The microRNA miR-134-5p induces calcium deposition by inhibiting histone deacetylase 5 in vascular smooth muscle cells.

Calcium deposition in vascular smooth muscle cells (VSMCs) is a form of ectopic ossification in blood vessels. It can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that the microRNA miR-134-5p potentiates inorganic phosphate (Pi)-induced calcium deposition in VSMCs by inhibiting histone deacetylase 5 (HDAC5). Using miRNA microarray analysis of Pi-treated rat VSMCs, we first selected miR-134-5p for further evaluation. Quantitative RT-PCR confirmed that miR-134-5p was increased in Pi-treated A10 cells, a rat VSMC line. Transfection of miR-134-5p mimic potentiated the Pi-induced increase in calcium contents. miR-134-5p increased the amounts of bone runt-related transcription factor 2 (RUNX2) protein and bone morphogenic protein 2 (BMP2) mRNA in the presence of Pi but decreased the expression of osteoprotegerin (OPG). Bioinformatic analysis showed that the HDAC5 3'untranslated region (3'UTR) was one of the targets of miR-134-5p. The luciferase construct containing the 3'UTR of HDAC5 was down-regulated by miR-134-5p mimic in a dose-dependent manner in VSMCs. Overexpression of HDAC5 mitigated the calcium deposition induced by miR-134-5p. Our results suggest that a Pi-induced increase of miR-134-5p may cause vascular calcification through repression of HDAC5.

Twist1 and Twist2 Contribute to Cytokine Downregulation following Chronic NOD2 Stimulation of Human Macrophages through the Coordinated Regulation of Transcriptional Repressors and Activators.

Proper regulation of microbial-induced cytokines is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, chronic NOD2 stimulation in macrophages decreases cytokines upon pattern recognition receptor (PRR) restimulation; cytokine attenuation to PRR stimulation is similarly observed in intestinal macrophages. The role for the transcriptional repressors Twist1 and Twist2 in regulating PRR-induced cytokine outcomes is poorly understood and has not been reported for NOD2. We found that Twist1 and Twist2 were required for optimal cytokine downregulation during acute and, particularly, chronic NOD2 stimulation of human macrophages. Consistently, Twist1 and Twist2 expression was increased after chronic NOD2 stimulation; this increased expression was IL-10 and TGF-ß dependent. Although Twist1 and Twist2 did not coregulate each other's expression, they cooperated to enhance binding to cytokine promoters after chronic NOD2 stimulation. Moreover, Twist1 and Twist2 contributed to enhance expression and promoter binding of the proinflammatory inhibitor c-Maf and the transcriptional repressor Bmi1. Restoring c-Maf and Bmi1 expression in Twist-deficient macrophages restored NOD2-induced cytokine downregulation. Furthermore, with chronic NOD2 stimulation, Twist1 and Twist2 contributed to the decreased expression and cytokine promoter binding of the transcriptional activators activating transcription factor 4, C/EBPα, Runx1, and Runx2. Knockdown of these transcriptional activators in Twist-deficient macrophages restored cytokine downregulation after chronic NOD2 stimulation. Finally, NOD2 synergized with additional PRRs to increase Twist1 and Twist2 expression and Twist-dependent pathways. Therefore, after chronic NOD2 stimulation Twist1 and Twist2 coordinate the regulation of both transcriptional activators and repressors, thereby mediating optimal cytokine downregulation.

Silencing of Long Non-Coding RNA NONHSAT009968 Ameliorates the Staphylococcal Protein A-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells.

BACKGROUND/AIMS: Osteomyelitis is defined as an inflammation of the bones and bone marrow. The inflammatory microenvironment attenuates the osteogenic differentiation capacity of stem cells and inhibits osteoblast-mediated bone formation, leading to net bone loss. However, the whole expression profile, function and side effect of long non-coding RNAs (lncRNAs) on osteogenic differentiation of stem cells in an inflammatory microenvironment of osteomyelitis are not known. METHODS: In the present study, human bone mesenchymal stem cells (hBMSCs) were treated with different concentrations of Staphylococcal protein A (SpA) to trigger an inflammatory microenvironment in vitro to partly duplicate the inflammatory microenvironment of osteomyelitis, which was confirmed using ELISA for detecting the inflammatory cytokines. The complete expression profiles of lncRNAs and mRNA during osteogenic differentiation of hBMSCs in an inflammatory microenvironment triggered by SpA were analyzed using a lncRNA microarray. LncRNA expression levels were verified by quantitative reverse transcription PCR analysis (qRT-PCR). The expression of NONHSAT009968 in hB-MSCs was silenced by infection with lentivirus expressing NONHSAT009968-shRNA. The expression of Runx2, OCN, OPN, COL1A1, and alkaline phosphatase (ALP) activity was detected by western blot. Alizarin red staining and ALP activity detection were carried out. RESULTS: The results of ELISA showed that SpA treatment induced secretion of inflammatory cytokines IL-1A, IL-6, and TNFA. The results of alizarin red staining and ALP detection showed that SpA treatment suppressed the osteogenic differentiation of hBMSCs. A total of 2033 lncRNAs were found with aberrant expression in SpA-treated hBMSCs compared to controls. Among these lncRNAs, 641 were down-regulated and 1392 were up-regulated. Based on the results of qRT-PCR, lncRNA NONHSAT009968 was chosen for further investigation. The results of alizarin red staining, ALP activity detection, and western blot detection of Runx2, OCN, OPN, COL1A1, and ALP indicated that NONHSAT009968 silencing ameliorates SpA-inhibited osteogenic differentiation in hBMSCs. CONCLUSION: Our present study provides a basis for future analyses of the role of lncRNAs in osteoblastic differentiation in an inflammatory environment triggered by SpA, and lncRNA NONHSAT009968 might be a new target for promoting osteoblast formation.

Inhibition of osteoclastogenesis by osteoblast-like cells genetically engineered to produce interleukin-10.

Bone destruction at inflamed joints is an important complication associated with rheumatoid arthritis (RA). Interleukin-10 (IL-10) may suppress not only inflammation but also induction of osteoclasts that play key roles in the bone destruction. If IL-10-producing osteoblast-like cells are induced from patient somatic cells and transplanted back into the destructive bone lesion, such therapy may promote bone remodeling by the cooperative effects of IL-10 and osteoblasts. We transduced mouse fibroblasts with genes for IL-10 and Runx2 that is a crucial transcription factor for osteoblast differentiation. The IL-10-producing induced osteoblast-like cells (IL-10-iOBs) strongly expressed osteoblast-specific genes and massively produced bone matrix that were mineralized by calcium phosphate in vitro and in vivo. Culture supernatant of IL-10-iOBs significantly suppressed induction of osteoclast from RANKL-stimulated Raw264.7 cells as well as LPS-induced production of inflammatory cytokine by macrophages. The IL-10-iOBs may be applicable to novel cell-based therapy against bone destruction associated with RA.

Depletion of Intestinal Microbiome Partially Rescues Bone Loss in Sickle Cell Disease Male Mice.

Osteoporosis or osteopenia are common clinical manifestations of sickle cell disease (SCD) with unclear mechanisms. Since senescence of circulating neutrophil can be modulated by signals derived from intestinal microbiome and neutrophils are abundant in bone marrow and can regulate osteoblasts and osteoclasts, we examined whether gut microbiome contributes to bone loss in SCD mice. SCD and their littermates control mice were treated with antibiotics to deplete gut microbiome. At the end of 7 weeks treatment, serum was collected for biochemistry marker measurements. Bone mass and remodeling were evaluated by dual beam X-ray absorptiometry, micro-computed tomography, and histomorphometry. Bone-related genes in tibia and barrier marker genes in the small intestine were analyzed by quantitative PCR. Antibiotic treatment rescued increased intestinal inflammatory cytokine marker genes (Tnfα, IL17, Ifnγ) expression, rescued decreased intestinal barrier marker genes (claudin 3 and claudin 15) expression, and rescued increased serum cytokines (IFNγ, IL27, IL10) in SCD mice. Antibiotic significantly improved decreased bone mass in SCD mice mainly through enhanced osteoblast function and increased osteoblast-related genes (Runx2 and Igf1) expression in SCD mice. Our findings support that increased bacteria load augments antigenic load traversing the impaired intestinal barrier through inflammation, leading to increased inflammatory cytokines, impaired osteoblast function, and bone loss in SCD mice.

Characterization of embryonic stem cell-differentiated fibroblasts as mesenchymal stem cells with robust expansion capacity and attenuated innate immunity.

BACKGROUND: Mesenchymal stem cells (MSCs) isolated from adult tissues (Ad-MSCs) have shown great promise for use in regenerative medicine. However, their poor in vitro expansion capacity and tissue scarcity have been major limitations. In this study, we demonstrate that mouse embryonic stem cells (mESCs) can differentiate into cells with MSC properties. METHODS: Using previously established methods that characterize Ad-MSCs, we analyzed mESC-differentiated fibroblasts (mESC-FBs), including plastic adherence, clonogenic growth, MSC marker expression, tri-lineage differentiation potential, and the capacity to express immunomodulators. RESULTS: Although previously characterized as mESC-differentiated fibroblasts (mESC-FBs), these cells exhibit major properties of Ad-MSCs. However, mESC-FBs also display unique features inherited from ESCs, including robust expansion capacity, senescence resistance, and attenuated innate immunity. In particular, mESC-FBs are insensitive to bacterial endotoxin (lipopolysaccharide, LPS) and do not express LPS-induced inflammatory molecules, in contrast to bone marrow (BM)-MSCs. We further demonstrate that mESC-FBs are resistant to the cytotoxicity associated with inflammatory cytokines, bacterial endotoxins (LPS and heat-killed bacteria), and macrophage-mediated inflammation. CONCLUSIONS: While it remains to be determined how the unique properties of mESC-FBs will affect their immunoregulatory activity under an in vivo condition, our findings demonstrate that ESCs could be used as an alternative source to generate a new class of ESC-MSCs with unique features potentially useful in regenerative medicine.

Epigenetic mechanisms underlying the aberrant catabolic and anabolic activities of osteoarthritic chondrocytes.

The development of disease-modifying pharmacologic therapy for osteoarthritis currently faces major obstacles largely because the pathogenetic mechanisms for the development of osteoarthritis remain unclear. Previous studies suggest that the alterations in expression of catabolic and anabolic genes in articular chondrocytes may be involved in the pathogenesis of osteoarthritis. However, the regulatory mechanisms for gene expression in osteoarthritic chondrocytes are largely unknown. The objective of this review is to highlight the recent studies on epigenetic regulation of gene expression in the development of osteoarthritis. The review will begin with current understanding of epigenetic mechanisms, especially the newly emerging areas including the regulatory role of non-coding RNAs in gene expression and crosstalk among the epigenetic mechanisms. The main content of this review focuses on the significance of epigenetic regulation of the expression of catabolic and anabolic genes in osteoarthritic chondrocytes, including the regulatory roles of various epigenetic mechanisms in the expression of genes for specific matrix-degrading proteinases, cytokines, and extracellular matrix proteins. Recent novel findings on the epigenetic regulation of specific transcription factor genes are particularly important for the understanding of osteoarthritis pathogenesis, as these transcription factors may act as upstream regulators of multiple catabolic and anabolic genes. In conclusion, these recent advances in epigenetic studies have shed light on the importance of epigenetic regulation of gene expression in the development of osteoarthritis, leading to a better understanding of the epigenetic mechanisms underlying the pathogenesis of osteoarthritis. This may promote the development of new epigenetics-based strategies for the treatment of osteoarthritis. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

A quantitative assay to study protein:DNA interactions, discover transcriptional regulators of gene expression, and identify novel anti-tumor agents.

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

Interactions between extracellular signal-regulated kinase 1/2 and p38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity.

RUNX2, a key transcription factor for osteoblast differentiation, is regulated by ERK1/2 and p38 MAP kinase-mediated phosphorylation. However, the specific contribution of each kinase to RUNX2-dependent transcription is not known. Here we investigate ERK and p38 regulation of RUNX2 using a unique P-RUNX2-specific antibody. Both MAP kinases stimulated RUNX2 Ser319 phosphorylation and transcriptional activity. However, a clear preference for ERK1 versus p38α/ß was found when the ability of these MAPKs to phosphorylate and activate RUNX2 was compared. Similarly, ERK1 preferentially bound to a consensus MAPK binding site on RUNX2 that was essential for the activity of either kinase. To assess the relative contribution of ERK1/2 and p38 to osteoblast gene expression, MC3T3-E1 preosteoblast cells were grown in control or ascorbic acid (AA)-containing medium ± BMP2/7. AA-induced gene expression, which requires collagen matrix synthesis, was associated with parallel increases in P-ERK and RUNX2-S319-P in the absence of any changes in P-p38. This response was blocked by ERK, but not p38, inhibition. Significantly, in the presence of AA, BMP2/7 synergistically stimulated RUNX2 S319 phosphorylation and transcriptional activity without affecting total RUNX2 and this response was totally dependent on ERK/MAPK activity. In contrast, although p38 inhibition partially blocked BMP-dependent transcription, it did not affect RUNX2 S319 phosphorylation, suggesting the involvement of other phosphorylation sites and/or transcription factors in this response. Based on this work, we conclude that extracellular matrix and BMP regulation of RUNX2 phosphorylation and transcriptional activity in osteoblasts is predominantly mediated by ERK rather than p38 MAPKs.

Immunologically restricted patients exhibit a pronounced inflammation and inadequate response to hypoxia in fracture hematomas.

For patients who are known to have an impaired immune system, bone healing is often impaired. Therefore, it has been suggested that an effectively functioning immune system will have an influence on the quality of bone healing. Here, we demonstrate that cells within the fracture hematoma of immunologically restricted patients (1) exhibit a disturbed osteogenic differentiation (normal SPP1 but diminished RUNX2 expression), (2) show a strong inflammatory reaction (high IL8 and CXCR4), and (3) react on local hypoxia (high expression of HIF1A) but with inadequate target gene responses (diminished LDHA and PGK1 expression). Thus, it is already within the early inflammatory phase of fracture healing that the local gene expression in fracture hematomas of immunologically restricted patients points toward a critical regeneration.

Immunolocalization and expression of Runx2 in tertiary dentinogenesis.

Runx2 is a new transcription factor that takes part in odontoblast differentiation. This study is aimed at investigating the immunolocalization and expression of Runx2 in the process of dental pulp injury and repair using immunohistochemical technique. In normal dental pulp, positive staining can hardly be detected. In experimental groups, strong positive staining was detected at the site of the impaired pulp after 1 day, while only weak Runx2 staining was detected 3 days after operation. Five days later, a large number of stellate cells in the root apex expressed Runx2, and after 7 days, followed by the reparative dentinogenesis, Runx2 expression vanished slowly, then totally disappeared. Taken together, the expression of Runx2 has temporal and spatial specificity during different phases in rat tertiary dentinogenesis.

Peripheral blood expression profiles of bone morphogenetic proteins, tumor necrosis factor-superfamily molecules, and transcription factor Runx2 could be used as markers of the form of arthritis, disease activity, and therapeutic responsiveness.

OBJECTIVE: To assess whether different forms of arthritis and disease activity could be distinguished by peripheral blood expression profiles of bone-regulatory factors including tumor necrosis factor (TNF)-superfamily [TNF-related apoptosis-inducing ligand (TRAIL), the Fas ligand (FasL), and the ligand for herpesvirus entry mediator (LIGHT)] and bone morphogenetic protein (BMP)-family members (BMP-2, BMP-4, BMP-6) as well as osteoblast differentiation gene Runx2. METHODS: Blood cells from healthy controls (n = 25) and patients at different disease stages with rheumatoid arthritis (RA; n = 49), osteoarthritis (OA; n = 17), or spondyloarthritis, including ankylosing spondylitis (AS; n = 27) or psoriatic arthritis (PsA; n = 23), were processed for quantitative polymerase chain reaction. Gene expression was assessed in comparison with control samples, correlated with clinical data of different forms of arthritis, and analyzed for discriminative efficacy between groups by receiver-operation characteristic (ROC) curves. Results were confirmed on diagnostic RA (n = 5) and AS (n = 8) samples. RESULTS: BMP-4, BMP-6, and Runx2 expressions were significantly decreased in patients with RA and OA versus controls. Patients with RA also had decreased FasL and LIGHT expression, while patients with AS had increased Runx2 expression. Negative correlation with disease activity was found for BMP-4, FasL, and Runx2 in RA and for Runx2 in PsA, while positive correlation was found for BMP-4 in PsA. Gene expression was higher in the therapy-resistant form of AS (for BMP-4, LIGHT, and Runx2) and in methotrexate-treated patients in RA (for BMP-2 and LIGHT). ROC curve analysis confirmed discrimination between groups, particularly decreased LIGHT and Runx2 for RA and increased Runx2 for AS. CONCLUSION: Our study identified BMP and Runx2 as possible biomarkers of bone metabolism in several forms of arthritis, while lower FasL and LIGHT were associated with RA. Correlation between gene expression and disease activity may be clinically useful in assessing therapeutic effectiveness and disease monitoring.