Runx2 Activity Research Articles

TDAG51 (T-Cell Death-Associated Gene 51) Is a Key Modulator of Vascular Calcification and Osteogenic Transdifferentiation of Arterial Smooth Muscle Cells.

Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.

Adhesion, intracellular signalling and osteogenic differentiation of mesenchymal progenitor cells and preosteoblasts on poly(epsilon)caprolactone films functionalized by peptides derived from fibronectin and/or BMP-9.

Biomaterials that can control the behaviour of stem cells play a major role in regenerative medicine and tissue engineering. We previously showed that poly(epsilon)caprolactone (PCL) films functionalized with adhesive peptides containing sequences of both cell binding domain (RGD) and synergistic site (PHSRN) of the fibronectin (pFibro) enhanced the osteoblastic commitment of C3H10T1/2 mesenchymal progenitor cells (C3H10T1/2 cells) induced by soluble BMP-9 or its derived peptide SpBMP-9. Here, the effect of PCL films functionalized with pFibro and/or SpBMP-9 or its negative peptide NSpBMP-9 on adhesion and intracellular signalling of C3H10T1/2 cells was determined. The differentiation of adherent C3H10T1/2 cells and MC3T3-E1 preosteoblasts into osteoblasts was also analysed with or without IGF-2, since this growth factor can favour the osteoblastic differentiation induced by BMP-9. We found that pFibro and SpBMP-9 co-functionalization on PCL films promoted the adhesion of C3H10T1/2 cells with well-organized focal adhesion points and FAK activation. In these mesenchymal progenitor cells, PCL-SpBMP-9 and PCL-pFibro/SpBMP-9 induced the activation and nuclear translocation of Smad 1/5 after 4h, and enhanced the protein expression of RUNX2 (3 d) and alkaline phosphatase (ALP) activity (7 d), two markers of osteoblast differentiation. No PPARγ, a marker of adipogenic differentiation, was detected. C3H10T1/2 cells attached to PCL-SpBMP-9 also contained more SOX9, a marker of chondroblastic lineage, compared with other experimental conditions. The use of inactive peptides NSpBMP-9 confirmed the specificity and effectiveness of SpBMP-9 on cell adhesion, intracellular signalling and osteoblastic differentiation. Adding IGF-2 only significantly improved the differentiation of MC3T3-E1 preosteoblasts into osteoblasts as shown by the increase in gene expression encoding Osterix (mRNA Sp7) and ALP (mRNA Alpl), probably because of the lack of serum in the medium. Therefore, material surface co-functionalized with pFibro and SpBMP-9 could be most useful for developing scaffolds with both osseointegrative and osteoinductive properties for bone application and tissue engineering strategy when combined with IGF-2 in serum free medium.

G9a is involved in the regulation of cranial bone formation through activation of Runx2 function during development.

The methyltransferase G9a was originally isolated as a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9) to a dimethylated state (H3K9me2). Recent studies have revealed that G9a has multiple functions in various cells, including osteoblasts. Here, we investigated G9a function during cranial bone formation. Crossing Sox9-cre with G9aflox/flox (fl/fl) mice generated conditional knockout mice lacking G9a expression in Sox9-positive neural crest-derived bone cells. Sox9-Cre/G9afl/fl mice showed severe hypo-mineralization of cranial vault bones, including defects in nasal, frontal, and parietal bones with opened fontanelles. Cell proliferation was inhibited in G9a-deleted calvarial bone tissues. Expression levels of bone marker genes, i.e., alkaline phosphatase and osteocalcin, were suppressed, whereas Runx2 expression was not significantly decreased in those tissues. In vitro experiments using G9a-deleted calvarial osteoblasts showed decreased cell proliferation after G9a deletion. In G9a-deleted osteoblasts, expression levels of fibroblast growth factor receptors and several cyclins were suppressed. Moreover, the expression of bone marker genes was decreased, whereas Runx2 expression was not altered by G9a deletion in vitro. G9a enhanced the transcriptional activity of Runx2, whereas siRNA targeting G9a inhibited the transcriptional activity of Runx2 in C3H10T1/2 mesenchymal cells. We confirmed the direct association of endogenous Runx2 with G9a. Chromatin immunoprecipitation experiments showed that G9a bound to Runx2-target regions in promoters in primary osteoblasts. Furthermore, Runx2 binding to the osteocalcin promoter was abrogated in G9-deleted osteoblasts. These results suggest that G9a regulates proliferation and differentiation of cranial bone cells through binding to and activating Runx2.

CHD9 upregulates RUNX2 and has a potential role in skeletal evolution

BackgroundChanges in gene regulation are widely recognized as an important driver of adaptive phenotypic evolution. However, the specific molecular mechanisms that underpin such changes are still poorly understood. Chromatin state plays an essential role in gene regulation, by influencing the accessibility of coding loci to the transcriptional machinery. Changes in the function of chromatin remodellers are therefore strong candidates to drive changes in gene expression associated with phenotypic adaptation. Here, we identify amino acid homoplasies in the chromatin remodeller CHD9, shared between the extinct marsupial thylacine and eutherian wolf which show remarkable skull convergence. CHD9 is involved in osteogenesis, though its role in the process is still poorly understood. We examine whether CHD9 is able to regulate the expression of osteogenic target genes and examine the function of a key substitution in the CHD9 DNA binding domain.ResultsWe examined whether CHD9 was able to upregulate its osteogenic target genes, RUNX2, Osteocalcin (OC) and ALP in HEK293T cells. We found that overexpression of CHD9 upregulated RUNX2, the master regulator of osteoblast cell fate, but not the downstream genes OC or ALP, supporting the idea that CHD9 regulates osteogenic progenitors rather than terminal osteoblasts. We also found that the evolutionary substitution in the CHD9 DNA binding domain does not alter protein secondary structure, but was able to drive a small but insignificant increase in RUNX2 activation. Finally, CHD9 was unable to activate an episomal RUNX2 promoter-reporter construct, suggesting that CHD9 requires the full chromatin complement for its function.ConclusionsWe provide new evidence to the role of CHD9 in osteogenic differentiation through its newly observed ability to upregulate the expression of RUNX2. Though we were unable to identify significant functional consequences of the evolutionary substitution in HEK293T cells, our study provides important steps forward in the functional investigation of protein homoplasy and its role in developmental processes. Mutations in coding genes may be a mechanism for driving adaptive changes in gene expression, and their validation is essential towards determining the functional consequences of evolutionary homoplasy.

Rapid bone repair with the recruitment of CD206+M2-like macrophages using non-viral scaffold-mediated miR-133a inhibition of host cells

microRNAs offer vast therapeutic potential for multiple disciplines. From a bone perspective, inhibition of miR-133a may offer potential to enhance Runx2 activity and increase bone repair. This study aims to assess the therapeutic capability of antagomiR-133a delivery from collagen-nanohydroxyapatite (coll-nHA) scaffolds following cell-free implantation in rat calvarial defects (7 mm diameter). This is, to the best of our knowledge, the first report of successful in vivo antagomiR uptake in host cells of fully immunocompetent animals without distribution to other off-target tissues. Our results demonstrate the localized release of antagomiR-133a to the implant site at 1 week post-implantation with increased calcium deposits already evident in the antagomiR-133a loaded scaffolds at this early timepoint. This was followed by an approximate 2-fold increase in bone volume versus antagomiR-free scaffolds and a significant 10-fold increase over the empty defect controls, after just 4 weeks. An increase in host CD206+ cells suggests an accelerated pro-remodeling response by M2-like macrophages accompanying bone repair with this treatment. Overall, this non-viral scaffold-mediated antagomiR-133a delivery platform demonstrates capability to accelerate bone repair in vivo – without the addition of exogenous cells – and underlines the role of M2 macrophage-like cells in directing accelerated bone repair. Expanding the repertoire of this platform to deliver alternative miRNAs offers exciting possibilities for a variety of therapeutic indications. Statement of SignificancemicroRNAs, small non-coding RNA molecules involved in gene regulation, may have potential as a new class of bone healing therapeutics as they can enhance the regenerative capacity of bone-forming cells. We developed a collagen-nanohydroxyapatite-microRNA scaffold system to investigate whether miR133a inhibition can enhance osteogenesis in rat MSCs and ultimately accelerate endogenous bone repair by host cells in vivo without pre-seeding cells prior to implantation. Overall, this off-the-shelf, non-viral scaffold-mediated antagomiR-133a delivery platform demonstrates capability to accelerate bone repair in vivo – without the requirement of exogenous cells – and highlights the role of CD206+M2 macrophage-like cells in guiding accelerated bone repair. Translating the repertoire of this platform to deliver alternative miRNAs offers exciting possibilities for a vast myriad of therapeutic indications.

NAD-dependent deacetylase sirtuin-1 mediates the mechanical stress-induced degeneration of articular cartilage in osteoarthritis: mechanical stress stimulates the expression of osteogenic transcription factor RUNX2 via the srituin-1 activation in chondrocytes

Purpose: Mechanical stress is the major pathologic factors associated with osteoarthritis (OA). It has been indicated that the NAD-dependent deacetylase sirtuin-1 (Sirt-1) has an important role in human aging. Recent report demonstrated that SIRT-1 insufficiency induced the shear (mechanical) stress-induced vascular calcification and artherosclerosis in cardiovascular tissues, suggesting that SIRT-1 may be of benefit in the protection against vascular calcification and maintenance of cardiovascular function against shear stress. In addition, it has been suggested that Sirt-1 promotes osteogenic and chondrogenic differentiation of mesenchymal stem cells. However, the impact of Sirt-1 in the osteoarthritic chondrocyte state still remains unclear. We postulated that Sirt-1 regulates a mechanical stress-induced hypertrophic chondrocyte lineage and osteophyte formation through the activation of osteogenic transcription factor Runx2 in osteoarthritic chondrocytes. Since it is well known that Runx2 is a promotor of the activation of cartilage matrix degrading enzyme, matrix metalloproteinase (MMP)-13, Sirt-1 may also influence the expression of MMP-13 in chondrocytes, consequently facilitating the deterioration of articular cartilage in OA. To verify the impact of Sirt-1 in the pathology of OA, we investigated the mechanical stress-induced expressions of Sirt-1, Runx2 and MMP-13, and their correlations in human chondrocytes in OA. Methods: i) The expressions of Sirt-1 and Runx2 were histologically analyzed in the in vitro OA model (mechanical stress-culture system). ii) Human chondrocytes were isolated from articular cartilage tissues from OA patients who underwent the knee joint replacement surgery. Levels of expressions of Runx2, Sirt-1 and MMP-13 were analyzed in the presence or absence of mechanical stress (25 gf/cm2, 2.5 kPa), in chondrocytes. The impact of Sirt-1 insufficiency in the chondrocyte activity was also examined in chondrocytes cultures that were treated with Sirt-1 inhibitor. Results: i) The expression of Sirt-1 was ubiquitously observed in chondrocytes, in contrast, Runx2 expressed in the only osteophyte region in the knee joint of OA model mice. ii) Mechanical stress up-regulated the expression of Runx2 in osteoarthritic chondrocytes. The mechanical stress-accelerated expression of Runx2 was inhibited by the pretreatment with Sirt1 inhibitor in osteoarthritic chondrocytes. Also, the mechanical stress-induced production of MMP-13 from chondrocytes was reduced by the pretreatment with Sirt1 inhibitor. Conclusions: Our study revealed that Sirt-1 inactivation trended to inhibit the mechanical stress-induced expressions of Runx2 and MMP-13 in chondrocytes. The Runx2 has been reported to have an important role as a promotor of activation of MMP-13 in chondrocytes (4). Thus, our findings suggest that the NAD-dependent deacetylase Sirt-1 may upregulate the osteophyte formation and the expression of cartilage degrading enzyme MMP-13 through the mechanism involving the acceleration of the osteogenic transcription factor Runx2 in OA cartilage tissues. Since it is well known that Sirt-1 activity is affected by several stresses as well as mechanical stress, Sirt-1 may be involved in the pathology of OA. Our study may provide a pathologic mechanism linking the NAD-dependent deacetylase Sirt-1 and its modulation of Runx2 in OA.

Evidence that TNF-β suppresses osteoblast differentiation of mesenchymal stem cells and resveratrol reverses it through modulation of NF-κB, Sirt1 and Runx2.

It has been established that inflammation plays an important role in bone formation and bone loss. Although a lot is known about the role of TNF-α in bone health, very little is understood about TNF-β, also called lymphotoxin. In this report, we examine the effect of TNF-β on osteogenic differentiation of mesenchymal stem cells (MSCs) and its modulation by resveratrol. Monolayer and high-density cultures of MSCs were treated with osteogenic induction medium with/without TNF-β, Sirt1 inhibitor nicotinamide (NAM), antisense oligonucleotides against Sirt1 (ASO) and/or Sirt1 stimulator resveratrol. We found that TNF-β inhibits, in a similar way to NAM or Sirt1-ASO, the early stage of osteogenic differentiation of MSCs and this was accompanied with downregulation of bone-specific matrix, β1-integrin, Runx2 and with upregulation of NF-κB phosphorylation and NF-κB-regulated gene products involved in the inflammatory, degradative processes and apoptosis. However, resveratrol reversed TNF-β- and NAM-suppressed MSCs osteogenesis by activation of Sirt1 and Runx2 that led to osteoblast differentiation. Furthermore, downregulation of Sirt1by mRNA inhibited the effect of resveratrol, highlighting the important impact of this enzyme in the TNF-β signaling pathway. Finally, resveratrol was able to manifest its effect both by suppression of TNF-β-induced NF-κB and through direct activation of the Sirt1 and Runx2 pathway. Thus, through these studies, we present a mechanism by which a T cell-derived cytokine, TNF-β can affect bone formation through modulation of MSCs differentiation that involves NF-κB, Sirt1, Runx2 and resveratrol reversed TNF-β-promoted impairments in MSCs osteogenesis.

Flame-sprayed strontium- and magnesium-doped hydroxyapatite on titanium implants for osseointegration enhancement

Strontium (Sr) and magnesium (Mg) have been reported to promote osteoblastic cell proliferation and to accelerate bone formation. However, the biofunctional effect on osseointegration caused by coating dental implants with hydroxyapatite (HA) incorporating both of these elements has not been investigated to date. The aim of this study was to examine bone formation and osseointegration in Sr- and Mg-doped HA-coated titanium implants. In this study, the flame-spraying technique was used to deposit Sr- and Mg-doped HA onto titanium discs and implants. The biocompatibility and alkaline phosphatase (ALP) activity of human embryonic palatal mesenchymal pre-osteoblasts on titanium discs was evaluated. In addition, the expression of the osteogenesis gene and the protein expression of Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osteocalcin (OCN) on titanium discs were determined by using real-time polymerase chain reaction assays, Western blot analysis, and enzyme-linked immunosorbent assays. Ultimately, the effects of Sr- and Mg-doped HA on the osseointegration of implants were examined in beagle dogs. The results showed that of the groups tested, the 5Sr5Mg-HA group exhibited the highest biocompatibility, ALP activity, and mRNA and protein expression of RUNX2, OPN, and OCN. Prominent new bone ingrowth with osteoblasts and osteoid tissue was found in the 5Sr5Mg-HA group at 4 weeks and 8 weeks. In addition, the highest bone-to-implant contact ratio was found in the 5Sr5Mg-HA group compared to other groups at 8 weeks in beagle dogs. The results show the potential of using 5Sr5Mg-HA coating on dental implants for enhanced osseointegration. Statement of SignificanceOsseointegration is assessed according to the extent of bone-to-implant contact and is an indication of implant success. This study is the first to report that flame-sprayed Sr- and Mg-doped HA coatings on titanium surfaces enhance osseointegration and new bone apposition in a beagle dog model. Therefore, the use of titanium implants incorporating Sr and Mg might be advantageous for patients with a low bone density.

SET7/9 promotes multiple malignant processes in breast cancer development via RUNX2 activation and is negatively regulated by TRIM21

Although the deregulation of lysine methyltransferase (su(var)-3–9, enhancer-of-zeste, trithorax) domain-containing protein 7/9 (SET7/9) has been identified in a variety of cancers, the potential role of SET7/9 and the molecular events in which it is involved in breast cancer remain obscure. Using the online Human Protein Atlas and GEO databases, the expression of SET7/9 was analyzed. Furthermore, we investigated the underlying mechanisms using chromatin immunoprecipitation-based deep sequencing (ChIP-seq) and quantitative ChIP assays. To explore the physiological role of SET7/9, functional analyses such as CCK-8, colony formation, and transwell assays were performed and a xenograft tumor model was generated with the human breast cancer cell lines MCF-7 and MDA-MB-231. Mass spectrometry, co-immunoprecipitation, GST pull-down, and ubiquitination assays were used to explore the mechanisms of SET7/9 function in breast cancer. We evaluated the expression of SET7/9 in different breast cancer cohorts and found that higher expression indicated worse survival times in these public databases. We demonstrated positive effects of SET7/9 on cell proliferation, migration, and invasion via the activation of Runt-related transcription factor 2 (RUNX2). We demonstrate that tripartite motif-containing protein 21 (TRIM21) physically associates with SET7/9 and functions as a major negative regulator upstream of SET7/9 through a proteasome-dependent mechanism and increased ubiquitination. Taken together, our data suggest that SET7/9 has a promoting role via the regulation of RUNX2, whereas TRIM21-mediated SET7/9 degradation acts as an anti-braking system in the progression of breast cancer.

MiR-873-3p targets HDAC4 to stimulate matrix metalloproteinase-13 expression upon parathyroid hormone exposure in rat osteoblasts.

Matrix metalloproteinase-13 (MMP-13) plays a predominant role in endochondral bone formation and bone remodeling. Parathyroid hormone (PTH) stimulates the expression of MMP-13 via Runx2, a bone transcription factor in rat osteoblastic cells (UMR106-01), and histone deacetylase 4 (HDAC4) acts as a corepressor of Runx2. Moreover, microRNAs (miRNAs) play an important role in regulating genes posttranscriptionally. Here, we hypothesized that PTH upregulates the miRNAs targeting HDAC4, which could lead to increased Runx2 activity and MMP-13 expression in rat osteoblastic cells. We identified several miRNAs that putatively target rat HDAC4 using bioinformatics tools. miR-873-3p was significantly upregulated by PTH in rat osteoblasts. miR-873-3p overexpression downregulated HDAC4 protein expression, increased Runx2 binding at the MMP-13 promoter, and increased MMP-13 messenger RNA expression in UMR106-01 cells. A luciferase reporter assay identified the direct targeting of miR-873-3p at the 3'-untranslated region of HDAC4. Thus, miR-873-3p targeted HDAC4 and relieved the corepressor effect of HDAC4 on Runx2 for MMP-13 expression in rat osteoblasts. This study advances our knowledge of posttranscriptional gene regulation occurring in bone and bone-related diseases and clarifies the role of miRNAs as diagnostic biomarkers.

Osteoporosis genome-wide association study variant c.3781 C>A is regulated by a novel anti-osteogenic factor miR-345-5p.

Upstream transcription factor family member 3 (USF3) c.3781C>A (rs1026364) in the 3'-untranslated region (3'-UTR) has been firmly associated with bone mineral density (BMD) in genome-wide association study (GWAS). However, the molecular mechanism by which it influences BMD and osteoporosis is unknown. Bioinformatics analyses suggested that the risk c.3781A allele creates a target site for hsa-miR-345-5p binding. Luciferase assay validated that the c.3781A allele displayed significantly lower luciferase activities than the c.3781C allele in the human osteoblast cell line hFOB1.19, osteosarcoma cell lines U-2OS and Saos-2, and embryonic kidney cell line 293T. Furthermore, hsa-miR-345-5p regulated USF3 expression on both messenger RNA and protein levels in hFOB1.19 and U937 cells with heterozygous A/C genotype. Transfection of hsa-miR-345-5p antagomiR in heterozygous hFOB1.19 cells significantly increased the expression of osteogenic marker genes RUNX2, OSTERIX, COL1A1, ALP, OPN, OCN, and alkaline phosphatase activity and matrix mineralization level. Importantly, we found that hsa-miR-345-5p also inhibits osteoblast maturation in cell lines U-2OS with hsa-miR-345-5p nonbinding C/C genotype by targeting RUNX3 and SMAD1. Our findings uncovered a novel pathogenetic mechanism of osteoporosis by GWAS variant c.3781C>A-mediated disruption of hsa-miR-345-5p binding at the 3'-UTR of USF3 and the functional role of hsa-miR-345-5p in osteogenic differentiation.

BMP9 Reduces Bone Loss in Ovariectomized Mice by Dual Regulation of Bone Remodeling.

Bone remodeling is dynamic and is tightly regulated through bone resorption dominated by osteoclasts and bone formation dominated by osteoblasts. Imbalances in this process can cause various pathological conditions, such as osteoporosis. Bone morphogenetic protein 9 (BMP9), a biomolecule produced and secreted by the liver, has many pharmacological effects, including anti-liver fibrosis, antitumor, anti-heart failure, and antidiabetic activities. However, the effects of BMP9 on the regulation of osteoblast and osteoclast functions and the underlying molecular mechanism(s) have not yet been investigated. In this study, BMP9 increased the expression of osteoblastogenic gene markers, such as ALP, Cola1, OCN, RUNX2, and OSX, and ALP activity in MC3T3-E1 cells by upregulating LGR6 and activating the Wnt/β-catenin pathway. BMP9 also suppressed receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation of bone marrow macrophages (BMMs) by inhibiting the Akt-NF-κB-NFATc1 pathway. More importantly, in an ovariectomy (OVX) mouse model, BMP9 attenuated bone loss and improved bone biomechanical properties in vivo by increasing bone-forming activity and suppressing bone resorption activity. Accordingly, our current work highlights the dual regulatory effects that BMP9 exerts on bone remodeling by promoting bone anabolic activity and inhibiting osteoclast differentiation in OVX mice. © 2020 American Society for Bone and Mineral Research.

Post-Translational Regulations of Transcriptional Activity of RUNX2.

Runt-related transcription factor 2 (RUNX2) is a key transcription factor for bone formation and osteoblast differentiation. Various signaling pathways and mechanisms that regulate the expression and transcriptional activity of RUNX2 have been thoroughly investigated since the involvement of RUNX2 was first reported in bone formation. As the regulation of Runx2 expression by extracellular signals has recently been reviewed, this review focuses on the regulation of post-translational RUNX2 activity. Transcriptional activity of RUNX2 is regulated at the post-translational level by various enzymes including kinases, acetyl transferases, deacetylases, ubiquitin E3 ligases, and prolyl isomerases. We describe a sequential and linear causality between post-translational modifications of RUNX2 by these enzymes. RUNX2 is one of the most important osteogenic transcription factors; however, it is not a suitable drug target. Here, we suggest enzymes that directly regulate the stability and/or transcriptional activity of RUNX2 at a post-translational level as effective drug targets for treating bone diseases.

Phosphoproteomic profiling reveals a defined genetic program for osteoblastic lineage commitment of human bone marrow-derived stromal stem cells.

Bone marrow–derived mesenchymal stem cells (MSCs) differentiate into osteoblasts upon stimulation by signals present in their niche. Because the global signaling cascades involved in the early phases of MSCs osteoblast (OB) differentiation are not well-defined, we used quantitative mass spectrometry to delineate changes in human MSCs proteome and phosphoproteome during the first 24 h of their OB lineage commitment. The temporal profiles of 6252 proteins and 15,059 phosphorylation sites suggested at least two distinct signaling waves: one peaking within 30 to 60 min after stimulation and a second upsurge after 24 h. In addition to providing a comprehensive view of the proteome and phosphoproteome dynamics during early MSCs differentiation, our analyses identified a key role of serine/threonine protein kinase D1 (PRKD1) in OB commitment. At the onset of OB differentiation, PRKD1 initiates activation of the pro-osteogenic transcription factor RUNX2 by triggering phosphorylation and nuclear exclusion of the histone deacetylase HDAC7.

Bmi1 Induces Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Under Inflammatory Environment

Bmi1 is a polycomb histone that regulates stem cells, but the role and mechanism of Bmi1 in bone marrow mesenchymal stem cells (BMSCs) differentiation has not been elucidated. Rat BMSCs were cultured in vitro and randomly divided into control group and inflammation group (treated with LPS). Bmi1 and Bmi1 siRNA were transfected into inflammatory BMSCs, followed by analysis of Bmi1 expression by Real time PCR, cell proliferation by MTT assay, Caspase3 activity, ALP activity, expression of Runx2, OP and PPARγ 2 by Real time PCR, as well as secretion of TNF-α and IL-1β by ELISA. In inflammatory environment, Bmi1 expression was significantly decreased, cell proliferation was significantly inhibited, along with increased Caspase3 activity, decreased ALP activity and the expression of Runx2 and OP, increased PPAR 2 expression and secretion of TNF-α and IL-1β (P < 0 05). Transfection of Bmi1 siRNA into inflammatory BMSCs further significantly aggravated the above changes (P < 0 05). Bmi1 plasmid transfected into inflammatory BMSCs significantly promoted Bmi1 expression and cell proliferation, decreased Caspase3 activity, increased ALP activity and expression of Runx2 and OP, decreased PPAR γ2 expression and TNF-α and IL-1β secretion (P < 0 05). Bmi1 expression is reduced in BMSCs under inflammation. Up-regulation of Bmi1 can inhibit the secretion of inflammatory factors, regulate the proliferation and apoptosis of BMSCs, and promote the proliferation and osteogenic differentiation of BMSCs.

Glutamine Depletion By Addicted Myeloma Cells Inhibits Osteoblastic Differentiation of Bone Marrow Mesenchymal Stromal Cells Limiting Asparagine Availability: A Possible New Mechanism for Myeloma Bone Disease

Metabolic alterations of cancer cells, aimed at sustaining their growth, may also influence the behavior of the tumor microenvironment. Our group has recently demonstrated that multiple myeloma (MM) is a highly glutamine(Gln)-addicted tumor that utilize huge amounts of Gln to fuel its metabolism through the enzyme glutaminase (GLS). For this reason, MM cells exhibits increased Gln uptake, mainly through the ASCT2 transporter. Interestingly, lower bone marrow (BM) plasma Gln concentration (down to a median value of 0.4 mM vs. a median value of 0.6 mM) was found in MM patients as compared with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). The main feature of MM BM microenvironment is the suppression of osteoblastic (OB) differentiation leading to the development of osteolytic bone lesions, the hallmark of MM. Most recently, it has been demonstrated that Gln metabolism is needed to sustain bone mass formation in murine models and that GLS inhibition decreases OB differentiation of human mesenchymal stromal cells (hMSCs). However, no information is yet available on the role of Gln depletion imposed by MM cell metabolism on OB differentiation into the BM. This topic has been investigated in the present study. Firstly, human MM cells were co-cultured with BM hMSCs, and Gln medium concentration was evaluated with mass spectrometry (MS), demonstrating a MM-induced depletion of the amino acid. Upon Gln depletion, MSC exhibited a sustained induction of Glutamine Synthetase (GS). On the contrary, when differentiated in osteogenic medium (D-MEM + 5% Fetal Bovine Serum, supplemented with 2 mM Gln, ascorbic acid and dexamethasone), GS was suppressed. Conversely, GLS (both KGA and GAC isoforms) and SLC38A2, the gene for the concentrative Gln transporter SNAT2, were induced. These data suggest that hMSCs differentiation in OBs is associated with an increased dependence upon extracellular Gln. Consistent with this conclusion, the activity of SNAT2 was absent in undifferentiated hMSCs but well detectable after 14 days of OB differentiation, when total Gln uptake was also increased. Under the same conditions, OB differentiation markers (RUNX2, COL1A1, ALPL expression and ALPL activity or staining) were significantly induced but their expression was blunted by incubation in low-Gln (0.4 mM) medium or in the presence of the SNAT2 inhibitor MeAIB. The incubation in Gln-free D-MEM suppressed the induction of GLS and SLC38A2 along with OB differentiation, which was restored by the supplementation of Non-Essential Amino Acids (NEAA). Among NEAA, only asparagine (Asn) was able to rescue OB differentiation in the absence of Gln. The determination of intracellular amino acids with MS indicated that OB differentiation was associated with the increase of cell Asn, without significant changes of Gln, glutamate (Glu) or aspartate (Asp). Asparagine Synthetase (ASNS), the Gln-dependent enzyme that accounts for Asn synthesis, was also found induced during OB differentiation of hMSCs. Gene Expression Profiles of primary BM hMSCs and OBs from bone biopsies of both healthy donors (n=7) and MM patients (n=16) indicated that GLS, ASNS, and SLC38A2 are more expressed in OBs, while the expression of GLUL, the gene for GS, is higher in undifferentiated hMSCs from healthy donors. Overall, these results indicate that (1) OB differentiation of hMSCs is Gln-dependent; (2) the partial Gln depletion, imposed by Gln-addicted MM cells in the BM microenvironment, contributes to the impairment of osteoblastic differentiation of hMSCs; (3) hindrance of differentiation may depend on the limited availability of intracellular Asn derived from Gln-dependent ASNS. These results support the evidence that Gln addiction of MM cells affects bone microenvironment leading to the inhibition of OB differentiation and, consequently, to the development of MM bone disease. Disclosures Giuliani: Janssen: Research Funding.

Collagen-derived dipeptide prolyl hydroxyproline directly binds to Foxg1 to change its conformation and inhibit the interaction with Runx2.

Collagen-derived dipeptide prolyl hydroxyproline (Pro-Hyp) is involved in the proliferation and differentiation of various types of cultured cells. To elucidate the mechanism underlying Pro-Hyp actions during osteoblast differentiation, we hypothesized that proteins binding to Pro-Hyp serve to mediate cellular signaling, affecting Runx2 expression. Recently, we performed the characterization of Foxg1, that it enhances Runx2 expression in the presence of Pro-Hyp. Our findings indicate that Pro-Hyp directly binds to the Foxg1 recombinant protein, which leads to the structural alteration of the Foxg1 protein. In addition, Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp, with Pro-Hyp disrupting the interaction between Foxg1 and Runx2. Collectively, our results indicate that the Pro-Hyp bound Foxg1 alters the structured conformation of Foxg1, resulting in conformational changes that lead to dissociation from Runx2. These novel findings suggest that during osteoblast differentiation, Pro-Hyp mediates Runx2 activity though directly binding to Foxg1 and increases Runx2 expression. Abbreviations: CPT: collagen peptide; GST: Glutathione S-transferase; PAGE: Polyacrylamide gel electrophoresis; PCR: Polymerase chain reaction; prolyl hydroxyproline: Pro-Hyp.

Overexpression of Heme Oxygenase-1 Promotes Osteoblast Differentiation of Osteoporosis Rat Bone Marrow Stromal Cells in Bone Morphogenetic Protein-Dependent Manner

BMSCs play a role in osteoporosis (OP) and their differentiation can lead to OP progression. Heme oxygenase-1 (HO-1) involves in many diseases, but the effect of HO-1 on osteoblast differentiation of BMSCs in OP rats remains unclear. SD rats were divided into control group and OP group. Rats BMSCs in OP group were cultured in vitro, HO-1 expression was up-regulated by HO-1 agonist hemin, and BMPR inhibitor LDN-19318 was added followed by analysis of HO-1 expression by real time PCR and ELISA, cell proliferation by MTT assay, apoptosis by Caspase 3 activity, BMP-2 expression by Western blot, ALP activity, expression of Runx2 and OC by real time PCR. In OP group, HO-1 expression was significantly decreased, cell proliferation was inhibited, Caspase 3 activity was increased along with decreased ALP activity and expression of Runx2, OC and BMP-2 compared to control (P < 0.05). Up-regulation of HO-1 expression significantly promoted cell proliferation, reduced Caspase 3 activity, increased ALP activity, and expression of Runx2, OC and BMP-2 (P < 0.05). However, inhibition of HO-1 significantly promoted bone differentiation after the addition of BMPR inhibitor LDN-193189 (P < 0.05). HO-1 expression is decreased in BMSCs of OP group rats. Up-regulation of HO-1 promoted BMSCs proliferation in OP rats in BMP-dependent manner, inhibited apoptosis, and promoted osteoblast differentiation.

Osteogenic gene markers are epigenetically reprogrammed during contractile-to-calcifying vascular smooth muscle cell phenotype transition

The understanding of vascular calcification-based mechanism is an urgent pending task in vascular biology and this prompted us to better address this issue by investigating whether DNA methylation mechanism might drive osteogenic marker genes modulation in primary human vascular smooth muscle cells (VSMCs) responding to calcium and phosphate levels overload up to 72 h. Firstly, our data shows this calcifying process recapitulates the molecular repertory of osteogenic biomarkers and specifically requiring RUNX2, Osterix and ALP, BSP genes activations along 72 h in vitro, and this behavior was validated here using other lineages. Conversely, both BMPs 4 and 7 were significantly overexpressed, maybe already as a mechanism in response to RUNX2 and Osterix genes activities identified earlier in response to the calcifying condition, and taken into maintain the calcifying phenotype of VSMCs. Additionally, survival signaling was maintained active and accompanied by a dynamic cytoskeleton rearrangement signaling requiring MAPK and AKT phosphorylations. Moreover, during the contractile-to-calcifying transition phenotype of VSMCs, epigenetic machinery was finely modulated, requiring the translocation of DNMT3B and TET2 into nucleus and this prompted us evaluating whether the profile of osteogenic-related gene promoters’ methylation might contribute with this process. By firstly estimating 5meC/5 hmeC ratio changes, we further specifically show the significance of the epigenetic modulation of Osterix and Bone sialoprotein related gene promoters, presenting a positive correlation between the epigenetic signature of their gene promoters and transcriptional patterns. Altogether, our results show for the first time the importance of epigenetic mechanism on modulating osteogenic gene markers reprogramming during calcifying VSMCs phenotype acquisition, which might drive the genesis of vascular ectopic calcification.

Symphytum officinale augments osteogenesis in human bone marrow-derived mesenchymal stem cells in vitro as they differentiate into osteoblasts.

Mesenchymal stem cells (MSCs) are multipotent stem cells possessing regenerative potential. Symphytum officinale (SO) is a medicinal plant and in homoeopathic literature, believed to accelerate bone healing. This study aimed to determine if homoeopathic doses of SO could augment osteogenesis in MSCs as they differentiate into osteoblasts in vitro. Bone marrow samples were obtained from patients who underwent bone grafting procedures (n = 15). MSCs were isolated, expanded and characterized by flow cytometry (CD90, CD105). Cytotoxicity of SO was evaluated by MTT assay. Osteogenic differentiation was induced in MSCs with β-glycerophosphate, ascorbic acid and dexamethasone over 2 weeks. Different homoeopathic doses of SO (MT, 3C, 6C, 12C and 30C) were added to the basic differentiation medium (BDM) and efficiency of MSCs differentiating into osteoblasts were measured by evaluating expression of Osteocalcin using flow cytometry, and alkaline phosphatase activity using ELISA. Gene expression analyses for osteoblast markers (Runx-2, Osteopontin and Osteocalcin) were evaluated in differentiated osteoblasts using qPCR. Flow cytometry (CD90, CD105) detected MSCs isolated from bone marrow (93-98%). MTT assay showed that the selected doses of SO did not induce any cytotoxicity in MSCs (24 hours). The efficiency of osteogenic differentiation (2 weeks) for different doses of Symphytum officinale was determined by flow cytometry (n = 10) for osteoblast marker, Osteocalcin, and most doses of Symphytum officinale enhanced osteogenesis. Interestingly, gene expression analysis for Runx-2 (n = 10), Osteopontin (n = 10), Osteocalcin (n = 10) and alkaline phosphatase activity (n = 8) also showed increased osteogenesis with the addition of Symphytum officinale to BDM, specially mother tincture. Our findings suggest that homoeopathic dose (specially mother tincture) of Symphytum officinale has the potential to enhance osteogenesis.