van Wijnen, Andre J

Bone marrow mesenchymal stem cells exist in a multipotential state, where osteogenic and adipogenic genomes are silenced in heterochromatin at the inner nuclear leaflet. Physical force, generated in the marrow space during dynamic exercise exerts control overexpression of differentiation. Mesenchymal stem cells experience mechanical force through their cytoskeletal attachments to substrate, inducing signaling that alters gene expression. The generated force is further transferred from the cytoskeleton to the nucleoskeleton through tethering of actin to Linker of Nucleus and Cytoskeleton (LINC) complexes. Forces exerted on LINC alter the shape and placement of the nucleus within the cell, and are ultimately transferred into the nucleus. LINC complexes transverse the nuclear membrane and connect to the internal nucleoskeleton that is made up of lamin filaments and actin. Force transfer through LINC thus causes structural rearrangements of the nuclear scaffolding upon which chromosomes are arranged. Gene availability is not only modulated through heterochromatin remodeling enzymes and active transcription factors but also by control of nucleoskeletal structure and nuclear enzymes that mediate actin polymerization in the nucleus. Nuclear actin structure may be affected by similar force-activated pathways as those controlling the cytoplasmic actin cytoskeleton and represent a critical determinant of mesenchymal stem cell lineage commitment. Impact statement Gene expression is controlled by nuclear structure which is modulated by both internal and external forces exerted on the nucleoskeleton. Extracellular forces experienced through the actin cytoskeleton are transmitted to the internal nucleoskeleton via Linker of Nucleus and Cytoskeleton (LINC) protein connections. LINC complexes directly alter nuclear shape and entry of molecules that regulate transcription. New mechanistic models indicate that nuclear actin is a dynamic component of the filamentous nucleoskeleton and modified by an intranuclear “actin toolbox”, a set of enzymes that regulate linear and branched polymerization of nuclear actin. External stimulation of both biomechanical and biochemical pathways alters nuclear actin structure and has profound effects on gene expression by controlling chromatin architecture and transcription factor access to gene targets. The available data indicate that nucleoskeletal control of gene expression is critical for self-renewal and mesenchymal lineage-allocation in stem cells.

ABSTRACTSkeletal regenerative medicine aims to repair or regenerate skeletal tissues using pharmacotherapies, cell‐based treatments, and/or surgical interventions. The field is guided by biological principles active during development, wound healing, aging, and carcinogenesis. Skeletal development and tissue maintenance in adults represent highly intricate biological processes that require continuous adjustments in the expression of cell type‐specific genes that generate, remodel, and repair the skeletal extracellular matrix. Errors in these processes can facilitate musculoskeletal disease including cancers or injury. The fundamental molecular mechanisms by which cell type‐specific patterns in gene expression are established and retained during successive mitotic divisions require epigenetic control, which we review here. We focus on epigenetic regulatory proteins that control the mammalian epigenome at the level of chromatin with emphasis on proteins that are amenable to drug intervention to mitigate skeletal tissue degeneration (e.g., osteoarthritis and osteoporosis). We highlight recent findings on a number of druggable epigenetic regulators, including DNA methyltransferases (e.g., DNMT1, DNMT3A, and DNMT3B) and hydroxylases (e.g., TET1, TET2, and TET3), histone methyltransferases (e.g., EZH1, EZH2, and DOT1L) as well as histone deacetylases (e.g., HDAC3, HDAC4, and HDAC7) and histone acetyl readers (e.g., BRD4) in relation to the development of bone or cartilage regenerative drug therapies. We also review how histone mutations lead to epigenomic catastrophe and cause musculoskeletal tumors. The combined body of molecular and genetic studies focusing on epigenetic regulators indicates that these proteins are critical for normal skeletogenesis and viable candidate drug targets for short‐term local pharmacological strategies to mitigate musculoskeletal tissue degeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1465–1474, 2019.

Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial‐to‐mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer‐compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.

ObjectiveTo assess the biologic effects of lidocaine on the viability, proliferation, and function of human adipose tissue–derived mesenchymal stromal/stem cells (MSCs) in vitro.MethodsAdipose‐derived MSCs from three donors were exposed to lidocaine at various dilutions (2 mg/mL to 8 mg/mL) and exposure times (0.5 to 4 hours). Cell number and viability, mitochondrial activity, and real‐time reverse‐transcriptase quantitative polymerase chain reaction (RT‐qPCR) were analyzed at 0 (immediate effects) or 24 and 48 hours (recovery effects) after treatment with lidocaine.ResultsTrypan blue staining showed that increasing concentrations of lidocaine decreased the number of observable viable cells. 3‐[4,5,dimethylthiazol‐2‐yl]‐5‐[3‐carboxymethoxy‐phenyl]‐2‐[4‐sulfophenyl]‐2H‐tetrazolium (MTS) assays revealed a concentration‐ and time‐ dependent decline of mitochondrial activity and proliferative ability. Gene expression analysis by RT‐qPCR revealed that adipose‐derived MSCs exposed to lidocaine express robust levels of stress response/cytoprotective genes. However, higher concentrations of lidocaine caused a significant downregulation of these genes. No significant differences were observed in expression of extracellular matrix (ECM) markers COL1A1 and DCN except for COL3A1 (P < .05). Levels of messenger RNA (mRNA) for proliferation markers (CCNB2, HIST2H4A, P < .001) and MKI67 (P < .001) increased at 24 and 48 hours. Expression levels of several transcription factors— including SP1, PRRX1, and ATF1—were modulated in the same manner. MSC surface markers CD44 and CD105 demonstrated decreased expression immediately after treatment, but at 24 and 48 hours postexposure, the MSC markers showed no significant difference among groups.ConclusionLidocaine is toxic to MSCs in a dose‐ and time‐ dependent manner. MSC exposure to high (4‐8 mg/mL) concentrations of lidocaine for prolonged periods can affect their biologic functions. Although the exposure time in vivo is short, it is essential to choose safe concentrations when applying lidocaine along with MSCs to avoid compromising the viability and potency of the stem cell therapy.

Abstract 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.

AbstractThe RUNX1 transcription factor has recently been shown to be obligatory for normal development. RUNX1 controls the expression of genes essential for proper development in many cell lineages and tissues including blood, bone, cartilage, hair follicles, and mammary glands. Compromised RUNX1 regulation is associated with many cancers. In this review, we highlight evidence for RUNX1 control in both invertebrate and mammalian development and recent novel findings of perturbed RUNX1 control in breast cancer that has implications for other solid tumors. As RUNX1 is essential for definitive hematopoiesis, RUNX1 mutations in hematopoietic lineage cells have been implicated in the etiology of several leukemias. Studies of solid tumors have revealed a context‐dependent function for RUNX1 either as an oncogene or a tumor suppressor. These RUNX1 functions have been reported for breast, prostate, lung, and skin cancers that are related to cancer subtypes and different stages of tumor development. Growing evidence suggests that RUNX1 suppresses aggressiveness in most breast cancer subtypes particularly in the early stage of tumorigenesis. Several studies have identified RUNX1 suppression of the breast cancer epithelial‐to‐mesenchymal transition. Most recently, RUNX1 repression of cancer stem cells and tumorsphere formation was reported for breast cancer. It is anticipated that these new discoveries of the context‐dependent diversity of RUNX1 functions will lead to innovative therapeutic strategies for the intervention of cancer and other abnormalities of normal tissues.

Abstract Background The use of mesenchymal stem cells (MSCs) in clinical applications for the treatment of musculoskeletal disease is steadily increasing in office-based practice. The so-called “first generation” of MSCs is defined as autologous stem cells that have undergone minimal manipulation and are used for a homologous purpose. Systematic reviews of the clinical trials completed to date of such MSCs enable practitioners to better understand what is currently known about the outcomes and side effects of such treatments. Study Design A systematic review of human clinical studies of office-based MSC therapy for the treatment of painful degenerative musculoskeletal conditions. Methods A search of the Ovid MEDLINE, EMBASE, and Scopus databases was conducted from 2006 through September 2016. Seven hundred sixty-one records were identified from database searching, and two records from reference review of included papers. Studies with human subjects that evaluated treatment of musculoskeletal disease with minimally manipulated MSCs were included. Results Eight studies were included in this review based on selection criteria. A total of 941 patients were included, 841 of whom received cellular products, and no significant adverse events were reported. Symptomatology generally improved, though no differences were seen over controls where present. Conclusion Support in the literature is strongest for the use of bone marrow aspirate concentrate (BMAC) injections for the treatment of knee pain, but applications of the use of BMAC and peripheral blood–derived MSCs for the treatment of hip pain, tendon pain, and disc pain have all been reported. Further research is required, with large randomized controlled trials.

ABSTRACT The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell‐released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV‐associated functional activities. Finally, a checklist is provided with summaries of key points.

ABSTRACT Developing novel approaches to treat skeletal disorders requires an understanding of how critical molecular factors regulate osteoblast differentiation and bone remodeling. We have reported that (1) retinoic acid receptor-related orphan receptor beta (Rorβ) is upregulated in bone samples isolated from aged mice and humans in vivo; (2) Rorβ expression is inhibited during osteoblastic differentiation in vitro; and (3) genetic deletion of Rorβ in mice results in preservation of bone mass during aging. These data establish that Rorβ inhibits osteogenesis and that strict control of Rorβ expression is essential for bone homeostasis. Because microRNAs (miRNAs) are known to play important roles in the regulation of gene expression in bone, we explored whether a predicted subset of nine miRNAs regulates Rorβ expression during both osteoblast differentiation and aging. Mouse osteoblastic cells were differentiated in vitro and assayed for Rorβ and miRNA expression. As Rorβ levels declined with differentiation, the expression of many of these miRNAs, including miR-219a-5p, was increased. We further demonstrated that miR-219a-5p was decreased in bone samples from old (24-month) mice, as compared with young (6-month) mice, concomitant with increased Rorβ expression. Importantly, we also found that miR-219a-5p expression was decreased in aged human bone biopsies compared with young controls, demonstrating that this phenomenon also occurs in aging bone in humans. Inhibition of miR-219a-5p in mouse calvarial osteoblasts led to increased Rorβ expression and decreased alkaline phosphatase expression and activity, whereas a miR-219a-5p mimic decreased Rorβ expression and increased osteogenic activity. Finally, we demonstrated that miR-219a-5p physically interacts with Rorβ mRNA in osteoblasts, defining Rorβ as a true molecular target of miR-219a-5p. Overall, our findings demonstrate that miR-219a-5p is involved in the regulation of Rorβ in both mouse and human bone. © 2018 American Society for Bone and Mineral Research.