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Fibrillin-1 is an extracellular glycoprotein present throughout the body. Mutations in fibrillin-1 cause a wide spectrum of type I fibrillinopathies, including Marfan syndrome characterized by clinical manifestations in adipose tissues, among others. This study addresses the hypothesis that fibrillin-1 regulates adipocyte development and plays a vital role in adipose tissue homeostasis. We employed two mouse models - Fbn1

Multidomain matrix-associated zinc extracellular proteases ADAMTS and ADAMTS-like proteins have important biological activities in cells and tissues. Beyond their traditional role in procollagen and von Willebrand factor processing and proteoglycan cleavage, ADAMTS/ADAMTSL likely participate in or at least have some role in ECM assembly as some of these proteins bind ECM proteins including fibrillins, fibronectin, and LTBPs. In this chapter, we present four biophysical techniques largely used for the characterization, multimerization, and interaction of proteins: surface plasmon resonance spectroscopy, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy.

ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs) proteins regulate tissue homeostasis and extracellular matrix (ECM)-related pathogenesis. Some ADAMTS proteins interact with or process multiple ECM proteins, including fibrillins, fibronectin, and collagens. Therefore, characterization and quantification of these ECM fiber systems is essential to understand their functional relationship with ADAMTS proteins. Here we describe unbiased methods to quantify various aspects of ADAMTS-related ECM fiber systems in cell culture and in tissues. We focus on cell counting, overall fiber intensity, fiber length, and focal adhesion analysis in cell culture, and on the quantification of immunohistochemical and immunofluorescent tissue sections. We use ImageJ/Fiji, a widely used Java-based open source software which provides efficient and customizable quantification methods for microscopy images.

Fibrillins are one of the major components of supramolecular fibrous structures in the extracellular matrix of elastic and nonelastic tissues, termed microfibrils. Microfibrils provide tensile strength in nonelastic tissues and scaffolds for the assembly of tropoelastin in elastic tissues, and act a regulator of growth factor bioavailability and activity in connective tissues. Mutations in fibrillins lead to a variety of connective tissue disorders including Marfan syndrome, stiff skin syndrome, dominant Weill-Marchesani syndrome, and others. Therefore, fibrillins are frequently studied to understand the pathophysiology of these diseases and to identify effective treatment strategies. Extraction of endogenous microfibrils from cells and tissues can aid in obtaining structural insights of microfibrils. Recombinant production of fibrillins is an important tool which can be utilized to study the properties of normal fibrillins and the consequences of disease causing mutations. Other means of studying the role of fibrillins in the context of various physiological settings is by knocking down the mRNA expression and analyzing its downstream consequences. It is also important to study the interactome of fibrillins by protein-protein interactions, which can be derailed in pathological situations. Interacting proteins can affect the assembly of fibrillins in cells and tissues or can affect the levels of growth factors in the matrix. This chapter describes important techniques in the field that facilitate answering relevant questions of fibrillin biology and pathophysiology.

Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1.

Finegoldia magna is a Gram-positive anaerobic commensal of the human skin microbiota, but also known to act as an opportunistic pathogen. Two primary virulence factors of F. magna are the subtilisin-like extracellular serine protease SufA and the adhesive protein FAF. This study examines the molecular mechanisms F. magna uses when colonizing or establishing an infection in the skin. FAF was found to be essential in the initial adherence of F. magna to human skin biopsies. In the upper layers of the epidermis FAF mediates adhesion through binding to galectin-7 - a keratinocyte cell marker. Once the bacteria moved deeper into the skin to the basement membrane layer, SufA was found to degrade collagen IV which forms the backbone structure of the basement membrane. It also degraded collagen V, whereby F. magna could reach deeper dermal tissue sites. In the dermis, FAF interacts with collagen V and fibrillin, which presumably helps the bacteria to establish infection in this area. The findings of this study paint a clear picture of how F. magna interacts with human skin and explain how it is such a successful opportunistic pathogen in chronic wounds and ulcers.

Fibrillins constitute the backbone of extracellular multifunctional assemblies present in elastic and non-elastic matrices, termed microfibrils. Assembly of fibrillins into microfibrils and their homoeostasis is poorly understood and is often compromised in connective tissue disorders such as Marfan syndrome and other fibrillinopathies. Using interaction mapping studies, we demonstrate that fibrillins require the complete gelatin-binding region of fibronectin for interaction, which comprises domains FNI6–FNI9. However, the interaction of fibrillin-1 with the gelatin-binding domain of fibronectin is not involved in fibrillin-1 network assembly mediated by human skin fibroblasts. We show further that the fibronectin network is essential for microfibril homoeostasis in early stages. Fibronectin is present in extracted mature microfibrils from tissue and cells as well as in some in situ microfibrils observed at the ultrastructural level, indicating an extended mechanism for the involvement of fibronectin in microfibril assembly and maturation.

Summary Mutations in the fibrillin-1 gene give rise to a number of heritable disorders, which are all characterized by various malformations of bone as well as manifestations in other tissues. However, the role of fibrillin-1 in the development and homeostasis of bone is not well understood. Here, we examined the role of fibrillin-1 in regulating osteoclast differentiation from primary bone-marrow-derived precursors and monocytic RAW 264.7 cells. The soluble N-terminal half of fibrillin-1 (rFBN1-N) strongly inhibited osteoclastogenesis, whereas the C-terminal half (rFBN1-C) did not. By contrast, when rFBN1-N was immobilized on calcium phosphate, it did not affect osteoclastogenesis but modulated osteoclast resorptive activity, which was evident by a larger number of smaller resorption pits. Using a panel of recombinant sub-fragments spanning rFBN1-N, we localized an osteoclast inhibitory activity to the 63 kDa subfragment rF23 comprising the N-terminal region of fibrillin-1. Osteoclastic resorption led to the generation of small fibrillin-1 fragments that were similar to those identified in human vertebral bone extracts. rF23, but not rFBN1-N, was found to inhibit the expression of cathepsin K, matrix metalloproteinase 9 and Dcstamp in differentiating osteoclasts. rFBN1-N, but not rF23, exhibited interaction with RANKL. Excess RANKL rescued the inhibition of osteoclastogenesis by rFBN1-N. By contrast, rF23 disrupted RANKL-induced Ca2+ signaling and activation of transcription factor NFATc1. These studies highlight a direct dual inhibitory role of N-terminal fibrillin-1 fragments in osteoclastogenesis, the sequestration of RANKL and the inhibition of NFATc1 signaling, demonstrating that osteoclastic degradation of fibrillin-1 provides a potent negative feedback that limits osteoclast formation and function.

ADAMTS-like proteins are related to ADAMTS metalloproteases by their similarity to ADAMTS ancillary domains. Here, we have characterized ADAMTSL5, a novel member of the superfamily with a unique modular organization that includes a single C-terminal netrin-like (NTR) module. Alternative splicing of ADAMTSL5 at its 5′ end generates two transcripts that encode different signal peptides, but the same mature protein. These transcripts differ in their translational efficiency. Recombinant ADAMTSL5 is a secreted, N-glycosylated 60 kDa glycoprotein located in the subcellular matrix, on the cell-surface, and in the medium of transfected cells. RT-PCR and western blot analysis of adult mouse tissues showed broad expression. Western blot analysis suggested proteolytic release of the NTR module in transfected cells as well as in some mouse tissues. Immunostaining during mouse organogenesis identified ADAMTSL5 in musculoskeletal tissues such as skeletal muscle, cartilage and bone, as well as in many epithelia. Affinity-chromatography demonstrated heparin-binding of ADAMTSL5 through its NTR-module. Recombinant ADAMTSL5 bound to both fibrillin-1 and fibrillin-2, and co-localized with fibrillin microfibrils in the extracellular matrix of cultured fibroblasts, but without discernible effect on microfibril assembly. ADAMTSL5 is the first family member shown to bind both fibrillin-1 and fibrillin-2. Like other ADAMTS proteins implicated in microfibril biology through identification of human and animal mutations, ADAMTSL5 could have a role in modulating microfibril functions.

Homocystinuria is a genetic disorder resulting in elevated levels of homocysteine in plasma and tissues. Some of the skeletal and ocular symptoms such as long bone overgrowth, scoliosis, and ectopia lentis overlap with symptoms seen in Marfan syndrome. Marfan syndrome is caused by mutations in the extracellular matrix protein fibrillin-1. We previously showed that fibrillin-1 is a target for homocysteine and that the deposition of homocysteinylated fibrillin-1 in the extracellular matrix is compromised. Since the assembly of fibrillin-1 is critically dependent on fibronectin, we analyzed the consequences of fibronectin homocysteinylation and its interaction with fibrillin-1. Cellular fibronectin and proteolytic fragments were homocysteinylated and tested in various interaction assays with recombinant fibrillin-1 and heparin. Fibronectin homocysteinylation consistently compromised the fibronectin-fibrillin-1 interaction, while the interaction with heparin was not affected. Fibronectin homocysteinylation, but not cysteinylation, reduced the fibronectin dimers to monomers as shown by Western blotting. ELISA analyses of homocysteinylated fibronectin with three monoclonal antibodies demonstrated structural changes in the disulfide-containing FNI domains FNI(2), FNI(4), and FNI(9). Using fluorescently labeled fibronectin, we studied the consequence of fibronectin homocysteinylation on assembly in cell culture. Modified fibronectin showed deficiencies in denovo matrix incorporation and initial assembly. In conclusion, we define here characteristic structural changes of fibronectin upon homocysteinylation that translate into functional deficiencies in the fibronectin-fibrillin-1 interaction and in fibronectin assembly. Since fibronectin is a major organizer of various extracellular protein networks, these structural and functional alterations may contribute to the pathogenesis of homocystinuria and Marfan syndrome.

Contains fulltext : 79597.pdf (Publisher’s version ) (Closed access) Micromechanical properties of single elastic fibers and fibrillin-microfibrils, isolated from equine ligamentum nuchae using chemical and enzymatic methods, were determined with atomic force microscopy (AFM). Young's moduli of single elastic fibers immersed in water, devoid of or containing fibrillin-microfibrils, were determined using bending tests. Bending freely suspended elastic fibers on a micro-channeled substrate by a tip-less AFM cantilever generated a force versus displacement curve from which Young's moduli were calculated. For single elastic fibers, Young's moduli in the range of 0.3-1.5 MPa were determined, values not significantly affected by the presence of fibrillin-microfibrils. To further understand the role of fibrillin-microfibrils in vertebrate elastic fibers, layers of fibrillin-microfibrils were subjected to nano-indentation tests. From the slope of the force versus indentation curves, Young's moduli ranging between 0.56 and 0.74 MPa were calculated. The results suggest that fibrillin-microfibrils are not essential for the mechanical properties of single vertebrate elastic fibers.

Since the discovery of epithelial hair follicle stem cells (eHFSCs) in the bulge of human hair follicles (HFs) an important quest has started: to define useful markers. In the current study, we contribute to this by critically evaluating corresponding published immunoreactivity (IR) patterns, and by attempting to identify markers for the in situ identification of human eHFSCs and their niche. For this, human scalp skin cryosections of at least five different individuals were examined, employing standard immunohistology as well as increased sensitivity methods. Defined reference areas were compared by quantitative immunohistochemistry for the relative intensity of their specific IR. According to our experience, the most useful positive markers for human bulge cells turned out to be cytokeratin 15, cytokeratin 19 and CD200, but were not exclusive, while beta1 integrin and Lhx2 IR were not upregulated by human bulge keratinocytes. Absent IR for CD34, connexin43 and nestin on human bulge cells may be exploited as negative markers. alpha6 integrin, fibronectin, nidogen, fibrillin-1 and latent transforming growth factor (TGF)-beta-binding protein-1 were expressed throughout the connective tissue sheath of human HFs. On the other hand, tenascin-C was upregulated in the bulge and may thus constitute a component of the bulge stem cell niche of human HFs. These immunophenotyping results shed further light on the in situ expression patterns of claimed follicular 'stem cell markers' and suggest that not a single marker alone but only the use of a limited corresponding panel of positive and negative markers may offer a reasonable and pragmatic compromise for identifying human bulge stem cells in situ.

Incisional hernias represent one of the most common complications after laparotomy. Specific pre-operative risk factors have not yet been identified. Recent studies indicate that changes in extracellular matrix components such as collagen I and collagen III may be involved in hernia development. In the present study we have evaluated the significance of fibrillin-1 in hernia development as one of the main components of the extracellular matrix. Tissue samples from non-scar skin and muscle fascia of 12 patients with incisional hernias as well as from the respective scar tissues were obtained. Corresponding tissue samples of 10 patients with normal postoperative wound healing served as controls. Distribution of fibrillin-1 was evaluated immunohistochemically. Differences in fibrillin-1 distribution in the non-scar tissues of muscle fascia have been found in patients with incisional hernia, compared to those without hernia. In scar regions of both patient groups, slight differences in the pattern of fibrillin-1 were observed. A tendency to a differential deposition of fibrillin-1 in skin samples, although hardly quantifiable, was observed as well. Our results suggest that fibrillin-1 is a relevant factor contributing to tissue stability. Disturbances in its deposition, even before scar formation, may be an important factor to the development of incisional hernias.

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.

The pathogenesis of fibrosis, especially involving post-translational modifications of collagen, is poorly understood. Lysyl hydroxylase 2 (long) (LH2 (long)) is thought to play a pivotal role in fibrosis by directing the collagen cross-link pattern. Here we show that LH2 (long) exerts a bimodal function on collagen synthesis in human dermal fibroblasts. Adenoviral-mediated overexpression of LH2 (long) resulted in a mRNA increase of collagen α1(I) but not of fibronectin and fibrillin-1. This was accompanied by a higher mRNA level of prolyl-4-hydroxylase but not of other ER proteins (Bip, Hsp47, LH1, LH3). The collagen mRNA increase led to an elevated collagen synthesis, which was higher in the fraction of extracellularly deposited, cell-associated collagen than in the medium. The cross-link pattern of cell-associated collagen showed an increase of the hydroxylysine-aldehyde-derived cross-link dihydroxylysinonorleucine and a decrease of the lysine-aldehyde-derived component hydroxylysinonorleucine. The helical lysyl hydroxylation of the procollagen molecule was unaltered. The increase of collagen synthesis in fibroblasts overexpressing LH2 (long) was independent from cross-linking as it was also observed in the presence of β-aminopropionitril, a cross-linking inhibitor. Together our data identify LH2 (long) as a bifunctional protein and underscores its potential role in the pathogenesis of fibrosis.

The microfibrillar protein fibrillin-1 is a component of the mesangial matrix. Defects in fibrillin-1 predisposes individuals to vascular damage in Marfan syndrome, but the role of fibrillin-1 in kidney disease is unknown. We hypothesized that fibrillin-1 is involved in hypertensive or diabetic glomerular disease. DOCA-salt hypertension or streptozotocin (STZ) diabetes led to a significant increase in glomerular fibrillin-1 deposition. To test the functional role of fibrillin-1, DOCA hypertension and STZ diabetes were induced in mice homozygous for a mutation leading to a fivefold lower expression of fibrillin-1 (mgR/mgR). Untreated male mgR/mgR mice usually die from aortic dissection during the first 4 mo of life. All DOCA-treated mgR/mgR mice died within 2 wk after onset of DOCA treatment. DOCA-treated heterozygous (mgR/+) and their wild-type littermates displayed similar blood pressure levels, but albuminuria was significantly lower in mgR/+ than in wild-type mice after DOCA treatment. Similarly, STZ diabetic mgR/mgR and mgR/+ developed lower albuminuria than wild-type mice despite higher blood glucose levels in mgR/mgR and mgR/+ compared with wild-type mice. Blood pressure, blood glucose, and albuminuria did not differ among untreated mgR/mgR, mgR/+, and wild-type mice, respectively. In diabetic mgR/+ and mgR/mgR, but not in wild-type mice, an induction of glomerular decorin expression was observed. Thus underexpression of fibrillin-1 predisposes individuals to lethal aortic dissection in the presence of hypertension. On the other hand, albuminuria as a parameter of microvascular damage in hypertension and diabetes was ameliorated in fibrillin-1-underexpressing mice, possibly due to a compensatory upregulation of decorin. We conclude that fibrillin-1 may contribute to glomerular damage in hypertensive and diabetic kidney disease.

Fibrillin-1 localization in the myocardium and the modulation of its expression in cardiac fibrosis were examined. In normal rat hearts, fibrillin-1 was abundant throughout the myocardium as thin fibers that crossed over the perimysium and around arteries. After cardiac fibrosis was induced in rats by either 14-day ANG II infusion or 21-day DOCA-salt treatment [a high endothelin-1 (ET-1) model], fibrillin-1 immunostaining was stronger in the interstitium (2.8-fold and 4.4-fold increases, respectively, in each model), extended between myocytes, and accumulated in microscopic scars and in the perivascular area of both ventricles. mRNA analysis confirmed its enhanced ventricular expression in both groups of rats (2.5-fold and 6.6-fold increments, respectively, in each model). In 1B normotensive and 2C hypertensive transgenic mice, two lines expressing an ANG II fusion protein in cardiac myocytes, strong fibrillin-1 immunoreactivity was observed in the interstitium and around arteries (3.7-fold and 7-fold increases, respectively). ANG II and transforming growth factor-β1 enhanced fibrillin-1 synthesis by cardiac fibroblasts. Some fibrillin-1 fragments interacted with RGD-dependent integrins, including α8β1-integrin, of cardiac fibroblasts but not necessarily through the RGD motif. Our findings illustrate that fibrillin-1 is an important constituent of the myocardium. In vitro and in vivo evidence suggests that ANG II can directly induce fibrillin-1 expression in cardiac fibroblasts. This protein can thus contribute to reactive and reparative processes.

Mutational defects in fibrillin-rich microfibrils give rise to a number of heritable connective tissue disorders, generally termed microfibrillopathies. To understand the pathogenesis of these microfibrillopathies, it is important to elucidate the supramolecular composition of microfibrils and their interaction properties with extracellular matrix components. Here we demonstrate that the proteoglycan perlecan is an associated component of microfibrils typically close to basement membrane zones. Double immunofluorescence studies demonstrate colocalization of fibrillin-1, the major backbone component of microfibrils, with perlecan in fibroblast cultures as well as in dermal and ocular tissues. Double immunogold labeling further confirms colocalization of perlecan to microfibrils in various tissues at the ultrastructural level. Extraction studies revealed that perlecan is not covalently associated with microfibrils. High affinity interactions between fibrillin-1 and perlecan were found by kinetic binding studies with dissociation constants in the low nanomolar range. A detailed mapping study of the interaction epitopes by solid phase binding assays primarily revealed interactions of perlecan domains I and II with a central region of fibrillin-1. Analysis of perlecan null embryos showed less microfibrils at the dermal-epidermal junction as compared with wild-type littermates. The data presented indicate a functional significance for perlecan in anchoring microfibrils to basement membranes and in the biogenesis of microfibrils.

The Marfan syndrome (MFS), a relatively common autosomal dominant disorder of connective tissue, is caused by mutations in the gene for fibrillin-1 (FBN1). Fibrillin-1 is the main component of the 10- to 12-nm microfibrils that together with elastin form elastic fibers found in tissues such as the aortic media. Recently, FBN1 mutations have been shown to increase the susceptibility of fibrillin-1 to proteolysis in vitro, and other findings suggest that up-regulation of matrix metalloproteinases (MMP), as well as fragmentation of microfibrils, could play a role in the pathogenesis of MFS. In the present work, we have investigated the influence of fibrillin-1 fragments on the expression of MMP-1, MMP-2, and MMP-3 in a cell culture system. Cultured human dermal fibroblasts were incubated with several different recombinant fibrillin-1 fragments. The expression level of MMP-1, MMP-2, and MMP-3, was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and the concentration of the corresponding proteins was estimated by quantitative Western blotting. Our results establish that treatment of cultured human dermal fibroblasts with recombinant fibrillin-1 fragments containing the arginine-glycine-aspartic acid (RGD) integrin-binding motif of fibrillin-1 induces up-regulation of MMP-1 and MMP-3. A similar effect was seen upon stimulation with a synthetic RGD peptide. The expression of MMP-2 was not influenced by treatment. Our results suggest the possibility that fibrillin fragments could themselves have pathogenic effects by leading to up-regulation of MMPs, which in turn may be involved in the progressive breakdown of microfibrils thought to play a role in MFS.

There is growing evidence that the two small leucine-rich proteoglycans biglycan and decorin regulate the assembly of connective tissues and alter cell behavior during development and pathological processes. In this study, we have used an experimental animal model of unilateral ureteral ligation and mice deficient in either biglycan or decorin. We discovered that pressure-induced injury to the wild-type kidneys led to overexpression of decorin, biglycan, fibrillin-1, and fibrillin-2. In contrast, in biglycan-deficient kidneys the overexpression of fibrillin-1 was markedly attenuated and this was associated with cystic dilatation of Bowman’s capsule and proximal tubules. Notably, we found that in ligated kidneys from decorin-null mice, fibrillin-1 expression was initially enhanced to the same extent as in wild-type animals. However, long-term obstruction resulted in down-regulation of fibrillin-1 and concurrent cystic dilatation of Bowman’s capsule in 33% of kidneys at 5 months after obstruction. In all of the genotypes, no differences in fibrillin-2 expression were observed. These in vivo data correlated with a significant induction of fibrillin-1 expression in renal fibroblasts and mesangial cells by recombinant biglycan and decorin. Our results indicate a novel role for decorin and biglycan during pressure-induced renal injury by stimulating fibrillin-1 expression.

Mutations in fibrillin-1 lead to Marfan syndrome and some related genetic disorders. Many of the more than 600 mutations currently known in fibrillin-1 eliminate or introduce cysteine residues in epidermal growth factor-like modules. Here we report structural and functional consequences of three selected cysteine mutations (R627C, C750G, and C926R) in fibrillin-1. The mutations have been analyzed by means of recombinant polypeptides produced in mammalian expression systems. The mRNA levels for the mutation constructs were similar to wild-type levels. All three mutated polypeptides were secreted by embryonic kidney cells (293) into the culture medium. Purification was readily feasible for mutants R627C and C750G, but not for C926R, which restricted the availability of this mutant polypeptide to selected analyses. The overall folds of the mutant polypeptides were indistinguishable from the wild-type as judged by the ultrastructural shape, CD analysis, and reactivity with a specific antibody sensitive for intact disulfide bonds. Subtle structural changes caused by R627C and C750G, however, were monitored by proteolysis and heat denaturation experiments. These changes occurred in the vicinity of the mutations either as short range effects (R627C) or both short and long range effects (C750G). Enhanced proteolytic susceptibility was observed for R627C and C750G to a variety of proteases. These results expand and further strengthen the concept that proteolytic degradation of mutated fibrillin-1 might be an important potential mechanism in the pathogenesis of Marfan syndrome and other disorders caused by mutations in fibrillin-1.

Fibrillin-1 is a microfibrillar extracellular matrix protein that was described to be a ligand for α8 integrin. α8 integrin is a matrix receptor specifically expressed in mesangial and smooth muscle cells of the kidney. In previous studies we detected glomerular expression of fibrillin-1. Moreover, fibrillin-1 promoted adhesion, migration, and proliferation of mesangial cells. We hypothesized that fibrillin-1 and α8 integrin might interact in the glomerulus, and thus, regulate mesangial cell properties. Our studies showed that fibrillin-1 and α8 integrin colocalize in the glomerular mesangium. Induction of experimental glomerulonephritis led to an increase of both fibrillin-1 and α8 integrin expression. In vitro studies revealed that mesangial cells deficient for α8 integrin adhere weaker to fibrillin-1 and migrate more easily on fibrillin-1 than wild-type mesangial cells. Baseline proliferation on fibrillin-1 is higher in α8 integrin-deficient mesangial cells, but the induction of proliferation is not different in α8 integrin-deficient and wild-type mesangial cells. We conclude that fibrillin-1 and α8 integrin interact, and thus, regulate mesangial cell adhesion and migration. The concomitant induction of both fibrillin-1 and α8 integrin in a self-limited model of glomerular injury points to a protective role of the interaction of fibrillin-1 with α8 integrin in the glomerulus resulting in reduced damage of the glomerular tuft as a consequence of firm adhesion of mesangial cells.

Mutations in the secreted glycoprotein ADAMTSL2 cause recessive geleophysic dysplasia (GD) in humans and Musladin–Lueke syndrome (MLS) in dogs. GD is a severe, often lethal, condition presenting with short stature, brachydactyly, stiff skin, joint contractures, tracheal-bronchial stenosis and cardiac valve anomalies, whereas MLS is non-lethal and characterized by short stature and severe skin fibrosis. Although most mutations in fibrillin-1 (FBN1) cause Marfan syndrome (MFS), a microfibril disorder leading to transforming growth factor-β (TGFβ) dysregulation, domain-specific FBN1 mutations result in dominant GD. ADAMTSL2 has been previously shown to bind FBN1 and latent TGFβ-binding protein-1 (LTBP1). Here, we investigated mice with targeted Adamtsl2 inactivation as a new model for GD (Adamtsl2−/− mice). An intragenic lacZ reporter in these mice showed that ADAMTSL2 was produced exclusively by bronchial smooth muscle cells during embryonic lung development. Adamtsl2−/− mice, which died at birth, had severe bronchial epithelial dysplasia with abnormal glycogen-rich inclusions in bronchial epithelium resembling the cellular anomalies described previously in GD. An increase in microfibrils in the bronchial wall was associated with increased FBN2 and microfibril-associated glycoprotein-1 (MAGP1) staining, whereas LTBP1 staining was increased in bronchial epithelium. ADAMTSL2 was shown to bind directly to FBN2 with an affinity comparable to FBN1. The observed extracellular matrix (ECM) alterations were associated with increased bronchial epithelial TGFβ signaling at 17.5 days of gestation; however, treatment with TGFβ-neutralizing antibody did not correct the epithelial dysplasia. These investigations reveal a new function of ADAMTSL2 in modulating microfibril formation, and a previously unsuspected association with FBN2. Our studies suggest that the bronchial epithelial dysplasia accompanying microfibril dysregulation in Adamtsl2−/− mice cannot be reversed by TGFβ neutralization, and thus might be mediated by other mechanisms., Summary: The extracellular protein ADAMTSL2 is a crucial regulator of microfibril composition in the extracellular matrix of bronchial smooth muscle cells and influences bronchial epithelial function.

Fibrillins form multifunctional microfibrils in most connective tissues. Deficiencies in fibrillin assembly can result in fibrillinopathies, such as Marfan syndrome. We demonstrate the presence of heparin/heparan sulfate binding sites in fibrillin-2 and -3. Multimerization of all three fibrillins drastically increased the apparent affinity of their interaction with heparin/heparan sulfate. Surprisingly, contrary to other reports heparin/heparan sulfate strongly inhibited homo- and heterotypic N-to-C-terminal fibrillin interactions. These data suggest that heparin/heparan sulfate controls the formation of microfibrils at the bead interaction stage.

Fibrillin proteins constitute the backbone of extra-cellular macromolecular microfibrils. Mutations in fibrillins cause heritable connective tissue disorders, including Marfan syndrome, dominant Weill-Marchesani syndrome, and stiff skin syndrome. Fibronectin provides a critical scaffold for microfibril assembly in cell culture models. Full length recombinant fibrillin-1 was expressed by HEK 293 cells, which deposited the secreted protein in a punctate pattern on the cell surface. Cocultured fibroblasts consistently triggered assembly of recombinant fibrillin-1, which was dependent on a fibronectin network formed by the fibroblasts. Deposition of recombinant fibrillin-1 on fibronectin fibers occurred first in discrete packages that subsequently extended along fibronectin fibers. Mutant fibrillin-1 harboring either a cysteine 204 to serine mutation or a RGD to RGA mutation which prevents integrin binding, did not affect fibrillin-1 assembly. In conclusion, we developed a modifiable recombinant full-length fibrillin-1 assembly system that allows for rapid analysis of critical roles in fibrillin assembly and functionality. This system can be used to study the contributions of specific residues, domains, or regions of fibrillin-1 to the biogenesis and functionality of microfibrils. It provides also a method to evaluate disease-causing mutations, and to produce microfibril-containing matrices for tissue engineering applications, for example, in designing novel vascular grafts or stents.

Purpose: Mutations in human fibrillin-1 and -2, which are major constituents of tissue microfibrils, can affect multiple ocular components, including the ciliary zonule, lens, drainage apparatus, cornea, and retina. However, the expression pattern of the three human fibrillins and an integral microfibrillar component, MAGP1, during human eye development is not known. Methods: We analyzed sections from human eyes at gestational weeks (GWs) 6, 8, and 11 and at 1 and 3 years of age with antibodies specific for each human fibrillin isoform or MAGP1, using immunofluorescence microscopy. Results: During embryonic development, each fibrillin isoform was detected in vascular structures bridging the ciliary body and the developing lens, hyaloid vasculature, and retina. In addition, they were present in the developing corneal basement membranes and lens capsule. MAGP1 codistributed with the fibrillin isoforms. In contrast, the juvenile zonule was composed of fibrillin-1 microfibrils containing MAGP1, but fibrillin-2 was absent and fibrillin-3 was only sparsely detected. Conclusions: Fibrillin-1, -2, and, unique to humans, fibrillin-3 are found in various ocular structures during human embryonic eye development, whereas fibrillin-1 dominates the postnatal zonule. We speculate that vasculature spanning the ciliary body and lens, which elaborates fibrillin-2 and -3, may provide an initial scaffold for fibrillin assembly and zonule formation. © 2014 The Association for Research in Vision and Ophthalmology, Inc.

Elastic fibers consist primarily of an amorphous elastin core associated with microfibrils, 10-12 nm in diameter, containing fibrillins and microfibril-associated glycoproteins (MAGPs). To investigate the interaction of MAGP-1 with tropoelastin and fibrillin-1, we expressed human MAGP-1 as a T7-tag fusion protein in Escherichia coli. Refolding of the purified protein produced a soluble form of MAGP-1 that displayed saturable binding to tropoelastin. Fragments of tropoelastin corresponding to the N-terminal, C-terminal, and central regions of the molecule were used to characterize the MAGP-1 binding site. Cleavage of tropoelastin with kallikrein, which cleaves after Arg(515) in the central region of the molecule, disrupted the interaction, suggesting that the separated N- and C-terminal fragments were insufficient to determine MAGP-1 binding to intact tropoelastin. In addition, no evidence of an interaction was observed between MAGP-1 and a tropoelastin construct consisting of domains 17-27 that brackets the kallikrein cleavage site, suggesting a complex mechanism of interaction between the two molecules. Binding of MAGP-1 was also tested with overlapping recombinant fibrillin-1 fragments. MAGP-1 bound to a region at the N terminus of fibrillin-1 in a calcium-dependent manner. In summary, these results suggest a model for the interaction of elastin with the microfibrillar scaffold.

Most extracellular proteins consist of various modules with distinct functions. Mutations in one common type, the calcium-binding epidermal growth factor-like module (cbEGF), can lead to a variety of genetic disorders. Here, we describe as a model system structural and functional consequences of two typical mutations in cbEGF modules of fibrillin-1 (N548I, E1073K), resulting in the Marfan syndrome. Large (80-120 kDa) wild-type and mutated polypeptides were recombinantly expressed in mammalian cells. Both mutations did not alter synthesis and secretion of the polypeptides into the culture medium. Electron microscopy after rotary shadowing and comparison of circular dichroism spectra exhibited minor structural differences between the wild-type and mutated forms. The mutated polypeptides were significantly more susceptible to proteolytic degradation by a variety of proteases as compared with their wild-type counterparts. Most of the sensitive cleavage sites were mapped close to the mutations, indicating local structural changes within the mutated cbEGF modules. Other cleavage sites, however, were observed at distances beyond the domain containing the mutation, suggesting longer range structural effects within tandemly repeated cbEGF modules. We suggest that proteolytic degradation of mutated fibrillin-1 may play an important role in the pathogenesis of Marfan syndrome and related disorders.

Fibrillins are the major constituents of extracellular microfibrils. How fibrillin molecules assemble into microfibrils is not known. Sequential extractions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing fibrillin-1. These results demonstrated that intermolecular disulfide bond formation is an initial step in the assembly process. To identify free cysteine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed. Results revealed that the first four cysteine residues in the unique N terminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thiol and is therefore a good candidate for intermolecular disulfide bonding in initial steps of the assembly process. Furthermore, evidence indicated that the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily available for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage site of the fibrillin-1 signal peptide and the N-terminal sequence of monomeric authentic fibrillin-1 from conditioned fibroblast medium were determined.

Aortic aneurysm and dissection account for about 2% of all deaths in industrialized countries; they are also components of several genetic diseases, including Marfan syndrome (MFS). The vascular phenotype of MFS results from mutations in fibrillin-1 (FBN1), the major constituent of extracellular microfibrils. Microfibrils, either associated with or devoid of elastin, give rise to a variety of extracellular networks in elastic and non-elastic tissues. It is believed that microfibrils regulate elastic fibre formation by guiding tropo-elastin deposition during embryogenesis and early post-natal life. Hence, vascular disease in MFS is thought to result when FBN1 mutations preclude elastic fibre maturation by disrupting microfibrillar assembly. Here we report a gene-targetting experiment in mice that indicates that fibrillin-1 microfibrils are predominantly engaged in tissue homeostasis rather than elastic matrix assembly. This finding, in turn, suggests that aortic dilation is due primarily to the failure by the microfibrillar array of the adventitia to sustain physiological haemodynamic stress, and that disruption of the elastic network of the media is a secondary event.

Velocity sedimentation experiments using authentic fibrillin-1 demonstrated sedimentation coefficients of s20,w0 = 5.1 +/- 0.1 in the Ca2+ form and s20,w0 = 6.2 +/- 0.1 in the Ca2+-free form. Calculations based on these results and the corresponding molecular mass predicted a shortening of fibrillin by approximately 25% and an increase in width of approximately 13-17% upon removal of Ca2+. These observations were confirmed by analysis of Ca2+-loaded and Ca2+-free rotary shadowed fibrillin molecules. Analysis of recombinant fibrillin-1 subdomain rF17, consisting primarily of an array of 12 Ca2+-binding epidermal growth factor (cbEGF)-like repeats, by analytical ultracentrifugation and rotary shadowing further confirmed Ca2+-dependent structural changes in the tertiary structure of fibrillin-1. Based on these results, the contribution of a single cbEGF-like repeat to the length of tandem arrays is predicted to be approximately 3 nm in the Ca2+ form. Ca2+-free forms demonstrated a decrease of 20-30% in length, indicating significant structural changes of these motifs when they occur in tandem. Circular dichroism measurements of rF17 in the presence and absence of Ca2+ indicated secondary structural changes within and adjacent to the interdomain regions that connect cbEGF-like repeats. The results presented here suggest a flexible structure for the Ca2+-free form of fibrillin which becomes stabilized, more extended, and rigid in the Ca2+ form.

The calcium-binding epidermal growth factor (cbEGF)-like domain is a structural motif that is present in many matrix proteins throughout the animal kingdom from invertebrates to mammals. This module has been demonstrated to bind calcium in the micromolar range. However, little is known about the functional consequences of calcium binding to proteins that contain this structural element. We used fibrillin-1, an extracellular matrix protein consisting of approximately 60% cbEGF-like motifs, as a model system to study stabilizing effects of calcium in protease degradation assays. Authentic human fibrillin-1 and recombinant human fibrillin-1 subdomains, spanning the whole molecule, showed significantly slower proteolytic degradation in the presence of CaCl2 than in the presence of EDTA, demonstrating that calcium stabilizes the structure of fibrillin-1 and protects the molecule against proteolytic degradation. Information about cleavage sites protected by calcium was obtained with a new recombinant subdomain, rF17 (Asp 952-Val 1527), comprising the longest stretch of cbEGF-like motifs in the center of the fibrillin-1 molecule. The most sensitive sites for trypsin and endoproteinase Glu-C were observed in cbEGF-like motifs 11 (Met 1034 and Asn 1046), 12 (Ser 1103), and 17 (Thr 1318). Since most of the currently known mutations in fibrillin-1 are found within cbEGF-like motifs and are predicted to disrupt calcium binding, we suggest that these mutations render fibrillin-1 more susceptible to proteolytic cleavage, and this might be one of the reasons why these mutations result in Marfan's syndrome.

Fibrillins are the major constituent of tissue microfibrils, which form the ocular zonule. In Marfan syndrome (MFS), FBN1 mutations lead to ectopia lentis. The goal of this work was to investigate zonule composition and formation in fibrillin-deficient and wild-type mice.Immunofluorescence staining of eyes from wild-type, Fbn1-deficient, and Fbn2-deficient mice, as well as other species, was performed using monospecific fibrillin 1 and fibrillin 2 antibodies. The zonule of Fbn1-deficient and Fbn2-deficient mice was studied by electron microscopy. Microfibril formation in vitro was evaluated by immunofluorescence microscopy of cultured nonpigmented ciliary epithelial cells and fibroblasts.A zonule was present in both Fbn1-deficient and Fbn2-deficient mouse eyes. Immunofluorescence demonstrated that the zonule of Fbn1-deficient mice, wild-type mice, rats, and hamsters contained fibrillin 2. The zonule of Fbn2(-/-) mice contained fibrillin 1. Fibrillin 1 and fibrillin 2 colocalized in microfibrils formed in human nonpigmented ciliary epithelium cultures. Like fibrillin 1, fibrillin 2 microfibril assembly was fibronectin dependent and initiated by cell surface punctate deposits that elongated to form microfibrils.These data suggest that fibrillin 1 assembly and fibrillin 2 assembly share similar mechanisms. Microfibril composition depends substantially on the local levels of fibrillin isoforms and is not highly selective in regard to the isoform. This raises the intriguing possibility that the zonule could be strengthened in MFS by inducing fibrillin 2 expression in ciliary epithelium. The presence of fibrillin 2 in the murine zonule and an intact zonule in Fbn1-knockout mice may limit the utility of rodent models for studying ectopia lentis in MFS.

Background: Latent transforming growth factor-β-binding protein 1 (LTBP-1) and fibrillin-1 were shown to colocalize and interact in the extracellular matrix of the skin and vasculature. This interaction may regulate transforming growth factor-β (TGF-β) activity. TGF-β is an important progression factor for glomerular diseases. We hypothesized that LTBP-1 and fibrillin-1 are coexpressed in the glomerulus and upregulated during glomerulonephritis. Methods: Acute anti-Thy1.1 glomerulonephritis was induced with a single intravenous injection (1 mg/kg body weight) of a monoclonal anti-Thy1.1 antibody in rats. Real-time RT-PCR and immunohistochemical analyses for LTBP-1 and fibrillin-1 were performed. Results: Induction of glomerular LTBP-1 mRNA was detected on day 2 of disease, while mRNA for fibrillin-1 was already upregulated 1 day after induction of disease. Both LTBP-1 and fibrillin-1 showed a mesangial distribution. An expansion of the LTBP-1 and fibrillin-1-positive mesangial area was seen on day 6 of disease, when transient matrix accumulation was most prominent. On day 12 of disease, glomerular LTBP-1 and fibrillin-1 immunoreactivities had returned to control levels. In serial sections, some colocalization of LTBP-1 and fibrillin-1 was detected in control as well as in nephritic glomeruli. Conclusion: Mesangial expression of LTBP-1 and fibrillin-1 is induced early in experimental nephritis and LTBP-1 and fibrillin-1 are partially colocalized in the nephritic glomerulus. An interaction of these molecules could stabilize latent TGF-β complexes and thus attenuate the activation of TGF-β during this self-limited glomerular disease.

Mutations in fibrillin-1 give rise to Marfan syndrome (MFS) characterized by vascular, skeletal, and ocular abnormalities. Fibrillins form the backbone of extracellular matrix microfibrils in tissues including blood vessels, bone, and skin. They are crucial for regulating elastic fiber biogenesis and growth factor bioavailability. To compare the molecular consequences of mutations causing the severe neonatal MFS with mutations causing the milder classical MFS, we introduced representative point mutations from each group in a recombinant human fibrillin-1 fragment. Structural effects were analyzed by circular dichroism spectroscopy and analytical gel filtration chromatography. Proteolytic susceptibility was probed with non-physiological and physiological proteases, including plasmin, thrombin, matrix metalloproteinases, and cathepsins. All mutant proteins showed a similar gross secondary structure and no differences in heat stability as compared with the wild-type protein. Proteins harboring neonatal mutations were typically more susceptible to proteolytic cleavage compared with those with classical mutations and the wild-type protein. Proteolytic neo-cleavage sites were found both in close proximity and distant to the mutations, indicating small but significant structural changes exposing cryptic cleavage sites. We also report for the first time that cathepsin K and V cleave non-mutated fibrillin-1 at several domain boundaries. Compared with the classical mutations and the wild type, the group of neonatal mutations more severely affected the ability of fibrillin-1 to interact with heparin/heparan sulfate, which plays a role in microfibril assembly. These results suggest differential molecular pathogenetic concepts for neonatal and classical MFS including enhanced proteolytic susceptibility for physiologically relevant enzymes and loss of function for heparin binding.

Although not often discussed, the ovaries of women with polycystic ovary syndrome (PCOS) show all the hallmarks of increased TGF-β activity, with increased amounts of fibrous tissue and collagen in the ovarian capsule or tunica albuginea and ovarian stroma. Recent studies suggest that PCOS could have fetal origins. Genetic studies of PCOS have also found linkage with a microsatellite located in intron 55 of the extracellular matrix protein fibrillin 3. Fibrillins regulate TGF-β bioactivity in tissues by binding latent TGF-β binding proteins. We therefore examined expression of fibrillins 1–3, latent TGF-β binding proteins 1–4, and TGF-β 1–3 in bovine and human fetal ovaries at different stages of gestation and in adult ovaries. We also immunolocalized fibrillins 1 and 3. The results indicate that TGF-β pathways operate during ovarian fetal development, but most important, we show fibrillin 3 is present in the stromal compartments of fetal ovaries and is highly expressed at a critical stage early in developing human and bovine fetal ovaries when stroma is expanding and follicles are forming. These changes in expression of fibrillin 3 in the fetal ovary could lead to a predisposition to develop PCOS in later life.—Hatzirodos, N., Bayne, R. A., Irving-Rodgers, H. F., Hummitzsch, K., Sabatier, L., Lee, S., Bonner, W., Gibson, M. A., Rainey, W. E., Carr, B. R., Mason, H. D., Reinhardt, D. P., Anderson, R. A., Rodgers, R. J. Linkage of regulators of TGF-β activity in the fetal ovary to polycystic ovary syndrome.

Microfibrils are supramolecular structures ubiquitously found in the extracellular matrix of elastic and nonelastic tissues. The three members of the cysteine-rich fibrillin family constitute the core of microfibrils. Mutations in fibrillin-1 and -2 lead to a number of heritable connective tissue disorders termed fibrillinopathies. Clinical symptoms affect blood vessels, bone, the eye, and other organ systems and highlight the importance of fibrillins in development and homeostasis of tissues and organs. Microfibrils have functional significance (1) in conferring mechanical stability and limited elasticity to tissues; (2) in the biogenesis and maintenance of the elastic fiber system; and (3) in the modulation of the activity of growth factors, including transforming growth factor-β and several bone morphogenetic proteins. In this chapter, we provide an overview of the structure, assembly, and functions of fibrillins and microfibrils and also the pathobiology associated with genetic aberrations in the microfibril system. Lessons learned from mouse models will be discussed as well as the emerging role of microfibrils and fibrillins in the regulation of growth factor bioavailability. Due to the large number of articles in the field, we repeatedly cite excellent review articles to which interested readers are referred to for more details.

Fibrillin proteins are the major components of extracellular microfibrils found in many connective tissues. Fibrillin-1 and fibrillin-2 are well studied and mutations in these proteins cause a number of fibrillinopathies including Marfan syndrome and congenital contractural arachnodactyly, respectively. Fibrillin-3 was more recently discovered and is much less well characterized. Fibrillin-1 is expressed throughout life, whereas fibrillins-2 and -3 are thought to be primarily present during development. Here, we report detailed fibrillin-3 expression patterns in early human development. A polyclonal antiserum against a C-terminal recombinant half of human fibrillin-3 was produced in rabbit. Anti-fibrillin-3 antibodies were affinity-purified and antibodies cross-reacting with the other fibrillins were removed by absorption resulting in specific anti-fibrillin-3 antibodies. Immunohistochemical analyses with these purified antibodies demonstrate that fibrillin-3 is temporally expressed in numerous tissues relatively evenly from the 6th to the 12th gestational week. Fibrillin-3 was found spatially expressed in perichondrium, perineurium, perimysium, skin, developing bronchi, glomeruli, pancreas, kidney, heart and testis and at the prospective basement membranes in developing epithelia and endothelia. Double immunohistochemical analyses showed that all fibrillins are globally expressed in the same organs, with a number of differences on the tissue level in cartilage, perichondrium and developing bronchi. These results suggest that fibrillin-3, compared to the other fibrillins, fulfills both overlapping and distinct functions in human development.

Wound healing and sclerosis are characterized by an increase of extracellular matrix proteins, which are characteristically expressed in the embryo-fetal period. We analyzed the expression of fibrillin-2, which is typically found in embryonic tissues, but only scarcely in adult skin. In wound healing and sclerotic skin diseases such as lipodermatosclerosis and scleroderma, a marked increase of fibrillin-2 expression was found by immunohistology. Double labelling of fibrillin-2 and tenascin-C, which is also expressed in wound healing and sclerosis, showed co-localization of both proteins. Solid-phase and slot blot-overlay assays showed a dose-dependent binding of the recombinant N-terminal half of fibrillin-2 (rFBN2-N) to tenascin-C. Real-time PCR showed an increase of the fibrillin-2 gene expression in cell culture triggered by typical mediators for fibroblast activation such as serum, IL-4, and TGF-beta. By contrast, prolonged hypoxia is not associated with changes in fibrillin-2 expression. Tenascin-C is an anti-adhesive substrate for fibroblasts, whereas fibrillin-2 stimulates cell attachment. Attachment assays using mixed substrates showed decreased cell attachment when tenascin-C and rFBN2-N were coated together, compared with the attachment to rFBN2-N alone. Fibrillins are involved in storage and activation of TGF-beta. Immunohistology with an antibody against the latency-associated peptide (LAP (TGF-beta1)) showed a marked increase of inactive LAP-bound TGF-beta1 in wound healing and sclerotic skin whereas normal skin showed only a weak expression. Double immunofluorescence confirmed a partial colocalization of both proteins. In conclusion, we show that a stimulation of the fibrillin-2 expression is a characteristic feature of fibroblasts present in wound healing and sclerosis, which may be involved in the alteration of cell attachment and storage of inactive TGF-beta in the matrix.

Homocystinuria caused by cystathionine-beta-synthase deficiency represents a severe form of homocysteinemias, which generally result in various degrees of elevated plasma homocysteine levels. Marfan syndrome is caused by mutations in fibrillin-1, which is one of the major constituents of connective tissue microfibrils. Despite the fundamentally different origins, both diseases share common clinical symptoms in the connective tissue such as long bone overgrowth, scoliosis, and ectopia lentis, whereas they differ in others. Fibrillin-1 contains approximately 13% cysteine residues and can be modified by homocysteine. We report here that homocysteinylation affects functional properties of fibrillin-1 and tropoelastin. We used recombinant fragments spanning the entire fibrillin-1 molecule to demonstrate that homocysteinylation, but not cysteinylation leads to abnormal self-interaction, which was attributed to a reduced amount of multimerization of the fibrillin-1 C terminus. The deposition of the fibrillin-1 network by human dermal fibroblasts was greatly reduced by homocysteine, but not by cysteine. Furthermore, homocysteinylation, but not cysteinylation of elastin-like polypeptides resulted in modified coacervation properties. In summary, the results provide new insights into pathogenetic mechanisms potentially involved in cystathionine-beta-synthase-deficient homocystinuria.

Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and nonelastic extracellular matrices. Proper assembly mechanisms are central to the formation and function of these microfibrils, and their properties are often compromised in pathological circumstances such as in Marfan syndrome and in other fibrillinopathies. Here, we have used human dermal fibroblasts to analyze the assembly of fibrillin-1 in dependence of other matrix-forming proteins. siRNA knockdown experiments demonstrated that the assembly of fibrillin-1 is strictly dependent on the presence of extracellular fibronectin fibrils. Immunolabeling performed at the light and electron microscopic level showed colocalization of fibrillin-1 with fibronectin fibrils at the early stages of the assembly process. Protein-binding assays demonstrated interactions of fibronectin with a C-terminal region of fibrillin-1, -2, and -3 and with an N-terminal region of fibrillin-1. The C-terminal half of fibrillin-2 and -3 had propensities to multimerize, as has been previously shown for fibrillin-1. The C-terminal of all three fibrillins interacted strongly with fibronectin as multimers, but not as monomers. Mapping studies revealed that the major binding interaction between fibrillins and fibronectin involves the collagen/gelatin-binding region between domains FNI6and FNI9.

Elastin is the polymeric protein responsible for the properties of extensibility and elastic recoil of the extracellular matrix in a variety of tissues. Although proper assembly of the elastic matrix is crucial for its durability, the process by which this assembly takes place is not well-understood. Recent data suggest the complex interaction of tropoelastin, the monomeric form of elastin, with a number of other elastic matrix-associated proteins, including fibrillins, fibulins, and matrix-associated glycoprotein (MAGP), is important to achieve the proper architecture of the elastic matrix. At the same time, it is becoming clear that self-assembly properties intrinsic to tropoelastin itself, reflected in a temperature-induced phase separation known as coacervation, are also important in this assembly process. In this study, using a well-characterized elastin-like polypeptide that mimics the self-assembly properties of full-length tropoelastin, the process of self-assembly is deconstructed into "coacervation" and "maturation" stages that can be distinguished kinetically by different parameters. Members of the fibrillin, fibulin, and MAGP families of proteins are shown to profoundly affect both the kinetics of self-assembly and the morphology of the maturing coacervate, restricting the growth of coacervate droplets and, in some cases, causing clustering of droplets into fibrillar structures.

Microfibrils are essential elements in elastic and nonelastic tissues contributing to homeostasis and growth factor regulation. Fibrillins form the core of these multicomponent assemblies. Various human genetic disorders, the fibrillinopathies, arise from mutations in fibrillins and are frequently associated with aberrant microfibril assembly. These disorders include Marfan syndrome, Weill–Marchesani syndrome, Beals syndrome, and others. Although homotypic and heterotypic fibrillin self-interactions are considered to provide critical initial steps, the detailed mechanisms for microfibril assembly are unknown. We show here that the C-terminal recombinant half of fibrillin-1 assembles into disulfide-bonded multimeric globular structures with peripheral arms and a dense core. These globules are similar to the beaded structures observed in microfibrils isolated from tissues. Only these C-terminal fibrillin-1 multimers interacted strongly with the fibrillin-1 N terminus, whereas the monomers showed very little self-interaction activity. The multimers strongly inhibited microfibril formation in cell culture, providing evidence that these recombinant assemblies can also interact with endogenous fibrillin-1. The C-terminal self-interaction site was fine-mapped to the last three calcium-binding EGF domains in fibrillin-1. These results suggest a new mechanism for microfibril formation where fibrillin-1 first oligomerizes via its C terminus before the partially or fully assembled bead-like structures can further interact with other beads via the fibrillin-1 N termini.

Fibrillin is a very large molecule whose primary structure is now known from the cloning and sequencing of 10 kb of cDNA. Immunohistochemical results suggest that one of the functions of fibrillin molecules is to contribute to the structure of the microfibril. The importance of fibrillin as a structural macromolecule has been demonstrated by the identification of the gene for fibrillin (FBN1) as the disease-causing gene in Marfan's syndrome. While it is clear that fibrillin contributes to the structure of the microfibril, it is not known whether fibrillin molecules self-assemble or whether fibrillin interacts with other molecules in order to form microfibrils. In order to investigate whether particular domains of fibrillin are important to the assembly of the microfibril and to specify domains that participate in interactions with other proteins, we have produced recombinant fibrillin 1 peptides in human cells and used them in studies described here. Additionally, new information regarding the 5' end of FBN1 has been obtained from studies investigating promoter activity, and potential proteolytic cleavage sites have been identified in the N- and C-terminal domains.

Decorin, a small leucine-rich proteoglycan, affects the synthesis of the elastic fiber component fibrillin-1 in the kidney via hitherto unknown mechanisms. Here, we show that decorin binds to and induces phosphorylation of insulin-like growth factor-I (IGF-I) receptor in renal fibroblasts. Inhibition of the IGF-I receptor tyrosine kinase and its downstream target phosphoinositide-3 kinase prevented decorin-mediated synthesis of fibrillin-1. Furthermore, decorin induced phosphorylation of phosphoinositide-dependent kinase 1, protein kinase B/Akt, mammalian target of rapamycin (mTOR), and p70 S6 kinase. Accordingly, the enhanced synthesis of fibrillin-1 was blocked by rapamycin, an inhibitor of mTOR. Notably, IGF-I, which signals through the same pathway, also stimulated fibrillin-1 synthesis. Systemic administration of rapamycin to mice subjected to unilateral ureteral obstruction, a model of renal fibrosis and increased fibrillin-1 synthesis, markedly reduced the number of interstitial fibroblasts and fibrillin-1 deposition. In streptozotocin-induced diabetes, IGF-I receptor was up-regulated in the kidneys from decorin-null mice. However, this could not compensate for the decorin deficiency, resulting ultimately in decreased fibrillin-1 content. This study provides evidence for the involvement of decorin and the IGF-I receptor/mTOR/p70 S6 kinase signaling pathway in the translational regulation of fibrillin-1.

Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.

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-beta) 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.

The microfibrillar protein fibrillin-1 is present in many organs, including the vasculature, eye, and dermis, and is thought to convey structural anchorage and elastic strength. Fibrillin-1 is also a component of the mesangial matrix. To assess the functional relevance of fibrillin-1 for cell–matrix interactions in the glomerulus, we studied the attachment, spreading, migration and proliferation of mesangial cells on fibrillin-1 and the regulation of fibrillin-1 in experimental anti-Thy1.1 nephritis displaying mesangial cell migration and proliferation in vivo . During the acute phase of experimental Thy1.1 nephritis, glomerular fibrillin-1 messenger ribonucleic acid expression and protein immunoreactivity were significantly induced as compared to controls. In a hexosaminidase-based adhesion assay, mesangial cells showed concentration-dependent attachment to fibrillin-1, similar to what was observed for fibronectin. The cell attachment was Arg–Gly–Asp dependent. Further, fibrillin-1 significantly promoted spreading and focal contact formation detected by immunostaining for vinculin. Mesangial cell migration, assessed by a transmigration assay, and proliferation, measured by a 5-bromo-2′-deoxy-uridine incorporation assay, were augmented by fibrillin-1. In diabetic mice underexpressing fibrillin-1, glomerular cell proliferation, determined by counting proliferating cell nuclear antigen-positive cells in renal sections, was significantly lower than in diabetic control mice. We conclude that fibrillin-1 promotes mesangial cell attachment, spreading, migration, and proliferation. We speculate that fibrillin-1 may thus contribute to mesangial hypercellularity during glomerular disease.

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-beta) 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.