Mechanotransduction in fibroblasts

Response to mechanical stress is important for tissue homeostasis, tissue architecture and muscle regeneration. All cells of an organism are subject to at least one of three types of mechanical stress: compression, shear stress or tension. The exact mechanisms how a cell senses mechanical stress and how it converts mechanical into chemical signals are still unknown and the elucidation of this process is the aim of my thesis.
Defective mechanotransduction can be observed in a diverse group of diseases called laminopathies, such as Emery-Dreifuss muscular dystrophy or dilated cardiomyopathy. LaminA and emerin, the proteins affected in these diseases, are part of a physical link that spans from the nuclear lamina to the extracellular matrix and might play a role in a cell’s sensation of and response to mechanical stress. Cells lacking either protein display reduced nuclear structural integrity, changes in transcriptional regulation, and defective nuclear mechanics and mechanotransduction.
In the present study the first microarray-analysis of stretched primary mouse embryo fibroblasts revealed that cells react to biaxial strain by upregulation of a very distinct group of around 30 genes within 1 hour of stretching. No transcripts were downregulated, whereas after 6 hours of strain a large group of genes was affected by mechanical stress and showed up as well as downregulation. Among them was tenascin-C as well as some other proteins involved with extracellular matrix function. This was confirmed by QPCR and Affymetrix chip analysis in 2 immortalized mouse embryo fibroblast cell lines.
We observed that biaxial strain leads to the activation of Erk, Rho/ROCK, and NfB pathways. However, though all three pathways were activated upon stretching, inhibition of Erk or ROCK did not decrease the early response to biaxial strain since transcripts induced after 1 hour of stretching were still upregulated in the presence of these inhibitors. Only inhibition of the NfB pathway blocked the stretch response of selected genes after one hour of cyclic stretching. Our results suggest that activation of NfB seem to depend on Ca2+-influx that is mediated by stretch-gated ion-channels in the cell membrane.
One of the genes responding to stretching is Egr3. It is an immediate early growth response gene which is induced by mitogenic stimulation and may play a role in muscle development. Its ability to activate tenascin-C, which possesses an Egr binding site in its promoter region, was examined by promotor-SEAP-reporter assays and by western blot.
The role of the LINC complex in mechanotransduction was analyzed by expressing dominant-negative forms of sun1 and nesprin in fibroblasts as well as in myogenic progenitors. Live imaging of mouse embryo fibroblasts revealed that nuclei are rotating in response to stretching. Although some nuclei
were slowly rotating with time when monitored at rest, stretching induced nuclear spinning. Upon disruption of this direct link by overexpression of truncated dominant-negative forms of sun1 or nesprin-1, nuclei remained in their position even in stretched cells. Contrary to our expectations disruption of the LINC complex did not inhibit the stress response as determined by transcript profiling of stretched cells.
To address the question whether a functional LINC complex plays a role in muscle development, we examined the differentiation of C2C12 myogenic progenitor cells in the presence and absence of dominant-negative sun and nesprin. C2C12 cells differentiate in the presence of 5% horse serum in the medium but not when stretched for 1 hour every 24 hours. When the direct link is disrupted cells continue to differentiate even when stretched. This indicates that sun and nesprin are necessary for the sensation of mechanical signals in differentiating C2C12 cells.