Extracellular Matrix Protein Fibulin-1 Research Articles

Calumenin-1 Interacts with Climp63 to Cooperatively Determine the Luminal Width and Distribution of Endoplasmic Reticulum Sheets.

SummaryThe ER is composed of distinct structures like tubules, matrices, and sheets, all of which are important for its various functions. However, how these distinct ER structures, especially the perinuclear ER sheets, are formed remains unclear. We report here that the ER membrane protein Climp63 and the ER luminal protein calumenin-1 (Calu1) collaboratively maintain ER sheet morphology. We show that the luminal length of Climp63 is positively correlated with the luminal width of ER sheets. Moreover, the lumen-only mutant of Climp63 dominant-negatively narrows the lumen of ER sheets, demonstrating that Climp63 acts as an ER luminal bridge. We also reveal that Calu1 specifically interacts with Climp63 and antagonizes Climp63 in terms of both ER sheet distribution and luminal width. Together, our data provide insight into how the structure of ER sheets is maintained and regulated.

Fibulin-1 is required for bone formation and Bmp-2-mediated induction of Osterix

The extracellular matrix protein Fibulin-1 (Fbln1) has been shown to be involved in numerous processes including cardiovascular and lung development. Here we have examined the role of Fbln1 in bone formation. Alizarin red staining of skulls from Fbln1-deficient mice showed reduced mineralization of both membranous and endochondral bones. MicroCT (μCT) analysis of the calvarial bones (i.e., frontal, parietal and interparietal bones collectively) indicated that bone volume in Fbln1 nulls at neonatal stage P0 were reduced by 22% (p=0.015). Similarly, Fbln1 null frontal bones showed a 16% (p=0.035) decrease in bone volume, with a reduction in the interfrontal bone, and a discontinuity in the leading edge of the frontal bone. To determine whether Fbln1 played a role in osteoblast differentiation during bone formation, qPCR was used to measure the effects of Fbln1 deficiency on the expression of Osterix (Osx), a transcription factor essential for osteoblast differentiation. This analysis demonstrated that Osx mRNA was significantly reduced in Fbln1-deficient calvarial bones at developmental stages E16.5 (p=0.049) and E17.5 (p=0.022). Furthermore, the ability of Bmp-2 to induce Osx expression was significantly diminished in Fbln1-deficient mouse embryo fibroblasts. Together, these findings indicate that Fbln1 is a new positive modulator of the formation of membranous bone and endochondral bone in the skull, acting as a positive regulator of Bmp signaling.

Metformin, but Not Rosiglitazone, Attenuates the Increasing Plasma Levels of a New Cardiovascular Marker, Fibulin-1, in Patients With Type 2 Diabetes

OBJECTIVE The extracellular matrix protein fibulin-1 is upregulated in the arterial wall in type 2 diabetes (T2D) and circulates in increased concentrations in diabetes. Metformin is an antidiabetic drug with beneficial cardiovascular disease effects in diabetes. We hypothesized that metformin would influence the increased level of plasma fibulin-1 in diabetes. RESEARCH DESIGN AND METHODS After a 4-week run-in period, 371 eligible patients with T2D were randomized to treatment groups in a factorial design including insulin alone (control), +metformin, +rosiglitazone, or +both metformin and rosiglitazone. Plasma fibulin-1 was analyzed at the beginning of the study and after 18 and 24 months. RESULTS Plasma fibulin-1 increased in all groups throughout the 2-year period; however, the increase was strongly attenuated among patients treated with metformin. A highly significant difference was observed when the mean change in plasma fibulin-1 was compared between metformin- and non-metformin-treated individuals both at 18 and 24 months of treatment, but rosiglitazone had no effect. Metformin and rosiglitazone alone reduced the HbA1c levels to comparable levels and in combination even further. CONCLUSIONS Metformin attenuates the increase in plasma fibulin-1 concentrations in T2D, independently of glycemic effects. Changes in fibulin-1 may reflect an important element in diabetic arteriopathy that can be influenced by metformin.

Plasma concentrations of extracellular matrix protein fibulin-1 are related to cardiovascular risk markers in chronic kidney disease and diabetes

BackgroundFibulin-1 is one of a few extracellular matrix proteins present in blood in high concentrations. We aimed to define the relationship between plasma fibulin-1 levels and risk markers of cardiovascular disease.MethodsPlasma fibulin-1 was determined in subjects with chronic kidney disease (n = 32; median age 62.5, inter-quartile range 51 – 73 years) and 60 age-matched control subjects. Among kidney disease patients serological biomarkers related to cardiovascular disease (fibrinogen, interleukin 6, C-reactive protein) were measured. Arterial applanation tonometry was used to determine central hemodynamic and arterial stiffness indices.ResultsWe observed a positive correlation of fibulin-1 levels with age (r = 0.38; p = 0.033), glycated hemoglobin (r = 0.80; p = 0.003), creatinine (r = 0.35; p = 0.045), and fibrinogen (r = 0.39; p = 0.027). Glomerular filtration rate and fibulin-1 were inversely correlated (r = −0.57; p = 0.022). There was a positive correlation between fibulin-1 and central pulse pressure (r = 0.44; p = 0.011) and central augmentation pressure (r = 0.55; p = 0.001). In a multivariable regression model, diabetes, creatinine, fibrinogen and central augmentation pressure were independent predictors of plasma fibulin-1.ConclusionIncreased plasma fibulin-1 levels were associated with diabetes and impaired kidney function. Furthermore, fibulin-1 levels were associated with hemodynamic cardiovascular risk markers. Fibulin-1 is a candidate in the pathogenesis of cardiovascular disease observed in chronic kidney disease and diabetes.

Serum Opacity Factor Is a Streptococcal Receptor for the Extracellular Matrix Protein Fibulin-1

The adhesion of bacteria to host tissues is often mediated by interactions with extracellular matrices. Herein, we report on the interactions of the group A streptococcus, Streptococcus pyogenes, with the extracellular matrix protein fibulin-1. S. pyogenes bound purified fibulin-1 in a dose-dependent manner. Genetic ablation of serum opacity factor (SOF), a virulence determinant of S. pyogenes, reduced binding by approximately 50%, and a recombinant peptide of SOF inhibited binding of fibulin-1 to streptococci by approximately 45%. Fibulin-1 bound to purified SOF2 in a dose-dependent manner with high affinity (K(d) = 1.6 nm). The fibulin-1-binding domain was localized to amino acid residues 457-806 of SOF2, whereas the fibronectin-binding domain is contained within residues 807-931 of SOF2, indicating that these two domains are separate and distinct. Fibulin-1 bound to recombinant SOF from M types 2, 4, 28, and 75 of S. pyogenes, indicating that the fibulin-1-binding domain is likely conserved among SOF from different serotypes. Mixed binding experiments suggested that gelatin, fibronectin, fibulin-1, and SOF form a quaternary molecular complex that enhanced the binding of fibulin-1. These data indicate that S. pyogenes can interact with fibulin-1 and that SOF is a major streptococcal receptor for fibulin-1 but not the only receptor. Such interactions with fibulin-1 may be involved in the adhesion of S. pyogenes to extracellular matrices of the host.

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.

Expression of Extracellular Matrix Proteins Fibulin-1 and Fibulin-2 by Human Corneal Fibroblasts

Purpose: The fibulins are a family of extracellular matrix (ECM) molecules that regulate the organ shape along with other growth factors and stromal cells. We report here the in vitro expression of ECM proteins fibulin-1 and fibulin-2 by human corneal fibroblasts. The ability of fibulin-1 to modulate cell motility was investigated. Methods: Fibulin-1 and fibulin-2 mRNA and proteins expression were analyzed in primary and immortalized human corneal fibroblasts (CHN) respectively by gene array, RT-PCR, and immunocytochemistry. The motility and adhesion of the cells transfected with fibulin-1 siRNA were analyzed on tissue culture polystyrene coated with Matrigel or ECM secreted by those fibroblasts. Results: (1) The microarray analysis shows the expression of fibulin-1, fibulin-2, and their binding partners (i.e., fibronectin, nidogen-1, aggrecan, fibrilin-1, endostatin, and laminin alpha-2 chain). Interestingly, a matrix metalloprotease, ADAMTS-1, for which fibulin-1 acts as a cofactor, was also detected in CHN. (2) The synthesis by CHN of fibulin-1 and 2 mRNA and proteins was confirmed respectively by RT-PCR and immunocytochemistry. (3) Transfection of CHN by fibulin-1 siRNA has no effect on cell adhesion but increases cell migration compared with that of the control cells. This observation suggests an important role of fibulin-1 on cell motility. Conclusions: The expression of fibulins and that of their binding partners by human corneal fibroblasts indicate the important role of these proteins in the organization of supramolecular ECM structures of cornea. The variation of their expression and the structural changes of fibulins remain to be determined in corneal pathology.

Expression and Functional Analysis of Fibulin-1 ( Fbln1) During Normal and Abnormal Placental Development of the Mouse

The extracellular matrix protein fibulin-1 (FBLN1) is an important component of blood vessel walls, as shown by the lethality of mice with homozygous targeted deletion of the Fbln1 gene. Here, we show that a murine placental overgrowth phenotype is associated with elevated Fbln1 transcript levels, suggesting that the gene and its product have a functional role in placentation. Fbln1 exhibits a specific expression pattern in the mouse placenta. Transcripts could not be detected prior to day 12. In subsequent stages, Fbln1 was expressed strongly in the spongiotrophoblast. Other sites of expression were endothelia of large fetal blood vessels, a tissue type reported to not express this gene. In addition, a subset of giant cells expressed the gene. This giant cell specific expression was strongly increased in hyperplastic placentas. Analysis of the placentation in fibulin null mice did not show any abnormality. Attempts to rescue the placental phenotypes of a congenic model of interspecies hybrid placental dysplasia (IHPD) by normalizing expression of Fbln1 proved that Fbln1 alone is not the key cause of phenotypes in these models of placental hyperplasia.

Fibulin-1 Acts as a Cofactor for the Matrix Metalloprotease ADAMTS-1

ADAMTS-1 is a metalloprotease that has been implicated in the inhibition of angiogenesis and is a mediator of proteolytic cleavage of the hyaluronan binding proteoglycans, aggrecan and versican. In an attempt to further understand the biological function of ADAMTS-1, a yeast two-hybrid screen was performed using the carboxyl-terminal region of ADAMTS-1 as bait. As a result, the extracellular matrix protein fibulin-1 was identified as a potential interacting molecule. Through a series of analyses that included ligand affinity chromatography, co-immunoprecipitation, pulldown assays, and enzyme-linked immunosorbent assay, the ability of these two proteins to interact was substantiated. Additional studies showed that ADAMTS-1 and fibulin-1 colocalized in vivo. Furthermore, fibulin-1 was found to enhance the capacity of ADAMTS-1 to cleave aggrecan, a proteoglycan known to bind to fibulin-1. We confirmed that fibulin-1 was not a proteolytic substrate for ADAMTS-1. Together, these findings indicate that fibulin-1 is a new regulator of ADAMTS-1-mediated proteoglycan proteolysis and thus may play an important role in proteoglycan turnover in tissues where there is overlapping expression.

Elevated expression and altered processing of fibulin-1 protein in human breast cancer.

The extracellular matrix protein fibulin-1 suppresses the motility and invasiveness of a variety of tumour cell types in vitro as well as the growth of fibrosarcoma tumours in nude mice. In this study, fibulin-1 protein expression in breast carcinoma specimens and normal breast tissue was evaluated immunohistologically. Fibulin-1 protein expression was also semiquantitatively assessed by immunoblot analysis in a collection of normal breast tissues (n=18), benign tumours (n=5) and breast carcinomas (n=39). In normal breast tissue, fibulin-1 protein expression predominated in the ductal epithelium and underlying myoepithelium, with weaker staining evident in the loose connective surrounding the ducts. Examination of breast carcinomas revealed that the tumour cells also expressed fibulin-1 protein. The level of mature fibulin-1 polypeptide (100 kDa) was higher in the breast carcinoma specimens as compared to normal breast tissue (Mann–Whitney U-test, P=0.0005). In addition to the mature fibulin-1 polypeptide, several smaller sized polypeptides of 55, 50 and 25 kDa were detected using monoclonal antibodies reactive towards an epitope located at the N-terminus of fibulin-1. The immunoreactive 50 kDa polypeptide was detected more frequently in breast carcinoma specimens than in normal breast tissue (χ2=17.22, P<0.0001). Furthermore, the ratio of the 50 kDa fragment to the mature fibulin-1 polypeptide correlated with the level of oestrogen receptor α (Spearman correlation coefficient, rs=0.49, P<0.003, n=36) and progesterone receptor (rs=0.43, P=0.008, n=36) expression in the tumour specimens. Taken together, these findings indicate that elevated expression and altered processing of fibulin-1 is associated with human breast cancer.

Perinatal lethality and endothelial cell abnormalities in several vessel compartments of fibulin-1-deficient mice.

The extracellular matrix protein fibulin-1 is a distinct component of vessel walls and can be associated with other ligands present in basement membranes, microfibrils, and elastic fibers. Its biological role was investigated by the targeted inactivation of the fibulin-1 gene in mice. This led to massive hemorrhages in several tissues starting at midgestation, ultimately resulting in the death of almost all homozygous embryos upon birth. Histological analysis demonstrated dilation and ruptures in the endothelial lining of various small vessels but not in that of larger vessels. Kidneys displayed a distinct malformation of glomeruli and disorganization of podocytes. A delayed development of lung alveoli suggested impairment in lung inflation. Immunohistology demonstrated the absence of fibulin-1 in its typical localizations but no aberrant patterns for several other extracellular matrix proteins. Electron microscopy revealed intact basement membranes but very irregular cytoplasmic processes of capillary endothelial cells in the organs that were most severely affected. Absence of fibulin-1 caused considerable blood loss but did not compromise blood clotting. The data indicate a strong but restricted abnormality in some endothelial compartments which, together with some kidney and lung defects, may be responsible for early death.

Association of extracellular matrix proteins fibulin-1 and fibulin-2 with fibronectin in bone marrow stroma.

Extracellular matrix (ECM) molecules, together with growth factors and stromal cells, regulate haematopoietic cell development in bone marrow (BM). We report here expression of ECM proteins fibulin-1 and fibulin-2 in mouse BM. In other tissues, fibulin-1 and fibulin-2 associate with fibronectin and other ECM proteins. Fibulin-2 has also been found to adhere to cells via beta3 integrins. We studied the association of fibulins with fibronectin in BM stroma. By confocal microscopy, fibulin-1 and fibulin-2 immunostainings were co-localized with fibronectin in the adherent layer of long-term BM cultures. In cell adhesion assays using recombinant proteins, mouse fibulin-2 adhered to human erythroid-megakaryocytic leukaemia cell line HEL. This adhesion was mediated by beta3 integrins. However, HEL cells did not adhere to human fibulin-2. We therefore studied a possible species-specific cell-adhesive activity of mouse fibulin-2 by using mouse megakaryocytes, obtained by culture of BM cells in the presence of thrombopoietin. These megakaryocytes did not adhere to mouse fibulin-2. Our findings suggested that the functional role of fibulin-1 and fibulin-2 in BM stroma is related to binding to the major cell adhesion protein fibronectin, whereas adhesion of mouse fibulin-2 to human cells containing the integrin beta3 chain is not related to an apparent physiological function of the protein.

The extracellular matrix proteins fibulin-1 and fibulin-2 in the early human embryo.

Fibulin-1 and fibulin-2, two recently identified extracellular matrix proteins with a homologous domain structure, are known to bind various extracellular ligands and calcium. In this study, they have been localized at the light microscopical level in human embryos of gestational weeks 4-10, using polyclonal antibodies. Identical localization patterns were observed for the two fibulins in most of the tissues. In the heart, the endocardial cushion tissue and endocardium, but not the myocardium, were stained, as were the basement membrane zones and adventitia of blood vessels. Staining was also observed in the perichondrium and calcifying regions of developing bones. Moreover, reactions occurred with the gut subepithelium and epithelial basement membranes of the skin. Differences in staining patterns, however, were observed in various neural structures. Fibulin-1 was prominent in the matrix of the leptomeningeal anlage, in basement membranes of the neuroepithelium and the perineurium of peripheral nerves. Fibulin-2 was detected primarily within the neuropithelium, spinal ganglia and peripheral nerves. The early embryonic expression of both fibulins indicates specific roles during organ development and, in particular, involvements in the differentiation of heart, skeletal and neuronal structures.

Integrin-Binding and Cell-Adhesion Studies of Fibulins Reveal a Particular Affinity for αIIbβ3

The extracellular matrix proteins fibulin-1 (variants C and D) and fibulin-2 occur in basement membranes and in vessel walls and are thus potential candidates for cellular interactions. Recombinant forms of these proteins were obtained from stably transfected kidney cell clones and examined for cell-adhesion activity and binding to five different purified integrins. The two variants of mouse fibulin-1 were inactive in all these assays. Mouse fibulin-2, however, bound to αIIbβ3 integrin almost as strongly as fibrinogen, while a lower activity was found for αVβ3 and almost none for α5β1 integrin. Synthetic SVPRGDLDG peptide, corresponding to the single RGD site of mouse fibulin-2, was a strong antagonist of αIIbβ3 integrin binding. Its affinity for αVβ3 and α5β1 integrins was, however, 10- to 50-fold lower compared to GRGDS. Mouse fibulin-2 also promoted adhesion of thrombin-stimulated platelets and of some established cell lines which could be inhibited by RGD peptides. Human fibulin-2, in which the RGD sequence is changed to RSS, bound less strongly to αIIbβ3 integrin and showed no cell-adhesion activity. Together these data suggest a potential role in hemostatic control for mouse fibulin-2 and possibly also for human fibulin-2.