Lamin A/C Is Dispensable to Mechanical Repression of Adipogenesis

Abstract

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.

Depletion of Sun1/2 induces heterochromatin accrual in mesenchymal stem cells during adipogenesis

“…This work supports previous observations 24, 55, 56 that inhibiting the LINC complex via siSun and dnKASH expression reduces overall nuclear structural integrity as indicated by reduced nuclear circularity or aspect ratio. Our observed increase in nuclear area and perimeter seen during Sun1/2 depletion is similar to that of our previous observations of nuclear morphology changes during Lamin A/C depletion 57 . Both Sun1/2 and Lamin A/C are important inner nuclear membrane nucleoskleton elements that associate with other structural proteins and elements such as chromatin.…”

“…Bone marrow derived MSCs (mdMSC) from 8-10 wk male C57BL/6 mice were isolated as described 57, 61 from multiple mouse donors and MSCs pooled, providing a heterogenous MSCs cell line. Briefly, tibial and femoral marrow were collected in RPMI-1640, 9% FBS, 9% HS, 100 μg/ml pen/strep and 12μM L-glutamine.…”

“…The mean lipid droplet intensity per cell was calculated by dividing the sum of lipid droplet stain intensity by the nuclei count per image. Exported images were used to quantify lipid droplet formation, Sun1, Sun2, mCherry, nuclear area, nuclear perimeter, and nuclear circularity via the custom-made MATLAB program previously published 57 . Cell Profiler (https://cellprofiler.org/) was used to count the number of H3K9me3 foci per cell and foci area.…”

“…During adipogenesis for example, inhibition of heterochromatin H3K27me3 via methyltransferase Ezh2 inhibition results in increased protein levels of Adipoq, Fabp4, and decreased osteogeneic gene expression 36 . We have recently reported that depletion of a Sun1/2 binding partner and a nuclear envelope protein, Lamin A/C, decreases MSC adipogenesis independent of mechanical stimulation 37 , suggesting that structural proteins at the INM may have mechanically independent roles to control MSC function.…”

“…Indeed, Sun1/2 elements provide organizational capacity to chromatin through associations with LEM domain proteins (LAP2, Emerin, MAN1 domain), direct links to chromatin 46,47 and, ultimately, regulation of the genome 50,51 . In mammalian cells, Sun1/2 proteins tether chromatin to the nuclear envelope through direct connections to Emerin 52 .…”