Abstract
Mechanical interactions of mesenchymal stem cells (MSC) with the environment play a significant role in controlling the diverse biological functions of these cells. Mechanical forces are transduced by integrins to the actin cytoskeleton that functions as a scaffold to switch mechanical signals into biochemical pathways. To explore the significance of cytoskeletal mechanisms in human MSC we modulated the actin cytoskeleton using the depolymerising drugs cytochalasin D (CytD) and latrunculin A (LatA), as well as the stabilizing drug jasplakinolide (Jasp) and examined the activation of the signalling molecules ERK and AKT during mechanical loading. All three drugs provoked significant changes in cell morphology and organisation of the cytoskeleton. Application of mechanical forces to β1-integrin receptors using magnetic beads without deformation of the cell shape induced a phosphorylation of ERK and AKT. Of the two drugs that inhibited the cytoskeletal polymerization, LatA completely blocked the activation of ERK and AKT due to mechanical forces, whereas CytD inhibited the activation of AKT but not of ERK. Activation of both signalling molecules by integrin loading was not affected due to cell treatment with the cytoskeleton stabilizing drug Jasp. To correlate the effects of the drugs on mechanically induced activation of AKT and ERK with parameters of MSC differentiation, we studied ALP activity as a marker for osteogenic differentiation and examined the uptake of fat droplets as marker for adipogenic differentiation in the presence of the drugs. All three drugs inhibited ALP activity of MSC in osteogenic differentiation medium. Adipogenic differentiation was enhanced by CytD and Jasp, but not by LatA. The results indicate that modulation of the cytoskeleton using perturbing drugs can differentially modify both mechanically induced signal transduction and MSC differentiation. In addition to activation of the signalling molecules ERK and AKT, other cytoskeletal mechanisms are involved in MSC differentiation.
Highlights
Mechanical forces in the microenvironment of adult stem cells play a decisive role in controlling the fate of these cells [1,2,3,4]
The forces that are required to regulate the differentiation of mesenchymal stem cells (MSC) to multiple lineages correlate with the mechanical properties of the specific tissue [5]
We demonstrate that application of three agents which modulate the actin cytoskeleton affects differentially the activation of ERK and AKT induced by a physical stress to integrins
Summary
Mechanical forces in the microenvironment of adult stem cells play a decisive role in controlling the fate of these cells [1,2,3,4]. The forces that are required to regulate the differentiation of mesenchymal stem cells (MSC) to multiple lineages correlate with the mechanical properties of the specific tissue [5]. Both 2D in vitro systems as well as 3D experiments demonstrated that soft matrix promoted fat cell differentiation whereas a rigid substrate facilitates osteogenic differentiation [5,6]. Due to the contractibility of the actin filaments, proteins associated with the cytoskeleton may be stretched which results in an unfolding and presenting of new binding sites [19] Such mechanisms can lead to an activation of signalling proteins by phosphorylation. We demonstrate how cytoskeleton perturbing drugs affect the activation of signalling molecules in combination with defined applications of physical loads to b1-integrins on the surface of MSC
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