Assembly of fibrillin microfibrils governs extracellular deposition of latent TGF beta.

Control of the bioavailability of the growth factor TGFbeta is essential for tissue formation and homeostasis, yet precisely how latent TGFbeta is incorporated into the extracellular matrix is unknown. Here, we show that deposition of a large latent TGFbeta complex (LLC), which contains latent TGFbeta-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 alpha5beta1 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 microfibril-associated 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 TGFbeta bioavailability.

Fibrillin-1 regulates the bioavailability of TGFbeta1.

We have discovered that fibrillin-1, which forms extracellular microfibrils, can regulate the bioavailability of transforming growth factor (TGF) beta1, a powerful cytokine that modulates cell survival and phenotype. Altered TGFbeta 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 TGFbeta1, thereby stimulating TGFbeta receptor-mediated Smad2 signaling. This altered TGFbeta1 bioavailability does not require intact cells, proteolysis, or the altered expression of TGFbeta1 or its receptors. Mass spectrometry revealed that a fibrillin-1 fragment containing the TGFbeta1-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 TGFbeta-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.

Substrate chemistry influences the morphology and biological function of adsorbed extracellular matrix assemblies.

In addition to mediating cell signalling events, native extracellular matrix (ECM) assemblies interact with other ECM components, act as reservoirs for soluble signalling molecules and perform structural roles. The potential of native ECM assemblies in the manufacture of biomimetic materials has not been fully exploited due, in part, to the effects of substrate interactions on their morphology. We have previously demonstrated that the ECM components, fibrillin and type VI collagen microfibrils, exhibit substrate dependent morphologies on chemically and topographically variable heterogeneous surfaces. Using both cleaning and coating approaches on silicon wafers and glass coverslips we have produced chemically homogeneous, topographically similar substrates which cover a large amphiphilic range. Extremes of substrate amphiphilicity induced morphological changes in periodicity, curvature and lateral spreading which may mask binding sites or disrupt domain structure. Biological functionality, as assayed by the ability to support cell spreading, was significantly reduced for fibrillin microfibrils adsorbed on highly hydrophilic substrates (contact angle 20.7 degrees) compared with less hydrophilic (contact angle 38.3 degrees) and hydrophobic (contact angle 92.8 degrees) substrates. With an appropriate choice of surface chemistry, multifunctional ECM assemblies retain their native morphology and biological functionality.