Fibulin-5, an integrin-binding matricellular protein: its function in development and disease.
ABSTRACT: Interactions between the extracellular matrix (ECM) and cells are critical in embryonic development, tissue homeostasis, physiological remodeling, and tumorigenesis. Matricellular proteins, a group of ECM components, mediate cell-ECM interactions. One such molecule, Fibulin-5 is a 66-kDa glycoprotein secreted by various cell types, including vascular smooth muscle cells (SMCs), fibroblasts, and endothelial cells. Fibulin-5 contributes to the formation of elastic fibers by binding to structural components including tropoelastin and fibrillin-1, and to cross-linking enzymes, aiding elastic fiber assembly. Mice deficient in the fibulin-5 gene (Fbln5) exhibit systemic elastic fiber defects with manifestations of loose skin, tortuous aorta, emphysematous lung and genital prolapse. Although Fbln5 expression is down-regulated after birth, following the completion of elastic fiber formation, expression is reactivated upon tissue injury, affecting diverse cellular functions independent of its elastogenic function. Fibulin-5 contains an evolutionally conserved arginine-glycine-aspartic acid (RGD) motif in the N-terminal region, which mediates binding to a subset of integrins, including alpha5beta1, alphavbeta3, and alphavbeta5. Fibulin-5 enhances substrate attachment of endothelial cells, while inhibiting migration and proliferation in a cell type- and context-dependent manner. The antagonistic function of fibulin-5 in angiogenesis has been demonstrated in vitro and in vivo; fibulin-5 may block angiogenesis by inducing the anti-angiogenic molecule thrompospondin-1, by antagonizing VEGF(165)-mediated signaling, and/or by antagonizing fibronectin-mediated signaling through directly binding and blocking the alpha5beta1 fibronectin receptor. The overall effect of fibulin-5 on tumor growth depends on the balance between the inhibitory property of fibulin-5 on angiogenesis and the direct effect of fibulin-5 on proliferation and migration of tumor cells. However, the effect of tumor-derived versus host microenvironment-derived fibulin-5 remains to be evaluated.
Project description:The fibulin family of extracellular matrix/matricellular proteins is composed of long fibulins (fibulin-1, -2, -6) and short fibulins (fibulin-3, -4, -5, -7) and is involved in protein-protein interaction with the components of basement membrane and extracellular matrix proteins. Fibulin-1, -2, -3, -4, and -5 bind the monomeric form of elastin (tropoelastin) in vitro and fibulin-2, -3, -4, and -5 are shown to be involved in various aspects of elastic fiber development in vivo. In particular, fibulin-4 and -5 are critical molecules for elastic fiber assembly and play a non-redundant role during elastic fiber formation. Despite manifestation of systemic elastic fiber defects in all elastogenic tissues, fibulin-5 null (Fbln5(-/-)) mice have a normal lifespan. In contrast, fibulin-4 null (Fbln4(-/-)) mice die during the perinatal period due to rupture of aortic aneurysms, indicating differential functions of fibulin-4 and fibulin-5 in normal development. In this review, we will update biochemical characterization of fibulin-4 and fibulin-5 and discuss their roles in elastogenesis and outside of elastogenesis based on knowledge obtained from loss-of-function studies in mouse and in human patients with FBLN4 or FBLN5 mutations. Finally, we will evaluate therapeutic options for matrix-related diseases.
Project description:Impaired elastogenesis and increased degradation of elastic fibers has been implicated in the pathogenesis of pelvic organ prolapse. Loss of the elastogenic organizer, fibulin-5 (FBLN5), leads to pelvic organ prolapse in mice. The objective of this study was to investigate the regulation of FBLN5 after surgical injury of the vaginal wall using the rat as a preclinical animal model. Both endogenous and recombinant FBLN5 were degraded after surgical injury. Estrogen did not alter the dramatic loss of vaginal FBLN5 in the acute phase after injury (12-48?h), but resulted in rescue of the poor recovery of FBLN5 levels in the late phase (7 d) of healing in ovariectomized animals. In contrast with estrogen, the general MMP inhibitor, actinonin, abrogated injury-induced degradation of FBLN5 significantly. Further, actinonin rescued the negative effects of injury on biomechanics, histomorphology, and elastic fibers. Control of excessive matrix degradation by local application of actinonin at the time of surgery may lead to improved elastic fiber regeneration and wound healing, thereby potentially enhancing pelvic floor recovery after reconstructive surgery for prolapse.
Project description:Pelvic organ prolapse (POP) is a common condition affecting almost half of women over the age of 50. The molecular and cellular mechanisms underlying this condition, however, remain poorly understood. Here we have reported that fibulin-5, an integrin-binding matricellular protein that is essential for elastic fiber assembly, regulated the activity of MMP-9 to maintain integrity of the vaginal wall and prevented development of POP. In murine vaginal stromal cells, fibulin-5 inhibited the ?1 integrin-dependent, fibronectin-mediated upregulation of MMP-9. Mice in which the integrin-binding motif was mutated to an integrin-disrupting motif (Fbln5RGE/RGE) exhibited upregulation of MMP-9 in vaginal tissues. In contrast to fibulin-5 knockouts (Fbln5-/-), Fbln5RGE/RGE mice were able to form intact elastic fibers and did not exhibit POP. However, treatment of mice with ?-aminopropionitrile (BAPN), an inhibitor of matrix cross-linking enzymes, induced subclinical POP. Conversely, deletion of Mmp9 in Fbln5-/- mice significantly attenuated POP by increasing elastic fiber density and improving collagen fibrils. Vaginal tissue samples from pre- and postmenopausal women with POP also displayed significantly increased levels of MMP-9. These results suggest that POP is an acquired disorder of extracellular matrix and that therapies targeting matrix proteases may be successful for preventing or ameliorating POP in women.
Project description:Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-β binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-β because TGF-β-free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-β-bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.
Project description:Pelvic organ prolapse is strongly associated with a history of vaginal delivery. The mechanisms by which pregnancy and parturition lead to failure of pelvic organ support, however, are not known. Recently, it was reported that mice with null mutations in lysyl oxidase-like 1 (LOXL1) develop pelvic organ prolapse. Elastin is a substrate for lysyl oxidase (LOX) and LOXL1, and LOXL1 interacts with fibulin-5 (FBLN5). Therefore, to clarify the potential role of elastic fiber assembly in the pathogenesis of pelvic organ prolapse, pelvic organ support was characterized in Fbln5-/- mice, and changes in elastic fiber homeostasis in the mouse vagina during pregnancy and parturition were determined. Pelvic organ prolapse in Fbln5-/- mice was remarkably similar to that in primates. The temporal relationship between LOX mRNA and protein, processing of LOXL1 protein, FBLN5 and tropoelastin protein, and desmosine content in the vagina suggest that a burst of elastic fiber assembly and cross linking occurs in the vaginal wall postpartum. Together with the phenotype of Fbln5-/- mice, the results suggest that synthesis and assembly of elastic fibers are crucial for recovery of pelvic organ support after vaginal delivery and that disordered elastic fiber homeostasis is a primary event in the pathogenesis of pelvic organ prolapse in mice.
Project description:Recent findings on the role of fibulin-5 (Fbln5) have provided substantial progress in understanding the molecular mechanism of elastic fiber assembly in vitro. However, little is known about differential roles of fibulins in the elastogenesis of blood vessels. Here, we generated double knockout mice for Fbln5 and Fbln2 (termed DKO) and examined the role of fibulins-2 and -5 in development and injury response of the blood vessel wall.Fibulin-2 is distinctly located in the subendothelial matrix, whereas fibulin-5 is observed throughout the vessel wall. All of the elastic laminae, including the internal elastic lamina (IEL), were severely disorganized in DKO mice, which was not observed in single knockout mice for Fbln2 or Fbln5. Furthermore, DKO vessels displayed upregulation of vascular adhesion molecules, tissue factor expression, and thrombus formation with marked dilation and thinning of the vessel wall after carotid artery ligation-injury.Fibulin-2 and fibulin-5 cooperatively function to form the IEL during postnatal development by directing the assembly of elastic fibers, and are responsible for maintenance of the adult vessel wall after injury. The DKO mouse will serve as a unique animal model to test the effect of vessel integrity during various pathological insults.
Project description:Mice deficient in Latent TGF? Binding Protein 4 (Ltbp4) display a defect in lung septation and elastogenesis. The lung septation defect is normalized by genetically decreasing TGF?2 levels. However, the elastic fiber assembly is not improved in Tgfb2(-/-) ;Ltbp4S(-/-) compared to Ltbp4S(-/-) lungs. We found that decreased levels of TGF?1 or TGF?3 did not improve lung septation indicating that the TGF? isoform elevated in Ltbp4S(-/-) lungs is TGF?2. Expression of a form of Ltbp4 that could not bind latent TGF? did not affect lung phenotype indicating that normal lung development does not require the formation of LTBP4-latent TGF? complexes. Therefore, the change in TGF?-level in the lungs is not directly related to Ltbp4 deficiency but probably is a consequence of changes in the extracellular matrix. Interestingly, combination of the Ltbp4S(-/-) mutation with a fibulin-5 null mutant in Fbln5(-/-) ;Ltbp4S(-/-) mice improves the lung septation compared to Ltbp4S(-/-) lungs. Large globular elastin aggregates characteristic for Ltbp4S(-/-) lungs do not form in Fbln5(-/-) ;Ltbp4S(-/-) lungs and EM studies showed that elastic fibers in Fbln5(-/-) ;Ltbp4S(-/-) lungs resemble those found in Fbln5(-/-) mice. These results are consistent with a role for TGF?2 in lung septation and for Ltbp4 in regulating fibulin-5 dependent elastic fiber assembly.
Project description:Fibulin-5, an extracellular matrix glycoprotein expressed in elastin-rich tissues, regulates vascular cell behaviour and elastic fibre deposition. Recombinant full-length human fibulin-5 supported primary human aortic SMC (smooth-muscle cell) attachment through alpha5beta1 and alpha4beta1 integrins. Cells on fibulin-5 spread poorly and displayed prominent membrane ruffles but no stress fibres or focal adhesions, unlike cells on fibronectin that also binds these integrins. Cell migration and proliferation were significantly lower on fibulin-5 than on fibronectin. Treatment of cells on fibulin-5 with a beta1 integrin-activating antibody induced stress fibres, increased attachment, migration and proliferation, and stimulated signalling of epidermal growth factor receptor and platelet-derived growth factor receptors alpha and beta. Fibulin-5 also modulated fibronectin-mediated cell spreading and morphology. We have thus identified the beta1 integrins on primary SMCs that fibulin-5 interacts with, and have shown that failure of fibulin-5 to activate these receptors limits cell spreading, migration and proliferation.
Project description:Evolution of elastic fibers is associated with establishment of the closed circulation system. Primary roles of elastic fibers are to provide elasticity and recoiling to tissues and organs and to maintain the structural integrity against mechanical strain over a lifetime. Elastic fibers are comprised of an insoluble elastin core and surrounding mantle of microfibrils. Elastic fibers are formed in a regulated, stepwise manner, which includes the formation of a microfibrillar scaffold, deposition and integration of tropoelastin monomers into the scaffold, and cross-linking of the monomers to form an insoluble, functional polymer. In recent years, an increasing number of glycoproteins have been identified and shown to be located on or surrounding elastic fibers. Among them, the short fibulins-3, -4 and -5 particularly drew attention because of their potent elastogenic activity. Fibulins-3, -4 and -5 are characterized by tandem repeats of calcium binding EGF-like motifs and a C-terminal fibulin module, which is conserved throughout fibulin family members. Initial biochemical characterization and gene expression studies predicted that fibulins might be involved in structural support and/or matrix-cell interactions. Recent analyses of short fibulin knockout mice have revealed their critical roles in elastic fiber development in vivo. We review recent findings on the elastogenic functions of short fibulins and discuss the molecular mechanism underlying their activity in vitro and in vivo.
Project description:Abstract The large arteries serve as compliant vessels that store energy during systole and return it during diastole. This function is made possible by the elastic fibers in the arterial wall that are assembled during late embryonic and early postnatal development from various proteins, including fibulin-5. Mice and humans with insufficient amounts of fibulin-5 have reduced arterial compliance as adults. Reduced compliance of the large arteries is correlated with hypertension, reduced cardiac function, and an increased risk of death from cardiac and cardiovascular disease. The goal of this study was to quantify arterial compliance, blood pressure, and left ventricular (LV) function from early postnatal development to young adulthood in fibulin-5 null (Fbln5-/-) mice to determine the effects of reduced arterial compliance during this critical period of elastic fiber assembly. We find that ascending aorta compliance is reduced as early as postnatal day (P) 7 and carotid artery compliance is reduced by P21 in Fbln5-/- mice. We did not find significant increases in systolic blood pressure by P60, but pulse pressures are increased by P21 in Fbln5-/- mice. LV systolic function, as measured by ejection fraction and fractional shortening, is unaffected in Fbln5-/- mice. However, LV diastolic function, as measured by tissue Doppler imaging, is compromised at all ages in Fbln5-/- mice. We propose that Fbln5-/- mice represent a suitable model for further studies to determine mechanistic relationships between arterial compliance and LV diastolic function.