Integrin activation and internalization on soft ECM as a mechanism of induction of stem cell differentiation by ECM elasticity

Abstract

The mechanism by which ECM elasticity induces lineage specification of stem cells has not been clearly understood. Integrins are well-documented mechanosensors that are positioned at the beginning of the sensing pathway. By using an antibody specifically recognizing the active conformation of β1 integrin, we observed that β1 integrin activation in bone marrow mesenchymal stem cells (BMMSCs) was induced by soft substrate to a significantly greater degree than by stiff substrate. In contrast, however, the level of cell surface integrin on soft substrate was significantly lower than that on stiff substrate. Soft substrate markedly enhanced the internalization of integrin, and this internalization was mediated mainly through caveolae/raft-dependent endocytosis. The inhibition of integrin internalization blocked the neural lineage specification of BMMSCs on soft substrate. Furthermore, soft substrate also repressed the bone morphogenetic protein (BMP)/Smad pathway at least partially through integrin-regulated BMP receptor endocytosis. A theoretical analysis based on atomic force microscopy (AFM) data indicated that integrin-ligand complexes are more easily ruptured on soft substrate; this outcome may contribute to the enhancement of integrin internalization on soft substrate. Taken together, our results suggest that ECM elasticity affects integrin activity and trafficking to modulate integrin BMP receptor internalization, thus contributing to stem cell lineage specification.integrin trafficking | mesencymal stem cells | neurogenic lineage | traction force M echanical environment plays an important role in regulating cellular function and behavior, including proliferation, migration, apoptosis, and differentiation (1-3). It has been shown recently that the mechanical properties (e.g., elasticity) of adhesion substrates modulate stem cell fate in both 2D (4, 5) and 3D (6) cultures. However, the mechanism by which mechanical properties of ECM affect the chemical signaling processes has not been clearly understood.Mechanical stimuli induce changes in focal adhesion (FA) protein activities and FA remodeling (7,8). The growth and elongation of FAs vary with changes in substrate stiffness, indicating that ECM elasticity regulates FA assembly (4). FA complexes consist of many signaling molecules (including Src, Cas, vinculin, and integrins), which can undergo tension-dependent conformational changes to affect kinase activity, phosphorylation site availability, intracellular localization, and/or ligand affinity (9-12). Among these molecules, integrins are necessary for most mechanosensing processes and are positioned at the beginning of the sensing pathway (13).We aimed to explore the mechanism by which stem cells sense ECM elasticity, especially the role of β1 integrin in bone marrow mesenchymal stem cells (BMMSCs) (14). Activation of β1 integrin in BMMSCs was significantly greater on softer than on stiffer substrate. Most importantly, the intracellular localization of β1 integrin varied with substrate elasticity, being pres...

Discoidin Domain Receptor 1 Protein Is a Novel Modulator of Megakaryocyte-Collagen Interactions

“…2D, panel i). The levels of activated integrin were determined using a ␤1 integrin antibody (clone HUTS-4) recognizing epitopes in the 355-425 region (hybrid domain), whose expression parallels the activity of ␤1 integrin (36). It is known that GPVI may form a dimer that has functional significance.…”

β1 integrin

“…A similar phenomenon was also observed by Wei and colleagues, who saw an inhibition of β1-integrin activation and decreased phosphorylation of FAK on soft substrate. 45 In contrast, the study by Du et al 44 indicated that the reliance on specific ECM elasticity for cell lineage specificity was due to increased activation and internalization of β1 integrin on soft ECM substrates. The differences seen in integrin activation in these studies suggests that integrin regulation of the observed phenotypes may be cell type-dependent in addition to being influenced by elasticity and hence these factors should also be considered when examining the role of integrin signaling in vitro.…”

“…Thus the elasticity of the growth environment may also be considered as a contributing factor to phenotypic differences observed in 2D vs. 3D assay conditions. Indeed, the importance of ECM elasticity has recently been noted for a variety of processes including transcription and replication, 41 and for the self-renewal and differentiation of stem cell populations in culture, [42][43][44] among others. Interestingly, the study by Kocgozlu et al 41 indicated that substrate rigidity affected both integrin and FAK activation, with a small window of optimal substrate elasticity for the activation of FAK to occur.…”

Schwann Cell Myelination Requires Integration of Laminin Activities

“…The laminin-induced increase in proliferation was found to partially depend upon a contribution for the b1-integrin-binding site (especially a7b1 and a6b1) in the LG domains and substantially depend upon the polymerization activity of laminins. The latter effect may be an indirect one due in part to reduced laminins and other BM component receptor-ligands and possibly to an alteration of BM rigidity, a property recently found to be involved in integrin activity and turnover (Du et al, 2011).…”