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Multipotent bone marrow mesenchymal stromal/stem cells (MSCs) respond to mechanical forces. MSCs perceive static and dynamic forces through focal adhesions, as well as cytoskeletal and intranuclear actin. Dynamic strain stimulates nuclear β-catenin (Ctnnb1) that controls gene expression and suppresses osteogenesis. The sensitivity of MSCs to external mechanical forces may be altered by cessation of proliferation, when MSCs begin to express extracellular matrix (ECM) proteins and generate cell/cell contact. Therefore, we assessed whether and how gene expression of proliferating versus quiescent MSCs responds to mechanical stimuli. We used RNA-seq and RT-qPCR to evaluate transcriptomes at 3 h after dynamic strain (200 cycles × 2 % for 20 min) once daily during a two-day time course in naïve (uninduced) MSCs. Transcriptomes of untreated MSCs show that cells become quiescent at day 2 when proliferation markers are downregulated, and ECM related genes are upregulated. On both day 1 and day 2, dynamic strain stimulates expression of oxidative stress related genes (e.g., Nqo1, Prl2c2, Prl2c3). Strikingly, in quiescent MSCs, we observe that dynamic strain suppresses multiple interferon (IFN) responsive genes (e.g., Irf7, Oasl2 and Isg15). IFN responsive genes are activated in MSCs depleted of β-catenin using siRNAs, indicating that β-catenin normally suppresses these genes. Our data indicate that the functional effects of dynamic strain and β-catenin on IFN responsive genes in MSCs are mechanistically coupled. Because dynamic strain and β-catenin reduce the osteogenic potential of MSCs, our findings suggest that IFN responsive genes are novel biomarkers and potential regulators of mechanical responses at early stages of lineage-commitment in post-proliferative MSCs.

Osteogenic differentiation of mesenchymal cells is controlled by epigenetic enzymes that regulate post-translational modifications of histones. Compared to acetyl or methyltransferases, the physiological functions of protein arginine methyltransferases (PRMTs) in osteoblast differentiation remain minimally understood. Therefore, we surveyed the expression and function of all nine mammalian PRMT members during osteoblast differentiation. RNA-seq gene expression profiling shows that Prmt1, Prmt4/Carm1 and Prmt5 represent the most prominently expressed PRMT subtypes in mouse calvarial bone and MC3T3 osteoblasts as well as human musculoskeletal tissues and mesenchymal stromal cells (MSCs). Based on effects of siRNA depletion, it appears that PRMT members have different functional effects: (i) loss of Prmt1 stimulates and (ii) loss of Prmt5 decreases calcium deposition of mouse MC3T3 osteoblasts, while (iii) loss of Carm1 is inconsequential for calcium deposition. Decreased Prmt5 suppresses expression of multiple genes involved in mineralization (e.g., Alpl, Ibsp, Phospho1) consistent with a positive role in osteogenesis. Depletion of Prmt1, Carm1 and Prmt5 has intricate but modest time-dependent effects on the expression of a panel of osteoblast differentiation and proliferation markers but does not change mRNA levels for select epigenetic regulators (e.g., Ezh1, Ezh2, Brd2 and Brd4). Treatment with the Class I PRMT inhibitor GSK715 enhances extracellular matrix mineralization of MC3T3 cells, while blocking formation of H3R17me2a but not H4R3me2a marks. In sum, Prmt1, Carm1 and Prmt5 have distinct biological roles during osteoblast differentiation, and different types histone H3 and H4 arginine methylation may contribute to the chromatin landscape during osteoblast differentiation.