Dudakovic, Amel

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.

ABSTRACTBromodomain (BRD) proteins are histone code interpreters that recognize acetylated lysines and link the dynamic state of chromatin with the transcriptional machinery. Here, we demonstrate that ablation of the Brd4 gene in primary mouse bone marrow–derived mesenchymal stem cells via a conditional Brd4fl/fl allele suppresses osteogenic lineage commitment. Remarkably, loss of Brd4 function also enhances expression of genes in engineered adenoviral vectors, including Cre recombinase and green fluorescent protein (GFP). Similarly, vector‐based expression of BMP2 mRNA and protein levels are enhanced upon Brd4 depletion in cells transduced with an adenoviral vector that expresses BMP2 (Ad‐BMP2). Importantly, Brd4 depletion in MC3T3‐E1 and human adipose‐derived mesenchymal stem cells (AMSCs) transduced with Ad‐BMP2 enhances osteogenic differentiation of naïve MC3T3‐E1 cells via paracrine mechanisms based on transwell and conditioned medium studies. Our studies indicate that Brd4 depletion enhances adenoviral transgene expression in mammalian cells, which can be leveraged as a therapeutic strategy to improve viral vector‐based gene therapies. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

ABSTRACT Use of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce conditional expression of Cre recombinase in transgenic laboratory mice. To excise β‐catenin fl/fl in 28‐day‐old male and female Prrx1‐CreER/β‐catenin fl/fl mice (C57BL/6), we utilized TAM at 150 mg/kg; despite β‐catenin knockout in MSC, we found a significant increase in trabecular and cortical bone volume in all genders. Because TAM was similarly anabolic in KO and control mice, we investigated a dose effect on bone formation by treating wild‐type mice (WT C57BL/6, 4 weeks) with TAM (total dose 0, 20, 40, 200 mg/kg via four injections). TAM increased bone in a dose‐dependent manner analyzed by micro–computed tomography (μCT), which showed that, compared to control, 20 mg/kg TAM increased femoral bone volume fraction (bone volume/total volume [BV/TV]) (21.6% ± 1.5% to 33% ± 2.5%; 153%, p < 0.005). With TAM 40 mg/kg and 200 mg/kg, BV/TV increased to 48.1% ± 4.4% (223%, p < 0.0005) and 58% ± 3.8% (269%, p < 0.0001) respectively, compared to control. Osteoblast markers increased with 200 mg/kg TAM: Dlx5 (224%, p < 0.0001), Alp (166%, p < 0.0001), Bglap (223%, p < 0.0001), and Sp7 (228%, p < 0.0001). Osteoclasts per bone surface (Oc#/BS) nearly doubled at the lowest TAM dose (20 mg/kg), but decreased to <20% control with 200 mg/kg TAM. Our data establish that use of TAM at even very low doses to excise a floxed target in postnatal mice has profound effects on trabecular and cortical bone formation. As such, TAM treatment is a major confounder in the interpretation of bone phenotypes in conditional gene knockout mouse models. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.