Collagen in normal lung

1.4.1.1 Collagen types and distribution

Extracellular matrix protein accumulation in alveolar walls is a key feature of the pathogenesis of pulmonary fibrosis as well as a potential therapeutic target. In order to appreciate the ways in which altered collagen metabolism may lead to the

development of lung fibrosis, our existing knowledge of the role played by collagen in normal lung and of the mechanisms underlying collagen biosynthesis and degradation is summarised below.

Collagens comprise the largest group of proteins in the lung and account for approximately 20% of its dry weight in the adult human lung (Bradley et al 1975; Hurst et al 1977). Together with other extracellular matrix proteins such as elastin, proteoglycans and fibronectin, collagens provide a three-dimensional structural framework which also plays roles in regulating cellular function including adhesion, migration and cell-matrix interactions (for a review see Schnaper and Kleinman 1993 (Schnaper, Kleinman, 1993).

The collagens consist of a family of at least 19 closely related proteins, denoted by Roman numerals, which share common structural properties. All are composed of three polypeptide «-chains, which may be identical or different, and which are linked by hydrogen bonding to form a right handed triple helix. At least 30 «-chains are now recognised, denoted by Arabic numerals and encoded by separate genes (for recent reviews see Kielty et al 1993; Mays, Laurent, 1994; Chambers, Laurent, 1996). The «-chains have a high glycine content and contain repeated triplets of the structure Gly- X-Y, where approximately 30% of the X residues are proline and 30% of the Y residues are hydroxyproline (Miller, 1985).

Collagen types I, II, III, V and XI are the fibril-forming interstitial collagens. Types I and III constitute approximately 90% of collagen in adult human lung, in a ratio of 2:1 (Rennard, Crystal, 1982; Kirk et al 1984). Type II collagen is found only in cartilage and is therefore restricted to the trachea and larger airways. Type V collagen is found in small amounts in basement membranes and in association with type I in the lung interstitium (Madri, Furthmayr, 1979) and type XI collagen is thought to be associated with type II collagen in the airways (Laurent, 1986).

1,4.1,2 Collagen turnover

al 1974), and continues to increase during growth to adulthood, with a five to ten fold increase in lung collagen concentration (Mays et al 1988). Moreover, collagens are not inert proteins, but are continuously synthesised and degraded throughout life. Synthesis and degradation rates are quite rapid, with a daily turnover rate of the order of 1 0 % in

adult rats and rabbits (Laurent, 1986). In vitro studies have shown that a proportion of collagen produced by lung fibroblasts is degraded intracellularly within minutes of synthesis (Bienkowski et al 1978b; Bienkowski et al 1978a). In vivo, the process occurs within 15 mins (McAnulty, Laurent, 1987). Since collagen deposition in the lung is determined by the balance between synthesis and degradation, changes in either or both of these processes may lead to net collagen deposition and fibrosis.

1,4,1,3 Collagen synthesis

The fibroblast is an important source of pulmonary collagen (Hance et al 1976) but other cell types including endothelial, alveolar epithelial and mésothélial cells can also produce collagen (see review Bienkowski 1991 (Bienkowski, 1996). The pathways involved in collagen biosynthesis are now well-described (see reviews by Nimni, Harkness, 1988 and Kielty et al 1993) and the principal steps, illustrated in figure 1.3, are outlined below.

Collagen production begins in the nucleus with transcription of collagen genes to type specific mRNA transcripts. Translation of mRNA produces pre-procollagen «-chains containing large extension peptides at both ends and an N-terminal hydrophobic signal sequence designating the molecules for secretion. The signal peptide is cleaved in the rough endoplasmic reticulum. There is growing evidence that several classes of molecular chaperones, including heat-shock protein (Hsp) 47 and glucose-regulated protein (Grp) 78 and 94, regulate procollagen processing in the endoplasmic reticulum (Nakai et al 1992; Ferreira et al 1994; Freyria et al 1995; Lamande et al 1995). These proteins play important roles in binding malfolded procollagen molecules, preventing their secretion and promoting correct folding.

Further post-translational modification occurs in the Golgi apparatus. This includes hydroxylation of proline and lysine residues in the X position by prolyl-4-hydroxylase

and lysyl hydroxylase. Some proline residues in the X position are hydroxylated in the 3-position of the pyrrole ring by prolyl-3-hydroyxlase. The process of proline hydroxylation, for which ascorbic acid is an essential cofactor, is almost specific to collagen. Elastin, lung surfactant apolipoprotein A and D, mannose-binding protein, C lq component of complement and acetylcholinesterase also contain hydroxyproline. However, their relative scarcity and the small amounts of hydroxyproline they contain has led to the measurement of hydroxyproline being used as an index of collagen content. Hydroxylysine residues are glycosylated by galactosyl transferase and glucosyl transferase.

Interchain and intrachain disulphide bond formation then initiates the formation of the triple helix. In the Golgi apparatus procollagen molecules are packed into secretory vesicles. As the molecules are secreted the C- and N- terminal peptides are cleaved by specific proteases to yield the triple helical collagen molecule. The triple helices spontaneously assemble to form fibrils, four molecules are required to initiate fibril formation. Electron microscopy studies have shown that collagen fibrils have highly tapered and symmetrical pointed tips, and grow by addition of further molecules in the C- to N-terminal direction (Kadler et al 1990). Electrostatic interactions initially hold the molecules together, but as they mature, lysyl oxidase catalyses the formation of aldehyde derivatives from some of the lysine and hydroxylysine residues, generating covalent bonds between the chains of neighbouring molecules and stabilising the fibrils (Ricard-Blum, Ville, 1988).

NUCLEUS