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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 ?1 integrin, we observed that ?1 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.

SUBMITTER: Du J

PROVIDER: S-EPMC3111285 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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

Du Jing J Chen Xiaofei X Liang Xudong X Zhang Guangyao G Xu Jia J He Linrong L Zhan Qingyuan Q Feng Xi-Qiao XQ Chien Shu S Yang Chun C

Proceedings of the National Academy of Sciences of the United States of America 20110518 23

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, howe ...[more]

PMID: 21593411

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Project description:The extracellular matrix plays a crucial role in controlling human mesenchymal stem cell (hMSC) biology including differentiation, and α5β1 integrin signaling plays an important role during osteogenic differentiation of hMSCs. Here, peptide-functionalized hydrogels were used to examine the role of α5β1 integrin signaling in inducing osteogenic differentiation in hMSCs. Further, the role of substrate elasticity was also studied. A thiolene chemistry was used to functionalize poly(ethylene glycol) hydrogels with a pendant peptide moieity, c(RRETAWA), as previous studies have shown that RRETAWA containing peptides bind to the α5β1 integrin with very high specificity. Notably, hMSC attachment to c(RRETAWA)-functionalized hydrogels was found to occur primarily through α5 integrins, as the number of attached cells was significantly reduced to ~20% upon blocking the α5 integrin during culture. To investigate the interplay between stiffness and c(RRETAWA) concentration, hydrogels were formulated with Young's moduli of ~2 kPa (soft) and ~25 kPa (stiff) and c(RRETAWA) concentrations of 0.1 mM and 1 mM. Stiff substrates led to ~3.5 fold higher hMSC attachment and ~3 fold higher cell area in comparison to soft substrates. hMSCs formed robust and larger focal adhesions on stiff substrates at 1 mM c(RRETAWA) compared to soft substrates. Alkaline phosphatase (ALP) activity in hMSCs cultured on stiff gels at 0.1 mM and 1 mM c(RRETAWA) was increased 2.5 and 3.5 fold, respectively after 14 days in growth media. hMSCs did not show an increase in ALP activity when cultured on soft gels. Further, gene expression of osteogenic related genes, core binding factor-1, osteopontin and Collagen-1a at day 14 in hMSCs cultured on stiff gels at 1 mM c(RRETAWA) were increased 10, 7 and 4 fold, respectively, while on soft gels, gene expression was at basal levels. Collectively, these results demonstrate that the combination of high substrate stiffness and α5β1 integrin signaling stimulated by c(RRETAWA) is sufficient to induce osteogenic differentiation of hMSCs without requiring the addition of soluble factors.

Project description:Biological cells embedded in fibrous matrices have been observed to form intercellular bands of dense and aligned fibers through which they mechanically interact over long distances. Such matrix-mediated cellular interactions have been shown to regulate various biological processes. This study aimed to explore the effects of elastic nonlinearity of the fibers contained in the extracellular matrix (ECM) on the transmission of mechanical loads between contracting cells. Based on our biological experiments, we developed a finite-element model of two contracting cells embedded within a fibrous network. The individual fibers were modeled as showing linear elasticity, compression microbuckling, tension stiffening, or both of the latter two. Fiber compression buckling resulted in smaller loads in the ECM, which were primarily directed toward the neighboring cell. These loads decreased with increasing cell-to-cell distance; when cells were >9 cell diameters apart, no such intercellular interaction was observed. Tension stiffening further contributed to directing the loads toward the neighboring cell, though to a smaller extent. The contraction of two neighboring cells resulted in mutual attraction forces, which were considerably increased by tension stiffening and decayed with increasing cell-to-cell distances. Nonlinear elasticity contributed also to the onset of force polarity on the cell boundaries, manifested by larger contractile forces pointing toward the neighboring cell. The density and alignment of the fibers within the intercellular band were greater when fibers buckled under compression, with tension stiffening further contributing to this structural remodeling. Although previous studies have established the role of the ECM nonlinear mechanical behavior in increasing the range of force transmission, our model demonstrates the contribution of nonlinear elasticity of biological gels to directional and efficient mechanical signal transfer between distant cells, and rehighlights the importance of using fibrous gels in experimental settings for facilitating intercellular communication. VIDEO ABSTRACT.

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

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

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