An abundant ECM surrounds bone cells. Osteoblasts, chondrocytes and perhaps osteocytes are responsible for the synthesis of this matrix. Mature bone is known to contain abundant type I and type III collagen, osteopontin, bone sialoprotein and osteocalcin. Components of the ECM tend to have the ability to bind several integrins, for example fibronectin binds «5(31 and seven other integrins. There is little data available on the amount of fibronectin present in mature human bone.
1.4.1 Collagen
To date there are nineteen proteins classified as collagens. Each is a triple helical protein trimer (Prockop and Kivirikko, 1995). Type I collagen is the most abundant collagen sub-type in human bone. Along with types II, III, V and XI this sub-type forms continuous triple helices that can form large fibrils (Ayad et al, 1994). The a l p l , a 2 p l, aS pi, alO pi and a l i p i integrins all bind to collagen in the ECM. The a i p i and a2pl integrins are the predominant collagen binding in osteoblasts (Bennett et al, 2001b) and are also important in platelets and epithelial cells integrins (Santoro and Zutter, 1995). The alO pi and a l l p l integrins have been recently identified and are present on chondrocytes and in foetal muscle respectively (Camper et al, 2001; Veiling et al, 1999). There have been several integrin binding
sites identified in type I collagen including several sites in the a 1 (1) chain such as the putative Asp-Glu-Gly-Ala (DGEA) motif. This sequence has been implicated in a2 p l binding to type I collagen (Staatz et al, 1990; 1991). The involvement of this sequence in a2 p l binding is controversial. Studies by Knight et al (1998) reported that peptide fragments containing the DGEA motif did not bind to the «2pi integrin. This study found that the Gly-Glu-Arg (GER) triplicate, present in type I collagen, was required for the binding of the a2 p l integrin. The GER sequence is similar to the binding motif for a i p i in collagen type IV (Eble et al, 1993). The crystal structure of a complex between the I domain of integrin a2 p i and a triple helical collagen peptide containing a critical GFOGER motif has been shown (Emsley et al, 2000).
The collagen binding integrins and the LFA-1 leukocyte integrin all contain a common feature, an A domain at the N terminus of the a sub-unit (Michishita et al,
1993; Tuckwell et al, 1995; Nolte et al, 1999). Binding at this site is cation dependent (Mn^^ or Mg^^ not Ca^^) (Tuckwell et al, 1995). Although both a i p i and a2 p i bind to collagen types I and IV, their relative affinities for the two types of collagen differ. The a l pi integrin binds to collagen type IV with higher affinity than to type I, whereas a2 p l binds to type I collagen with higher affinity (Kern et al,
1993). It has been suggested that the a i p i and a2pl collagen integrin receptors could have distinct signalling pathways. In a review by Heino, (2000) it was proposed that a i p i integrin-mediated signalling could lead to cell proliferation and a reduction in collagen synthesis, whereas a2pl signalling could lead to collagen production and also collagenase gene expression. It is also suggested that in 3D collagen gel cultures interaction of a2pl with collagen leads to activation of the p38/MAPK signalling pathway. It is likely that the cell signalling pathways linked to collagen binding are a great deal more complicated but the observations made to date in other systems may be applicable to osteoblasts and bone.
repeating amino acids termed type I, type II and type III repeats (Peterson et al, 1983; Ruoslahti, 1988). Fibronectin has a number of receptor binding sites, including several for integrins and also binding sites for other extracellular proteins. Knockout mice for fibronectin or the a5 integrin receptor resulted in the death of embryos early in development (Hynes et al, 1992) and studies have reported the importance of fibronectin and integrin interaction in this process (reviewed in Miyamoto et al,
1998).
Integrins are thought to bind to specific sequences in fibronectin. The most widely researched of these sequences has been the Arg-Gly-Asp (RGD) motif, located in a type III repeat (Ruoslahti, 1988; 1996). Many other components of the ECM contain this motif, for example fibronectin, vitronectin, osteopontin, collagens, thrombospondin, fibrinogen, and von Willebrand factor. Some integrins bind to the RGD sequence of a single adhesion protein only, whereas others recognise groups of them (Ruoslahti and Pierschbacher, 1987). In addition, fibronectin contains the ‘synergy’ site 1 that is also required for aSpi binding (Obara et al, 1988) (shown in figure 1(f)). Studies suggest that the p sub-unit binds the RGD sequence and the a sub-unit binds the synergy sequence (Obara et al, 1988; Kimizuka et al, 1991; Aota
et al, 1994). Several integrins bind to plasma fibronectin, including aSpi, a4 p i, aS pi, avp3 and avps. The experiments in this thesis used three commercially available fibronectin fragments produced by proteolysis of plasma fibronectin and these are also indicated in figure 1(f).
Figure 1(f) - Fibronectin and proteolytic fragments