Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

Abstract

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound ␣ 5 and ␤ 1 integrin subunits but not ␣ v or ␤ 3 . C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B Ͼ T Ͼ C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of ␣ 5 ␤ 1 integrin bound to Fn, and differentiation was inhibited by anti-␣ 5 , but not anti-␣ v , antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications. INTRODUCTIONThe adhesion of cells to their substrate through an extracellular matrix provides signals that influence their ability to survive, proliferate, and express specific developmental phenotypes (Menko and Boettiger, 1987;Werb et al., 1989;Adams and Watt, 1990;Streuli et al., 1991;Zhu et al., 1996;Chen et al., 1997). One of the early examples was the development of in vitro culture conditions that permitted the differentiation of avian myogenic cells into contracting myotubes (Hauschka and Konigsberg, 1966;Bischoff and Holtzer, 1968). The critical element for this system was the precoating of tissue culture surfaces with rat tail collagen. This general principle of providing an appropriate substrate to permit the expression of developmental phenotypes has been applied to a wide variety of cells. These include systems that allow the maintenance of neurons and outgrowth of growth cones (Westerfield, 1987) and the recapitulation of the stages of mammary gland development and involution (Li et al., 1987;Barcellos-Hoff et al., 1989). These findings indicate that critical elements of the message directing the expression of a differentiated phenotype are encoded in the extracellular matrix.Cells interact with extracellular matrices primarily through integrins, a widely expressed family of cell surface receptors (Hynes, 1987), and integrin binding to its extracellular ligand is responsible for the downstream effects of the matrix on cell function. For example, in the muscle differentiation syste...

Growth substrate induced functional changes elucidated by FTIR and Raman spectroscopy in in–vitro cultured human keratinocytes

“…The as shown previously [19]. This also indicates that the Ag/SnO 2 coating on the reflective surface of MirrIR substrates plays some role in promotion of cellular attachment, which may be as a result of surface roughness on the nanometer scale [46][47][48].…”

“…This ultimately results in increasing proliferation and motility, which is also dependent on the charge distribution presented by the ECM coating molecule to the adhering cell [18][19][20][21][22][23]25]. Ultimately the reaction of the cell to such influences are complex, being the cumulative effect of the increased or decreased regulation of a number of stimulatory pathways in the cell, with the end physiological change in any given cell being dependent on the cell and substrate ECM [50,51].…”

“…Noninvasive spectroscopic measurements of cellular function in in-vitro cultured cell lines require substrates such as quartz, ZnSe and MirrIR (Ag/SnO 2 coated glass for FTIRM from Kevley Technologies) etc. It has been demonstrated that surfaces and scaffolds for cell culture can induce changes in cellular adhesion and motility [17,18], in their proliferation and differentiation [16,19], and in gene expression [20], ultimately influencing the fate of the cell [21]. Much of the interaction of adherent cells with their culture substrates is dependent on the surface chemistry [18,22] and surface energy [23].…”

“…Much of the interaction of adherent cells with their culture substrates is dependent on the surface chemistry [18,22] and surface energy [23]. When substrates are coated with biocompatible molecules to effect or enhance cellular proliferation, the conformation of the molecule has also been shown to produce changes in the level of response [19].…”

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

“…First indications also exist that downstream cell signaling is impacted by the conformation of Fn Lan et al, 2005). When adsorbed from solution to biomaterial surfaces, its conformation is for example impacted by the underlying surface chemistry (Altroff et al, 2003;Antia et al, 2006;Baugh and Vogel, 2004;Garcia et al, 1999;Halter et al, 2005) which can then alter integrin binding . The conformations Fn assumes on synthetic surfaces, however, are rather distinct from those of native Fn in soluble and fibrillar forms (reviewed in Antia et al, 2006;Baugh and Vogel, 2004;Halter et al, 2005).…”