Effect of N-Vinyl-2-Pyrrolidone (NVP), a Bromodomain-Binding Small Chemical, on Osteoblast and Osteoclast Differentiation and Its Potential Application for Bone Regeneration.

The human skeleton is a dynamic and remarkably organized organ system that provides mechanical support and performs a variety of additional functions. Bone tissue undergoes constant remodeling; an essential process to adapt architecture/resistance to growth and mechanical needs, but also to repair fractures and micro-damages. Despite bone's ability to heal spontaneously, certain situations require an additional stimulation of bone regeneration, such as non-union fractures or after tumor resection. Among the growth factors used to increase bone regeneration, bone morphogenetic protein-2 (BMP2) is certainly the best described and studied. If clinically used in high quantities, BMP2 is associated with various adverse events, including fibrosis, overshooting bone formation, induction of inflammation and swelling. In previous studies, we have shown that it was possible to reduce BMP2 doses significantly, by increasing the response and sensitivity to it with small molecules called "BMP2 enhancers". In the present study, we investigated the effect of N-Vinyl-2-pyrrolidone (NVP) on osteoblast and osteoclast differentiation in vitro and guided bone regeneration in vivo. We showed that NVP increases BMP2-induced osteoblast differentiation and decreases RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, in a rabbit calvarial defect model, the histomorphometric analysis revealed that bony bridging and bony regenerated area achieved with NVP-loaded poly (lactic-co-glycolic acid (PLGA) membranes were significantly higher compared to unloaded membranes. Taken together, our results suggest that NVP sensitizes BMP2-dependent pathways, enhances BMP2 effect, and inhibits osteoclast differentiation. Thus, NVP could prove useful as "osteopromotive substance" in situations where a high rate of bone regeneration is required, and in the management of bone diseases associated with excessive bone resorption, like osteoporosis.

Mesenchymal stem cell expression of SDF-1ß synergizes with BMP-2 to augment cell-mediated healing of critical-sized mouse calvarial defects.

Bone has the potential for spontaneous healing. This process, however, often fails in patients with comorbidities. Tissue engineering combining functional cells, biomaterials and osteoinductive cues may provide alternative treatment strategies. We have recently demonstrated that stromal cell-derived factor-1ß (SDF-1ß) works in concert with bone morphogenetic protein-2 (BMP-2) to potentiate osteogenic differentiation of bone marrow-derived mesenchymal stem/stromal cells (BMSCs). Here, we test the hypothesis that SDF-1ß overexpressed in Tet-Off-SDF-1ß BMSCs, delivered on acellular dermal matrix (ADM), synergistically augments BMP-2-induced healing of critical-sized mouse calvarial defects. BMSC therapies alone showed limited bone healing, which was increased with co-delivery of BMP-2. This was further enhanced in Tet-Off-SDF-1ß BMSCs + BMP-2. Only limited BMSC retention on ADM constructs was observed after 4 weeks in vivo, which was increased with BMP-2 co-delivery. In vitro cell proliferation studies showed that supplementing BMP-2 to Tet-Off BMSCs significantly increased the cell number during the first 24 h. Consequently, the increased cell numbers decreased the detectable BMP-2 levels in the medium, but increased cell-associated BMP-2. The data suggest that SDF-1ß provides synergistic effects supporting BMP-2-induced, BMSC-mediated bone formation and appears suitable for optimization of bone augmentation in combination therapy protocols. Copyright © 2015 John Wiley & Sons, Ltd.

Pharmacological activation of aldehyde dehydrogenase 2 promotes osteoblast differentiation via bone morphogenetic protein-2 and induces bone anabolic effect.

Aldehyde dehydrogenases (ALDHs) are a family of enzymes involved in detoxifying aldehydes. Previously, we reported that an ALDH inhibitor, disulfiram caused bone loss in rats and among ALDHs, osteoblast expressed only ALDH2. Loss-of-function mutation in ALDH2 gene is reported to cause bone loss in humans which suggested its importance in skeletal homeostasis. We thus studied whether activating ALDH2 by N-(1, 3-benzodioxol-5-ylmethyl)-2, 6-dichlorobenzamide (alda-1) had osteogenic effect. We found that alda-1 increased and acetaldehyde decreased the differentiation of rat primary osteoblasts and expressions of ALDH2 and bone morphogenetic protein-2 (BMP-2). Silencing ALDH2 in osteoblasts abolished the alda-1 effects. Further, alda-1 attenuated the acetaldehyde-induced lipid-peroxidation and oxidative stress. BMP-2 is essential for bone regeneration and alda-1 increased its expression in osteoblasts. We then showed that alda-1 (40mg/kg dose) augmented bone regeneration at the fracture site with concomitant increase in BMP-2 protein compared with control. The osteogenic dose (40mg/kg) of alda-1 attained a bone marrow concentration that was stimulatory for osteoblast differentiation, suggesting that the tissue concentration of alda-1 matched its pharmacologic effect. In addition, alda-1 promoted modeling-directed bone growth and peak bone mass achievement, and increased bone mass in adult rats which reiterated its osteogenic effect. In osteopenic ovariectomized (OVX) rats, alda-1 reversed trabecular osteopenia with attendant increase in serum osteogenic marker (procollagen type I N-terminal peptide) and decrease in oxidative stress. Alda-1 has no effect on liver and kidney function. We conclude that activating ALDH2 by alda-1 had an osteoanabolic effect involving increased osteoblastic BMP-2 production and decreased OVX-induced oxidative stress.

Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation.

Iron overload results in significant morbidity and mortality in ß-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in ß-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

[Evaluation of osteogenic effects of BMP2 up-regulator E40071 in vitro].

Bone morphogenetic protein 2(BMP2) plays a key role in bone development and reestablishment. In the study, we screened up-regulators of BMP2 among 20 000 compounds through a cell-based high throughput screening model and a positive compound E40071 [2-(4-(5-methyl-3-phenylpyrazolo[1,5-a]pyrimidin-7-yl) piperazin-1-yl)ethan-1-ol] was found as the positive hit. The EC(50) value of E40071 was 2.73 µmol·L(-1). In vitro, E40071 upregulated the m RNA levels of BMP2 and the downstream transcription factors, Runx2 and Osx in MC3T3-E1(subclone 14). Protein expression of Runx2 was up-regulated by E40071 through induction of Smad1/5/8 phosphorylation. The alkaline phosphatase(ALP) activity was increased by E40071. Moreover, E40071 promoted the mineralization of MC3T3-E1(subclone 14) by Alizarin red S staining. In addition, E40071 markedly inhibited osteoclast differentiation of mice macrophage Raw264.7 induced by RANKL and reduced the expression of osteoclast differentiation markers, including MMP9 and NFATc1. The results suggest that E40071 is able to promote bone formation activity of osteoblasts and inhibit differentiation of osteoclasts.

Bone morphogenetic protein 2 stimulates noncanonical SMAD2/3 signaling via the BMP type 1A receptor in gonadotrope-like cells: implications for FSH synthesis.

FSH is an essential regulator of mammalian reproduction. Its synthesis by pituitary gonadotrope cells is regulated by multiple endocrine and paracrine factors, including TGFß superfamily ligands, such as the activins and inhibins. Activins stimulate FSH synthesis via transcriptional regulation of its ß-subunit gene (Fshb). More recently, bone morphogenetic proteins (BMPs) were shown to stimulate murine Fshb transcription alone and in synergy with activins. BMP2 signals via its canonical type I receptor, BMPR1A (or activin receptor-like kinase 3 [ALK3]), and SMAD1 and SMAD5 to stimulate transcription of inhibitor of DNA binding proteins. Inhibitor of DNA binding proteins then potentiate the actions of activin-stimulated SMAD3 to regulate the Fshb gene in the gonadotrope-like LßT2 cell line. Here, we report the unexpected observation that BMP2 also stimulates the SMAD2/3 pathway in these cells and that it does so directly via ALK3. Indeed, this novel, noncanonical ALK3 activity is completely independent of ALK4, ALK5, and ALK7, the type I receptors most often associated with SMAD2/3 pathway activation. Induction of the SMAD2/3 pathway by ALK3 is dependent upon its own previous activation by associated type II receptors, which phosphorylate conserved serine and threonine residues in the ALK3 juxtamembrane glycine-serine-rich domain. ALK3 signaling via SMAD3 is necessary for the receptor to stimulate Fshb transcription, whereas its activation of the SMAD1/5/8 pathway alone is insufficient. These data challenge current dogma that ALK3 and other BMP type I receptors signal via SMAD1, SMAD5, and SMAD8 and not SMAD2 or SMAD3. Moreover, they suggest that BMPs and activins may use similar intracellular signaling mechanisms to activate the murine Fshb promoter in immortalized gonadotrope-like cells.

Synergistic and sequential effects of BMP-2, bFGF and VEGF on osteogenic differentiation of rat osteoblasts.

In the present study, the effects of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) on regulation of rat osteoblast (ROB) maturation in vitro were investigated. It was found that the proliferation, differentiation and mineralization of ROBs were all dose-dependently increased at particular times in the case of treatment with only one growth factor. To investigate the effects of combined treatment, ROBs were treated with either a single application of a relatively high dose of each growth factor, or binary/triple combined applications of relatively low doses of the growth factors. Osteogenic differentiation was significantly promoted in the triple combination treatment of BMP-2, VEGF and bFGF compared with the single or binary combination treatments. The optimal timing of the triple combination to enhance osteogenesis was also tested. When bFGF and VEGF were added in the early stage, and BMP-2 and VEGF were added in the late stage, osteogenic differentiation of ROBs could be enhanced more effectively. These results could be used to construct bone tissue engineering scaffolds that release growth factors sequentially.

Role of matrix metalloproteinase-10 in the BMP-2 inducing osteoblastic differentiation.

Fibrodysplasia ossificans progressiva (FOP) is a skeletal disorder with progressive heterotopic ossification in skeletal muscle. A mutation causing constitutive activation in a bone morphogenetic protein (BMP) type 1 receptor [ALK2(R206H)] is found in most patients with FOP. However, the details in the heterotopic ossification of muscle in FOP and the role of matrix metalloproteinase-10 (MMP-10) in bone remain to be fully elucidated. In the present study, we investigated the role of MMP-10 in the differentiation of mouse myoblastic C2C12 cells into osteoblasts. MMP-10 was extracted as a factor, whose expression was most extensively enhanced by ALK2 (R206H) transfection in C2C12 cells. MMP-10 significantly augmented the levels of Osterix, type 1 collagen, alkaline phosphatase (ALP) and osteocalcin mRNA as well as ALP activity enhanced by BMP-2 in C2C12 cells. Moreover, a reduction in endogenous MMP-10 levels by siRNA significantly decreased the levels of Runx2, Osterix, type 1 collagen, ALP and osteocalcin mRNA enhanced by BMP-2 in these cells. In addition, MMP-10 increased the phosphorylation of Smad1/5/8 as well as enhanced the levels of Smad6 and Smad7 mRNA induced by BMP-2. In conclusion, the present study first demonstrated that MMP-10 promotes the differentiation of myoblasts into osteoblasts by interacting with the BMP signaling pathway. MMP-10 may play some important role in the heterotopic ossification of muscle in FOP.

Honokiol stimulates osteoblastogenesis by suppressing NF-κB activation.

Magnolia officinalis, a component of Asian herbal teas, has long been employed in traditional Japanese and Chinese medicine to treat numerous maladies. Honokiol, a biphenolic compound, is now considered to be one of the major active ingredients of Magnolia extract, and is under intense investigation for its anti-angiogenic, anti-inflammatory, anti-tumor and neuroprotective properties. Biochemically, honokiol has been recognized to modulate the nuclear factor κ B (NF-κB) signal transduction pathway suggesting that it possesses anti-inflammatory properties. Inflammation is intimately associated with bone turnover and skeletal deterioration and consequently, anti-inflammatory drugs may hold significant promise as bone protective agents to stem bone loss in osteoporotic conditions. We and others have demonstrated that suppression of NF-κB blunts osteoclastic bone resorption, but promotes osteoblastic bone formation. Indeed previous studies have demonstrated the anti-osteoclastogenic effects of honokiol, however, activities on osteoblast differentiation and activity have yet to be investigated. In this study, we show that honokiol is a potent inducer of in vitro osteoblast differentiation by virtue of its capacity to suppress basal and tumor necrosis factor alpha (TNFα)-induced NF-κB activation and to alleviate the suppressive action of TNFα on bone morphogenetic protein (BMP)-2-induced Smad activation. Our data confirm that honokiol may have considerable promise as a dual anabolic/anti-catabolic agent for the amelioration of multiple osteoporotic diseases.

8-Prenylkaempferol accelerates osteoblast maturation through bone morphogenetic protein-2/p38 pathway to activate Runx2 transcription.

AIMS: In this study, we investigated the effect of 8-prenylkaempferol (8-PK), a prenyl-flavonoid isolated from Sophora flavescens, on osteoblast differentiation and maturation. MAIN METHODS: MC3T3-E1 cells were exposed to 8-PK and the cytotoxicity was assayed. Osteoblast differentiation and maturation were evaluated by analyzing alkaline phosphatase (ALP) activity and cell mineralization, respectively. RT-PCR and Western blot were executed to determine the effects of 8-PK on osteoblast differentiation-related gene expression and signaling pathway. KEY FINDINGS: 8-PK significantly promoted ALP activity, up-regulated mRNA expressions of osteocalcin, osteopontin, and type I collagen, and induced bone nodules formation. Induction of differentiation by 8-PK was associated with increased bone morphogenetic protein (BMP)-2 expression, and sequentially up-regulated the phosphorylations of Smad1/5/8 and p38, and increased the nuclear translocation of runt-related transcription factor 2 (Runx2). Addition of BMP-2 antagonist noggin blocked 8-PK and recombinant mouse BMP-2-induced ALP activity, reconfirming that BMP-2 production is required in 8-PK-mediated osteoblast differentiation. Noggin also abrogated 8-PK evoked phosphorylations of Smad1/5/8 and p38, suggesting that BMP-2 signaling is required for p38 activation in 8-PK-treated cells. Application of p38 inhibitor SB203580 repressed not only 8-PK-mediated activation of ALP, but also the nuclear translocation of Runx2 and bone nodules formation. SIGNIFICANCE: The present results suggested that BMP-2/p38/Runx2 pathways were involved in 8-PK-induced differentiation/maturation of MC3T3-E1 osteoblasts and firstly demonstrated that 8-PK might be a promising agent for inducing osteogenesis.

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell-based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post-translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression-based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP-1. Among them, we selected SVAK-3, a compound that showed a dose-dependent potentiation of BMP-2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP-1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP-2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored.