Date Published: July 29, 2013
Publisher: Public Library of Science
Author(s): Petra Müller, Anne Langenbach, Alexander Kaminski, Joachim Rychly, Laurent Kreplak.
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.
Mechanical forces in the microenvironment of adult stem cells play a decisive role in controlling the fate of these cells –. Within the tissues stem cells are constantly subjected to external forces and are able to adjust to their changes. 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 . 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 , . Similarly, to maintain stem cells in the state of pluripotency and self-renewal a defined mechanical environment is required . The main cellular components that mediate mechanical forces from the extracellular matrix outside the cells into the cell interior are integrin receptors that bind to proteins of the extracellular matrix and are able to transmit forces by physical interacting with the actin cytoskeleton –. The backbone of the cytoskeleton is F-actin, which clusters to form filaments. The filaments can be bundled and cross-linked by actin-binding proteins to form a network . This actin filamentous network is highly dynamic. Cells are able to sense the mechanical properties of the adhesive substrate through a balance between the cytoskeletal contractibility facilitated by actomyosin and the resistant forces of the extracellular matrix , . The dynamic behaviour of the actin cytoskeleton forms the basis for a number of cellular functions including migration or division . With the progress in stem cell research it became obvious that the actin cytoskeleton is a central modulator that controls function and modulates differentiation . The structural organization of the cytoskeletal network determines the cell shape which was found to regulate the fate of stem cells. Evidence exists that differentiation to chondrocytes requires a more rounded phenotype which can be facilitated by a pellet culture or encapsulation of the cells , . When used the technique of micropatterning, round MSC differentiated to adipocytes, whereas spread cells developed to osteoblasts . In addition to sensing mechanical forces, the cytoskeleton forms a structure to transform mechanical forces into biochemical signals. 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 . Such mechanisms can lead to an activation of signalling proteins by phosphorylation. In addition, forces can be transduced from the cell surface to the nucleus via the actin cytoskeleton by a direct mechanocoupling . This process propagates the mechanical signal much faster through the cytoplasm and induces biochemical events in the nucleus. Despite the central role of the actin cytoskeleton in mechanically induced signalling and biological responses in mesenchymal stem cells, little is known about the effects of modulation of the actin cytoskeleton in these cells by known drugs that impair or stabilize actin polymerization. We demonstrate how cytoskeleton perturbing drugs affect the activation of signalling molecules in combination with defined applications of physical loads to β1-integrins on the surface of MSC.
The study was aimed to modify the actin cytoskeleton of MSC without impairing the survival of the cells. Therefore, loss of cell adhesion and reduced metabolic activity tested in the MTT test were used to reveal the critical concentration for the application of the drugs to manipulate the actin network but maintain cell survival (data not shown). First we were interested in the effects of the three pharmacological agents on the cell shape (Fig. 1). DMSO in the culture medium, which was required to dissolve the drugs, did not affect cell morphology. CytD induced a marked change in cell shape. Cells converted from spindle shaped to more round cells with similar length and width. LatA induced a broader cell shape, obviously visible at the higher concentration of 0.1 µM. Jasp did not provoke an obvious change in the cell morphology at a concentration of 0.01 µM, but some cells became retracted. Higher concentrations impaired the cell survival.
As a control result we demonstrated that a short time mechanical load to β1-integrin induced an activation of ERK and AKT in MSC. In addition, in earlier studies we have shown that this integrin load provokes signalling events like an intracellular calcium signal and a physical anchorage of integrins to the cytoskeleton , . Recently, the actin-binding protein filamin A was identified as molecular link between integrin and the cytoskeleton . Strain increases β-integrin binding to filamin A and facilitates mechanotransduction. Thus, these results together support the mechanical coupling between integrins and the cytoskeleton as well as reveal further signalling events when mechanically loading of integrins on the surface of cells without obvious changes in the cell shape. We now 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. While the actin inhibiting drugs CytD and LatA reduced the phosphorylation of the signalling proteins to different extents and in dependence on the type of the protein, the actin stabilizing agent Jasp did not affect the mechanically induced activation of both ERK and AKT. CytD induced dramatic changes in cell morphology which was accompanied by a partly fragmentation of the cytoskeletal filaments and loss of a colocalization between vinculin and actin. This correlates with an impaired activation of AKT due to CytD treatment, but despite the distinct loss of structural organization of the cytoskeleton, CytD had surprisingly no obvious effect on ERK activation. Inhibition of activation of AKT by CytD was also shown during cyclically stretching of mouse embryonic stem cells on a flexible culture plate . With our approach to apply a mechanical load and to explore the effect of drugs on the cytoskeleton we could now establish the direct link to the integrins as transducers of the mechanical load. The finding that ERK activation was not inhibited by CytD in our experiments correlates with studies using other cells including cardiomyocytes, in which stretch was applied using a silastic membrane . The authors found that pretreatment with CytD did not affect ERK activation, but prevented its nuclear translocation. Activation of ERK by integrins may be facilitated by alternative pathways, e. g. the signalling proteins Fyn and Shc can be involved instead of the focal adhesion kinase FAK . However, our result that LatA completely blocked the phosphorylation of ERK due to a mechanical integrin load indicates that the actin cytoskeleton is required for the activation of ERK in this context. Concerning the mechanisms, how LatA and CytD perturb the actin cytoskeleton, it is known that both drugs sequester actin monomers to prevent polymerization . CytD caps the barbed end of actin filaments, whereas LatA binds to actin at the nucleotide binding cleft and in vitro forms a nonpolymerizable complex with G-actin , . More specifically both drugs inhibit the movement of mDia, which belongs to the class of formins, on actin filaments . The speed of the mDia movement correlates with actin elongation rates. Mechanical stimuli regulate the activation of mDia by modulating the concentration of G-actin . The differences we observed between LatA and CytD concerning the effect on ERK activation may be explained by additional activities. For example, LatA is able to prevent specific binding of thymosin-β4 to the actin cytoskeleton, which in complex with profilin regulates the dynamics of the cytoskeleton , . In contrast to both inhibitors of actin polymerization, the actin stabilizing drug Jasp did not affect activation of both AKT and ERK due to mechanical integrin stress. Jasp stabilizes actin filaments and is a potent inducer of actin polymerization . In our experiments Jasp induced a ring like distribution of actin around the nucleus and strong filaments directed to the filopodia. As shown in the fluorescent image, cells may be retracted, which is supported by a previous study at higher concentrations of Jasp . However, despite these distinct alterations in the structural organization of the actin cytoskeleton by Jasp, the integrin mediated mechanically induced activation of both signalling molecules was not affected.
Our results provided insights into the role of the actin cytoskeleton in integrin mediated mechanically induced signalling pathways in MSC. Two actin depolymerizing drugs and one actin stabilizing agent affected differentially the mechanically induced activation of AKT and ERK. This indicates that various mechanisms associated with the cytoskeleton are involved in the mechanical control of signalling events. In addition to the effects on activation of signalling molecules, the actin perturbing drugs affected parameters of osteogenic and adipogenic differentiation of MSC. Adipogenic differentiation can be promoted by cytoskeletal drugs and osteogenic differentiation was inhibited by drugs. Together, actin filament perturbing drugs are suitable to explore molecular mechanisms in the biological response of MSC. In addition, non-toxic cytoskeleton modulating drugs are promising candidates to regulate cellular functions of stem cells.