Lynn Y. Sakai

Dysregulation of TGF-β activation contributes to pathogenesis in Marfan syndrome

Marfan syndrome is an autosomal dominant disorder of connective tissue caused by mutations in fibrillin-1 (encoded by FBN1 in humans and Fbn1 in mice), a matrix component of extracellular microfibrils. A distinct subgroup of individuals with Marfan syndrome have distal airspace enlargement, historically described as emphysema, which frequently results in spontaneous lung rupture (pneumothorax; refs. 1–3). To investigate the pathogenesis of genetically imposed emphysema, we analyzed the lung phenotype of mice deficient in fibrillin-1, an accepted model of Marfan syndrome. Lung abnormalities are evident in the immediate postnatal period and manifest as a developmental impairment of distal alveolar septation. Aged mice deficient in fibrillin-1 develop destructive emphysema consistent with the view that early developmental perturbations can predispose to late-onset, seemingly acquired phenotypes. We show that mice deficient in fibrillin-1 have marked dysregulation of transforming growth factor-β (TGF-β) activation and signaling, resulting in apoptosis in the developing lung. Perinatal antagonism of TGF-β attenuates apoptosis and rescues alveolar septation in vivo. These data indicate that matrix sequestration of cytokines is crucial to their regulated activation and signaling and that perturbation of this function can contribute to the pathogenesis of disease.

Evidence for a critical contribution of haploinsufficiency in the complex pathogenesis of Marfan syndrome

Marfan syndrome is a connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1). A dominant-negative mechanism has been inferred based upon dominant inheritance, mulitimerization of monomers to form microfibrils, and the dramatic paucity of matrix-incorporated fibrillin-1 seen in heterozygous patient samples. Yeast artificial chromosome-based transgenesis was used to overexpress a disease-associated mutant form of human fibrillin-1 (C1663R) on a normal mouse background. Remarkably, these mice failed to show any abnormalities of cellular or clinical phenotype despite regulated overexpression of mutant protein in relevant tissues and developmental stages and direct evidence that mouse and human fibrillin-1 interact with high efficiency. Immunostaining with a human-specific mAb provides what we believe to be the first demonstration that mutant fibrillin-1 can participate in productive microfibrillar assembly. Informatively, use of homologous recombination to generate mice heterozygous for a comparable missense mutation (C1039G) revealed impaired microfibrillar deposition, skeletal deformity, and progressive deterioration of aortic wall architecture, comparable to characteristics of the human condition. These data are consistent with a model that invokes haploinsufficiency for WT fibrillin-1, rather than production of mutant protein, as the primary determinant of failed microfibrillar assembly. In keeping with this model, introduction of a WT FBN1 transgene on a heterozygous C1039G background rescues aortic phenotype.

Fibrillin-1 and Fibulin-2 Interact and Are Colocalized in Some Tissues

Microfibrils 10-12 nm in diameter are found in elastic and non-elastic tissues with fibrillin as a major component. Little is known about the supramolecular structure of these microfibrils and the protein interactions it is based on. To identify protein binding ligands of fibrillin-1, we tested binding of recombinant fibrillin-1 peptides to different extracellular matrix proteins in solid phase assays. Among the proteins tested, only fibulin-2 showed significant binding to rF11, the N-terminal half of fibrillin-1, in a calcium-dependent manner. Surface plasmon resonance demonstrated high affinity binding with a Kd = 56 nM. With overlapping recombinant fibrillin-1 peptides, the binding site for fibulin-2 was narrowed down to the N terminus of fibrillin-1 (amino acid positions 45-450). Immunofluorescence in tissues demonstrated colocalization of fibrillin and fibulin-2 in skin, perichondrium, elastic intima of blood vessels, and kidney glomerulus. Fibulin-2 was not present in ocular ciliary zonules, tendon, and the connective tissue around kidney tubules and lung alveoli, which all contain fibrillin. Immunogold labeling of fibulin-2 on microfibrils in skin was found preferentially at the interface between microfibrils and the amorphous elastin core, suggesting that in vivo the interaction between fibrillin-1 and fibulin-2 is regulated by cellular expression and deposition as well as by protein-protein interactions.

Regulation of limb patterning by extracellular microfibrils

To elucidate the contribution of the extracellular microfibril–elastic fiber network to vertebrate organogenesis, we generated fibrillin 2 (Fbn2)–null mice by gene targeting and identified a limb-patterning defect in the form of bilateral syndactyly. Digit fusion involves both soft and hard tissues, and is associated with reduced apoptosis at affected sites. Two lines of evidence suggest that syndactily is primarily due to defective mesenchyme differentiation, rather than reduced apoptosis of interdigital tissue. First, fusion occurs before appearance of interdigital cell death; second, interdigital tissues having incomplete separation fail to respond to apoptotic clues from implanted BMP-4 beads. Syndactyly is associated with a disorganized matrix, but with normal BMP gene expression. On the other hand, mice double heterozygous for null Fbn2 and Bmp7 alleles display the combined digit phenotype of both nullizygotes. Together, these results imply functional interaction between Fbn2-rich microfibrils and BMP-7 signaling. As such, they uncover an unexpected relationship between the insoluble matrix and soluble factors during limb patterning. We also demonstrate that the Fbn2- null mutation is allelic to the recessive shaker-with-syndactyly (sy) locus on chromosome 18.

Fibrillins 1 and 2 Perform Partially Overlapping Functions during Aortic Development

Fibrillin-rich microfibrils are extracellular assemblies that impart structural properties to the connective tissue. To elucidate the contribution of fibrillin-rich microfibrils to organogenesis, we have examined the vascular phenotype of a newly created strain of mice that completely lacks fibrillin-1 and the consequences of combined deficiency of fibrillins 1 and 2 on tissue formation. The results demonstrated that fibrillins 1 and 2 perform partially overlapping functions during aortic development. Fbn1-/- mice died soon after birth from ruptured aortic aneurysm, impaired pulmonary function, and/or diaphragmatic collapse. Analysis of the neonatal Fbn1-/- aorta documented a disorganized and poorly developed medial layer but normal levels of elastin cross-links. Transcriptional profiling revealed that aneurysm progression in Fbn1 null mice is accompanied by unproductive up-regulation of gene products normally involved in tissue repair and vascular integrity, such as plasminogen activator inhibitor-1, activin A, and cysteine-rich angiogenic protein 61. In contrast to Fbn1-/- mice, Fbn2 null mice had a well developed and morphologically normal aortic wall. However, virtually all Fbn1-/-;Fbn2-/- embryos and about half of the Fbn1+/-;Fbn2-/- embryos died in utero and displayed a significantly more severe vascular phenotype than Fbn1-/- mice. Consistent with a specialized function of fibrillin-2, electron microscopy visualized ultrastructurally different microfibrils in Fbn1 null compared with control cell cultures. Collectively, these data demonstrate that involvement of fibrillin-2 in the initial assembly of the aortic matrix overlaps in part with fibrillin-1 and that continued fibrillin-1 deposition is absolutely required for the maturation and function of the vessel during neonatal life.

The Prodomain of BMP-7 Targets the BMP-7 Complex to the Extracellular Matrix

Biochemical and biophysical methods are used to show that BMP-7 is secreted as a stable complex consisting of the processed growth factor dimer noncovalently associated with its two prodomain propeptide chains and that the BMP-7 complex is structurally similar to the small transforming growth factor β (TGFβ) complex. Because the prodomain of TGFβ interacts with latent TGFβ-binding proteins, a family of molecules homologous to the fibrillins, the prodomain of BMP-7 was tested for binding to fibrillin-1 or to LTBP-1. The BMP-7 prodomain and BMP-7 complex, but not the separated growth factor dimer, interact with N-terminal regions of fibrillin-1. This interaction may target the BMP-7 complex to fibrillin microfibrils in the extracellular matrix. Immunolocalization of BMP-7 in tissues like the kidney capsule and skin reveals co-localization with fibrillin. However, BMP-7 immunolocalization in other tissues known to be active sites for BMP-7 signaling is not apparent, suggesting that immunolocalization of BMP-7 in certain tissues represents specific extracellular storage sites. These studies suggest that the prodomains of TGFβ-like growth factors are important for positioning and concentrating growth factors in the extracellular matrix. In addition, they raise the possibility that prodomains of other TGFβ-like growth factors interact with fibrillins and/or LTBPs and are also targeted to the extracellular matrix.

Targeting of Bone Morphogenetic Protein Growth Factor Complexes to Fibrillin

Both latent transforming growth factor-β (TGF-β)-binding proteins fibrillins are components of microfibril networks, and both interact with members of the TGF-β family of growth factors. Interactions between latent TGF-β-binding protein-1 and TGF-β and between fibrillin-1 and bone morphogenetic protein-7 (BMP-7) are mediated by the prodomain of growth factor complexes. To extend this information, investigations were performed to test whether stable complexes are formed by additional selected TGF-β family members. Using velocity sedimentation in sucrose gradients as an assay, complex formation was demonstrated for BMP-7 and growth and differentiation factor-8 (GDF-8), which are known to exist in prodomain/growth factor complexes. Comparison of these results with complex formation by BMP-2, BMP-4 (full-length and shortened propeptides), BMP-10, and GDF-5 allowed us to conclude that all, except for BMP-2 and the short BMP-4 propeptides, formed complexes with their growth factors. Using surface plasmon resonance, binding affinities between fibrillin and all propeptides were determined. Binding studies revealed that the N-terminal end of fibrillin-1 serves as a universal high affinity docking site for the propeptides of BMP-2, -4, -7, and -10 and GDF-5, but not GDF-8, and located the BMP/GDF binding site within the N-terminal domain in fibrillin-1. Rotary shadowing electron microscopy of molecules of BMP-7 complex bound to fibrillin-1 confirmed these findings and also showed that prodomain binding targets the growth factor to fibrillin. Immunolocalization of BMP-4 demonstrated fibrillar staining limited to certain tissues, indicating tissue-specific targeting of BMP-4. These data implicate the fibrillin microfibril network in the extracellular control of BMP signaling and demonstrate differences in how prodomains target their growth factors to the extracellular space.

Mutations in Fibrillin-1 Cause Congenital Scleroderma: Stiff Skin Syndrome

Stiff skin syndrome, an autosomal dominant congenital form of scleroderma, is caused by mutations in the domain of fibrillin-1 that mediates integrin binding. Variation in Rare Disease Linked to Common Skin Disorder In the epic words of British physician and researcher Dr. William Harvey, “Nature is nowhere accustomed more openly to display her secret mysteries than in cases where she shows traces of her workings apart from the beaten path; nor is there any better way to advance the proper practice of medicine than to give our minds to the discovery of the usual law of nature by the careful investigation of cases of rarer forms of disease. For it has been found in almost all things, that what they contain of useful or of applicable nature, is hardly perceived unless we are deprived of them, or they become deranged in some way.” Such is the case for rare genetic diseases, which have provided the framework to understand some of the most devastating common diseases from just the simple permutation of a gene. Scleroderma, which literally means a pathological hardening of the skin, manifests as a complex phenotype. To better understand the etiology of scleroderma, Loeys and colleagues investigate four families with more than 10 affected individuals with a rare congenital form called stiff skin syndrome—which together account for more than 25% of the total cases currently documented in the literature—and identify the key mutations in a gene that encodes the connective tissue protein fibrillin-1. Fibrosis is not only seen in the context of systemic connective tissue disorders, but rather is often the major signature of the inflammatory burden in many common disorders, providing incentive to understand the factors critical in the initiation and maintenance of profibrotic programs. Mutations causing stiff skin syndrome cluster within a single domain of fibrillin-1 that mediates integin binding. Low amounts of an activated protein kinase that is triggered by ligand-integrin interactions provided further evidence for altered cell matrix interactions. This associates with evidence for activation of the TGFβ signaling cascade, a mechanism to instruct cells to deposit collagens and other matrix elements both during normal wound healing and in various fibrotic states. These data are consistent with a model in which integrins provide a means for cells to sample the matrix and to adjust their synthetic repertoire accordingly. Loss of this feedback would culminate in fibrosis. The results garnered from these patients with this rare disease prompted the authors to examine skin biopsies from five patients with common scleroderma, which surprisingly revealed that each scleroderma patient showed all of the abnormalities seen in stiff skin syndrome. These findings reinforce the power of studying rare genetic disorders to inform the underlying causes of common diseases, but also unveil a potential avenue for therapeutic intervention in common disorders that include fibrosis. The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence–encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor–β (TGFβ). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFβ concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

Cell adhesion and integrin binding to recombinant human fibrillin-1

Fibrillin‐1 is a major constituent of tissue microfibrils that occur in most connective tissues, either in close association with or independent of elastin. To test possible cell‐adhesive functions of this protein, we used recombinant human fibrillin‐1 polypeptides produced in a mammalian expression system in cell attachment and solid‐phase integrin binding assays. Fibrillin‐1 polypeptides containing the single RGD sequence located in the fourth 8‐cysteine domain, mediated distinct cell adhesion of a variety of cell lines and bound to purified integrin αVβ3. Integrins αIIbβ3, α5β1, α2β1 and α1β1 did not interact with any of the recombinant fibrillin‐1 peptides. Our results indicate a novel role for fibrillin‐1 in cellular interactions mediated via an RGD motif that is appropriately exposed for recognition by integrin αVβ3.

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 β (TGFβ) 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 TGFβ 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 TGFβ and BMP complexes to fibrillins. Hence, the emerging view is that fibrillin-rich microfibrils are molecular integrators of structural and instructive signals, with TGFβ and BMPs as the nodal points that convert extracellular inputs into discrete and context-dependent cellular responses.