We have discovered that fibrillin-1, which forms extracellular microfibrils, can regulate the bioavailability of transforming growth factor (TGF) β1, a powerful cytokine that modulates cell survival and phenotype. Altered TGFβ signaling is a major contributor to the pathology of Marfan syndrome (MFS) and related diseases. In the presence of cell layer extracellular matrix, a fibrillin-1 sequence encoded by exons 44–49 releases endogenous TGFβ1, thereby stimulating TGFβ receptor–mediated Smad2 signaling. This altered TGFβ1 bioavailability does not require intact cells, proteolysis, or the altered expression of TGFβ1 or its receptors. Mass spectrometry revealed that a fibrillin-1 fragment containing the TGFβ1-releasing sequence specifically associates with full-length fibrillin-1 in cell layers. Solid-phase and BIAcore binding studies showed that this fragment interacts strongly and specifically with N-terminal fibrillin-1, thereby inhibiting the association of C-terminal latent TGFβ-binding protein 1 (a component of the large latent complex [LLC]) with N-terminal fibrillin-1. By releasing LLC from microfibrils, the fibrillin-1 sequence encoded by exons 44–49 can contribute to MFS and related diseases.

Newly deposited microfibrils strongly colocalise with fibronectin in primary fibroblasts. Microfibril formation is grossly inhibited by fibronectin depletion, but rescued by supplementation with exogenous cellular fibronectin. As integrin receptors are key determinants of fibronectin assembly, we investigated whether they also influenced microfibril deposition. Analysis of β1-integrin-receptor-null fibroblasts, blockage of cell surface integrin receptors that regulate fibronectin assembly and disruption of Rho kinase all result in suppressed deposition of both fibronectin and microfibrils. Antibody activation of β1 integrins in fibronectin-depleted cultures is insufficient to rescue microfibril assembly. In fibronectinRGE/RGE mutant mouse fibroblast cultures, which do not engage α5β1 integrin, extracellular assembly of both fibronectin and microfibrils is markedly reduced. Thus, pericellular microfibril assembly is regulated by fibronectin fibrillogenesis.

SummaryControl of the bioavailability of the growth factor TGF is essential for tissue formation and homeostasis, yet precisely how latent TGF is incorporated into the extracellular matrix is unknown. Here, we show that deposition of a large latent TGF complex (LLC), which contains latent TGF-binding protein 1 (LTBP-1), is directly dependent on the pericellular assembly of fibrillin microfibrils, which interact with fibronectin during higher-order fibrillogenesis. LTBP-1 formed pericellular arrays that colocalized with microfibrils, whereas fibrillin knockdown inhibited fibrillar LTBP-1 and/or LLC deposition. Blocking 51 integrin or supplementing cultures with heparin, which both inhibited microfibril assembly, disrupted LTBP-1 deposition and enhanced Smad2 phosphorylation. Full-length LTBP-1 bound only weakly to N-terminal pro-fibrillin-1, but this association was strongly enhanced by heparin. The microfibrilassociated glycoprotein MAGP-1 (MFAP-2) inhibited LTBP-1 binding to fibrillin-1 and stimulated Smad2 phosphorylation. By contrast, fibulin-4, which interacted strongly with full-length LTBP-1, did not induce Smad2 phosphorylation. Thus, LTBP-1 and/or LLC deposition is dependent on pericellular microfibril assembly and is governed by complex interactions between LTBP-1, heparan sulfate, fibrillin-1 and microfibril-associated molecules. In this way, microfibrils control TGF bioavailability.

Hearing impairment is a common human condition, but we know little about the molecular basis of cochlear function. Shaker-with-syndactylism (sy) is a classic deaf mouse mutant and we show here that a second allele, sy(ns), is associated with abnormal production of endolymph, the fluid bathing sensory hair cells. Using a positional candidate approach, we demonstrate that mutations in the gene encoding the basolateral Na-K-Cl co-transporter Slc12a2 (Nkcc1, mBSC2) cause the deafness observed in sy and sy(ns) mice. This finding provides the molecular basis of another link in the chain of K+recycling in the cochlea, a process essential for normal cochlear function.

Fibrillins are large, cysteine-rich glycoproteins that form microfibrils and play a central role in elastic fibrillogenesis. Fibrillin-1 and fibrillin-2, encoded by FBN1 on chromosome 15q21.1 and FBN2 on chromosome 5q23-q31, are highly similar proteins. The finding of mutations in FBN1 and FBN2 in the autosomal dominant microfibrillopathies Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), respectively, has highlighted their essential role in the development and homeostasis of elastic fibres. MFS is characterized by cardiovascular, skeletal and ocular abnormalities, and CCA by long, thin, flexed digits, crumpled ears and mild joint contractures. Although mutations arise throughout FBN1, those clustering within exons 24-32 are associated with the most severe form of MFS, so-called neonatal MFS. All the mutations described in CCA occur in the "neonatal region" of FBN2. Both MFS and CCA are thought to arise via a dominant negative mechanism. The analysis of mouse mutations has demonstrated that fibrillin-1 microfibrils are mainly engaged in tissue homeostasis rather than elastic matrix assembly. In the current investigation, we have analysed the classical mouse mutant shaker-with-syndactylism using a positional candidate approach and demonstrated that loss-of-function mutations outside the "neonatal region" of Fbn2 cause syndactyly in mice. These results suggest that phenotypes distinct from CCA may result in man as a consequence of mutations outside the "neonatal region" of FBN2.

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