Substrate adhesion of rat hepatocytes: mechanism of attachment to collagen substrates

Abstract

Attachment of rat hepatocytes to collagen, which occurs without the aid of fibronectin, was found to be a time-dependent reaction characterized by an initial lag phase of 10-20 min before stable attachment bonds began to form. Increasing the density of molecules in the collagen substrates enhanced the rate of cell attachment. The hepatocytes attached essentially equally well to all the collagen types tested (types I, II, III, IV and V). The initial rate of cell attachment was more rapid to native collagen than to denatured collagen or alpha 1(I) chains, apparently indicating different affinities of the cells for these substrates. However, if cells were incubated for 60 min or more, efficient attachment occurred to the alpha 1(I) chain and to all cyanogen-bromide-treated peptides tested (alpha 1-CB2, alpha 1-CB3, alpha 1-CB4, alpha 1-CB5, alpha 1-CB6A, alpha 1-CB7, alpha 1-CB8, alpha 2-CB2, alpha 2-CB3 and alpha 2-CB4) but not to the aminopropeptide of type I procollagen. A low but significant degree of attachment also took place to substrates made of synthetic peptides with the collagen-like structures (Gly-Ala-Pro)n, (Gly-Pro-Pro)n and (Gly-Pro-Hyp)n, whereas no attachment was observed to polyproline. We suggest that the cell-binding sites in collagen have a simple structure and occur in multiple copies along the collagen molecule. Addition of collagen in solution inhibited initial cell attachment, an effect that persisted longer on substrates made of alpha 1(I) chain than on denatured collagen. The collected data are interpreted in terms of a model for cell-to-collagen adhesion where the formation of stable attachment bonds requires the binding of several low-affinity receptors, clustered at the site of adhesion, to collagen molecules in the substrate.