G-protein coupled receptor 5C (GPRC5C) is required for osteoblast differentiation and responds to EZH2 inhibition and multiple osteogenic signals.

Osteoblast differentiation is epigenetically suppressed by the H3K27 methyltransferase EZH2, and induced by the morphogen BMP2 and transcription factor RUNX2. These factors also regulate distinct G protein coupled receptors (GPRCs; e.g., PTH1R, GPR30/GPER1). Because GPRCs transduce many physiological stimuli, we examined whether BMP2 or EZH2 inhibition (i.e., GSK126) regulates other GPRC genes in osteoblasts. RNA-seq screening of >400 mouse GPRC-related genes showed that many GPRCs are downregulated during osteogenic differentiation. The orphan receptor GPRC5C, along with a small subset of other GPRCs, is induced by BMP2 or GSK126 during Vitamin C dependent osteoblast differentiation, but not by all-trans retinoic acid. ChIP-seq analysis revealed that GSK126 reduces H3K27me3 levels at the GPRC5C gene locus in differentiating MC3T3-E1 osteoblasts, consistent with enhanced GPRC5C mRNA expression. Loss of function analyses revealed that shRNA-mediated depletion of GPRC5C decreases expression of bone markers (e.g., BGLAP and IBSP) and mineral deposition in response to BMP2 or GSK126. GPRC5C mRNA was found to be reduced in the osteopenic bones of KLF10 null mice which have compromised BMP2 signaling. GPRC5C mRNA is induced by the bone-anabolic activity of 17ß-estradiol in trabecular but not cortical bone following ovariectomy. Collectively, these findings suggest that GPRC5C protein is a key node in a pro-osteogenic axis that is normally suppressed by EZH2-mediated H3K27me3 marks and induced during osteoblast differentiation by GSK126, BMP2, and/or 17ß-estradiol. Because GPRC5C protein is an understudied orphan receptor required for osteoblast differentiation, identification of ligands that induce GPRC5C signaling may support therapeutic strategies to mitigate bone-related disorders.

Brd4 Inactivation Increases Adenoviral Delivery of BMP2 for Paracrine Stimulation of Osteogenic Differentiation as a Gene Therapeutic Concept to Enhance Bone Healing.

Bromodomain (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.

Differences in Cytotoxicity of Lidocaine, Ropivacaine, and Bupivacaine on the Viability and Metabolic Activity of Human Adipose-Derived Mesenchymal Stem Cells.

PURPOSE: We evaluated biological effects of distinct local anesthetics on human adipose-derived mesenchymal stem cells when applied to reduce periprocedural pain during mesenchymal stem cell injections. METHODS AND MATERIALS: Metabolic activity (MTS assay), viability (Live/Dead stain), and gene expression (quantitative real-time reverse-transcriptase polymerase chain reaction) were measured in mesenchymal stem cells incubated with various concentrations of lidocaine, ropivacaine, or bupivacaine during a 12-hr time course. RESULTS: Cell viability and metabolic activity decreased in a dose, time, and substance-specific manner after exposure to lidocaine, ropivacaine, and bupivacaine, with ropivacaine being the least cytotoxic. Cell viability decreases after brief exposure (<1.5 hrs) at clinically relevant concentrations (eg, 8 mg/ml of lidocaine, 2.5 mg/ml of ropivacaine or bupivacaine). Mesenchymal stem cells exposed to local anesthetics change their expression of mRNA biomarkers for stress response (EGR1, EGR2), proliferation (MKI67, HIST2H4A), ECM (COL1A1, COL3A1), and cell surface marker (CD105). CONCLUSIONS: Local anesthetics are cytotoxic to clinical-grade human mesenchymal stem cells in a dose-, time-, and agent-dependent manner and change expression of ECM, proliferation, and cell surface markers. Lidocaine and bupivacaine are more cytotoxic than ropivacaine. Single-dose injections of local anesthetics may affect the biological properties of mesenchymal stem cells in vitro but may not affect the effective dose of MSCs in a clinical setting.

Hypothermia and nutrient deprivation alter viability of human adipose-derived mesenchymal stem cells.

PURPOSE: Adipose-derived mesenchymal stem cells (MSCs) are attractive biological agents in regenerative medicine. To optimize cell therapies, it is necessary to determine the most effective delivery method for MSCs. Therefore, we evaluated the biological properties of MSCs after exposure to various temperatures to define optimal storage conditions prior to therapeutic delivery of MSCs. DESIGN: Prospective observational study. METHODS AND MATERIALS: Adherent and non-adherent MSCs were incubated at multiple temperatures (i.e., 4, 23 and 37 °C) in Lactated Ringers (LR) solution lacking essential cell growth ingredients, or in culture media which is optimized for cell growth. Cells were assessed either after the temperature changes (4 h) or after recovery (24 h). Metabolic activity of MSCs, cell number and expression of representative mRNA biomarkers were evaluated to assess the biological effects of temperature. We monitored changes in mRNAs expression related to cytoprotective- or stress-related responses (e.g., FOS, JUN, ATF1, ATF4, EGR1, EGR2, MYC), proliferation (e.g., HIST2H4, CCNB2), and extracellular matrix production (ECM; e.g., COL3A1, COL1A1) by quantitative real time reverse-transcriptase polymerase chain reaction (RT-qPCR) analysis. RESULTS: Our study demonstrates that storing MSCs in Lactated Ringers (LR) solution for 4 h decreases cell number and metabolic activity. The number of viable MSCs decreased significantly when cultured at physiological temperature (37 °C) and severe hypothermia (4 °C), while cells grown at ambient temperature (23 °C) exhibited the least detrimental effects. There were no appreciable biological differences in mRNA markers for proliferation or ECM deposition at any of the temperatures. However, biomarkers related to cytoprotective- or stress-responses were selectively elevated depending on temperature or media type (i.e., LR versus standard media). CONCLUSION: The biological impact of nutrient-free media and temperature changes after 4 h exposure persists after a 24 h recovery period. Hence, storage temperature and media conditions should be optimized to improve effective dosing of MSCs.

The epigenetic reader Brd4 is required for osteoblast differentiation.

Transcription networks and epigenetic mechanisms including DNA methylation, histone modifications, and noncoding RNAs control lineage commitment of multipotent mesenchymal progenitor cells. Proteins that read, write, and erase histone tail modifications curate and interpret the highly intricate histone code. Epigenetic reader proteins that recognize and bind histone marks provide a crucial link between histone modifications and their downstream biological effects. Here, we investigate the role of bromodomain-containing (BRD) proteins, which recognize acetylated histones, during osteogenic differentiation. Using RNA-sequencing (RNA-seq) analysis, we screened for BRD proteins (n = 40) that are robustly expressed in MC3T3 osteoblasts. We focused functional follow-up studies on Brd2 and Brd4 which are highly expressed in MC3T3 preosteoblasts and represent "bromodomain and extra terminal domain" (BET) proteins that are sensitive to pharmacological agents (BET inhibitors). We show that small interfering RNA depletion of Brd4 has stronger inhibitory effects on osteoblast differentiation than Brd2 loss as measured by osteoblast-related gene expression, extracellular matrix deposition, and alkaline phosphatase activity. Similar effects on osteoblast differentiation are seen with the BET inhibitor +JQ1, and this effect is reversible upon its removal indicating that this small molecule has no lasting effects on the differentiation capacity of MC3T3 cells. Mechanistically, we find that Brd4 binds at known Runx2 binding sites in promoters of bone-related genes. Collectively, these findings suggest that Brd4 is recruited to osteoblast-specific genes and may cooperate with bone-related transcription factors to promote osteoblast lineage commitment and maturation.