Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.
The nucleus, central to cellular activity, relies on both direct mechanical input as well as its molecular transducers to sense external stimuli and respond by regulating intra-nuclear chromatin organization that determines cell function and fate. In mesenchymal stem cells of musculoskeletal tissues, changes in nuclear structures are emerging as a key modulator of their differentiation and proliferation programs. In this review we will first introduce the structural elements of the nucleoskeleton and discuss the current literature on how nuclear structure and signaling are altered in relation to environmental and tissue level mechanical cues. We will focus on state-ofthe-art techniques to apply mechanical force and methods to measure nuclear mechanics in conjunction with DNA, RNA, and protein visualization in living cells. Ultimately, combining realtime nuclear deformations and chromatin dynamics can be a powerful tool to study mechanisms of how forces affect the dynamics of genome function.
Mechanical signals regulate adipogenic differentiation of mesenchymal stem cells (MSCs). Critical to the mechano-regulation of MSCs, Linker of the Nucleoskeleton and Cytoskeleton (LINC) complexes are integral to both nucleo-cytoskeletal signal transduction and structural integrity of the nucleus. The LINC complex is made of Nesprin proteins that associate with the cytoskeleton on the outer nuclear membrane (ONM) and Sun proteins that bound to nuclear lamina and chromatin at the inner nuclear membrane (INM). In addition to their role in the LINC complex function, depletion of Sun1/2 effects chromosomal tethering to the nuclear envelope, nuclear morphology, and chromatin organization. Suggesting that Sun1/2 proteins may regulate chromatin organization and adipogenic differentiation independent of the LINC complex mediated nucleo-cytoskeletal connectivity. To test this hypothesis Sun1/2 depletion was compared to expression of a dominant-negative KASH (dnKASH) domain to decouple nucleus from cytoskeleton by inhibiting Nesprin-SUN association. Sun1/2 depletion inhibited fat droplet formation and production of adipogenic proteins such as Adipoq, which were supported by RNA-seq showing decreased adipogensis. In contrast dnKASH responded oppositely, increasing fat droplet formation, Adipoq and adipogenic gene expression. At the chromatin level, Sun1/2 depletion increased H3K9me3 levels, increased H3K9me3 foci count, and enrichment on Adipoq. No increase of H3K9me3 levels, foci count, or increased H3K9me3 enrichment on Adipoq was found during dnKASH expression. We conclude that physically decoupling of the LINC complex via dnKASH accelerates adipogenesis and that depletion of Sun1/2 increases heterochromatin accrual and inhibits adipogenesis independent of the LINC complex function.
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