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Phenotypic drug discovery (PDD) continues to fuel the research and development pipelines with first-in-class therapeutic modalities, but success rates critically depend on the quality of the underlying model system. Here, we employed a stem cell-based approach for the target-agnostic, yet pathway-centric discovery of small-molecule cytokine signaling activators to act as morphogens during development and regeneration. Unbiased screening identified triazolo[1,5- c ]quinazolines as a new-in-class in vitro and in vivo active amplifier of the bone morphogenetic protein (BMP) pathway. Cellular BMP outputs were stimulated via enhanced and sustained availability of BMP-Smad proteins, strictly dependent on a minimal BMP input. Holistic target deconvolution unveiled a unique mechanism of dual targeting of casein kinase 1 and phosphatidyl inositol 3-kinase isoforms as key effectors for efficient amplification of osteogenic BMP signaling. This work underscores the asset of PDD to discover unrecognized polypharmacology signatures, in this case significantly expanding the chemical and druggable space of BMP modulators.

Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues owing to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2 (also known as ACVR1). From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2, compared with other receptors of the BMP/TGF-β signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D-transgenic mouse line, which provides a model of heterotopic ossification (HO), as well as an inducible ACVR1R206H-knockin mouse, which serves as a genetically and physiologically faithful FOP model. In both models, saracatinib was well tolerated and potently inhibited the development of HO, even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in FOP treatment, offering an accelerated path to clinical proof-of-efficacy studies and potentially significant benefits to individuals with this devastating condition.

GDF11 has been reported to play a critical role in rejuvenating hypertrophy heart, skeletal muscle, and blood vessel regeneration in aged mice. Whether GDF11 can regulate autophagy in cardiomyocytes remains largely unknown. Thus, the purpose of the present study was to investigate the effects of GDF11 on cardiomyocyte autophagy induced by hypoxia, in addition to the underlying mechanisms. By using the MTT assay, Flow cytometry assay, LIVE/DEAD® Viability/Cytotoxicity Kit Stains and TUNEL assay, we found that exogenous GDF11 decreased apoptosis caused by prolonged hypoxia in cardiomyocytes. The expression of GDF11 was decreased obviously both in the cardiac tissue of myocardial infarction mice and the hypoxia treated cardiomyocytes. Protein levels of cleaved caspase-3, p-AMPK, SQSTM1, LC3B-I/II and GDF11 were detected by western blot. Autophagosomes and autolysosomes were identified by confocal laser microscopy after transfecting with the mRFP-eGFP-LC3 plasmids. Antibody against GDF11 (anti-GDF11) was used to inhibit the function of GDF11. At the molecular level, exogenous GDF11 increased AMPK function and enhanced autophagy activity. Anti-GDF11 inhibited autophagy and aggravated hypoxia-induced apoptosis in cardiomyocytes. Thus, GDF11 might be a potential target for myocardial infarction therapy., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Obesity has increased to pandemic levels and enhanced understanding of adipose regulation is required for new treatment strategies. Although bone morphogenetic proteins (BMPs) influence adipogenesis, the effect of BMP antagonists such as Noggin is largely unknown. The aim of the study was to define the role of Noggin, an extracellular BMP inhibitor, in adipogenesis., Methods: We generated adipose-derived progenitor cells and a mouse model with adipocyte-specific Noggin deletion using the Adiponectin Cre transgenic mouse, and determined the adipose phenotype of Noggin-deficiency., Results: Our studies showed that Noggin is expressed in progenitor cells but declines in adipocytes, possibly allowing for lipid accumulation. Correspondingly, adipocyte-specific Noggin deletion in vivo promoted age-related obesity in both genders with no change in food intake. Although the loss of Noggin caused white adipose tissue hypertrophy, and whitening and impaired function in brown adipose tissue in both genders, there were clear gender differences with the females being most affected. The females had suppressed expression of brown adipose markers and thermogenic genes including peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1alpha) and uncoupling protein 1 (UCP1) as well as genes associated with adipogenesis and lipid metabolism. The males, on the other hand, had early changes in a few BAT markers and thermogenic genes, but the main changes were in the genes associated with adipogenesis and lipid metabolism. Further characterization revealed that both genders had reductions in VO 2 , VCO 2, and RER, whereas females also had reduced heat production. Noggin was also reduced in diet-induced obesity in inbred mice consistent with the obesity phenotype of the Noggin-deficient mice., Conclusions: BMP signaling regulates female and male adipogenesis through different metabolic pathways. Modulation of adipose tissue metabolism by select BMP antagonists may be a strategy for long-term regulation of age-related weight gain and obesity., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Psoralen and angelicin are two effective compounds isolated from psoraleae, a traditional Chinese medicine. They have a wide range of applications for bone disease treatment and immune modulation. In this study, we explored their new applications for the treatment of periodontal diseases. This study aimed to investigate the effects of psoralen and angelicin on Porphyromonas gingivalis growth and P. gingivalis -derived lipopolysaccharide (Pg-LPS)-induced inflammation, and further to evaluate their effects on osteogenesis. Finally, the effects of angelicin on a mouse model of periodontitis were also investigated. The results showed that psoralen and angelicin had beneficial dose-dependent effects regarding the inhibition of planktonic P. gingivalis and biofilms of P. gingivalis . There were no significant differences in the viability of monocyte-like THP-1 cells and human periodontal ligament cells (hPDLCs) treated with either psoralen or angelicin compared to the untreated control cells. Psoralen and angelicin also markedly decreased the mRNA expression and release of inflammatory cytokines (interleukin [IL]-1β and IL-8) by THP-1 cells in a dose-dependent manner. They significantly enhanced the alkaline phosphatase (ALP) activity of hPDLCs and up-regulated the expression of osteogenic proteins (runt-related transcription factor 2 [RUNX2], distal-less homeobox 5 [DLX5], and osteopontin [OPN]). Angelicin significantly attenuated alveolar bone loss and inflammation response in the mice with periodontitis. In conclusion, our data demonstrated that psoralen and angelicin could inhibit the growth of planktonic P. gingivalis and P. gingivalis biofilm. It is also the first report on the anti-inflammatory effect of psoralen and angelicin against Pg-LPS. They also had an osteogenesis-potentiating effect on hPDLCs. The in vivo study also indicated the effect of angelicin regarding protection against periodontitis. Our study highlighted the potential ability of psoralen and angelicin to act as novel natural agents to prevent and treat periodontitis.

Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While the genetic basis for about 30% of cases is known, the causative genes for the diverse presentations of the remainder of cases are unknown. The recently discovered cranial suture stem cell population affords an opportunity to identify early signaling pathways that contribute to craniosynostosis. We previously demonstrated that enhanced BMP signaling in neural crest cells (caA3 mutants) leads to premature cranial suture fusion resulting in midline craniosynostosis. Since enhanced mTOR signaling in neural crest cells leads to craniofacial bone lesions, we investigated the extent to which mTOR signaling is involved in the pathogenesis of BMP-mediated craniosynostosis by affecting the suture stem cell population. Our results demonstrate a loss of suture stem cells in the caA3 mutant mice by the newborn stage. We have found increased activation of mTOR signaling in caA3 mutant mice during embryonic stages, but not at the newborn stage. Our study demonstrated that inhibition of mTOR signaling via rapamycin in a time specific manner partially rescued the loss of the suture stem cell population. This study provides insight into how enhanced BMP signaling regulates suture stem cells via mTOR activation., (© 2018 Wiley Periodicals, Inc.)

Objective: To evaluate the dose-response relationship of exercise loading in the cartilage-subchondral bone (SB) unit in surgically-induced post-traumatic osteoarthritis (PTOA) of the knee., Design: Destabilized medial meniscus (DMM) surgery was performed on the right knee of 12-week-old male Wistar rats, and sham surgery was performed on the contralateral knee. Four weeks after the surgery, the animals were subjected to moderate (12 m/min) or intense (21 m/min) treadmill exercises for 30 min/day, 5 days/week for 4 weeks. PTOA development in articular cartilage and SB was examined using histological and immunohistochemical analyses, micro-computed tomography (micro-CT) analysis, and biomechanical testing at 8 weeks after surgery. Gremlin-1 was injected to determine the role of bone morphogenetic protein (BMP) signaling on PTOA development following moderate exercise., Results: Moderate exercise increased BMP-2, BMP-4, BMP-6, BMP receptor 2, pSmad-5, and inhibitor of DNA binding protein-1 expression in the superficial zone chondrocytes and suppressed cartilage degeneration, osteophyte growth, SB damage, and osteoclast-mediated SB resorption. However, intense exercise had little effect on BMP expression and even caused progression of these osteoarthritis (OA) changes. Gremlin-1 injection following moderate exercise caused progression of the PTOA development down to the level of the non-exercise DMM-operated knee., Conclusions: Exercise regulated cartilage-SB PTOA development in DMM-operated knees in a dose-dependent manner. Our findings shed light on the important role of BMP expression in superficial zone chondrocytes in attenuation of PTOA development following physiological exercise loading. Further studies to support a mechanism by which BMPs would be beneficial in preventing PTOA progression are warranted., (Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Lack of bone formation-related health problems are a major problem for the aging population in the modern world. As a part of the ongoing trend of developing natural substances that attenuate osteoporotic bone loss conditions, dioxinodehydroeckol (DHE) from edible brown alga Ecklonia cava was tested for its effects on osteoblastogenic differentiation in MC3T3-E1 pre-osteoblasts. DHE was observed to successfully enhance osteoblast differentiation, as indicated by elevated cell proliferation, alkaline phosphatase activity, intracellular cell mineralization, along with raised levels of osteoblastogenesis indicators at the concentration of 20 μM. Results suggested a possible intervening of DHE on the bone morphogenetic protein (BMP) signaling pathway, according to elevated protein levels of BMP-2, collagen-I, and Smads. In addition, the presence of DHE was also able to raise the phosphorylated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) levels which are also activated by the BMP signaling pathway. In conclusion, DHE is suggested to be a potential bioactive compound against bone loss that could enhance osteoblastogenesis with a suggested BMP pathway interaction., Competing Interests: The authors declare no conflict of interest.

Bone differs from other tissues in its capacity to self-repair after a fracture. The low bone mass and structural deterioration of bone associated with osteoporosis increases the risk of fragility fracture compared with healthy individuals. The intention of this article is to review the complex process of fracture repair and essential requirements for a successful fracture healing response summarized as the "diamond concept" in terms of aging and osteoporosis. The current preclinical and clinical evidence for a beneficial or harmful influence of anti-osteoporosis medications such as bisphosphonates, parathyroid hormone (PTH), strontium ranelate and antibodies of Wnt-inhibiting signaling proteins on bone healing is presented and discussed. Literature suggests that there are no detrimental consequences of such therapeutics on fracture repair processes. Following a fragility fracture, it seems that early start of preventive anti-osteoporotic treatment right after surgery does not delay the union of the fracture, except perhaps in the case of very rigidly fixed fracture requiring direct bone healing. There is some promising experimental and clinical evidence for possible enhancement of the bone repair process via administration of systemic agents. Further well designed studies in humans are necessary to accumulate more evidence on the positive effects and to translate this knowledge into valid therapeutic applications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Development of anti-angiogenic therapy including the vascular endothelial growth factor (VEGF) antibodies and VEGF-tyrosine kinase receptors has been a major landmark in cancer therapy leading improvement in survival in several cancers. While anti-angiogenic therapy is effective in some settings, resistance often develops owing to evasive, alternative pathways. Novel targets for anti-angiogenic therapy are urgently required to provide treatment alternatives in patients whose tumors are unresponsive to approved anti-angiogenic agents; one such pathway is the bone morphogenetic proteins (BMP 9 and BMP 10) that activate the type I activin receptor-like kinase-1 (ALK1), which has been implicated in the development of functional vasculature. Dalantercept (ACE-041) is a novel anti-angiogenic agent, which is a soluble form of ALK1, and acts as a ligand trap for BMP 9 and BMP 10, inhibiting their interaction with ALK1, which further disrupts the process of vascular development. This review will discuss the preclinical and clinical development of dalantercept as a novel anti-angiogenic therapy in treating a variety of cancers and its distinct safety profile compared to other anti-VEGF agents. We will also discuss the ongoing and completed studies of dalantercept, including combination studies with other VEGF-directed therapies.

Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-β (TGF-β) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

Objective: Serum sclerostin levels have been reported to be inversely associated with serum 25OHD levels, but the effect of vitamin D and calcium supplementation on serum sclerostin levels is unknown. This study was carried out to determine whether vitamin D and calcium supplementation altered serum sclerostin levels in healthy older adults., Design: We measured serum sclerostin levels at baseline and after 2 years in 279 men and women who participated in a placebo-controlled vitamin D (700 IU/day) and calcium (500 mg/day) intervention trial carried out in men and women aged ≥65 years., Method: Serum sclerostin levels were measured using the MesoScale Discovery chemiluminescence assay., Results: In the men, sclerostin levels increased over 2 years by 4.11±1.81 ng/l (13.1%) in the vitamin D plus calcium-supplemented group and decreased by 3.16±1.78 ng/l (10.9%) in the placebo group (P=0.005 for difference in change). Adjustments for the season of measurement, baseline physical activity levels, baseline serum sclerostin levels, and total body bone mineral content did not substantially alter the changes. In the women, there was no significant group difference in change in serum sclerostin levels either before or after the above-mentioned adjustments. In both the sexes, vitamin D and calcium supplementation significantly increased serum ionized calcium levels and decreased parathyroid hormone levels., Conclusion: Men and women appear to have different serum sclerostin responses to vitamin D and calcium supplementation. The reason for this difference remains to be determined.

Fatty acid metabolism plays an important role in brain development and function. Mutations in acyl-CoA synthetase long-chain family member 4 (ACSL4), which converts long-chain fatty acids to acyl-CoAs, result in nonsyndromic X-linked mental retardation. ACSL4 is highly expressed in the hippocampus, a structure critical for learning and memory. However, the underlying mechanism by which mutations of ACSL4 lead to mental retardation remains poorly understood. We report here that dAcsl, the Drosophila ortholog of ACSL4 and ACSL3, inhibits synaptic growth by attenuating BMP signaling, a major growth-promoting pathway at neuromuscular junction (NMJ) synapses. Specifically, dAcsl mutants exhibited NMJ overgrowth that was suppressed by reducing the doses of the BMP pathway components, accompanied by increased levels of activated BMP receptor Thickveins (Tkv) and phosphorylated mothers against decapentaplegic (Mad), the effector of the BMP signaling at NMJ terminals. In addition, Rab11, a small GTPase involved in endosomal recycling, was mislocalized in dAcsl mutant NMJs, and the membrane association of Rab11 was reduced in dAcsl mutant brains. Consistently, the BMP receptor Tkv accumulated in early endosomes but reduced in recycling endosomes in dAcsl mutant NMJs. dAcsl was also required for the recycling of photoreceptor rhodopsin in the eyes, implying a general role for dAcsl in regulating endocytic recycling of membrane receptors. Importantly, expression of human ACSL4 rescued the endocytic trafficking and NMJ phenotypes of dAcsl mutants. Together, our results reveal a novel mechanism whereby dAcsl facilitates Rab11-dependent receptor recycling and provide insights into the pathogenesis of ACSL4-related mental retardation.

Background: Although enamel matrix derivative (EMD) has demonstrated the ability to promote angiogenesis and osteogenesis both in vitro and in vivo, the specific elements within the EMD compound responsible for these effects remain unknown., Methods: Nine different protein pools from a commercially produced EMD were collected based on molecular weight. Six of these pools, along with the complete EMD unfractionated compound and positive and negative controls, were tested for their ability to induce bone formation in a calvarial induction assay. Immunocytochemistry of phosphorylated SMAD1/5/8 (phospho-SMAD), osterix, and vascular endothelial growth factor A (VEGF-A) was carried out at selected time points. Finally, proteomic analysis was completed to determine the specific protein-peptide content of the various osteoinductive pools., Results: One of the lower-molecular-weight pools tested, pool 7, showed bone induction responses significantly greater than those of the other pools and the complete EMD compound and was concentration dependent. Dynamic bone formation rate analysis demonstrated that pool 7 was optimally active at the 5- to 10-μg concentration. It was demonstrated that EMD and pool 7 induced phospho-SMAD, osterix, and VEGF-A, which is indicative of increased bone morphogenetic protein (BMP) signaling. Proteomic composition analysis demonstrated that pool 7 had the highest concentration of the biologically active amelogenin-leucine-rich amelogenin peptide and ameloblastin 17-kDa peptides., Conclusions: These studies demonstrate that the low-molecular-weight protein pools (7 to 17 kDa) within EMD have greater osteoinductive potential than the commercially available complete EMD compound and that the mechanism of action, in part, is through increased BMP signaling and increased osterix and VEGF-A. With this information, selected components of EMD can now be formulated for optimal osteo- and angio-genesis.

Background: A number of medications are approved for treatment of osteoporosis. As mode of action usually is anti-catabolic/anti-resorptive or anabolic, it is of interest to know whether these drugs affect not only normal bone remodeling, but also fracture healing., Objective: The purpose of this paper is to give a short overview of the potential effect of various anti-osteoporotic medication on fracture healing., Methods: A narrative literature review was performed to describe the current knowledge., Results: Anti-catabolic/anti-resorptive drugs: for bisphosphonates, the most common class of drugs in this group, experimental studies have shown a larger and stronger callus and delayed remodeling but no evidence of delayed healing. A human monoclonal antibody to RANKL is another anti-catabolic drug, with the only report to date showing enhanced healing in an animal model. Strontium ranelate is a drug where both anti-catabolic and a weak anabolic effect have been proposed, with experimental data ranging from no effect to significant increase in both callus volume and strength. Anabolic drugs: PTH has demonstrated accelerated healing of various experimental fractures and of distal radius and pelvic fractures in humans. While the exact mechanism is not fully understood, PTH results in increased recruitment and differentiation of chondrocytes and enhancement of endochondral ossification. A monoclonal antibody to block sclerostin is another potential anabolic pathway, where animal data have shown increase in bone mass and strength. The potential effect on fracture healing is yet to be studied., Conclusion: There are still large gaps in the understanding of the potential effect of anti-osteoporotic drugs on fracture healing, although based on present knowledge a recent or present fracture should not be considered as a contraindication to such treatment.

Introduction: During root formation, Smad-4 plays a key role during the epithelial-mesenchymal interactions and the Hertwig's epithelial root sheath (HERS) apical proliferation. The root formation and eruption of rat molars is impeded by alendronate treatment due to the inhibition of bone resortion by this drug. The present study aimed to examine the structures affected in the developing root and immunodetect the presence of Smad-4 in rats treated with alendronate., Methods: Newborn Wistar rats were daily injected 2.5 mg/kg alendronate (ALN) during 9, 12 and 30 days. The controls (CON) were injected with saline. The maxillae were fixed and embedded in paraffin or Spurr resin. Paraffin sections were incubated in Smad-4 antibody that was labelled with DAB. The ultrathin sections were examined in a transmission electron microscope., Results: In ALN, a short portion of root dentine was formed; the epithelial diaphragm (ED) and the dental follicle (DF) were disorganized by the contact of bone trabeculae. The (CON) molar roots developed normally. Smad-4 labelling was detected in the cytoplasm of fibroblasts and cementoblasts adjacent to the cementum in CON; in ALN group, few ED cells presented weak immunolabelling. Ultrastructurally, the ED and DF appeared disrupted due to the presence of thin bone trabeculae between its cells. It resulted in the lack of apical proliferation of HERS and, consequently, arrest of root formation., Conclusion: The immunodetection of Smad-4 in the DF cells of ALN specimens indicates that the signalling for the differentiation of these cells into cementum-forming fibroblasts and cementoblasts occurs, despite the impairment of root elongation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Unlabelled: We determined whether suppression of sclerostin levels by estrogen treatment was mediated by anti-resorptive effect. Raloxifene, but not bisphosphonates, suppressed circulating sclerostin concentration, suggesting that sclerostin may mediate the action of estrogen on bone metabolism, independently of their anti-resorptive effects., Introduction: Circulating sclerostin concentrations are higher in postmenopausal than in premenopausal women, and estrogen treatment suppresses sclerostin levels in both men and women. We determined whether anti-resorptives may suppress the circulating sclerostin levels., Methods: We conducted a retrospective observational study. Eighty postmenopausal women were treated with raloxifene for 19.4 ± 7.7 months (n = 16), bisphosphonates for 19.2 ± 6.7 months (n = 32), or were untreated (n = 32) for 17.1 ± 4.6 months. Plasma sclerostin concentrations were measured before and after treatment., Results: Plasma sclerostin levels after treatment were significantly lower in the raloxifene than in the control group (55.8 ± 23.4 pmol/l vs. 92.1 ± 50.4 pmol/l, p = 0.046), but were similar between the bisphosphonate and control groups. Relative to baseline, raloxifene treatment markedly reduced plasma sclerostin concentration (-40.7 ± 22.8%, p < 0.001), with respect to both control (-7.5 ± 29.1%) and bisphosphonate (-3.1 ± 35.2%) groups. Changes in bone-specific alkaline phosphatase and osteocalcin levels showed reverse associations with sclerostin concentration changes in the raloxifene (γ = -0.505, p = 0.017) and control (γ = -0.410, p = 0.020) groups., Conclusions: Raloxifene, but not bisphosphonates, significantly suppressed circulating sclerostin concentration, suggesting that sclerostin may mediate the action of estrogen on bone metabolism, independently of their anti-resorptive effects.

Unlabelled: Sclerostin is expressed by osteocytes and inhibits bone formation by osteoblasts. In this study, serum sclerostin was positively correlated with either lumbar spinal bone mineral density or T-score. Furthermore, serum sclerostin was increased after 6 months treatment with risedronate, whereas remained unchanged after 6 months teriparatide treatment., Introduction: The primary aim of this study was the evaluation of serum sclerostin levels in postmenopausal women and their association with bone mineral density (BMD) and bone turnover markers. The secondary aim was the evaluation of treatment with either teriparatide (TPTD) or risedronate (RIS) on serum sclerostin levels in women with postmenopausal osteoporosis., Methods: Women with postmenopausal osteoporosis, assigned to receive either TPTD (TPTD group, n = 13) or RIS (RIS group, n = 36) for 6 months, and non-osteoporotic early postmenopausal women (NOEP group, n = 13) were recruited. Main outcome measure was serum sclerostin levels., Results: Serum sclerostin was higher in the NOEP group at baseline compared with either TPTD group (p = 0.007) or RIS group (p = 0.049). Sclerostin was positively correlated with both lumbar spinal (LS) BMD (r = 0.353; p = 0.005) and T-score (r = 0.501; p < 0.001) and negatively correlated with intact parathyroid hormone (r = -0.343; p = 0.024) at baseline. Multiple regression analysis showed that either LS BMD (Beta = 0.653; p = 0.018) or T-score (Beta = 0.711; p = 0.005) were independent predictors of serum sclerostin levels. No significant correlation was observed between serum sclerostin and bone turnover markers or estradiol at baseline. Sclerostin was significantly increased 6 months post-treatment in RIS group (p = 0.002), whereas remained statistically unaffected in the TPTD group., Conclusions: Serum sclerostin is decreased in women with postmenopausal osteoporosis compared with non-osteoporotic early postmenopausal women and is positively correlated to either LS BMD or LS T-score. Furthermore, serum sclerostin was increased after 6 months treatment with RIS, whereas remained essentially unchanged after 6 months TPTD treatment.

Objective: We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explored whether reducing macrophage intracellular iron levels via pharmacological suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis., Methods and Results: To suppress hepcidin, increase expression of the iron exporter ferroportin, and reduce macrophage intracellular iron, we used a small molecule inhibitor of bone morphogenetic protein (BMP) signaling, LDN 193189 (LDN). LDN (10 mg/kg IP b.i.d.) was administered to mice, and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to apolipoprotein E-/- mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; P=0.03), reduced Oil Red O-positive lipid area by 50% (n=8; P=0.02), and decreased total plaque area by 43% (n=8; P=0.001). LDN suppressed liver hepcidin transcription and increased macrophage ferroportin, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1, and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects., Conclusions: These data suggest that pharmacological manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.

Background and Purpose: Sclerostin is produced by osteocytes and is an inhibitor of bone formation. Thus, inhibition of sclerostin by a monoclonal antibody increases bone formation and improves fracture repair. Sclerostin expression is upregulated in unloaded bone and is downregulated by loading. We wanted to determine whether an anti-sclerostin antibody would stimulate metaphyseal healing in unloaded bone in a rat model., Methods: 10-week-old male rats (n = 48) were divided into 4 groups, with 12 in each. In 24 rats, the right hind limb was unloaded by paralyzing the calf and thigh muscles with an injection of botulinum toxin A (Botox). 3 days later, all the animals had a steel screw inserted into the right proximal tibia. Starting 3 days after screw insertion, either anti-sclerostin antibody (Scl-Ab) or saline was given twice weekly. The other 24 rats did not receive Botox injections and they were treated with Scl-Ab or saline to serve as normal-loaded controls. Screw pull-out force was measured 4 weeks after insertion, as an indicator of the regenerative response of bone to trauma., Results: Unloading reduced the pull-out force. Scl-Ab treatment increased the pull-out force, with or without unloading. The response to the antibody was similar in both groups, and no statistically significant relationship was found between unloading and antibody treatment. The cancellous bone at a distance from the screw showed changes in bone volume fraction that followed the same pattern as the pull-out force., Interpretation: Scl-Ab increases bone formation and screw fixation to a similar degree in loaded and unloaded bone.

Objective: Age-related bone loss is associated with significant changes in bone remodeling characterized by decreased trabecular and periosteal bone formation relative to bone resorption, resulting in bone fragility and increased risk of fractures. Prevention or reversal of age-related decrease in bone mass and increase in bone fragility has been based on inhibition of bone resorption using anticatabolic drugs. The current challenge is to promote osteoblastogenesis and bone formation to prevent age-related bone deterioration., Methods: A limited number of approved therapeutic molecules are available to activate bone formation. Therefore, there is a need for anabolic drugs that promote bone matrix apposition at the endosteal, endocortical, and periosteal envelopes by increasing the number of osteoblast precursor cells and/or the function of mature osteoblasts. In this study, we review current therapeutics promoting bone formation and anabolic molecules targeting signaling pathways involved in osteoblastogenesis, based on selected full-text articles searched on Medline search from 1990 to 2010., Results and Discussion: We present current therapeutic approaches focused on intermittent parathyroid hormone and Wnt signaling agonists/antagonists. We also discuss novel approaches for prevention and treatment of defective bone formation and bone loss associated with aging and osteoporosis. These strategies targeting osteoblastic cell functions may prove to be useful in promoting bone formation and improving bone strength in the aging population.

Aims: Thiazolidinediones (TZDs) are associated with a higher risk of bone fracture in women compared with men. The aim of the present study was to investigate whether TZDs could influence osteocyte behaviour and contribute to the skeletal phenotype observed in TZD-treated patients., Methods: The murine MLO-Y4 cell line was used as a source of osteocytes. These cells were cultured for 24 h with 0, 10(-8) m, 10(-7) m, 10(-6) m, 10(-5) m or 10(-4) m of pioglitazone, rosiglitazone or troglitazone in the presence or absence of 17beta-oestradiol. The extent of osteocyte apoptosis was assessed, as was the expression of the bone formation inhibitor sclerostin and receptor activator for nuclear factor kappaB ligand (RANKL) also., Results: In the absence of 17beta-oestradiol, pioglitazone, rosiglitazone and troglitazone induced osteocyte apoptosis dose-dependently even at the lowest concentration of 10(-8) m. Furthermore, the expression of sclerostin but not RANKL was significantly increased in TZD-treated cultures compared with untreated cultures. The presence of 17beta-oestradiol significantly reduced TZD-induced osteocyte apoptosis and also sclerostin up-regulation., Conclusions: These findings therefore raise the potential concern of using TZDs in post-menopausal women where the lack of oestrogen would not prevent osteocyte apoptosis and sclerostin up-regulation and may aggravate the reduction in bone mass in these patients.

Previous studies revealed that bone morphogenetic protein (BMP) induces commitment to the adipocyte lineage in pluripotent stem cells. The present study explored the role of endogenous BMP activity in 3T3-L1 preadipocytes. The expression of phospho-Smad1/5/8 was monitored because BMP transmits its signal through Smad1/5/8 phosphorylation. Phosphorylated Smad1/5/8 was higher in proliferating preadipocytes, and lower in differentiating adipocytes after removing differentiation inducers. Reporter assays revealed that dorsomorphin predominantly inhibits the BMP pathway but not the structurally related TGF-beta/activin pathway. The addition of dorsomorphin to the culture medium prior to treatment with differentiation inducers impaired lipid accumulation in 3T3-L1 cells. The present study indicated that activation of BMP signaling in preadipocytes is required for these cells to initiate the adipogenic program.

The feature of osteoconductivity, and expression of inductive BMP and transcription factors (Runx2 and Osterix) for osteoblast differentiation, which was related to conductive bone formation, were observed in experimentally created defects in rat femoral and parietal bones filled with beta-tricalcium phosphate (beta-TCP) or carbonate apatite (CAP). Femoral cortical bone defects were repaired by conductive bone formed by osteoblasts differentiated around beta-TCP and CAP, and immunohistochemical observation revealed that the osteoblasts expressed BMPs, Runx2, and Osterix. However, the repair in parietal bone defects was incomplete despite the beta-TCP and CAP filling. Only cells, which differentiated around beta-TCP or CAP, and formed conductive bone expressed BMPs, Runx2, and Osterix. These findings revealed that the osteoconductivity of calcium phosphate materials required the expression of BMPs as the prerequisite for Runx2 and Osterix expression. Therefore, it is suggested that when calcium phosphate ceramics are used as bone substitute materials, BMPs are essential for osteoconductivity.

Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

Background: Previous reports have indicated that statins could prevent bone loss in ovariectomized (OVX) rats and increase the expressions of osteogenic genes in cultured osteoblasts. In this study, we hypothesized that simvastatin might increase osteoblast number and protein expressions of osteogenic markers localized in bones in concomitance with the prevention of bone loss in OVX rats., Materials and Methods: Fifty-four 3-month-old OVX and sham-operated (SHAM) female Sprague-Dawley rats were used. Simvastatin (10-20 mg kg(-1) day(-1)) was administrated orally for 6 weeks. Trabecular volume, osteoblast number and osteogenic proteins including BMP2, collagen type I and osteocalcin on bone sections obtained from lumbar vertebral body, distal femur and proximal tibia were measured., Results: The results showed that SHAM rats had significantly less trabecular bone volume and osteoblast number than that of OVX rats 6 weeks after operation. Oral simvastatin treatment (10-20 mg kg(-1) day(-1)) increased bone volume and osteoblast number in the distal femurs, proximal tibiae and vertebrae of OVX rats. Furthermore, the osteoblastic cells with immuno-stained BMP2, collagen type I and osteocalcin in vertebral bones were significantly increased by simvastatin treatment (20 mg kg(-1) day(-1)) in OVX rats., Conclusions: This study demonstrates that simvastatin enhances the production of osteogenic proteins in bone and this effect may contribute to the prevention of bone loss in OVX rats.

Background and Purpose: Inhibition of proteasome has been emerging as a promising approach in pathway-directed cancer therapy. Bone morphogenetic protein (BMP) signalling, which is known to be regulated by the ubiquitin-proteasome pathway in osteoblasts, plays a crucial role in the suppression of gastrointestinal carcinogenesis. Here we sought to elucidate the anti-mitogenic effect of a proteasome inhibitor in relation to BMP signalling in colon cancer., Experimental Approach: The effects of the proteasome inhibitor MG-132 on proliferation of SW1116 and HT-29 colon cancer cells were determined by [(3)H]-thymidine incorporation and colony-formation assay. The involvement of BMP signalling in the action of MG-132 was elucidated by western blot, real-time PCR, immunofluorescence and RNA interference., Key Results: MG-132 significantly suppressed the proliferation of colon cancer SW1116 and HT-29 cells. In this regard, MG-132 activated BMP signalling and this was manifested as an increase in Smad1/5/8 phosphorylation and upregulation of p21(Waf1/Cip1) and p27(Kip1) expression. Knockdown of BMP receptor II abolished Smad1/5/8 phosphorylation, the induction of p21(Waf1/Cip1) and p27(Kip1) and inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 upregulated the expression of BMP1 and BMP2, which are secreted members of the BMP superfamily. Moreover, the expression of Smad6, an intracellular inhibitor of BMP signalling, was suppressed by MG-132., Conclusions and Implications: These findings suggest that inhibition of proteasome suppresses the proliferation of colon cancer cells via activation of BMP signalling. They also demonstrate a novel aspect of proteasome function in the regulation of colon cancer cell proliferation.

Bone morphogenetic protein-7 (BMP7) is a member of the BMP-subfamily of perhaps a dozen proteins within the TGFbeta-superfamily of cysteine-knot fold cytokine-growth factors. BMP7 has pivotal functions during renal and eye development. In adult organisms, BMP7 is heavily expressed in kidney, specifically in podocytes, distal tubules and collecting ducts. The activity of BMP7 is reduced by inhibitors including some members of the dan-cerberus group and CTGF but can be enhanced by endoglin and KCP. Renal BMP7 disappears early in fibrogenic renal diseases which may facilitate progression. Exogenous administration of rhBMP7 or transgenic overexpression reduces renal fibrogenesis and apoptosis as well as transdifferentiation of epithelial cells. BMP7 improves maintenance of nephron function and structural integrity. These antifibrogenic activities result from inhibition of the nuclear translocation of TGFbeta-activated smad3 by smad6 downstream of BMP7-activated smad5. Although at present the beneficial effects of BMP7 have only been studied in rodent models of chronic renal diseases, there is promise for therapeutic utility of rhBMP7 or small molecule BMP7 agonists in patients.

Purpose: Current medulloblastoma therapy, surgery, radiation, and chemotherapy, is unacceptably toxic. However, 13-cis retinoic acid (RA) and SAHA, a histone deacetylase inhibitor, have each been shown to induce apoptosis in medulloblastoma cultures and mouse models. Both drugs cross the blood brain barrier, have been given safely to children, and achieve brain concentrations that are at or near therapeutic levels. Retinoic acid acts by transcriptionally activating bone morphogenetic protein-2 (BMP-2) and SAHA facilitates transcriptional activity through chromatin accessibility. We tested the hypothesis that these drugs additively induce BMP-2 transcription and apoptosis., Experimental Design: RA + SAHA induction of BMP-2 transcription and apoptosis in medulloblastoma cultures was evaluated. Subsequently the response of mouse medulloblastomas to these two agents in the presence and absence of cisplatin was evaluated., Results: BMP-2 transcription multiplied 3-fold with addition of RA to culture, and 7-fold with both agents. The IC50 of SAHA was reduced by 40% when low dose RA was added. Interestingly, a p38 MAP kinase inhibitor that partially blocks RA-induced apoptosis did not inhibit the activity of RA + SAHA. Flank D283 tumors in athymic mice had slower growth in the RA + SAHA arm than single drug or control arms. Intracranial tumors in ND2:SmoA1 mice treated with RA + SAHA + cisplatin showed a 4-fold increase in apoptosis over controls, and a 2-fold increase over animals receiving only SAHA or RA + SAHA., Conclusions: RA + SAHA additively induce BMP-2 transcription and medulloblastoma apoptosis. The combination may act through a p38 MAPK independent mechanism. Efficacy increased with cisplatin, which has implications for clinical trial design.

Osteoporosis is a reduction in skeletal mass due to an imbalance between bone resorption and bone formation. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. Therefore, components involved in BMP activation are good targets for the development of anti-osteoporosis drugs. In this study, imperatorin and bergapten, two coumarin derivatives, were shown to enhance alkaline phosphatase (ALP) activity, type I collagen synthesis and bone nodule formation in primary cultured osteoblasts. Imperatorin and bergapten increased mRNA levels of BMP-2 using quantitative RT-PCR, whereas the BMP-2 antagonist noggin attenuated the increase of ALP activity induced by imperatorin and bergapten, indicating that BMP-2 expression is required for the action of imperatorin and bergapten in osteoblastic maturation. Both imperatorin and bergapten enhanced the phosphorylation of SMAD (transcription factors activated by TGF-beta) 1/5/8, p38 and extracellular signal-regulated protein (ERK). Pretreatment of osteoblasts with p38 inhibitor (SB203580) or mitogen-activated protein kinase inhibitor (PD98059) or transfected with dominant negative mutant of p38 or ERK antagonized the elevation of BMP-2 expression and ALP activity induced by imperatorin and bergapten. Local administration of imperatorin or bergapten into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the BMP-2 immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provide evidence that coumarin derivatives increase BMP-2 expression and enhance bone formation in rat via the p38 and ERK-dependent signaling pathway.

Anti-bone resorption properties of the Korean herbal formulation, Gami-Honghwain (HJ), which comprises Carthamus tinctorius L. seed and hominis placenta, were investigated. We demonstrate that the production of PGE2 is inhibited by 20-100 microg/ml HJ in nontransformed osteoblastic cells (MC3T3-E1 cells), indicating that HJ inhibits PGE2 production. The effect of HJ on the proliferation and osteoblastic differentiation in MC3T3-E1 was also studied. HJ dose-dependently increased DNA synthesis (significant at 20-100 microg/ml), and increased alkaline phosphatase (ALP) and prolyl hydroxylase activities of MC3T3-E1 cells (20-100 microg/ml), while anti-estrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1 which was induced by HJ. These results indicate that HJ directly stimulates cell proliferation and differentiation of osteoblasts. Also, when we assessed the effects of HJ on osteoblastic differentiation in MC3T3-E1, HJ enhanced ALP activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the HJ was observed at relatively low doses (significant at 20-100 microg/ml and maximal at 100 microg/ml). Northern blot analysis showed that the HJ (60 microg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. HJ (100 microg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that HJ has anabolic effect on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases.

Bone morphogenetic protein-2 (BMP-2) plays a key role in both vascular development and pathophysiological processes. However, the effect of high glucose on BMP-2 expression remains unclear. Here we show in human umbilical vein endothelial cells that high glucose increased BMP-2 expression, associated with NF-kappaB activation, whereas pioglitazone suppressed BMP-2 expression and NF-kappaB65 activation induced by high glucose. Furthermore, the increase in MDA levels and decrease in activities of total superoxide dismutase of cell culture stimulated by high glucose were reversed by pioglitazone treatment. Our findings indicate that BMP-2 expression induced by high glucose might be closely related to NF-kappaB activation, and both effects can be suppressed by pioglitazone., ((c) 2008 S. Karger AG, Basel)

Carrageenans are highly sulfated polysaccharides that are widely used as food additives in the Western diet, in order to improve the texture of processed foods. Although native and degraded carrageenans induce colonic ulcerations, polyps, and colorectal tumors in animal models, very little is known about the effects of carrageenan on human colonocytes. We evaluated effects of lambda-carrageenan (lambdaCGN) on the normal human colonocyte cell line NCM460, using a concentration of 1 mug/ml, about less than one tenth the average daily exposure to carrageenan in the Western diet. We measured secreted bone morphogenetic protein-4 (BMP4) in spent media and quantified its expression by quantitative RT-PCR. Wnt-related genes were measured by an oligonucleotide array. Cellular beta-catenin was quantified by ELISA. We found a marked decline in secreted BMP4 (P < 0.001) following exposure of NCM460 cells to lambdaCGN for 24 hr. Quantitative RT-PCR for BMP4 transcripts revealed 24% and 45% inhibition of expression on days 2 and 4. cDNA gene expression array of Wnt signaling pathway target genes demonstrated significant changes, including 4.5-fold induction of Wnt 9A and suppression of Dickkopf 3 and RHOU genes. Measurement of beta-catenin by ELISA revealed concomitant accumulation with increases of 67.8%, 61.6%, and 73.9% on days 1, 2, and 4, compared to untreated controls. We conclude that treatment of normal human colonocytes with lambdaCGN activated the Wnt/beta-Catenin cascade and suppressed the expression and secretion of BMP4, inducing significant changes in cellular pathways that are associated with both sporadic and juvenile polyps. CGN may influence development of intestinal polyps in vivo by these mechanisms.

Background and Objective: We have previously reported that mechanical strain applied at a 1% level to an osteoblastic cell line induces the transcription of prostaglandin D2 synthase and increases the levels of prostaglandin D2 and its Delta12prostaglandin J2 metabolite. Mechanical strain also induces the expression of peroxisome proliferator-activated receptor gamma-1 and bone nodule formation. We hypothesized that mechanical load induces bone formation via Delta12prostaglandin J2-dependent synthesis of bone morphogenetic proteins. Our goal was to investigate the molecular events involved in osteogenesis induced by mechanical loading and Delta12prostaglandin J2, namely the induction of bone morphogenetic proteins and peroxisome proliferator-activated receptor gamma-1, a nuclear receptor for Delta12prostaglandin J2., Material and Methods: Osteoblast monolayers were stretched for 1 h with a 1-h resting period and stretched for another hour at 1 Hz with 1% elongation. Cells were collected 0, 1, 6 and 16 h after stretching. Cyclooxygenase inhibitors and Delta12prostaglandin J2 were added in some experiments. Relative quantitative reverse transcriptase-polymerase chain reaction was used to examine whether the mRNA of bone morphogenetic protein-2, -4, -6, -7 and peroxisome proliferator-activated receptor gamma-1 was induced. Immunohistochemistry was used to evaluate bone morphogenetic protein expression in cells., Results: Mechanical strain significantly increased the mRNA expression of bone morphogenetic protein-2, -6, -7 and of peroxisome proliferator-activated receptor gamma-1, but not of bone morphogenetic protein-4. In stretched cells, bone morphogenetic protein-2 and peroxisome proliferator-activated receptor gamma-1 expression was blocked by cyclooxygenase inhibitors, but restored by exogenous Delta12prostaglandin J2. Delta12Prostaglandin J2 significantly enhanced bone nodule formation and bone morphogenetic protein-2 expression when added alone to resting osteoblasts., Conclusion: These results suggest that the osteoblastic biomechanical pathways that trigger bone formation involve cyclooxygenase and prostaglandin D2 synthase activation, induction of Delta12prostaglandin J2 and its nuclear receptor, peroxisome proliferator-activated receptor gamma-1, and increased expression of bone morphogenetic protein-2. These data suggest that the Delta12prostaglandin J2/peroxisome proliferator-activated receptor gamma-1/bone morphogenetic protein-2 pathway plays an important role in osteogenesis.

Simvastatin, a cholesterol synthesis inhibitor, enhances BMP2 expression in osteoblasts. The purpose of the present study was to examine whether simvastatin stimulates bone regeneration when combined with calcium sulfate as a carrier. Critical-sized bone defects in rat calvaria were treated with calcium sulfate or with combination of 1 mg simvastatin and calcium sulfate. In the combination group, although the least amount of bone formation with intense soft tissue inflammation was observed at 2 and 4 weeks, remarkable bone formation was evident at 8 weeks. Conclusively, the combination of simvastatin and calcium sulfate stimulated bone regeneration in spite of the inflammatory response.

Background and Objective: Simvastatin, a cholesterol-lowering drug, also stimulates oral bone growth when applied topically, without systemic side-effects. However, the mechanisms involved in vivo are not known. We hypothesized that bone morphogenetic protein-2, nitric oxide synthase, and cyclooxygenase-2 are involved, based on prior in vitro evidence., Material and Methods: A rat bilateral mandible model, where 0.5 mg of simvastatin in methylcellulose gel was placed on one side and gel alone on the other, was used to quantify nitric oxide, cyclooxygenase-2 and bone morphogenetic protein-2 (via tissue extraction, enzyme activity or immunoassay), and to analyze the bone formation rate (via undecalcified histomorphometry). Cyclooxygenase-2 and nitric oxide synthase inhibitors (NS-398 and L-NAME, respectively) were administered intraperitoneally., Results: Simvastatin was found to stimulate local bone morphogenetic protein-2, nitric oxide and the regional bone formation rate (p < 0.05), whereas NS-398 inhibited bone morphogenetic protein-2 and reduced the bone formation rate (p < 0.05)., Conclusion: These data suggest an association between simvastatin-induced bone morphogenetic protein-2 and bone formation in the mandibular microenvironment, and the negative effect of cyclooxygenase-2 inhibitors on bone growth.

The aim of this study was to evaluate and compare the effects of two commercial mineral trioxide aggregate (MTA) cements (ProRoot MTA and MTA Angelus) on transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP)-2 levels produced by cultured human gingival fibroblasts (HGFs). Human gingival tissues were obtained from individuals with healthy periodontium. HGFs were grown at 37 degrees C in humidified atmosphere of 5% CO(2) in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, penicillin, and streptomycin. After 24 and 72 hours of exposure to the MTA products, HGF viability was determined by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay. TGF-beta1 and BMP-2 levels in cell-free culture media were determined by enzyme-linked immunosorbent assay. Cell viability of the test groups was significantly lower than that of control at 24 and 72 hours (p < 0.05) but showed an increase at 72 hours (p < 0.05). Both test groups showed increased TGF beta-1 levels at 72 hours (p < 0.05), whereas the MTA Angelus group displayed higher TGF beta-1 levels than control and ProRoot MTA groups at 24 and 72 hours (p < 0.05). At 24 hours, BMP-2 levels of the ProRoot group were significantly higher than that of MTA Angelus (p < 0.05). Both test materials increased the BMP-2 levels within time (p < 0.05) and displayed similar levels at 72 hours (p > 0.05). These results suggest that both MTA products are capable of stimulating HGF to produce BMP-2, whereas the stimulatory effect for TGF beta-1 is material dependent.

In this study, we examined the effects of TNF-alpha on Bone morphogenetic protein (BMP-2), Smads (which play intracellular signaling of BMPs) expression and alkaline phosphatase (ALP) activity of human dental pulp (HDP) cells to clarify the mechanism of tertiary dentin formation. The quantity of RT-PCR product for BMP-2 from the HDP cells stimulated by TNF-alpha is increased. However, ALP activity was not increased on the cells incubated with TNF-alpha. On the other hand, ALP activity was significantly increased on HDP cells treated with Ammonium Pyrrolidinedithiocarbamate (PDTC, NF- kappaB inhibitor) groups and combined supplementation of TNF-alpha and PDTC groups. Furthermore, we examined the effect of TNF-alpha and PDTC on Smad7 expression using RT-PCR and western blot analysis. Smad7 expression in HDP cells was increased by TNF-alpha, but decreased by PDTC treatment. These results suggest that NF- kappaB and Smad7 play an important role in the down regulation of ALP activity by TNF-alpha on HDP cells.

Enamel proteins, particularly amelogenin, have been associated with other functions in addition to regulating enamel biomineralization. Extracts of enamel proteins are currently being used to regenerate periodontal tissues, and new studies suggest that enamel proteins might have chondrogenic and osteogenic properties. In this study, we wanted to determine the effect, if any, of purified recombinant amelogenin and ameloblastin on the adhesion, proliferation, and differentiation of periodontal ligament cells in vitro. Immortomouse-derived periodontal ligament (PDL) cells were grown under permissive and differentiation conditions in the presence of different concentrations of mouse recombinant amelogenin, recombinant ameloblastin, or both. Cells were collected after 4 h to determine attachment, after 24 h to determine proliferation, and after 7, 14, 21 and 28 d to determine differentiation using reverse transcription-polymerase chain reaction (RT-PCR). Both amelogenin and ameloblastin had a small, but statistically significant, effect on increasing the cell attachment and proliferation of PDL cells. Both amelogenin and ameloblastin modulated bone morphogenetic protein (BMP) expression, down-regulated the expression of collagen type I, and induced the de novo expression of osteocalcin. Amelogenin also induced the expression of bone sialoprotein. These results suggest that amelogenin, as well as ameloblastin, might have some 'growth factor' activity during periodontium development and regeneration.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are potent inhibitors of cholesterol biosynthesis. Cholesterol-lowering therapy using statins significantly reduces the risk of coronary heart disease. However, extensive use of statins leads to increases of other undesirable as well as beneficial effects, so-called pleiotropic effects. With respect to these effects, statins augment the expression of bone morphogenetic protein-2, a potent simulator of osteoblast differentiation and its activity, and promote mineralization by cultured osteoblasts, indicating that statins have an anabolic effect on bone. Chronic administration of statins in ovariectomized (OVX) rats modestly increases bone mineral density (BMD) of cancellous bone but not of compact bone. In clinical studies, there are conflicting results regarding the clinical benefits of this therapy for the treatment of osteoporosis. Observational studies suggest an association between statin use and reduction in fracture risk. Clinical trials reported no effect of statin treatment on BMD in hip and spine, and on bone turnover. Statins also may influence oral osseous tissues. Administration of statins in combination with osteoporosis therapy appears to improve alveolar bone architecture in the mandibles of OVX rats with maxillary molar extraction. Statins continue to be considered as potential therapeutic agents for patients with osteoporosis and possibly with periodontal disease. Development of new statins that are more specific and potent for bone metabolism will greatly increase the usefulness of these drugs for the treatment of bone diseases.

It has been shown that Emdogain Gel (Emd-Gel) containing enamel matrix proteins promotes biomineralization, such as osteogenesis and cementogenesis, during the regeneration of periodontal tissues. However, the growth factors involved in these activities of Emd-Gel remain unclear. In this study, Emd-Gel was fractionated into 22 sub-fractions by size exclusion chromatography. The osteoinductive factors, TGF-beta and BMP, were examined by a specific luciferase reporter gene assay. In the unfractionated Emd-Gel, TGF-beta-like activity was detected, while BMP activity was not. In contrast, in the fractionated Emd-Gel samples, TGF-beta-like activity was detected from fractions 8 to 13, and BMP-like activity was detected from fractions 4 to 6. Also, it was confirmed that the BMP-like activity in Emd-Gel was inhibited by authentic TGF-beta1 and TGF-beta-like activity. These results indicate that Emd-Gel contains both TGF-beta- and BMP-like growth factors that contribute to the induction of biomineralization during periodontal regeneration.

Background and Aims: For human use, it is necessary to sterilize bone morphogenetic proteins (BMPs), in order to reduce the risk of infections and associated complications. We compared the effects of ethylene oxide and gamma irradiation in the sterilization of native reindeer BMP extract with regard to bone induction in the Balb/C mouse thigh muscle pouch model., Materials and Methods: BMP extract, sterilized with ethylene oxide gas (Steri-Vac 4XL, temperature 29 degrees C, exposure time 4 h, ethylene oxide concentration 860 mg/l), or gamma irradiation at doses of 3.15 MRad was administered in implants containing 5 or 10 mg of BMP extract with collagen carrier. Non-sterilized collagen implants served as controls. New bone formation was evaluated based on the incorporation of Ca45 and radiographically three weeks after implantation., Results: The collagen was not able to induce new bone visible in radiographs. The mean Ca45 incorporation in the gamma sterilized group containing 5 mg of BMP extract was 30% (p = 0.04) and that containing 10 mg of BMP extract was 60% (p = 0.02) higher than seen in the corresponding ethylene oxide sterilized groups. The mean new bone areas were 45% higher in the gamma sterilized groups than in the corresponding ethylene oxide sterilized groups, but the differences were not significant. The mean optical density of new bone in the gamma sterilized group containing 5 mg of BMP extract was 75% (p = 0.00) and in that containing 10 mg of BMP extract was 70% (p = 0.00) higher than seen in the corresponding ethylene oxide sterilized groups., Conclusion: Native reindeer BMP extract is more sensitive to the effects of ethylene oxide gas sterilization than gamma irradiation. These results suggest that gamma irradiation is recommendable for the sterilization of BMP extracts.

Calcitriol (1alpha,25-dihydroxycholecalciferol) seems to play an important role in the complex control of prostate cell growth. It inhibits proliferation and induces differentiation and apoptosis in prostate cancer cells. However, the molecular mechanisms of the antiproliferative activity of calcitriol are not completely understood. The expression of prostate-derived factor (PDF), a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to be associated with proapoptotic and antimitotic activities. We show that calcitriol induces PDF expression in LNCaP human prostate cancer cells in a concentration- and time-dependent manner. In LNCaP cells, the suppression of cell growth by calcitriol is accompanied by stimulation of PDF mRNA and protein synthesis. Human recombinant PDF inhibits LNCaP cell growth. We do not detect any effect of PDF-specific antibody on the basal growth of LNCaP cells, but this antibody partially reverses the suppression of LNCaP cell growth by calcitriol, suggesting that the effect of calcitriol on cell growth is at least partially mediated by PDF. In PC-3 cells, which are less responsive to the growth-inhibitory effect of calcitriol, it has no effect on PDF expression. We do not detect an effect of recombinant PDF on SMAD phosphorylation in LNCaP cells, but ERK1/2 kinases are transiently phosphorylated in response to PDF, which suggests that in LNCaP cells PDF may exert its action through pathway alternative to the classical TGF-beta signaling pathway. The present study describes the regulation of PDF, the proapoptotic protein of the TGF-beta superfamily, by calcitriol in androgen-responsive prostate cancer cells. This is a new link between calcitriol and growth factors of TGF-beta superfamily in the control of prostate cell growth., ((c) 2004 Wiley-Liss, Inc.)

The induction, growth, and differentiation of epithelial lung buds are regulated by the interaction of signals between the lung epithelium and its surrounding mesenchyme. Fibroblast growth factor-10 (FGF-10), which is expressed in the mesenchyme near the distal tips, and bone morphogenetic protein 4 (BMP4), which is expressed in the most distal regions of the epithelium, are important molecules in lung morphogenesis. In the present study, we used two in vitro systems to examine the induction, growth, and differentiation of lung epithelium. Transfilter cultures were used to determine the effect of diffusible factors from the distal lung mesenchyme (LgM) on epithelial branching, and FGF-10 bead cultures were used to ascertain the effect of a high local concentration of a single diffusible molecule on the epithelium. Embryonic tracheal epithelium (TrE) was induced to grow in both culture systems and to express the distal epithelial marker surfactant protein C at the tips nearest the diffusible protein source. TrE cultured on the opposite side of a filter to LgM branched in a pattern resembling intact lungs, whereas TrE cultured in apposition to an FGF-10 bead resembled a single elongating epithelial bud. Examination of the role of BMP4 on lung bud morphogenesis revealed that BMP4 signaling suppressed expression of the proximal epithelial genes Ccsp and Foxj1 in both types of culture and upregulated the expression of Sprouty 2 in TrE cultured with an FGF-10 bead. Antagonizing BMP signaling with Noggin, however, increased expression of both Ccsp and Foxj1.

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.

Objective: An in vitro experiment study using rat disc cells was carried out to determine the effect of bone morphogenetic protein-2 (BMP-2) on extracellular matrix production, other BMPs, and BMP receptors (BMPRs)., Methods: Cells from the anulus fibrosus and transition zone were harvested and cultured. When the cells reached 80% confluence, BMP-2 was added to reach a final concentration of 200 ng/mL. Three days later, the culture media were collected for the assay of sulfated glycosaminoglycans (sGAG) and collagen types I and II. The cells were harvested for RNA extraction to determine the genes expressed. All experiments were performed at least three times to ensure repeatability., Results: BMP-2 significantly increased aggrecan and collagen type II mRNA expression 8.30 and 4.61 times, respectively, and decreased versican mRNA expression 0.54 times as compared with control. Collagen type I production and mRNA level were not changed. BMP-2 significantly increased transforming growth factor-beta1 (TGFbeta1) and BMP-7 mRNA expression 2.32 and 2.45 times, respectively, compared with control. There was no significant change in BMP-6 mRNA expression. BMPR type IB and II mRNA expressions were increased and BMPR type 1A mRNA expression was decreased, but none of these differences was significant., Conclusions: The results of this study show that in rat intervertebral disc cells, BMP-2 increases aggrecan and collagen type II mRNA expression and decreases versican gene expression. BMP-2 also up-regulates mRNA expression for BMP-7 and TGFbeta but has no significant effect on the BMPRs.

Background: Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor cell proliferation and, following clonal expansion of these cells, promotion of differentiation along the osteogenic lineage., Objectives: We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors., Methods: The cell populations were assessed for mineralization potential after long-term culture in media containing beta-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogenic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin, osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7., Results: As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP, osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH., Conclusion: The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers.

We investigated the dynamics of bone morphogenetic protein (BMP) and their receptor mRNA expression in relation to combined treatment with all trans-retinoic acid (ATRA) and chemotherapy in four patients with acute promyelocytic leukemia (APL). Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the bone marrow cells at diagnosis showed strong expression of BMP-2, -4, and -7, and their receptors RIA, RIB, and RII, parallel to the expression of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) fusion gene transcripts. Therapeutic clearance of the tumor molecular marker corresponded to the absence of BMP expression, suggesting the possible role of BMPs as markers of the minimal residual disease (MRD) in APL.

Statins increase bone morphogenetic protein-2 (BMP-2) mRNA expression and subsequently increase new bone formation in vitro. However, the action of statins on the BMP-2 mRNA regulation of cartilage matrix synthesis by chondrocytes is unknown. We evaluated regulation of BMP-2, aggrecan, and type II collagen (COL2) mRNA and (35)S-labeled proteoglycan (PG) synthesis by mevastatin using cultured chondrocytes obtained from articular cartilage of fetal rats. Expression of BMP-2, aggrecan, and COL2 mRNAs were increased in the presence of 2 microM mevastatin on day 2. However, longer (10 day) culture in the presence of the drug decreased the expression of these mRNAs. PG synthesis was increased 3 days after treating the cells with mevastatin, which was also decreased with longer (10 day) mevastatin treatment. These results suggest that mevastatin increases mRNA expression of BMP-2, aggrecan, and COL2 as well as PG synthesis by fetal rat chondrocytes early in the treatment period. We suggest that statins have implications for fracture and cartilage repair.