The Melatonin Osteoporosis Prevention Study (MOPS) demonstrated that nightly melatonin resulted in a time‐dependent decrease in equilibrium ratios of serum osteoclasts and osteoblasts in ...perimenopausal women. This study examines mechanisms related to the ratios of osteoblasts and osteoclasts using coculture models (transwell or layered) of human mesenchymal stem cell (MSC) and human peripheral blood monocytes (PBMCs). Human MSC/PBMC cocultures exposed to melatonin in osteogenic (OS+) medium for 21 days induced osteoblast differentiation and mineralization; however, only in layered cocultures did melatonin inhibit osteoclastogenesis. Melatonin effects were mediated through MT2 melatonin receptors, MEK1/2, and MEK5. In layered but not transwell cocultures, melatonin increased OPG:RANKL ratios by inhibiting RANKL, suggesting that contact with osteoclasts during osteoblastogenesis inhibits RANKL secretion. Melatonin modulated expression of ERK1/2, ERK5, β1 integrin, GLUT4, and IRβ that was dependent upon the type of coculture; however, in both cultures, melatonin increased RUNX2 and decreased PPARγ expression, indicating a role for metabolic processes that control osteogenic vs adipogenic cell fates of MSCs. Furthermore, melatonin also has osteoblast‐inducing effects on human adipose‐derived MSCs. In vivo, one‐year nightly melatonin (15 mg/L) given to neu female mice in their drinking water increased pErk1/2, pErk5, Runx2, and Opg and Rankl levels in bone consistent with melatonin's already reported bone‐enhancing effects. Finally, analysis of daily logs from the MOPS demonstrated a significant improvement in mood and perhaps sleep quality in women receiving melatonin vs placebo. The osteoblast‐inducing, bone‐enhancing effects of melatonin and improvement in quality of life suggest that melatonin is a safe and effective bone loss therapy.
Actin structure contributes to physiologic events within the nucleus to control mesenchymal stromal cell (MSC) differentiation. Continuous cytochalasin D (Cyto D) disruption of the MSC actin ...cytoskeleton leads to osteogenic or adipogenic differentiation, both requiring mass transfer of actin into the nucleus. Cyto D remains extranuclear, thus intranuclear actin polymerization is potentiated by actin transfer: we asked whether actin structure affects differentiation. We show that secondary actin filament branching via the Arp2/3 complex is required for osteogenesis and that preventing actin branching stimulates adipogenesis, as shown by expression profiling of osteogenic and adipogenic biomarkers and unbiased RNA‐seq analysis. Mechanistically, Cyto D activates osteoblast master regulators (e.g., Runx2, Sp7, Dlx5) and novel coregulated genes (e.g., Atoh8, Nr4a3, Slfn5). Formin‐induced primary actin filament formation is critical for Arp2/3 complex recruitment: osteogenesis is prevented by silencing of the formin mDia1, but not its paralog mDia2. Furthermore, while inhibition of actin, branching is a potent adipogenic stimulus, silencing of either mDia1 or mDia2 blocks adipogenic gene expression. We propose that mDia1, which localizes in the cytoplasm of multipotential MSCs and traffics into the nucleus after cytoskeletal disruption, joins intranuclear mDia2 to facilitate primary filament formation before mediating subsequent branching via Arp2/3 complex recruitment. The resulting intranuclear branched actin network specifies osteogenic differentiation, while actin polymerization in the absence of Arp2/3 complex‐mediated secondary branching causes adipogenic differentiation. Stem Cells 2017;35:1624–1635
In mesenchymal stem cells, cytochalasin D treatment causes rapid cofilin/importin‐9 dependent transfer of G‐actin into the nucleus where intranuclear actin polymerization supports differentiation. End‐on‐end actin fiber formation facilitiates access of Pparg to adipogenic gene targets, while branching of actin polymers is critical for osteogenic differentiation. When secondary actin branching is prevented by inhibition of Arp2/3, adipogenesis predominates.•
Epigenetic mechanisms regulate osteogenic lineage differentiation of mesenchymal stromal cells. Histone methylation is controlled by multiple lysine demethylases and is an important step in ...controlling local chromatin structure and gene expression. Here, we show that the lysine-specific histone demethylase Kdm1A/Lsd1 is abundantly expressed in osteoblasts and that its suppression impairs osteoblast differentiation and bone nodule formation in vitro. Although Lsd1 knockdown did not affect global H3K4 methylation levels, genome-wide ChIP-Seq analysis revealed high levels of Lsd1 at gene promoters and its binding was associated with di- and tri-methylation of histone 3 at lysine 4 (H3K4me2 and H3K4me3). Lsd1 binding sites in osteoblastic cells were enriched for the Runx2 consensus motif suggesting a functional link between the two proteins. Importantly, inhibition of Lsd1 activity decreased osteoblast activity in vivo. In support, mesenchymal-targeted knockdown of Lsd1 led to decreased osteoblast activity and disrupted primary spongiosa ossification and reorganization in vivo. Together, our studies demonstrate that Lsd1 occupies Runx2-binding cites at H3K4me2 and H3K4me3 and its activity is required for proper bone formation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bone marrow adipocytes (BMAd) have recently been implicated in accelerating bone metastatic cancers, such as acute myelogenous leukemia and breast cancer. Importantly, bone marrow adipose tissue ...(BMAT) expands with aging and obesity, two key risk factors in multiple myeloma disease prevalence, suggesting that BMAds may influence and be influenced by myeloma cells in the marrow. Here, we provide evidence that reciprocal interactions and cross-regulation of myeloma cells and BMAds play a role in multiple myeloma pathogenesis and treatment response. Bone marrow biopsies from patients with multiple myeloma revealed significant loss of BMAT with myeloma cell infiltration of the marrow, whereas BMAT was restored after treatment for multiple myeloma. Myeloma cells reduced BMAT in different preclinical murine models of multiple myeloma and
using myeloma cell-adipocyte cocultures. In addition, multiple myeloma cells altered adipocyte gene expression and cytokine secretory profiles, which were also associated with bioenergetic changes and induction of a senescent-like phenotype.
, senescence markers were also increased in the bone marrow of tumor-burdened mice. BMAds, in turn, provided resistance to dexamethasone-induced cell-cycle arrest and apoptosis, illuminating a new possible driver of myeloma cell evolution in a drug-resistant clone. Our findings reveal that bidirectional interactions between BMAds and myeloma cells have significant implications for the pathogenesis and treatment of multiple myeloma. Targeting senescence in the BMAd or other bone marrow cells may represent a novel therapeutic approach for treatment of multiple myeloma. SIGNIFICANCE: This study changes the foundational understanding of how cancer cells hijack the bone marrow microenvironment and demonstrates that tumor cells induce senescence and metabolic changes in adipocytes, potentially driving new therapeutic directions.
Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained ...underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.
Osteogenic differentiation is initiated by transcriptional and post-transcriptional epigenetic mechanisms.
Inhibition of H3K27 methyltransferase EZH2 enhances osteogenic commitment of human mesenchymal progenitors, and its depletion in mouse mesenchymal cells causes multiple skeletal abnormalities.
EZH2 is required for skeletal patterning and bone formation.
EZH2-dependent epigenetic mechanisms control osteogenesis both in vitro and in vivo.
Vitamin C deficiency disrupts the integrity of connective tissues including bone. For decades this function has been primarily attributed to Vitamin C as a cofactor for collagen maturation. Here, we ...demonstrate that Vitamin C epigenetically orchestrates osteogenic differentiation and function by modulating chromatin accessibility and priming transcriptional activity. Vitamin C regulates histone demethylation (H3K9me3 and H3K27me3) and promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers and super-enhancers near bone-specific genes. This epigenetic circuit licenses osteoblastogenesis by permitting the expression of all major pro-osteogenic genes. Osteogenic cell differentiation is strictly and continuously dependent on Vitamin C, whereas Vitamin C is dispensable for adipogenesis. Importantly, deletion of 5hmC-writers, Tet1 and Tet2, in Vitamin C-sufficient murine bone causes severe skeletal defects which mimic bone phenotypes of Vitamin C-insufficient Gulo knockout mice, a model of Vitamin C deficiency and scurvy. Thus, Vitamin C's epigenetic functions are central to osteoblastogenesis and bone formation and may be leveraged to prevent common bone-degenerating conditions.
The biological interface between an orthopedic implant and the surrounding host tissue may have a dramatic effect upon clinical outcome. Desired effects include bony ingrowth (osseointegration), ...stimulation of osteogenesis (osteoinduction), increased vascularization, and improved mechanical stability. Implant loosening, fibrous encapsulation, corrosion, infection, and inflammation, as well as physical mismatch may have deleterious clinical effects. This is particularly true of implants used in the reconstruction of load-bearing synovial joints such as the knee, hip, and the shoulder. The surfaces of orthopedic implants have evolved from solid-smooth to roughened-coarse and most recently, to porous in an effort to create a three-dimensional architecture for bone apposition and osseointegration. Total joint surgeries are increasingly performed in younger individuals with a longer life expectancy, and therefore, the postimplantation lifespan of devices must increase commensurately. This review discusses advancements in biomaterials science and cell-based therapies that may further improve orthopedic success rates. We focus on material and biological properties of orthopedic implants fabricated from porous metal and highlight some relevant developments in stem-cell research. We posit that the ideal primary and revision orthopedic load-bearing metal implants are highly porous and may be chemically modified to induce stem cell growth and osteogenic differentiation, while minimizing inflammation and infection. We conclude that integration of new biological, chemical, and mechanical methods is likely to yield more effective strategies to control and modify the implant-bone interface and thereby improve long-term clinical outcomes.
Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and ...low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.
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.
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