Adrian Shuttleworth

Mesenchymal stem cell migration is regulated by fibronectin through α5β1-integrin-mediated activation of PDGFR-β and potentiation of growth factor signals

Cell migration during vascular remodelling is regulated by crosstalk between growth factor receptors and integrin receptors, which together coordinate cytoskeletal and motogenic changes. Here, we report extracellular matrix (ECM)-directed crosstalk between platelet-derived growth factor receptor (PDGFR)-β and α5β1-integrin, which controls the migration of mesenchymal stem (stromal) cells (MSCs). Cell adhesion to fibronectin induced α5β1-integrin-dependent phosphorylation of PDGFR-β in the absence of growth factor stimulation. Phosphorylated PDGFR-β co-immunoprecipitated with α5-integrin and colocalised with α5β1-integrin in the transient tidemarks of focal adhesions. Adhesion to fibronectin also strongly potentiated PDGF-BB-induced PDGFR-β phosphorylation and focal adhesion kinase (FAK) activity, in an α5β1-integrin-dependent manner. PDGFR-β-induced phosphoinositide 3-kinase (PI3K) and Akt activity, actin reorganisation and cell migration were all regulated by fibronectin and α5β1-integrin. This synergistic relationship between α5β1-integrin and PDGFR-β is a fundamental determinant of cell migration. Thus, fibronectin-rich matrices can prime PDGFR-β to recruit mesenchymal cells at sites of vascular remodelling.

Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils.

The α1(VIII) and α2(VIII) Chains of Type VIII Collagen Can Form Stable Homotrimeric Molecules

Type VIII collagen is a short chain collagen. Two chains have been described, α1(VIII) and α2(VIII), but the chain composition of type VIII collagen is far from resolved. To address this question, we have expressed full-length α1(VIII) and α2(VIII) chains in an in vitro translation system supplemented with semipermeabilized cells. Both chains gave a translation product of ∼80 kDa that could be shown to produce a chymotrypsin/trypsin-resistant product of ∼60 kDa, indicating that both chains could form homotrimers. Hydroxylation of proline residues was a prerequisite for stable trimer formation. The melting temperature for the α1(VIII) homotrimer was 45 °C, whereas that for α2(VIII) was 42 °C. The ability of both chains of type VIII collagen to form stable triple helices suggests that there may be different forms of this collagen and that cells may modulate the chain composition in response to different biological conditions.

Defining elastic fiber interactions by molecular fishing: an affinity purification and mass spectrometry approach.

Deciphering interacting networks of the extracellular matrix is a major challenge. We describe an affinity purification and mass spectrometry strategy that has provided new insights into the molecular interactions of elastic fibers, essential extracellular assemblies that provide elastic recoil in dynamic tissues. Using cell culture models, we defined primary and secondary elastic fiber interaction networks by identifying molecular interactions with the elastic fiber molecules fibrillin-1, MAGP-1, fibulin-5, and lysyl oxidase. The sensitivity and validity of our method was confirmed by identification of known interactions with the bait proteins. Our study revealed novel extracellular protein interactions with elastic fiber molecules and delineated secondary interacting networks with fibronectin and heparan sulfate-associated molecules. This strategy is a novel approach to define the macromolecular interactions that sustain complex extracellular matrix assemblies and to gain insights into how they are integrated into their surrounding matrix.

Type VIII collagen: heterotrimeric chain association

Two chains, alpha1(VIII) and alpha2(VIII), have been described for type VIII collagen. Early work suggested that these chains were present in a 2:1 ratio, although recent work has shown that homotrimers can form and predominate in some tissues. In order to address the question of whether the alpha1(VIII) and alpha2(VIII) chains could co-polymerise we made a shortened alpha1(VIII) chain and expressed this with full length alpha2(VIII) chain in an in vitro translation system supplemented with semi-permeabilised cells. Heterotrimers containing either two or one alpha2(VIII) were evident. Interestingly, a point mutation in the NC1 domain of the alpha1(VIII) chain abrogated trimer formation. In addition we were able to demonstrate chain association of the alpha1(X) chain of type X collagen with the shortened alpha1(VIII) chain. Variations in chain association were seen when altered ratios of message were used. These results demonstrate the importance of the NC1 domain in chain association and suggest that gene expression regulates the composition and function of type VIII collagen by varying chain composition.

Collagen type I

This chapter focuses on extracellular protein collagen type I, which is the major fibrillar collagen of bones, tendon, and skin and provides these and many other tissues with tensile strength. Collagen type I forms rope-like structures in tendon and sheet-like structures in skin, and in bones, it is reinforced with calcium hydroxyapatite. It is synthesized primarily as a heterotrimeric procollagen, which is processed extracellularly by N- and C-proteinases to give a triple-helical molecule that can assemble with a stagger of 234 amino acids into cross-banded fibrils with a 67 nm periodicity. These fibrils are stabilized by intermolecular cross-links derived from specific lysine/hydroxylysine residues in both the nonhelical (telopeptide) and helical domains. The isolation methods and gene structure of type-I collagen are presented in this chapter.

Matrix Gla protein

Publisher Summary This chapter focuses on the structure and functions of matrix Gla protein, which is a vitamin K-dependent protein initially isolated from bovine bones and associated with organic matrixes. The protein is shown to be expressed in many tissues, including cartilage and most visceral organs. It is the only known vitamin K-dependent protein that lacks a propeptide. Studies indicate that matrix Gla protein may act as an inhibitor of calcification in arteries. It is a single-chain polypeptide, which contains five Gla residues and is stabilized by one intrachain disulfide bond. Despite its high content of hydrophilic amino acids, it is exceptionally water-insoluble and requires 4 M guanidine-HC1 for extraction. The two forms of matrix Gla protein isolated so far contain 79 and 83 residues and lack 5 and 1 amino acids from the predicted carboxylic acid terminus. Residues 2–12 encode one α-helical portion, which shares no homology with other proteins. The chapter presents information on the gene structure, isolation, and the structural and functional sites of matrix Gla protein.

Collagen type X

This chapter describes the molecular structure, functions, and primary structure of collagen type X. This collagen is synthesized predominantly by hypertrophic chondrocytes during endochondral bone formation and has a restricted tissue distribution in the calcifying cartilage that is eventually replaced by a bone. It is a short-chain collagen that is both temporally and spatially regulated during fetal development. This molecule is a homotrimer comprising three identical α1(X) chains. It has a short triple helix approximately 132 nm in length, a small NH-terminal domain, and a large carboxylic acid-terminal globular domain. It is deposited in the cartilage matrix without apparent processing, and although its macromolecular organization is not determined in vivo, it may form a hexagonal-type lattice as in the case of type-VIII collagen. The chapter presents information on the gene structure and the structural and functional sites of type X collagen.

10 – Cartilage matrix protein

This chapter focuses on the cartilage matrix protein, which is a major component of the extracellular matrix of nonarticular cartilage. It is found in tracheal, nasal septal, xiphisternal, auricular, and epiphyseal cartilage but not in articular cartilage or extracts of intervertebral discs. Recent research suggests that the cartilage matrix protein may act as a marker for postmitotic chondrocytes. The cartilage matrix protein is a 54 kDa protein that occurs in cartilage as a disulfide-bonded multimer. The protein is normally isolated as a homotrimer. The cartilage matrix protein contains two repeating sequences of 190 amino acids that are homologous to the A-type repeats of von Willebrand factor, separated by a six-cysteine epidermal growth factor repeat. The primary gene structure and isolation methods of the cartilage matrix protein are presented in the chapter.

Collagen type VI

This chapter focuses on collagen type VI, which is essentially a glycoprotein with a short collagenous central domain and is present in most, possibly all, connective tissues. It assembles into a unique supramolecular structure of 5-nm-diameter microfibrils with a periodicity of approximately 100 nm. Mutations in the genes COL6A1, COL6A2, and COL6A3, which encode three distinct α chains, are linked to the autosomal dominant disorder of Bethlem myopathy and are characterized by contractures of multiple joints and generalized muscular weakness and wasting. Type VI collagen is synthesized largely as a heterotrimer comprising three genetically distinct α1(VI), α2(VI), and α3(VI) chains—although there are alternative, less stable assemblies comprising either α3(VI) or α1(VI)/α2(VI) chains. The chapter describes the structural and functional sites of type VI collagen.