Sen, Buer

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.

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.

ABSTRACT During bone marrow stromal cell (BMSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. β-catenin plays a critical role in the Wnt signaling pathway to facilitate downstream effects on gene expression. We show that β-catenin was additive with cytoskeletal signals to prevent adipogenesis, and β-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. β-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with β-catenin knockdown enhancing lineage commitment. Chromatin immunoprecipitation (ChIP)-sequencing demonstrated binding of β-catenin to the promoter of enhancer of zeste homolog 2 (EZH2), a key component of the polycomb repressive complex 2 (PRC2) complex that catalyzes histone methylation. Knockdown of β-catenin reduced EZH2 protein levels and decreased methylated histone 3 (H3K27me3) at osteogenic loci. Further, when EZH2 was inhibited, β-catenin's anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to β-catenin's effects on BMSCs to preserve multipotentiality. © 2020 American Society for Bone and Mineral Research.

AbstractNuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actin-lamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentiation and suppressed adipogenic differentiation. Hence, mDia2 is a potent tool to query intranuclear actin-lamin nucleoskeletal structure, and its presence serves to retain multipotent stromal cells in an undifferentiated state.

Abstract Strategies for musculoskeletal tissue regeneration apply adult mesenchymal stem/stromal cells (MSCs) that can be sourced from bone marrow- and lipo-aspirates. Adipose tissue-derived MSCs are more easily harvested in the large quantities required for skeletal tissue-engineering approaches, but are generally considered to be less osteogenic than bone marrow MSCs. Therefore, we tested a new molecular strategy to improve their osteogenic lineage-differentiation potential using the fungal metabolite cytochalasin D (CytoD). We show that CytoD, which may function by redistributing the intracellular location of β-actin (ACTB), is a potent osteogenic stimulant as reflected by significant increases in alkaline phosphatase activity, extracellular matrix mineralization, and osteoblast-related gene expression (e.g., RUNX2, ALPL, SPARC, and TGFB3). RNA sequencing analyses of MSCs revealed that acute CytoD treatment (24 hours) stimulates a broad program of osteogenic biomarkers and epigenetic regulators. CytoD decreases mRNA and protein levels of the Polycomb chromatin regulator Enhancer of Zeste Homolog 2 (EZH2), which controls heterochromatin formation by mediating trimethylation of histone 3 lysine 27 (H3K27me3). Reduced EZH2 expression decreases cellular H3K27me3 marks indicating a global reduction in heterochromatin. We conclude that CytoD is an effective osteogenic stimulant that mechanistically functions by blocking both cytoplasmic actin polymerization and gene-suppressive epigenetic mechanisms required for the acquisition of the osteogenic phenotype in adipose tissue-derived MSCs. This finding supports the use of CytoD in advancing the osteogenic potential of MSCs in skeletal regenerative strategies.

Abstract Actin structure contributes to physiologic events within the nucleus to control mesenchymal stromal cell (MSC) differentiation. Continuous cytochalasin D (Cyto D) disruption of the MSC actin cytoskeleton leads to osteogenic or adipogenic differentiation, both requiring mass transfer of actin into the nucleus. Cyto D remains extranuclear, thus intranuclear actin polymerization is potentiated by actin transfer: we asked whether actin structure affects differentiation. We show that secondary actin filament branching via the Arp2/3 complex is required for osteogenesis and that preventing actin branching stimulates adipogenesis, as shown by expression profiling of osteogenic and adipogenic biomarkers and unbiased RNA-seq analysis. Mechanistically, Cyto D activates osteoblast master regulators (e.g., Runx2, Sp7, Dlx5) and novel coregulated genes (e.g., Atoh8, Nr4a3, Slfn5). Formin-induced primary actin filament formation is critical for Arp2/3 complex recruitment: osteogenesis is prevented by silencing of the formin mDia1, but not its paralog mDia2. Furthermore, while inhibition of actin, branching is a potent adipogenic stimulus, silencing of either mDia1 or mDia2 blocks adipogenic gene expression. We propose that mDia1, which localizes in the cytoplasm of multipotential MSCs and traffics into the nucleus after cytoskeletal disruption, joins intranuclear mDia2 to facilitate primary filament formation before mediating subsequent branching via Arp2/3 complex recruitment. The resulting intranuclear branched actin network specifies osteogenic differentiation, while actin polymerization in the absence of Arp2/3 complex-mediated secondary branching causes adipogenic differentiation.