The fibrillin microfibril/elastic fibre network: A critical extracellular supramolecular scaffold to balance skin homoeostasis.

Supramolecular networks composed of fibrillins (fibrillin-1 and fibrillin-2) and associated ligands form intricate cellular microenvironments which balance skin homoeostasis and direct remodelling. Fibrillins assemble into microfibrils which are not only indispensable for conferring elasticity to the skin, but also control the bioavailability of growth factors targeted to the extracellular matrix architecture. Fibrillin microfibrils (FMF) represent the core scaffolds for elastic fibre formation, and they also decorate the surface of elastic fibres and form independent networks. In normal dermis, elastic fibres are suspended in a three-dimensional basket-like lattice of FMF intersecting basement membranes at the dermal-epidermal junction and thus conferring pliability to the skin. The importance of FMF for skin homoeostasis is illustrated by the clinical features caused by mutations in the human fibrillin genes (FBN1, FBN2), summarized as "fibrillinopathies." In skin, fibrillin mutations result in phenotypes ranging from thick, stiff and fibrotic skin to thin, lax and hyperextensible skin. The most plausible explanation for this spectrum of phenotypic outcomes is that FMF regulate growth factor signalling essential for proper growth and homoeostasis of the skin. Here, we will give an overview about the current understanding of the underlying pathomechanisms leading to fibrillin-dependent fibrosis as well as forms of cutis laxa caused by mutational inactivation of FMF-associated ligands.

TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators.

TGFB1 (transforming growth factor beta 1) is a potent cytokine playing a driving role in development, fibrosis and cancer. It is synthesized as prodomain-growth factor complex that requires tethering to LTBP (latent transforming growth factor beta binding protein) for efficient secretion into the extracellular space. Upon release, this large latent complex is sequestered by anchorage to extracellular matrix (ECM) networks, from which the mature growth factor needs to be activated in order to reach its receptors and initiate signaling. Here, we uncovered a novel intracellular secretion pathway by which the latent TGFB1 complex reaches the plasma membrane and is released from fibroblasts, the key effector cells during tissue repair, fibrosis and in the tumor stroma. We show that secretion of latent TGFB1, but not of other selected cytokines or of bulk cargo, is regulated by fibroblast-ECM communication through ILK (integrin linked kinase) that restricts RHOA activity by interacting with ARHGAP26/GRAF1. Latent TGFB1 interacts with GORASP2/GRASP55 and is detected inside MAP1LC3-positive autophagosomal intermediates that are secreted by a RAB8A-dependent pathway. Interestingly, TGFB1 secretion is fully abrogated in human and murine fibroblasts and macrophages that lack key components of the autophagic machinery. Our data demonstrate an unconventional secretion mode of TGFB1 adding another level of control of its bioavailability and activity in order to effectively orchestrate cellular programs prone to dysregulation as seen in fibrosis and cancer.

Structural characterization of twisted gastrulation provides insights into opposing functions on the BMP signalling pathway.

Twisted gastrulation (Tsg) and chordin are secreted glycoproteins that function together as BMP (bone morphogenetic protein) antagonists to regulate BMP growth factor signalling. Chordin binds to BMPs, preventing them from interacting with their receptors and Tsg is known to strengthen this inhibitory complex. Tsg also acts as a BMP agonist by promoting cleavage of chordin by tolloid-family proteinases. Here we explore the structural mechanism through which Tsg exerts this dual activity. We have characterized the nanoscale structure of human Tsg using in-solution biomolecular analysis and show that Tsg is a globular monomer with a flattened cross shape. Tsg has a high proportion of N-linked glycans, in relation to its molecular weight, which supports a role in solubilising BMPs. Tsg binds with high affinity to the C-terminal region of chordin and was also able to inhibit BMP-7 signalling directly but did not have an effect on BMP-4 signalling. Although both Tsg and mammalian tolloid are involved in chordin cleavage, no interaction could be detected between them using surface plasmon resonance. Together these data suggest that Tsg functions as a BMP-agonist by inducing conformational change in chordin making it more susceptible to tolloid cleavage and as a BMP-antagonist either independently or via a chordin-mediated mechanism. Following single cleavage of chordin by tolloids, Tsg continues to strengthen the inhibitory complex, supporting a role for partially cleaved chordin in BMP regulation.

Characterization of Microfibrillar-associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-binding Protein Involved in Elastic Fiber Formation.

MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization.

The role of chordin fragments generated by partial tolloid cleavage in regulating BMP activity.

Chordin-mediated regulation of bone morphogenetic protein (BMP) family growth factors is essential in early embryogenesis and adult homoeostasis. Chordin binds to BMPs through cysteine-rich von Willebrand factor type C (vWC) homology domains and blocks them from interacting with their cell surface receptors. These domains also self-associate and enable chordin to target related proteins to fine-tune BMP regulation. The chordin-BMP inhibitory complex is strengthened by the secreted glycoprotein twisted gastrulation (Tsg); however, inhibition is relieved by cleavage of chordin at two specific sites by tolloid family metalloproteases. As Tsg enhances this cleavage process, it serves a dual role as both promoter and inhibitor of BMP signalling. Recent developments in chordin research suggest that rather than simply being by-products, the cleavage fragments of chordin continue to play a role in BMP regulation. In particular, chordin cleavage at the C-terminus potentiates its anti-BMP activity in a type-specific manner.

Nanoscale structure of the BMP antagonist chordin supports cooperative BMP binding.

Bone morphogenetic proteins (BMPs) orchestrate key cellular events, such as proliferation and differentiation, in development and homeostasis. Extracellular antagonists, such as chordin, are essential regulators of BMP signaling. Chordin binds to BMPs blocking interaction with receptors, and cleavage by tolloid proteinases is thought to relieve this inhibition. A model has been previously proposed where chordin adopts a horseshoe-like arrangement enabling BMP binding cooperatively by terminal domains (1). Here, we present the nanoscale structure of human chordin using electron microscopy, small angle X-ray scattering, and solution-based biophysical techniques, which together show that chordin indeed has a compact horseshoe-shaped structure. Chordin variants were used to map domain locations within the chordin molecule. The terminal BMP-binding domains protrude as prongs from the main body of the chordin structure, where they are well positioned to interact with the growth factor. The spacing provided by the chordin domains supports the principle of a cooperative BMP-binding arrangement that the original model implied in which growth factors bind to both an N- and C-terminal von Willebrand factor C domain of chordin. Using binding and bioactivity assays, we compared full-length chordin with two truncated chordin variants, such as those produced by partial tolloid cleavage. Cleavage of either terminal domain has little effect on the affinity of chordin for BMP-4 and BMP-7 but C-terminal cleavage increases the efficacy of chordin as a BMP-4 inhibitor. Together these data suggest that partial tolloid cleavage is insufficient to ablate BMP inhibition and the C-terminal chordin domains play an important role in BMP regulation.