has widespread association with fibrillin-containing microfibrils in developing aorta and other tissues. We have now shown that full-length human recombinant LTBP-2 specifically binds to the amino-terminal region of fibrillin-1, but not to fibrillin-2, in solid phase assays and overlay blotting. The binding was enhanced by the inclusion of 2 mM Ca 2+ ions in the assay buffer and abolished by 5 mM EDTA indicating that the interaction was directly or indirectly Ca 2+ ion dependent. The K d for the interaction was calculated from the specific binding curve as 9.4 nM. A recombinant carboxyl-terminal fragment of LTBP-2 was shown to a) bind the amino-terminal fragment of fibrillin-1 and b) block completely the binding of full length LTBP-2 to fibrillin-1. This result indicates that the major fibrillin-1 binding site resides close to the carboxyl-terminus of LTBP-2. Further competitive binding studies showed that an analogous carboxyl terminal fragment of LTBP-1 was able to block the binding of LTBP-2 to fibrillin-1 and that the C-terminal fragment of LTBP-2 could block the interaction of the LTBP-1 fragment with the fibrillin. Thus the binding site for LTBP-2 on fibrillin-1 appears to be the same or in close proximity to that for LTBP-1. Immunohistochemical analysis of developing human aorta showed distinctive but extensively overlapping distributions for LTBPs-1 and -2. Both LTBPs showed extensive co-localization with fibrillin-1 and elastic lamellae but LTBP-2 had extensive signal throughout the medial layer whereas LTBP-1 showed strong localization only in the outer medial layer. The finding indicates that there is a possibility for LTBP-2 to compete with LTBP-1 for binding to fibrillin-containing microfibrils throughout the aortic wall but particularly in the outer medial region where the LTBP-1 is predominantly located. Overall, the results support the concept that that LTBP-2 may be an indirect negative modulator for storage of the large latent TGF-β complex on microfibrils in aorta and other fibrillin-rich tissues.

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.

Immunohistochemical Characterization of Elastic System Fibers in Rat Molar Periodontal Ligament

Among elastic system fibers, oxytalan fibers are known as a ubiquitous component of the periodontal ligament, but the localization and role of elastin-containing fibers, i.e., elastic and elaunin fibers, has yet to be clarified. In this study, we immunohistochemically investigated the localization of elastin and fibrillin, major proteins of elastin-containing fibers in the periodontal ligament of rat lower first molars. At the light microscope level, distribution of elastin-positive fibers was not uniform but often concentrated in the vicinity of blood vessels in the apical region of the ligament. In contrast, fibrillin-positive fibers were more widely distributed throughout the ligament, and the pattern of their distribution was comparable to the reported distribution of oxytalan fibers. At the ultrastructural level, assemblies or bundles of abundant fibrillin-containing microfibrils were intermingled with a small amount of elastin. This observation indicated that elastin-positive fibers observed under the light microscope were elaunin fibers. No mature elastic fibers, however, were found in the ligament. These results show that the major components of elastic system fibers in the periodontal ligament of the rat mandibular first molar were oxytalan and elaunin fibers, suggesting that the elastic system fibers play a role in the mechanical protection of the vascular system.

Development of a new in vitro model of elastic fiber assembly in human pigmented epithelial cells

Objectives: We developed an in vitro model of elastic fiber assembly that provides a comparison of the efficiency of different tropoelastin molecules to organize into fibers. Design and methods: Recombinant tropoelastin was added to ARPE-19 cell culture medium. The elastic fiber assembly was evaluated by immunofluorescence staining, the quantitative analysis of cross-linking amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. Results: We confirmed that ARPE-19 cells express fibrillin-containing microfibrils and lysyl oxidase, but they do not express tropoelastin. Immunofluorescence staining showed a dose- and time-dependent increase in the extracellular matrix. The quantity of cross-linking amino acids and matrix-associated tropoelastin also increased together with the matrix-associated elastin. Moreover, the analysis of a radioimmunoprecipitation assay (RIPA) buffer-soluble fraction indicated that tropoelastin interacted with microfibrils and cross-linked elastin was detected as a super molecular complex. Conclusion: These observations indicate that this in vitro model is especially useful for the analysis of mechanisms of elastic fiber formation.

MAGP-2 Has Multiple Binding Regions on Fibrillins and Has Covalent Periodic Association with Fibrillin-containing Microfibrils

The interactions of microfibril-associated glycoprotein (MAGP)-2 have been investigated with fibrillins and fibrillin-containing microfibrils. Solid phase binding assays were conducted with recombinant fragments covering fibrillin-1 and most of fibrillin-2. MAGP-2, and its structure relative MAGP-1, were found to bind two fragments spanning the N-terminal half of fibrillin-1 and an N-terminal fragment of fibrillin-2. Blocking experiments indicated that MAGP-2 had a binding site(s) close to the N terminus of the fibrillin-1 molecule that was distinct from that for MAGP-1 and an additional, more central binding site(s) that may be shared by the two MAGPs. Immunogold labeling of developing nuchal ligament tissue showed that MAGP-2 had regular covalent and periodic (about 56 nm) association with fibrillin-containing microfibrils of elastic fibers in this tissue. Further analysis of isolated microfibrils indicated that MAGP-2 was attached at two points along the microfibril substructure, "site 1" on the "beads" and "site 2" at the "shoulder" of the interbead region close to where the two "arms" fuse. In contrast, MAGP-1 was located only on the beads. Comparison of the MAGP-2 binding data with known fibrillin epitope maps of the microfibrils showed that site 1 correlated with the N-terminal MAGP-2 binding region, and site 2 correlated with the second, more central, MAGP-2 binding region on the fibrillin-1 molecule. Of particular note, immunolabeling at site 2 was markedly decreased, relative to that at site 1, on extended microfibrils with bead-to-bead periods over 90 nm, suggesting that site 2 may move toward the beads when the microfibril is stretched. The study points to MAGP-2 being an integral component of some populations of fibrillin-containing microfibrils. Moreover, the identification of multiple MAGP-binding sequences on fibrillins supports the concept that MAGPs may function as molecular cross-linkers, stabilizing fibrillin monomers in folded conformation within or between the microfibrils, and thus MAGPs may be implicated in the modulation of the elasticity of these structures.

Domains in Tropoelastin That Mediate Elastin Depositionin Vitro and in Vivo

Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities. However, the specific protein-protein interactions that facilitate elastin polymerization have not been defined. To identify domains in the tropoelastin molecule important for the assembly process, we utilized an in vitro assembly model to map sequences within tropoelastin that facilitate its association with fibrillin-containing microfibrils in the extracellular matrix. Our results show that an essential assembly domain is located in the C-terminal region of the molecule, encoded by exons 29-36. Fine mapping studies using an exon deletion strategy and synthetic peptides identified the hydrophobic sequence in exon 30 as a major functional element in this region and suggested that the assembly process is driven by the propensity of this sequence to form beta-sheet structure. Tropoelastin molecules lacking the C-terminal assembly domain expressed as transgenes in mice did not assemble nor did they interfere with assembly of full-length normal mouse elastin. In addition to providing important information about elastin assembly in general, the results of this study suggest how removal or alteration of the C terminus through stop or frameshift mutations might contribute to the elastin-related diseases supravalvular aortic stenosis and cutis laxa.

Spontaneous spinal cerebrospinal fluid leaks and minor skeletal features of Marfan syndrome: a microfibrillopathy.

Spontaneous spinal cerebrospinal fluid (CSF) leaks are increasingly recognized as a cause of postural headaches. The authors examined a group of patients suffering from spontaneous spinal CSF leaks who also had minor skeletal features of Marfan syndrome for abnormalities of fibrillin-containing microfibrils. Patients with spontaneous CSF leaks were evaluated for the clinical characteristics of connective tissue disorders. Skin biopsies were obtained in three patients with skeletal manifestations that constitute part of the Marfan syndrome phenotype. Cultured fibroblasts were studied for fibrillin-1 synthesis and incorporation into the extracellular matrix (ECM) by performing quantitative metabolic labeling and immunohistochemical analysis. Among 20 consecutive patients found to have spinal CSF leaks, four (20%) exhibited minor skeletal features of Marfan syndrome, but lacked any ocular or cardiovascular abnormalities. The mean age of these patients (30 years) was lower than that of the 16 patients without skeletal abnormalities (44 years; p = 0.01). Abnormalities in fibrillin-1 metabolism and immunostaining were detected in all three patients with the skeletal abnormalities who underwent examination, but not in a control patient without these skeletal manifestations. Twenty percent of patients who experience spontaneous spinal CSF leaks have minor skeletal features of Marfan syndrome. The authors demonstrated abnormalities in fibrillin-1 protein deposition in all patients examined, but only one person was found to have a fibrillin-1 abnormality typically found in classic Marfan syndrome. The results indicate that there is a heterogeneous involvement of other components of ECM microfibrils at the basis of this cerebrospinal manifestation. In addition, the authors identified a connective-tissue etiological factor in a group of disorders not previously classified as such.

Versican Interacts with Fibrillin-1 and Links Extracellular Microfibrils to Other Connective Tissue Networks

Fibrillin-containing microfibrils are polymeric structures that are difficult to extract from connective tissues. Proteolytic digestion of tissues has been utilized to release microfibrils for study. Few of the molecules that connect microfibrils to other elements in the matrix have been identified. In this study, electron microscopic immunolocalization of anti-versican antibodies in tissues and in extracted microfibrils demonstrated that the C-terminal region of versican is found associated with fibrillin microfibrils. Extraction of microfibrils followed by treatment of microfibrils under dissociating conditions suggested that the versican C terminus is covalently bound to microfibrils. Binding assays using recombinant fibrillin-1 polypeptides and recombinant lectican lectin domains indicated that the versican lectin domain binds to specific fibrillin-1 polypeptides. The versican lectin domain also bound to molecules comigrating with authentic fibrillin-1 monomers in an assay using cell culture medium. In assays using microfibrils, the versican lectin domain demonstrated preferential binding compared with other lecticans. Binding was calcium-dependent. The binding site for versican in microfibrils is most likely within a region of fibrillin-1 between calcium-binding epidermal growth factor-like domains 11 and 21. Human mutations in this region can result in severe forms of the Marfan syndrome ("neonatal" Marfan syndrome). The connection between versican and fibrillin microfibrils may be functionally significant, particularly in cardiovascular tissues.

Molecular interactions of biglycan and decorin with elastic fiber components: biglycan forms a ternary complex with tropoelastin and microfibril-associated glycoprotein 1.

The interactions of the dermatan sulfate proteoglycans biglycan and decorin have been investigated with the elastic fiber components, tropoelastin, fibrillin-containing microfibrils, and microfibril-associated glycoproteins (MAGP) 1 and 2. Both proteoglycans were found to bind tropoelastin and fibrillin-containing microfibrils but not MAGPs 1 and 2 in solid phase binding assays. The specificity of the binding of biglycan and decorin to tropoelastin was confirmed by co-immunoprecipitation experiments and by the blocking of the interactions with elastin-derived peptides. Isolated core proteins from biglycan and decorin bound to tropoelastin more strongly than the intact proteoglycans, and there were no differences in the tropoelastin binding characteristics of distinct glucuronate-rich and iduronate-rich glycoforms of biglycan. These findings indicated that the binding sites were contained in the protein cores of the proteoglycans rather than the glycosaminoglycan side chains. Scatchard analysis showed that biglycan bound more avidly than decorin to tropoelastin with K(d) values estimated as 1.95 x 10(-7) m and 5.3 x 10(-7) m, respectively. In blocking experiments each proteoglycan showed extensive inhibition of binding of the other to tropoelastin but was most effective at blocking its own binding. This result suggested that biglycan and decorin had closely spaced but distinct binding sites on tropoelastin. Addition of the elastin-binding protein MAGP-1 to the assays enhanced the binding of biglycan to tropoelastin but had no effect on the decorin-tropoelastin interaction. Co-immunoprecipitation experiments showed that MAGP-1 interacted with biglycan but not decorin in the solution phase. The results indicated that biglycan specifically formed a ternary complex with tropoelastin and MAGP-1. Overall the study supports the concept that biglycan may have a specific role in the elastinogenic phase of 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.

Expression of fibrillins and other microfibril-associated proteins in human bone and osteoblast-like cells

Fibrillin-containing microfibrils are structural components of extracellular matrices of a diverse range of tissues, including bone. Their importance in bone biology is illustrated by the skeletal abnormalities manifest in the congenital disorder, Marfan syndrome, which results from mutations in the fibrillin-1 gene. We investigated the expression of fibrillins and other microfibril-associated proteins in human bone and bone-derived osteoblasts. Analysis of RNA extracted from cancellous bone showed expression of mRNAs encoding fibrillin-1 and -2, MAGP-1 and -2, LTBP-2, and MP78/70 (Big-h3). In demineralized normal mature bone, fibrillin-1 was immunolocalized to fibrils within the bone matrix and pericellularly to cells lining the endosteal surfaces of trabecular bone, some osteocytes, and cells associated with blood vessels. LTBP-2 was also identified at the endosteal surface and within the bone matrix in a lamellar fashion. In addition, primary osteoblast-like cells cultured from human trabecular bone (obtained from patients at joint replacement surgery) were found to express abundant mRNA for fibrillins and associated glycoproteins. Moreover, using western blot analysis, fibrillin-1 protein was shown to be secreted into the medium and to be deposited into the cell layer. Immunofluorescence staining of the cell layer visualized fibrillin-1 in the matrix as a three-dimensional network of fine filaments. Expression of fibrillin-1 by osteoblast-like cells was constitutive, and a number of skeletally active agents had little effect on mRNA or protein levels. These results show that human osteoblasts from mature bone express fibrillins and other microfibril-associated proteins, and suggest a role for these molecules in adult human bone.

Role of the latent transforming growth factor beta binding protein 1 in fibrillin-containing microfibrils in bone cells in vitro and in vivo.

Latent transforming growth factor beta-binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor beta (TGF-beta) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67-467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGF beta 1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded.

Characterization of an in vitro model of elastic fiber assembly.

Elastic fibers consist of two morphologically distinct components: elastin and 10-nm fibrillin-containing microfibrils. During development, the microfibrils form bundles that appear to act as a scaffold for the deposition, orientation, and assembly of tropoelastin monomers into an insoluble elastic fiber. Although microfibrils can assemble independent of elastin, tropoelastin monomers do not assemble without the presence of microfibrils. In the present study, immortalized ciliary body pigmented epithelial (PE) cells were investigated for their potential to serve as a cell culture model for elastic fiber assembly. Northern analysis showed that the PE cells express microfibril proteins but do not express tropoelastin. Immunofluorescence staining and electron microscopy confirmed that the microfibril proteins produced by the PE cells assemble into intact microfibrils. When the PE cells were transfected with a mammalian expression vector containing a bovine tropoelastin cDNA, the cells were found to express and secrete tropoelastin. Immunofluorescence and electron microscopic examination of the transfected PE cells showed the presence of elastic fibers in the matrix. Biochemical analysis of this matrix showed the presence of cross-links that are unique to mature insoluble elastin. Together, these results indicate that the PE cells provide a unique, stable in vitro system in which to study elastic fiber assembly.