Fibrillin-rich Microfibrils Research Articles

A comparative study of the elastic fibre system within the mouse and human cornea

The cornea relies on its organised extracellular matrix for maintaining transparency and biomechanical strength. Studies have identified an elastic fibre system within the human posterior cornea, thought to allow for slight deformations in response to internal pressure fluctuations within the eye. However, the type of elastic fibres that exist within the cornea and their roles remain elusive. The aim of this study was to compare the distribution and organisation of the elastic fibres within the posterior peripheral mouse and human cornea, and elucidate how these fibres integrate with the trabecular meshwork, whilst characterising the distribution of their main likely components (fibrillin-1, elastin and type VI collagen) in different parts of the cornea and adjacent sclera.We identified key differences in the elastic fibre system between the human and mouse cornea. True elastic fibres (containing elastin) were identified within the human posterior peripheral cornea. Elastic fibres appeared to present as an extensive network throughout the mouse corneal stroma, but as fibrillin-rich microfibril bundles rather than true elastic fibres. However, tropoelastin staining indicated the possibility that true elastic fibres had yet to develop in the young mice studied. Differences were also apparent within the anatomy of the trabecular meshwork. The human trabecular meshwork appeared to insert between the corneal stroma and Descemet's membrane, with elastic fibres continuing into the stroma from the trabecular meshwork anterior to Descemet's membrane. Within the mouse cornea, no clear insertion point of the trabecular meshwork was seen, instead the elastic fibres within the trabecular meshwork continued into Descemet's membrane, with the trabecular meshwork joining posterior to Descemet's membrane.

A new in vitro assay to test UVR protection of dermal extracellular matrix components by a flat spectrum sunscreen

The efficacy of topical sunscreens is currently assessed by crude, costly and time consuming in vivo assays. We have previously demonstrated that components of the dermal extracellular matrix (ECM), rich in UV-absorbing amino acids, are susceptible to damage by solar simulated radiation (SSR) in vitro. Here we developed an in vitro method to test the ability of sunscreens to protect fibrillin-rich microfibrils (FRM) and fibronectin, key components of the dermal ECM from UV-induced damage. Solutions of FRM or fibronectin were irradiated without protection, in the presence of a vehicle or a commercially-available flat-spectrum sunscreen. The effect of SSR on molecular structure was determined by atomic force microscopy (FRM) and SDS-PAGE (fibronectin). Following irradiation, FRM periodicity became bi-modally distributed (peaks: 40nm & 59nm) compared to the unimodal distribution in unexposed controls (peak: 50nm). Irradiation in the presence of flat-spectrum sunscreen protected against this change, maintaining the unimodal distribution. SSR induced significant aggregation of fibronectin (p=0.005), which was abrogated by sunscreen. These results demonstrate that this in vitro assay system is sufficiently sensitive to act as an initial/additional screen of sunscreen efficacy. We conclude that sunscreen can reduce UV-mediated damage of key dermal ECM in vitro and thereby prevent remodelling associated with photoageing.

Thyroid Hormones Enhance Mitochondrial Function in Human Epidermis

Since it is unknown whether thyroid hormones (THs) regulate mitochondrial function in human epidermis, we treated organ-cultured human skin, or isolated cultured human epidermal keratinocytes, with triiodothyronine (100 pmol/L) or thyroxine (100 nmol/L). Both THs significantly increased protein expression of the mitochondrially encoded cytochrome C oxidase I (MTCO1), complex I activity, and the number of perinuclear mitochondria. Triiodothyronine also increased mitochondrial transcription factor A (TFAM) protein expression, and thyroxine stimulated complex II/IV activity. Increased mitochondrial function can correlate with increased reactive oxygen species production, DNA damage, and accelerated tissue aging. However, THs neither raised reactive oxygen species production or matrix metalloproteinase-1, -2 and -9 activity nor decreased sirtuin1 (Sirt1) immunoreactivity. Instead, triiodothyronine increased sirtuin-1, fibrillin-1, proliferator-activated receptor-gamma 1-alpha (PGC1α), collagen I and III transcription, and thyroxine decreased cyclin-dependent kinase inhibitor 2A (p16(ink4)) expression in organ-cultured human skin. Moreover, TH treatment increased intracutaneous fibrillin-rich microfibril and collagen III deposition and decreased mammalian target of rapamycin (mTORC1/2) expression exvivo. This identifies THs as potent endocrine stimulators of mitochondrial function in human epidermis, which down-regulates rather than enhance the expression of skin aging-related biomarkers exvivo. Therefore, topically applied THs deserve further exploration as candidate agents for treating skin conditions characterized by reduced mitochondrial function.

Geographical ancestry is a key determinant of epidermal morphology and dermal composition

Geographical ancestry plays a key role in determining the susceptibility of human skin to external insults and dermatological disease. Despite this, studies of skin from individuals of diverse geographical ancestry focus primarily on epidermal pigmentation. Few reports characterize the gross morphology and composition of the dermis and dermal-epidermal junction (DEJ). To characterize epidermal morphology and dermal composition in skin from individuals of diverse geographical ancestry. Immunohistochemical techniques were used to assess epidermal morphology and protein composition of the DEJ and dermal extracellular matrix in photoprotected skin from young African, Eurasian and Far East Asian individuals (n=7 per group; age 18-30years). The epidermis of African skin was thicker, with deeper rete ridges and a more convoluted DEJ than Eurasian and Far East Asian skin. Compared with Eurasians, protein composition of the DEJ was collagen VII poor in African and Far East Asian skin (P<0·001 and P<0·01, respectively); the dermis of African skin was enriched in fibrillar collagens (P<0·05), but was relatively elastin poor (P<0·05). African dermis was abundant in fibrillin-rich microfibrils and fibulin-5 (P<0·001 and P<0·001, respectively) compared with Eurasian and Far East Asian skin. We demonstrate that fundamental differences exist in skin structure and composition in individuals of diverse geographical ancestry. Disparate environmental pressures encountered by ancestral human populations living at different latitudes may have driven adaptations in skin structure and composition. Further research into the functional significance and clinical consequences of these differences is warranted.

In vitro and in vivo studies with tetra‐hydro‐jasmonic acid (LR2412) reveal its potential to correct signs of skin ageing

LR2412, a synthetic derivative of jasmonic acid, improved the reconstruction and homeostasis of our organotypic skin models. The need for efficient 'anti-ageing' treatments, in particular for the management of photoaged skin, prompted us to investigate this new ingredient for its potential to correct signs of skin ageing in vitro and in vivo and to identify its mode of action. In vitro, penetration of LR2412 was evaluated using a Franz diffusion cell on excised human skin. Its exfoliating properties and interactions with the stratum corneum were studied using electron microscopy and X-ray diffraction. Experiments were performed on a human reconstructed skin model. In vivo, the effects of LR2412 on steroid-induced skin atrophy, a clinical skin ageing model, were assessed vs. vehicle. A patch test study evaluated its effect on deposition of fibrillin-rich microfibrils in the papillary dermis in clinically photoaged volunteers. A clinical study on the appearance of crow's feet wrinkles was conducted over 3 months of daily application. Penetration studies revealed that LR2412 reaches viable epidermis and superficial dermis, which are skin targets of anti-ageing actives. Within the upper layers of the stratum corneum LR2412 accelerates desquamation and improves the mechanical properties. At the dermal-epidermal junction of reconstructed skin, collagen IV, laminin-5 and fibrillin were stimulated. In vivo, LR2412 reversed steroid-induced atrophy. The patch test model confirms the deposition of fibrillin-rich microfibrils, then an in use clinical study revealed that it reduced facial wrinkles. The in vitro and in vivo data demonstrate that based on its multiple interactions within human skin, LR2412 has potential to partially correct the signs of ageing in intrinsically and photoaged skin.

Exogenous Activation of BMP‐2 Signaling Overcomes TGFβ‐Mediated Inhibition of Osteogenesis in Marfan Embryonic Stem Cells and Marfan Patient‐Specific Induced Pluripotent Stem Cells

Marfan syndrome (MFS) is a hereditary disease caused by mutations in the gene encoding Fibrillin-1 (FBN1) and characterized by a number of skeletal abnormalities, aortic root dilatation, and sometimes ectopia lentis. Although the molecular pathogenesis of MFS was attributed initially to a structural weakness of the fibrillin-rich microfibrils within the extracellular matrix, more recent results have documented that many of the pathogenic abnormalities in MFS are the result of alterations in TGFβ signaling. Mutations in FBN1 are therefore associated with increased activity and bioavailability of TGF-β1, which is suspected to be the basis for phenotypical similarities of FBN1 mutations in MFS and mutations in the receptors for TGFβ in Marfan syndrome-related diseases. We have previously demonstrated that unique skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells (MFSiPS) derived independently from MFS patient fibroblasts. In this study, we aimed to determine further the biochemical features of transducing signaling(s) in MFS stem cells and MFSiPS cells highlighting a crosstalk between TGFβ and BMP signaling. Our results revealed that enhanced activation of TGFβ signaling observed in MFS cells decreased their endogenous BMP signaling. Moreover, exogenous BMP antagonized the enhanced TGFβ signaling in both MFS stem cells and MFSiPS cells therefore, rescuing their ability to undergo osteogenic differentiation. This study advances our understanding of molecular mechanisms underlying the pathogenesis of bone loss/abnormal skeletogenesis in human diseases caused by mutations in FBN1.

Rôle de l'élastine et des microfibrilles de la paroi artérielle au cours du développement et du vieillissement : complémentarité ou opposition ?

Large arteries allow the vascular system to be more than a simple route in which the blood circulates within the organism. The elastic fibers present in the wall endow these vessels with elasticity and are responsible for the smoothing of the blood pressure and flow, which are delivered discontinuously by the heart. This function is very important to ensure appropriate hemodynamics. Elastic fibers are composed of elastin (90%) and fibrillin-rich microfibrils (10%) which provide the vessels with elasticity and are also signals able to bind to relatively specific cell membrane receptors. Stimulation of the high affinity elastin receptor by elastin peptides or tropoelastin--the elastin precursor--triggers an increase in intracellular free calcium in vascular cells, especially endothelial cells, associated with attachment, migration or proliferation. Similar effects of the stimulation of endothelial cells by microfibrils or fibrillin-1 fragments, which bind to integrins, have been demonstrated. This dual function--mechanical and in signaling--makes the elastic fibers an important actor of the development and ageing processes taking place in blood vessels. An alteration of the elastin (Eln) or fibrillin (Fbn) gene products leads to severe genetic pathologies of the cardiovascular system, such as supravalvular aortic stenosis, or Williams Beuren syndrome--in which elastin deficiency induces aortic stenoses--or Marfan syndrome, in which on the contrary fibrillin-1 deficiency promotes the appearance of aortic aneurysms. Genetically-engineered mouse models of these pathologies (such as Eln+/- mice and Fbn-1+/mgΔ mice, Eln+/-Fbn-1+/- mice) have permitted a better understanding of the pathogenesis of these syndromes. In particular, it has been shown that elastin and fibrillin-1 roles can be complementary in some aspects, while they can be opposed in some other situations. For instance, the double heterozygosity in elastin and fibrillin-1 leads to increased arterial wall stress--compared to the level induced by one of these two deficiencies alone--while the decrease in diameter induced by Eln deficiency is partly compensated by an additional deficiency in Fbn-1. Also, it is now clear that early modifications of elastin or fibrillin-1 availability can alter the normal signaling action of these proteins and lead to long term modifications of the vascular physiology and ageing processes.

Ultrastructural localization of fibrillin-1 and fibrillin-2 in oxytalan fibers in periodontal ligament of Japanese Macaca fuscata monkey

Although oxytalan fibers are a ubiquitous component of the periodontal ligament, their composition and function remain to be fully clarified. The purpose of this study was to investigate the localization of fibrillin-1 and fibrillin-2, large cysteine-rich glycoproteins, in monkey molar periodontal ligament by means of immunohistochemistry and electron microscopic immunogold labeling. Fibers positive for both fibrillin-1 and -2 were observed throughout the periodontal ligament, and their pattern of distribution was basically the same as that of oxytalan fibers. Double immunogold electron microscopy revealed that fibrillin-1 co-localized with fibrillin-2 on the microfibrils of the oxytalan fibers. The results suggest that oxytalan fibers in monkey periodontal ligament are made up of fibrillin-rich microfibrils. These results should contribute to a better understanding of the role of oxytalan fibers in development and tissue remodeling in periodontal ligament.

Microfibril-associated Glycoprotein-1, an Extracellular Matrix Regulator of Bone Remodeling

MAGP1 is an extracellular matrix protein that, in vertebrates, is a ubiquitous component of fibrillin-rich microfibrils. We previously reported that aged MAGP1-deficient mice (MAGP1Delta) develop lesions that are the consequence of spontaneous bone fracture. We now present a more defined bone phenotype found in MAGP1Delta mice. A longitudinal DEXA study demonstrated age-associated osteopenia in MAGP1Delta animals and muCT confirmed reduced bone mineral density in the trabecular and cortical bone. Further, MAGP1Delta mice have significantly less trabecular bone, the trabecular microarchitecture is more fragmented, and the diaphyseal cross-sectional area is significantly reduced. The remodeling defect seen in MAGP1Delta mice is likely not due to an osteoblast defect, because MAGP1Delta bone marrow stromal cells undergo osteoblastogenesis and form mineralized nodules. In vivo, MAGP1Delta mice exhibit normal osteoblast number, mineralized bone surface, and bone formation rate. Instead, our findings suggest increased bone resorption is responsible for the osteopenia. The number of osteoclasts derived from MAGP1Delta bone marrow macrophage cells is increased relative to the wild type, and osteoclast differentiation markers are expressed at earlier time points in MAGP1Delta cells. In vivo, MAGP1Delta mice have more osteoclasts lining the bone surface. RANKL (receptor activator of NF-kappaB ligand) expression is significantly higher in MAGP1Delta bone, and likely contributes to enhanced osteoclastogenesis. However, bone marrow macrophage cells from MAGP1Delta mice show a higher propensity than do wild-type cells to differentiate to osteoclasts in response to RANKL, suggesting that they are also primed to respond to osteoclast-promoting signals. Together, our findings suggest that MAGP1 is a regulator of bone remodeling, and its absence results in osteopenia associated with an increase in osteoclast number.

Cell Adhesion to Tropoelastin Is Mediated via the C-terminal GRKRK Motif and Integrin αVβ3

Elastin fibers are predominantly composed of the secreted monomer tropoelastin. This protein assembly confers elasticity to all vertebrate elastic tissues including arteries, lung, skin, vocal folds, and elastic cartilage. In this study we examined the mechanism of cell interactions with recombinant human tropoelastin. Cell adhesion to human tropoelastin was divalent cation-dependent, and the inhibitory anti-integrin alpha(V)beta(3) antibody LM609 inhibited cell spreading on tropoelastin, identifying integrin alpha(V)beta(3) as the major fibroblast cell surface receptor for human tropoelastin. Cell adhesion was unaffected by lactose and heparin sulfate, indicating that the elastin-binding protein and cell surface glycosaminoglycans are not involved. The C-terminal GRKRK motif of tropoelastin can bind to cells in a divalent cation-dependent manner, identifying this as an integrin binding motif required for cell adhesion.

Biogenesis and function of fibrillin assemblies

Fibrillin-1 and fibrillin-2 are large cysteine-rich glycoproteins that serve two key physiological functions: as supporting structures that impart tissue integrity and as regulators of signaling events that instruct cell performance. The structural role of fibrillins is exerted through the temporal and hierarchical assembly of microfibrils and elastic fibers, whereas the instructive role reflects the ability of fibrillins to sequester transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) complexes in the extracellular matrix. Characterization of fibrillin mutations in human patients and in genetically engineered mice has demonstrated that perturbation of either function manifests in disease. More generally, these studies have indicated that fibrillins are integral components of a broader biological network of extracellular, cell surface, and signaling molecules that orchestrate morphogenetic and homeostatic programs in multiple organ systems. They have also suggested that the relative composition of fibrillin-rich microfibrils imparts contextual specificity to TGFbeta and BMP signaling by concentrating the ligands locally so as to regulate cell differentiation within a spatial context during organ formation (positive regulation) and by restricting their bioavailability so as to modulate cell performance in a timely fashion during tissue remodeling/repair (negative regulation). Correlative evidence suggests functional coupling of the cell-directed assembly of microfibrils and targeting of TGFbeta and BMP complexes to fibrillins. Hence, the emerging view is that fibrillin-rich microfibrils are molecular integrators of structural and instructive signals, with TGFbeta and BMPs as the nodal points that convert extracellular inputs into discrete and context-dependent cellular responses.

Microfibril‐associated glycoprotein‐1 binds multiple active TGF‐beta family members, affecting homeostasis in multiple organ systems

Microfibril‐associated glycoprotein‐1 (MAGP‐1) is a small molecular weight extracellular matrix component of the fibrillin‐rich microfibril that forms the scaffold for elastic fibers. Surprisingly, mice null for MAGP‐1 have normal elastic fiber morphology and their elastic tissues function normally. The complex phenotype of mice null for MAGP‐1 includes increased body weight and size due to excess body fat, an altered wound healing response in bone and skin, and a bleeding diathesis. The obesity and bone phenotype is intriguing given the localization of obesity and osteoporosis candidate genes to human chromosome 1, which also houses the MAGP‐1 gene (mfap2). These phenotypic traits are consistent with transforming growth factor‐beta (TGF‐beta) dysregulation, and we have determined that MAGP‐1 binds TGF‐beta and multiple BMPs with high affinity. Further, these traits are opposite to those associated with mutations in MAGP‐1's binding partner fibrillin‐1, and many fibrillin‐1 mutations result in enhanced TGF‐beta signaling. Together, our data implicates MAGP‐1 as an important modulator of microfibril‐mediated growth factor signaling.

Immunohistochemical Investigation of Mid-Dermal Elastolysis With a History of Erythema

Elastic fibers are essential extracellular matrix macromolecules comprising an elastin core surrounded by fibrillin-rich microfibrils. Fibulin-5, a microfibril, has been identified as one of the secreted extracellular matrix proteins that shows function as a scaffold for elastic fibers. However, the distribution of fibulin-5 in the skin is not clear. We report a case of a 43-year-old woman with erythema and subsequent wrinkling that met the clinical and histological criteria for mid-dermal elastolysis. We investigate the mechanism by which this disease occurs. The distribution of elastin, CD68, matrix metalloproteinase (MMP)-9, and fibulin-5 was examined immunohistochemically from both erythematous and wrinkled skin. There were numerous CD68 and MMP-9-producing histiocytes and giant cells in the erythematous lesions. Faint fibrillar staining of fibulin-5 was found in the deep dermis. In the wrinkled skin, there were few CD68 histiocytes or giant cells. Elastin immunoreactivity disappeared from the mid-dermis. Fibulin-5 colocalized in the lower dermis, shorter than in the erythema. Mid-dermal elastolysis may be initiated by MMP-9 produced by histiocytes and giant cells through its degradation of elastic fibers. In the lower dermis of the wrinkled skin, the fragmented expression of fibulin-5 was associated with the incomplete reproduction of the elastic fibers.

Changes in the Molecular Packing of Fibrillin Microfibrils During Extension Indicate Intrafibrillar and Interfibrillar Reorganization in Elastic Response

Fibrillin-rich microfibrils are the major structural components of the extracellular matrix that provide elasticity in a majority of connective tissues. The basis of elastic properties lies in the organization of fibrillin molecules, which, unfortunately, is still poorly understood. An X-ray diffraction study of hydrated fibrillin-rich microfibrils from zonular filaments has been conducted to give an insight into the molecular structure of microfibrils in intact tissue. A series of measurements was taken during controlled tissue extension to observe alterations in the lateral packing of microfibrils. Computer-generated simulated patterns were used to fit the experimental X-ray scattering data and to obtain the fibril diameter and lateral distance between the fibrils. The results suggest a nonlinear correlation between external strain and decrease in fibril diameter and lateral spacing. This was accompanied by a nonlinear increase in axial periodicity and a structure with a 160-nm periodicity, which is reported here for the first time using X-ray diffraction. These changes may reflect the unraveling of fibrillin from the complex folded arrangement into a linear structure. This finding supports a pleating model where fibrillin molecules are highly folded within the microfibrils; more importantly, the connection is made between the interaction of individual microfibrils and the change in their suprafibrillar coherent organization during extension. We suggest that the intermediate states observed in our study reflect sequential unfolding of fibrillin and can explain the process of its reversible unraveling.

Deficiency in Microfibril-associated Glycoprotein-1 Leads to Complex Phenotypes in Multiple Organ Systems

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight component of the fibrillin-rich microfibril. Gene-targeted inactivation of MAGP-1 reveals a complex phenotype that includes increased body weight and size due to excess body fat, an altered wound healing response in bone and skin, and a bleeding diathesis. Elastic tissues rich in MAGP-1-containing microfibrils develop normally and show normal function. The penetrance of MAGP-1-null phenotypes is highly variable and mouse strain-dependent, suggesting the influence of modifier genes. MAGP-1 was found to bind active transforming growth factor-beta (TGF-beta) and BMP-7 with high affinity, suggesting that it may be an important modulator of microfibril-mediated growth factor signaling. Many of the phenotypic traits observed in MAGP-1-deficient mice are consistent with loss of TGF-beta function and are generally opposite those associated with mutations in fibrillin-1 that result in enhanced TGF-beta signaling. Increased body size and fat deposition in MAGP-1-mutant animals are particularly intriguing given the localization of obesity traits in humans to the region on chromosome 1 containing the MAGP-1 gene.

Processing and assembly of fibrillin-1

Fibrillins are extracellular matrix proteins of ˜350 kD. They have a complex multidomain structure and are the major components of microfibrillar structures in both elastic and nonelastic tissues (Sakai et al. 1986; Kielty & Shuttleworth 1995). Mutations in the FBN-1 gene cause Marfan Syndrome, which is a heritable connective tissue disease with an incidence of 1:5000 (Lee et al. 1991). Marfan Syndrome is associated with shortened life expectancy due to aortic dissection, as well as range of skeletal and ocular abnormalities (Maslen et al. 1991). The disease may be due to reduced levels of fibrillin-rich microfibrils and/or the presence of structurally or functionally abnormal microfibrils. The mechanism of fibrillin assembly into microfibrils is poorly understood, largely due to the large size and complexity of the protein, and this in turn has hindered a full understanding of the mechanism of the disease. To facilitate study of the secretion and assembly of fibrillin-1 (Fib-1), a minigene construct has been assembled containing the N-and C-terminus and 2 blocks of cysteine rich repeats (exons 1–15 and exons 50–65). This cDNA has been linked at the C-terminus to a green fluorescent protein (GFP) tag in order to visualize the fibrillin minigene construct as it is translocated through the cell and secreted into the extracellular matrix.

Fibrillin‐rich microfibrils: Structural determinants of morphogenetic and homeostatic events

Fibrillin-rich microfibrils are specialized extracellular matrix assemblies that endow connective tissues with mechanical stability and elastic properties, and that participate in the regulation of organ formation, growth and homeostasis. Their physiological importance is underscored by the complex spectrum of clinical manifestations associated with mutations of fibrillin-1 and fibrillin-2 in Marfan syndrome (MFS) and congenital contractural arachnodactyly, respectively. Early evidence suggested that fibrillin-1 mutations in MFS lead to loss of tissue integrity by perturbing microfibril assembly and function. Recent studies in genetically targeted mice have however revealed that fibrillin-1 and fibrillin-2 mutations perturb signaling events mediated by TGF-beta superfamily members. As such, these studies have established a new biological paradigm whereby fibrillin-rich microfibrils are structural networks that specify the local concentration and timely release of signaling molecules during morphogenesis and tissue remodeling. This review summarizes our current understanding of the role of fibrillin-rich microfibrils in development and disease, as well as exciting new applications in the clinical management of MFS and related connective tissue disorders.

Extracellular microfibrils in development and disease.

Fibrillins are the structural components of extracellular microfibrils that impart physical properties to tissues, alone or together with elastin as elastic fibers. Genetic studies in mice have revealed that fibrillin-rich microfibrils are also involved in regulating developmental programs and homeostatic processes through the modulation of TGF-beta/BMP signaling events. A new paradigm has thus emerged whereby the spatiotemporal organization of microfibrils dictates both the cellular activities and physical properties of connective tissues. These observations have paved the way to novel therapeutic approaches aimed at counteracting the life-threatening complications in human conditions caused by dysfunctions of fibrillin-rich microfibrils.

Loss of Fibulin-2 Expression Is Associated with Breast Cancer Progression

Fibulin-2, an extracellular matrix protein expressed by normal epithelia, was found to be down-regulated in several breast cancer cell lines. Fibulin-2 protein expression was also decreased in breast cancer tissue samples as evaluated by immunohistochemistry. Reintroduction of Fibulin-2 into breast cancer cell lines that do not express Fibulin-2 reduced cancer cell motility and invasion in vitro but had no effect on cell growth and adhesion properties. Together with evidence that Fibulin-2 contributes to wound healing and inhibits smooth muscle cell migration, our findings suggest that loss of Fibulin-2 expression may facilitate migration and invasion in breast cancer.

Marfan Syndrome-causing Mutations in Fibrillin-1 Result in Gross Morphological Alterations and Highlight the Structural Importance of the Second Hybrid Domain

Mutations in fibrillin-1 result in Marfan syndrome, which affects the cardiovascular, skeletal and ocular systems. The multiorgan involvement and wide spectrum of associated phenotypes highlights the complex pathogenesis underlying Marfan syndrome. To elucidate the genotype to phenotype correlations, we engineered four Marfan syndrome causing mutations into a fibrillin-1 fragment encoded by exons 18-25, a region known to interact with tropoelastin. Biophysical and biochemical approaches, including small angle x-ray scattering, analytical ultracentrifugation, and circular dichroism, were used to study the impact of these mutations upon the structure and function of the protein. Mutations G880S, C862R, and C908R, situated within the second hybrid domain, disrupted the ratio of alpha-helix to beta-sheet leading to a more compact conformation. These data clearly demonstrate the importance of the previously uncharacterized hybrid domain in fibrillin-1 structure. In contrast, mutation K1023N situated within the linker region between the third eight cysteine motif and cbEGF 11 markedly extended the length of the fragment. However, none of the mutations affected tropoelastin binding. The profound effects of all four mutations on fragment conformation suggest that they contribute to the pathogenesis of Marfan syndrome by disrupting protein folding and its assembly into fibrillin-rich microfibrils.