Paradise, Christopher R.

Skeletal development is controlled by epigenetic mechanisms that regulate chromatin compaction and suppress access to gene regulatory sequences. Post‐translational modifications in the N‐termini of histone 3 (H3), including tri‐methylated H3 lysine 9 (H3K9Me3) or lysine 27 (H3K27Me3) represent repressive chromatin marks that are recognized by a family of heterochromatin‐associated Chromobox (Cbx) proteins (i.e., Cbx1 to Cbx8). We show using RNA‐seq analysis that each of these Cbx isoforms is actively expressed in bone and osteoblasts with variable expression of distinct members (between 1 and 100 reads per million). The most prominently expressed Cbx member is Cbx3, which encodes a member of the heterochromatin protein 1 (HP1) family that interacts with H3K9me3. Cbx3 is known to be important for normal development while promoting neurogenesis, as well as renal and smooth muscle development. Therefore, we postulated that CBx3 may perform a similar role in osteoblastogenesis. We tested this hypothesis by both transient and stable knock‐down of Cbx3 by respectively siRNA or shRNA. RNA interference in MC3T3 osteoblasts effectively decreases Cbx3 mRNA levels by 2 to 5 fold, while increasing expression of bone‐related markers by RT‐qPCR (e.g., Alpl/alkaline phosphatase, Ibsp/bone sialoprotein, Bglap/osteocalcin, and Col1a1/Collagen Type I α1 chain). In addition, Cbx3 depletion increases mineral deposition as measured by Alizarin Red staining. Thus, Cbx3 normally appears to suppress osteoblastic differentiation, unlike its essential function in the neuronal differentiation. Because Cbx3 recognizes H3K9me3, we examined whether Cbx3 controls the levels of this histone mark. Loss of Cbx3 did not affect total H3K9me3 levels unless Cbx1 and Cbx5 were simultaneously depleted by siRNA. We propose that Cbx3 may suppress osteoblastogenesis by controlling select loci with H3K9me3 marks.

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.

AbstractTranscription 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.

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.

Objective This study aims to (1) determine and validate living cartilage allograft transplantation as a novel source for viable osteochondral allograft (OCA) tissues and (2) perform histologic and viability comparisons of living donor cartilage tissues to currently available clinical-grade standard processed grafts. Design Using healthy cartilage from well-preserved contralateral compartments in 27 patients undergoing total knee arthroplasty (TKA) and 10 clinical-grade OCA specimens obtained immediately following operative implantation, standard and living donor OCA quality was evaluated at the time of harvest and following up to 3 weeks of storage on the basis of macroscopic International Cartilage Repair Society grade, histology, and viability. Results Osteochondral samples demonstrated a consistent decrease in viability and histologic quality over the first 3 weeks of storage at 37°C, supporting the utility of an OCA paradigm shift toward early implantation, as was the clinical standard up until recent adoption of transplantation at 14 to 35 days following donor procurement. Samples from the 10 clinical-grade OCAs, implanted at an average of 23 days following graft harvest demonstrated a mean viable cell density of 45.6% at implantation, significantly lower ( P < 0.01) than the 93.6% viability observed in living donor allograft tissues. Conclusions Osteochondral tissue viability and histologic quality progressively decreases with ex vivo storage, even when kept at physiologic temperatures. Currently available clinical OCAs are stored for 2 to 5 weeks prior to implantation and demonstrate inferior viability to that of fresh osteochondral tissues that can be made available through the use of a living donor cartilage program.

ABSTRACTArthrofibrosis is a common complication following total knee arthroplasty caused by pathologic fibroblast activation and excessive collagen deposition around a synovial joint leading to debilitating loss of motion. Treatment options are limited because the pathologic mechanisms remain to be characterized. Dysregulation of the inflammatory cascade may lead to communication between myofibroblasts and immune cells triggering tissue metaplasia, and excessive collagen deposition described clinically as arthrofibrosis. We explored the novel use of celecoxib (selective cyclooxygenase‐2 [COX‐2] inhibitor) to disrupt the downstream effects of the post‐traumatic inflammatory cascade and inhibit scar tissue formation in a validated rabbit model of arthrofibrosis combined with new parameters for quantifying the stiffness of the posterior capsule. Biomechanical and molecular analyses, of contracted rabbit knee posterior capsule tissue after COX‐2 inhibition revealed increased maximal passive extension and down‐regulation of collagen messenger RNA compared with controls. Histopathologic examination suggested a trend of decreased quantities of dense fibrous connective tissue with COX‐2 inhibition. These data may suggest that inhibiting the inflammatory cascade could potentially reduce pathologic myofibroblast activation, thereby reducing scar tissue formation and increasing the range of motion in arthrofibrotic joints. Implementing a multi‐modal pharmacologic approach may simultaneously target numerous cellular components contributing to the complex process of arthrofibrogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2609–2620, 2019

ABSTRACTOsteogenic 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.

AbstractAdipose-derived mesenchymal stem cells (AMSCs) offer potential as a therapeutic option for clinical applications in musculoskeletal regenerative medicine because of their immunomodulatory functions and capacity for trilineage differentiation. In preparation for a phase I clinical trial using AMSCs to treat patients with osteoarthritis, we carried out preclinical studies to assess the safety of human AMSCs within the intra-articular joint space. Culture-expanded human AMSCs grown in human platelet-lysate were delivered via intra-articular injections into normal healthy rabbit knees and knees at risk for the development of osteoarthritis after bilateral medial anterior hemimeniscectomy. Treatment outcomes and safety were evaluated by assessing the general health, function, and behavior of the animals. Joint tissues were analyzed by x-ray, magnetic resonance imaging, and histopathology. Intra-articular AMSC therapy was well tolerated in this study. We did not observe adverse systemic reactions, nor did we find evidence of damage to intra-articular joint tissues. Thus, the data generated in this study show a favorable safety profile for AMSCs within the joint space in support of a phase I clinical trial evaluating the clinical utility of AMSCs to treat osteoarthritis.