Rigidity of Collagen Fibrils Controls Collagen Gel-induced Down-regulation of Focal Adhesion Complex Proteins Mediated by α2β1 Integrin

Abstract

Previous studies have shown that collagen gel overlay induced selective proteolysis of focal adhesion complex proteins in Madin-Darby canine kidney (MDCK) cells. In this study, we examined whether morphological and biochemical changes were present in cells cultured on collagen gel. We found that focal adhesion complex proteins, including focal adhesion kinase (FAK), talin, paxillin, and p130cas , but not vinculin, were decreased within 1 h when MDCK cells were cultured on collagen gel. Collagen gel-induced selective decrease of focal adhesion proteins was observed in all lines of cells examined, including epithelial, fibroblastic, and cancer cells. Matrigel also induced selective down-regulation of focal adhesion proteins. However, cells cultured on collagen gel-or matrigel-coated dishes did not show any changes of focal adhesion proteins. These data suggest that the physical nature of the gel, i.e. the rigidity, is involved in the expression of focal adhesion proteins. The collagen gel-induced down-regulation of focal adhesion complex proteins was caused by reduction of protein synthesis and activation of proteases such as calpain. Overexpression of a dominant negative mutant of discoidin domain receptor 1 (DDR1) or FAK-related non-kinase (FRNK) did not prevent collagen gel-induced down-regulation of the focal adhesion complex protein, whereas an anti-␣2␤1 integrin-neutralizing antibody completely blocked it. Taken together, our results indicate that the rigidity of collagen gel controls the expression of focal adhesion complex proteins, which is mediated by ␣ 2 ␤ 1 integrin but not DDR1. Adhesion of cells to the extracellular matrix (ECM)1 is a crucial event in multi-cellular organisms for the modulation of cellular processes such as cell growth, differentiation, and apoptosis (1-4). The integration of intracellular signaling and the structure of ECM elicited by ECM-integrin engagement may be mediated by focal adhesion complex proteins (5). Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase located close to focal adhesions and may play a central role in integrating signals from ECM-integrin and growth factors (6 -8). FAK also plays important roles in the assembly of several signaling proteins to focal adhesions via interactions with a number of cellular proteins, including Src, Grb2, phosphatidylinostiol-3 kinase, paxillin, Crk, talin, and p130 cas (6, 9 -13). Recent data have shown that FAK plays important roles in cell cycle progression (14), migration (8, 9, 15), adhesion (12), and the prevention of apoptosis (16, 17).Three-dimensional collagen gel has been used as a cell culture vehicle for the study of morphogenesis by us and many laboratories (18 -20). Collagen fibrils can transduce signals through integrins and the receptor tyrosine kinase discoidin domain receptor 1 (DDR1) (21). Interaction of ECM with its transmembrane receptor integrins causes subsequent cascades of protein-protein interaction and modification at the focal adhesion complex site and the recruitment of several cy...

MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

“…The present study confirms and extends our previous studies to demonstrate that MT1-MMP stimulates ERK activation in 3-D collagen, which is indispensable for proliferation of cancer cells. It is well documented that the interaction between collagen fibrils and integrin α 2 β 1 generates the growth-suppressive signals in normal epithelial or cancer cells cultured in 3-D collagen [7,[20][21][22], which can be abrogated by pericellular collagenolysis [14,15]. We represent that MT1-MMP induces c-Src activation in 3-D collagen by modulating extracellular microenvironment, which subsequently activates paxillin to scaffold signaling molecules for ERK pathway and thus leads to escape cells from growth-suppressive signals.…”

“…In contrast, cultivation in three-dimensional (3-D) collagen suppresses the proliferation of numerous cell types. This growth-suppressive signal may be mediated by integrins, as integrin α 2 β 1 mediates fibril collagen-induced inhibition of cell growth [6] and 3-D collagen induces down-regulation of cell-matrix adhesion proteins such as FAK, talin, and paxillin, which is blocked by integrin α 2 β 1 -neutralizing antibody [7]. Recent study has demonstrated that reduction of cell-matrix adhesion in 3-D ECM causes growth inhibition [8].…”

“…Recent study has demonstrated that reduction of cell-matrix adhesion in 3-D ECM causes growth inhibition [8]. A normal cell culture dish is non-flexible with a rigidity of more than 1 gigapascals, whereas 3-D collagen is a flexible substrate with the rigidity of 30-100 pascals [7]. Therefore, when cells are plated on culture dish or 2-D collagen in the presence of serum, formation of robust cell-matrix adhesions is induced, which may function as a scaffold for signaling to promote cell growth, which masks the effects of growth factors.…”

An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions

“…Thus, by means of the collagenase treatment, we were able to dissolve the collagen gel and remove it from cell lysates. In previous studies, researchers often lysed the cells without TCA and collagenase treatment for WB (Wang et al 2003;Engler et al 2006;Tsutsumi et al 2009;Wu et al 2009). We refer to this approach as ''the conventional method'' in this study.…”

“…A recent study reported that substrate stiffness affects cell behavior. For example, the rigidity of a collagen matrix regulates collective motility and the expression of adhesion proteins (Wang et al 2003;Haga et al 2005). Furthermore, stiffer substrates may enhance cancer progression (Paszek et al 2005;Levental et al 2009).…”