Inhibition of Ezh2 redistributes bivalent domains within transcriptional regulators associated with WNT and Hedgehog pathways in osteoblasts.

Bivalent epigenomic regulatory domains containing both activating histone 3 lysine 4 (H3K4me3) and repressive lysine 27 (H3K27me3) trimethylation are associated with key developmental genes. These bivalent domains repress transcription in the absence of differentiation signals but maintain regulatory genes in a poised state to allow for timely activation. Previous studies demonstrated that enhancer of zeste homolog 2 (Ezh2), a histone 3 lysine 27 (H3K27) methyltransferase, suppresses osteogenic differentiation and that inhibition of Ezh2 enhances commitment of osteoblast progenitors in vitro and bone formation in vivo. Here, we examined the mechanistic effects of Tazemetostat (EPZ6438), an Food and Drug Administration approved Ezh2 inhibitor for epithelioid sarcoma treatment, because this drug could potentially be repurposed to stimulate osteogenesis for clinical indications. We find that Tazemetostat reduces H3K27me3 marks in bivalent domains in enhancers required for bone formation and stimulates maturation of MC3T3 preosteoblasts. Furthermore, Tazemetostat activates bivalent genes associated with the Wingless/integrated (WNT), adenylyl cyclase (cAMP), and Hedgehog (Hh) signaling pathways based on transcriptomic (RNA-seq) and epigenomic (chromatin immunoprecipitation [ChIP]-seq) data. Functional analyses using selective pathway inhibitors and silencing RNAs demonstrate that the WNT and Hh pathways modulate osteogenic differentiation after Ezh2 inhibition. Strikingly, we show that loss of the Hh-responsive transcriptional regulator Gli1, but not Gli2, synergizes with Tazemetostat to accelerate osteoblast differentiation. These studies establish epigenetic cooperativity of Ezh2, Hh-Gli1 signaling, and bivalent regulatory genes in suppressing osteogenesis. Our findings may have important translational ramifications for anabolic applications requiring bone mass accrual and/or reversal of bone loss.

AdipoRon reduces TGFß1-mediated collagen deposition in vitro and alleviates knee stiffness in vivo.

Arthrofibrosis, which causes joint motion restrictions, is a common complication following total knee arthroplasty (TKA). Key features associated with arthrofibrosis include myofibroblast activation, knee stiffness, and excessive scar tissue formation. We previously demonstrated that adiponectin levels are suppressed within the knee tissues of patients affected by arthrofibrosis and showed that AdipoRon, an adiponectin receptor agonist, exhibited anti-fibrotic properties in human mesenchymal stem cells. In this study, the therapeutic potential of AdipoRon was evaluated on TGFß1-mediated myofibroblast differentiation of primary human knee fibroblasts and in a mouse model of knee stiffness. Picrosirius red staining revealed that AdipoRon reduced TGFß1-induced collagen deposition in primary knee fibroblasts derived from patients undergoing primary TKA and revision TKA for arthrofibrosis. AdipoRon also reduced mRNA and protein levels of ACTA2, a key myofibroblast marker. RNA-seq analysis corroborated the anti-myofibrogenic effects of AdipoRon. In our knee stiffness mouse model, 6 weeks of knee immobilization, to induce a knee contracture, in conjunction with daily vehicle (DMSO) or AdipoRon (1, 5, and 25 mg/kg) via intraperitoneal injections were well tolerated based on animal behavior and weight measurements. Biomechanical testing demonstrated that passive extension angles (PEAs) of experimental knees were similar between vehicle and AdipoRon treatment groups in mice evaluated immediately following immobilization. Interestingly, relative to vehicle-treated mice, 5 mg/kg AdipoRon therapy improved the PEA of the experimental knees in mice that underwent 4 weeks of knee remobilization following the immobilization and therapy. Together, these studies revealed that AdipoRon may be an effective therapeutic modality for arthrofibrosis.

Effect of Hypoxia Preconditioning on the Regenerative Capacity of Adipose Tissue Derived Mesenchymal Stem Cells in a Model of Renal Artery Stenosis.

Atherosclerotic renal artery stenosis (ARAS) is associated with irreversible parenchymal renal disease and regenerative stem cell therapies may improve renal outcomes. Hypoxia preconditioning (HPC) may improve the regenerative functions of adipose tissue-derived mesenchymal stem cells (AMSC) by affecting DNA 5-hydroxymethylcytosine (5hmC) marks in angiogenic genes. Here, we investigated using a porcine ARAS model, whether growth of ARAS AMSCs in hypoxia (Hx) versus normoxia (Nx) would enhance renal tissue repair, and comprehensively analyze how HPC modifies DNA hydroxymethylation compared to untreated ARAS and healthy/normal pigs (n=5 each). ARAS pigs exhibited elevated serum cholesterol, serum creatinine and renal artery stenosis, with a concomitant decrease in renal blood flow (RBF) and increased blood pressure (BP) compared to healthy pigs. Renal artery injection of either autologous Nx or Hx AMSCs improved diastolic BP, reduced kidney tissue fibrosis, and inflammation (CD3+ T-cells) in ARAS pigs. In addition, renal medullary hypoxia significantly lowered with Nx but not Hx AMSC treatment. Mechanistically, levels of epigenetic 5hmC marks (which reflect gene activation) estimated using DNA immunoprecipitation technique were elevated in profibrotic and inflammatory genes in ARAS compared with normal AMSCs. HPC significantly reduced 5hmC levels in cholesterol biosynthesis and oxidative stress response pathways in ARAS AMSCs. Thus, autologous AMSCs improve key renovascular parameters and inflammation in ARAS pigs, with HPC mitigating pathological molecular effects on inflammatory and profibrotic genes which may play a role in augmenting regenerative capacity of AMSCs.

The epigenetic regulator BRD4 is required for myofibroblast differentiation of knee fibroblasts.

Arthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition. Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra-terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA-seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT-qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFß1-induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleled by siRNA-mediated depletion of BRD4. Together, these data reveal that BRD4-mediated epigenetic events support TGFß1-mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis.

Human outgrowth knee fibroblasts from patients undergoing total knee arthroplasty exhibit a unique gene expression profile and undergo myofibroblastogenesis upon TGFß1 stimulation.

Arthrofibrosis is characterized by excessive extracellular matrix (ECM) deposition that results in restricted joint motion after total knee arthroplasties (TKAs). Currently, treatment options are limited. Therefore, an in vitro model of knee-related myofibroblastogenesis is valuable to facilitate investigation of the arthrofibrotic process, diagnostic and therapeutic options. In this study, we obtained intraoperative posterior capsule (PC), quadriceps tendon (QT), and suprapatellar pouch (SP) tissues from the knees of four patients undergoing primary TKAs for osteoarthritis. From these tissues, we isolated primary cells by the outgrowth method and subsequently characterized these cells in the absence and presence of the pro-myofibroblastic cytokine, transforming growth factor beta 1 (TGFß1). Light microscopy of knee outgrowth cells revealed spindle-shaped cells, and immunofluorescence (IF) analysis demonstrated staining for the fibroblast-specific markers TE-7 and vimentin (VIM). These knee outgrowth fibroblasts differentiated readily into myofibroblasts as reflected by enhanced α-smooth muscle actin (ACTA2) mRNA and protein expression and increased mRNA expression of collagen type 1 (COL1A1) and type 3 (COL3A1) with collagenous matrix deposition in the presence of TGFß1. Outgrowth knee fibroblasts were more sensitive to TGFß1-mediated myofibroblastogenesis than adipose-derived mesenchymal stromal/stem cells (MSCs). While outgrowth knee fibroblasts isolated from three anatomical regions in four patients exhibited similar gene expression, these cells are distinct from other fibroblastic cell types (i.e., Dupuytren's fibroblasts) as revealed by RNA-sequencing. In conclusion, our study provides an in vitro myofibroblastic model of outgrowth knee fibroblasts derived from patients undergoing primary TKA that can be utilized to study myofibroblastogenesis and assess therapeutic strategies for arthrofibrosis.

Inhibition of the epigenetic suppressor EZH2 primes osteogenic differentiation mediated by BMP2.

Bone-stimulatory therapeutics include bone morphogenetic proteins (e.g. BMP2), parathyroid hormone, and antibody-based suppression of WNT antagonists. Inhibition of the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is both bone anabolic and osteoprotective. EZH2 inhibition stimulates key components of bone-stimulatory signaling pathways, including the BMP2 signaling cascade. Because of high costs and adverse effects associated with BMP2 use, here we investigated whether BMP2 dosing can be reduced by co-treatment with EZH2 inhibitors. Co-administration of BMP2 with the EZH2 inhibitor GSK126 enhanced differentiation of murine (MC3T3) osteoblasts, reflected by increased alkaline phosphatase activity, Alizarin Red staining, and expression of bone-related marker genes (e.g. Bglap and Phospho1). Strikingly, co-treatment with BMP2 (10 ng/ml) and GSK126 (5 µm) was synergistic and was as effective as 50 ng/ml BMP2 at inducing MC3T3 osteoblastogenesis. Similarly, the BMP2-GSK126 co-treatment stimulated osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells, reflected by induction of key osteogenic markers (e.g. Osterix/SP7 and IBSP). A combination of BMP2 (300 ng local) and GSK126 (5 µg local and 5 days of 50 mg/kg systemic) yielded more consistent bone healing than single treatments with either compound in a mouse calvarial critical-sized defect model according to results from µCT, histomorphometry, and surgical grading of qualitative X-rays. We conclude that EZH2 inhibition facilitates BMP2-mediated induction of osteogenic differentiation of progenitor cells and maturation of committed osteoblasts. We propose that epigenetic priming, coupled with bone anabolic agents, enhances osteogenesis and could be leveraged in therapeutic strategies to improve bone mass.

Molecular pathology of human knee arthrofibrosis defined by RNA sequencing.

Arthrofibrosis is an abnormal histopathologic response, is debilitating for patients, and poses a substantial unsolved clinical challenge. This study characterizes molecular biomarkers and regulatory pathways associated with arthrofibrosis by comparing fibrotic and non-fibrotic human knee tissue. The fibrotic group encompasses 4 patients undergoing a revision total knee arthroplasty (TKA) for arthrofibrosis (RTKA-A) while the non-fibrotic group includes 4 patients undergoing primary TKA for osteoarthritis (PTKA) and 4 patients undergoing revision TKA for non-arthrofibrotic and non-infectious etiologies (RTKA-NA). RNA-sequencing of posterior capsule specimens revealed differences in gene expression between each patient group by hierarchical clustering, principal component analysis, and correlation analyses. Multiple differentially expressed genes (DEGs) were defined in RTKA-A versus PTKA patients (i.e., 2059 up-regulated and 1795 down-regulated genes) and RTKA-A versus RTKA-NA patients (i.e., 3255 up-regulated and 3683 down-regulated genes). Our findings define molecular and pathological markers of arthrofibrosis, as well as novel potential targets for risk profiling, early diagnosis and pharmacological treatment of patients.

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.

Molecular pathology of adverse local tissue reaction caused by metal-on-metal implants defined by RNA-seq.

Total hip arthroplasty (THA) alleviates hip pain and improves joint function. Current implant design permits long-term survivorship of THAs, but certain metal-on-metal (MoM) articulations can portend catastrophic failure due to adverse local tissue reactions (ALTR). Here, we identified biological and molecular differences between periacetabular synovial tissues of patients with MoM THA failure undergoing revision THA compared to patients undergoing primary THA for routine osteoarthritis (OA). Analysis of tissue biopsies by RNA-sequencing (RNA-seq) revealed that MoM patient samples exhibit significantly increased expression of immune response genes but decreased expression of genes related to extracellular matrix (ECM) remodeling. Thus, interplay between local tissue inflammation and ECM degradation may account for the pathology and compromised clinical outcomes in select patients with MoM implants. We conclude that adverse responses of host tissues to implant materials result in transcriptomic modifications in patients with MoM implants that permit consideration of strategies that could mitigate ECM damage.

Enhancer of zeste homolog 2 (Ezh2) controls bone formation and cell cycle progression during osteogenesis in mice.

Epigenetic mechanisms control skeletal development and osteoblast differentiation. Pharmacological inhibition of the histone 3 Lys-27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) in WT mice enhances osteogenesis and stimulates bone formation. However, conditional genetic loss of Ezh2 early in the mesenchymal lineage (i.e. through excision via Prrx1 promoter-driven Cre) causes skeletal abnormalities due to patterning defects. Here, we addressed the key question of whether Ezh2 controls osteoblastogenesis at later developmental stages beyond patterning. We show that Ezh2 loss in committed pre-osteoblasts by Cre expression via the osterix/Sp7 promoter yields phenotypically normal mice. These Ezh2 conditional knock-out mice (Ezh2 cKO) have normal skull bones, clavicles, and long bones but exhibit increased bone marrow adiposity and reduced male body weight. Remarkably, in vivo Ezh2 loss results in a low trabecular bone phenotype in young mice as measured by micro-computed tomography and histomorphometry. Thus, Ezh2 affects bone formation stage-dependently. We further show that Ezh2 loss in bone marrow-derived mesenchymal cells suppresses osteogenic differentiation and impedes cell cycle progression as reflected by decreased metabolic activity, reduced cell numbers, and changes in cell cycle distribution and in expression of cell cycle markers. RNA-Seq analysis of Ezh2 cKO calvaria revealed that the cyclin-dependent kinase inhibitor Cdkn2a is the most prominent cell cycle target of Ezh2 Hence, genetic loss of Ezh2 in mouse pre-osteoblasts inhibits osteogenesis in part by inducing cell cycle changes. Our results suggest that Ezh2 serves a bifunctional role during bone formation by suppressing osteogenic lineage commitment while simultaneously facilitating proliferative expansion of osteoprogenitor cells.

The cancer-related transcription factor RUNX2 modulates expression and secretion of the matricellular protein osteopontin in osteosarcoma cells to promote adhesion to endothelial pulmonary cells and lung metastasis.

Osteosarcomas are bone tumors that frequently metastasize to the lung. Aberrant expression of the transcription factor, runt-related transcription factor 2 (RUNX2), is a key pathological feature in osteosarcoma and associated with loss of p53 and miR-34 expression. Elevated RUNX2 may transcriptionally activate genes mediating tumor progression and metastasis, including the RUNX2 target gene osteopontin (OPN/SPP1). This gene encodes a secreted matricellular protein produced by osteoblasts to regulate bone matrix remodeling and tissue calcification. Here we investigated whether and how the RUNX2/OPN axis regulates lung metastasis of osteosarcoma. Importantly, RUNX2 depletion attenuates lung metastasis of osteosarcoma cells in vivo. Using next-generation RNA-sequencing, protein-based assays, as well as the loss- and gain-of-function approaches in selected osteosarcoma cell lines, we show that osteopontin messenger RNA levels closely correlate with RUNX2 expression and that RUNX2 controls the levels of secreted osteopontin. Elevated osteopontin levels promote heterotypic cell-cell adhesion of osteosarcoma cells to human pulmonary microvascular endothelial cells, but not in the presence of neutralizing antibodies. Collectively, these findings indicate that the RUNX2/OPN axis regulates the ability of osteosarcoma cells to attach to pulmonary endothelial cells as a key step in metastasis of osteosarcoma cells to the lung.

A multi-chamber tissue culture device for load-dependent parallel evaluation of tendon explants.

BACKGROUND: Injuries in the musculoskeletal system, such as tendon and ligament ruptures, are challenging to manage and often require surgical reconstructions with limited long-term success. Thus, characterizations of these tissues are urgently needed to better understand cellular mechanisms that regulate tissue homeostasis and healing. Explant culturing systems allow for ex vivo analysis of tissues in an environment that mimics the native microenvironment in vivo. METHODS: Collaborative efforts within our institution facilitated the establishment of a novel explant culturing system. Tissue specimens cultured in single wells, with individual applied loading and/or biological environment, allowed characterization of tissue cultured under a variety of biological loading conditions. Quantitative PCR analysis for selected gene markers was our primary outcome. RESULTS: Data were stratified for analysis by either culture environment or loading condition. Our gene expression results show that specimens clustered by culture condition may differ in molecular markers related to ECM production (e.g., Col1a1, Adamts4) and/or organization (e.g., Tnc, Dnc). In contrast, loading condition did significantly alter the median gene expression levels of tissues in comparison to unloaded control samples, although gene expression values related to ECM degradation (e.g., Mmp1, Mmp10) were altered in tendons cultured under tension in the device. CONCLUSION: Our study demonstrates promising utility of a novel explant culturing system for further characterization of musculoskeletal tissues such as native tendons and ligaments, as well as pathologic fibrotic tissues resulting from arthrofibrosis or Dupuytren's disease.

In Vivo Survival of Mesenchymal Stromal Cell-Enhanced Decellularized Nerve Grafts for Segmental Peripheral Nerve Reconstruction.

PURPOSE: Adipose-derived mesenchymal stromal cells (MSCs) have emerged as promising tools for peripheral nerve reconstruction. There is a paucity of information regarding the ultimate survivorship of implanted MSCs or whether these cells remain where they are placed. The aim of the present study was to track the in vivo distribution and survival of MSCs seeded on a decellularized nerve allograft reconstruction of a peripheral nerve defect using luciferase-based bioluminescence imaging (BLI). METHODS: To determine the in vivo survivability of MSCs, autologous Lewis rat MSCs were stably labeled with luciferase by lentiviral particles. Labeled cells were dynamically seeded onto a Sprague Dawley decellularized rat nerve allograft and used to bridge a 10-mm sciatic nerve defect. The MSC survival was determined by performing in vivo BLI to detect living cells. Twelve animals were examined at 24 hours after implantation, 3, 7, 9, 11, and 14 days, and at daily intervals thereafter if signals were still present. RESULTS: Labeled MSCs could be detected for up to 29 days. Gradually diminishing BLI signals were observed within the first week following implantation. Implanted MSCs were not detected anywhere other than the site of surgery. CONCLUSIONS: The MSCs seeded on decellularized nerve allografts can survive in vivo but have finite survival after implantation. There was no evidence of migration of MSCs to surrounding tissues. CLINICAL RELEVANCE: The findings support a therapeutic approach that combines MSCs with a biological scaffold for peripheral nerve surgery. It provides understanding of the viability and distribution of implanted MSCs, which is a prerequisite before clinical translation can be considered.

Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2.

Osteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone-related genes, including those encoding extracellular matrix proteins or factors that control cell-cell, and cell-matrix interactions. Cell-cell communication in the many skeletal pericellular micro-niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by "A Disintegrin And Metalloproteinase" (ADAM) proteins. These cell membrane-associated metalloproteinases support proteolytic release ("shedding") of protein ectodomains residing at the cell surface. We analyzed microarray and RNA-sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα-signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (-0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic-nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2-Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.

Anabolic and Antiresorptive Modulation of Bone Homeostasis by the Epigenetic Modulator Sulforaphane, a Naturally Occurring Isothiocyanate.

Bone degenerative pathologies like osteoporosis may be initiated by age-related shifts in anabolic and catabolic responses that control bone homeostasis. Here we show that sulforaphane (SFN), a naturally occurring isothiocyanate, promotes osteoblast differentiation by epigenetic mechanisms. SFN enhances active DNA demethylation viaTet1andTet2and promotes preosteoblast differentiation by enhancing extracellular matrix mineralization and the expression of osteoblastic markers (Runx2,Col1a1,Bglap2,Sp7,Atf4, andAlpl). SFN decreases the expression of the osteoclast activator receptor activator of nuclear factor-κB ligand (RANKL) in osteocytes and mouse calvarial explants and preferentially induces apoptosis in preosteoclastic cells via up-regulation of theTet1/Fas/Caspase 8 and Caspase 3/7 pathway. These mechanistic effects correlate with higher bone volume (∼20%) in both normal and ovariectomized mice treated with SFN for 5 weeks compared with untreated mice as determined by microcomputed tomography. This effect is due to a higher trabecular number in these mice. Importantly, no shifts in mineral density distribution are observed upon SFN treatment as measured by quantitative backscattered electron imaging. Our data indicate that the food-derived compound SFN epigenetically stimulates osteoblast activity and diminishes osteoclast bone resorption, shifting the balance of bone homeostasis and favoring bone acquisition and/or mitigation of bone resorptionin vivo Thus, SFN is a member of a new class of epigenetic compounds that could be considered for novel strategies to counteract osteoporosis.

Enhancer of Zeste Homolog 2 Inhibition Stimulates Bone Formation and Mitigates Bone Loss Caused by Ovariectomy in Skeletally Mature Mice.

Perturbations in skeletal development and bone degeneration may result in reduced bone mass and quality, leading to greater fracture risk. Bone loss is mitigated by bone protective therapies, but there is a clinical need for new bone-anabolic agents. Previous work has demonstrated that Ezh2 (enhancer of zeste homolog 2), a histone 3 lysine 27 (H3K27) methyltransferase, suppressed differentiation of osteogenic progenitors. Here, we investigated whether inhibition of Ezh2 can be leveraged for bone stimulatory applications. Pharmacologic inhibition and siRNA knockdown of Ezh2 enhanced osteogenic commitment of MC3T3 preosteoblasts. Next generation RNA sequencing of mRNAs and real time quantitative PCR profiling established that Ezh2 inactivation promotes expression of bone-related gene regulators and extracellular matrix proteins. Mechanistically, enhanced gene expression was linked to decreased H3K27 trimethylation (H3K27me3) near transcriptional start sites in genome-wide sequencing of chromatin immunoprecipitations assays. Administration of an Ezh2 inhibitor modestly increases bone density parameters of adult mice. Furthermore, Ezh2 inhibition also alleviated bone loss in an estrogen-deficient mammalian model for osteoporosis. Ezh2 inhibition enhanced expression of Wnt10b and Pth1r and increased the BMP-dependent phosphorylation of Smad1/5. Thus, these data suggest that inhibition of Ezh2 promotes paracrine signaling in osteoblasts and has bone-anabolic and osteoprotective potential in adults.

Histone H4 Methyltransferase Suv420h2 Maintains Fidelity of Osteoblast Differentiation.

Osteogenic lineage commitment and progression is controlled by multiple signaling pathways (e.g., WNT, BMP, FGF) that converge on bone-related transcription factors. Access of osteogenic transcription factors to chromatin is controlled by epigenetic regulators that generate post-translational modifications of histones ("histone code"), as well as read, edit and/or erase these modifications. Our understanding of the biological role of epigenetic regulators in osteoblast differentiation remains limited. Therefore, we performed next-generation RNA sequencing (RNA-seq) and established which chromatin-related proteins are robustly expressed in mouse bone tissues (e.g., fracture callus, calvarial bone). These studies also revealed that cells with increased osteogenic potential have higher levels of the H4K20 methyl transferase Suv420h2 compared to other methyl transferases (e.g., Suv39h1, Suv39h2, Suv420h1, Ezh1, Ezh2). We find that all six epigenetic regulators are transiently expressed at different stages of osteoblast differentiation in culture, with maximal mRNAs levels of Suv39h1 and Suv39h2 (at day 3) preceding maximal expression of Suv420h1 and Suv420h2 (at day 7) and developmental stages that reflect, respectively, early and later collagen matrix deposition. Loss of function analysis of Suv420h2 by siRNA depletion shows loss of H4K20 methylation and decreased expression of bone biomarkers (e.g., alkaline phosphatase/Alpl) and osteogenic transcription factors (e.g., Sp7/Osterix). Furthermore, Suv420h2 is required for matrix mineralization during osteoblast differentiation. We conclude that Suv420h2 controls the H4K20 methylome of osteoblasts and is critical for normal progression of osteoblastogenesis. J. Cell. Biochem. 118: 1262-1272, 2017. © 2016 Wiley Periodicals, Inc.

Wnt/ß-Catenin Signaling Activates Expression of the Bone-Related Transcription Factor RUNX2 in Select Human Osteosarcoma Cell Types.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over-expression of the runt-related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/ß-catenin signaling is over-activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/ß-catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/ß-catenin pathway controls the tumor-related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of ß-catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, ß-catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of ß-catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3ß inhibitor SB216763. Furthermore, small interfering RNA (siRNA)-mediated depletion of ß-catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/ß-catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662-3674, 2017. © 2017 Wiley Periodicals, Inc.

Proteomic Analysis of Exosomes and Exosome-Free Conditioned Media From Human Osteosarcoma Cell Lines Reveals Secretion of Proteins Related to Tumor Progression.

Osteosarcomas are the most prevalent bone tumors in pediatric patients, but can also occur later in life. Bone tumors have the potential to metastasize to lung and occasionally other vital organs. To understand how osteosarcoma cells interact with their micro-environment to support bone tumor progression and metastasis, we analyzed secreted proteins and exosomes from three human osteosarcoma cell lines. Exosome isolation was validated by transmission electron microscopy (TEM) and immuno-blotting for characteristic biomarkers (CD63, CD9, and CD81). Exosomal and soluble proteins (less than 100 kDa) were identified by mass spectrometry analysis using nanoLC-MS/MS and classified by functional gene ontology clustering. We identified a secretome set of >3,000 proteins for both fractions, and detected proteins that are either common or unique among the three osteosarcoma cell lines. Protein ontology comparison of proteomes from exosomes and exosome-free fractions revealed differences in the enrichment of functional categories associated with different biological processes, including those related to tumor progression (i.e., angiogenesis, cell adhesion, and cell migration). The secretome characteristics of osteosarcoma cells are consistent with the pathological properties of tumor cells with metastatic potential. J. Cell. Biochem. 118: 351-360, 2017. © 2016 Wiley Periodicals, Inc.

Statin and Bisphosphonate Induce Starvation in Fast-Growing Cancer Cell Lines.

Statins and bisphosphonates are increasingly recognized as anti-cancer drugs, especially because of their cholesterol-lowering properties. However, these drugs act differently on various types of cancers. Thus, the aim of this study was to compare the effects of statins and bisphosphonates on the metabolism (NADP⁺/NADPH-relation) of highly proliferative tumor cell lines from different origins (PC-3 prostate carcinoma, MDA-MB-231 breast cancer, U-2 OS osteosarcoma) versus cells with a slower proliferation rate like MG-63 osteosarcoma cells. Global gene expression analysis revealed that after 6 days of treatment with pharmacologic doses of the statin simvastatin and of the bisphosphonate ibandronate, simvastatin regulated more than twice as many genes as ibandronate, including many genes associated with cell cycle progression. Upregulation of starvation-markers and a reduction of metabolism and associated NADPH production, an increase in autophagy, and a concomitant downregulation of H3K27 methylation was most significant in the fast-growing cancer cell lines. This study provides possible explanations for clinical observations indicating a higher sensitivity of rapidly proliferating tumors to statins and bisphosphonates.