Regulation of the Methylation and Expression Levels of the BMPR2 Gene by SIN3a as a Novel Therapeutic Mechanism in Pulmonary Arterial Hypertension.

BACKGROUND: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the BMPR2 (bone morphogenetic protein receptor type 2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of SIN3a (switch-independent 3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. METHODS: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells. Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific adeno-associated virus serotype 1 or a lentivirus encoding for human SIN3a in vivo. RESULTS: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. It is interesting that we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. It is interesting that SIN3a overexpression inhibited human pulmonary arterial smooth muscle cells proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA-sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 (DNA methyltransferase 1) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2) while promoting the expression of the DNA demethylase TET1 (ten-eleven translocation methylcytosine dioxygenase 1). Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF (CCCTC-binding factor) binding to the BMPR2 promoter. Last, we identified intratracheal delivery of adeno-associated virus serotype human SIN3a to be a beneficial therapeutic approach in PAH by attenuating pulmonary vascular and right ventricle remodeling, decreasing right ventricle systolic pressure and mean pulmonary arterial pressure, and restoring BMPR2 expression in rodent models of PAH. CONCLUSIONS: All together, our study unveiled the protective and beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in human pulmonary arterial smooth muscle cells. Our study also identified lung-targeted SIN3a gene therapy using adeno-associated virus serotype 1 as a new promising therapeutic strategy for treating patients with PAH.

The Wnt Signaling Pathway Is Differentially Expressed during the Bovine Herpesvirus 1 Latency-Reactivation Cycle: Evidence That Two Protein Kinases Associated with Neuronal Survival, Akt3 and BMPR2, Are Expressed at Higher Levels during Latency.

Sensory neurons in trigeminal ganglia (TG) of calves latently infected with bovine herpesvirus 1 (BoHV-1) abundantly express latency-related (LR) gene products, including a protein (ORF2) and two micro-RNAs. Recent studies in mouse neuroblastoma cells (Neuro-2A) demonstrated ORF2 interacts with ß-catenin and a ß-catenin coactivator, high-mobility group AT-hook 1 (HMGA1) protein, which correlates with increased ß-catenin-dependent transcription and cell survival. ß-Catenin and HMGA1 are readily detected in a subset of latently infected TG neurons but not TG neurons from uninfected calves or reactivation from latency. Consequently, we hypothesized that the Wnt/ß-catenin signaling pathway is differentially expressed during the latency and reactivation cycle and an active Wnt pathway promotes latency. RNA-sequencing studies revealed that 102 genes associated with the Wnt/ß-catenin signaling pathway were differentially expressed in TG during the latency-reactivation cycle in calves. Wnt agonists were generally expressed at higher levels during latency, but these levels decreased during dexamethasone-induced reactivation. The Wnt agonist bone morphogenetic protein receptor 2 (BMPR2) was intriguing because it encodes a serine/threonine receptor kinase that promotes neuronal differentiation and inhibits cell death. Another differentially expressed gene encodes a protein kinase (Akt3), which is significant because Akt activity enhances cell survival and is linked to herpes simplex virus 1 latency and neuronal survival. Additional studies demonstrated ORF2 increased Akt3 steady-state protein levels and interacted with Akt3 in transfected Neuro-2A cells, which correlated with Akt3 activation. Conversely, expression of Wnt antagonists increased during reactivation from latency. Collectively, these studies suggest Wnt signaling cooperates with LR gene products, in particular ORF2, to promote latency.IMPORTANCE Lifelong BoHV-1 latency primarily occurs in sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. RNA sequencing studies revealed 102 genes associated with the Wnt/ß-catenin signaling pathway are differentially regulated during the latency-reactivation cycle. Two protein kinases associated with the Wnt pathway, Akt3 and BMPR2, were expressed at higher levels during latency but were repressed during reactivation. Furthermore, five genes encoding soluble Wnt antagonists and ß-catenin-dependent transcription inhibitors were induced during reactivation from latency. These findings are important because Wnt, BMPR2, and Akt3 promote neurogenesis and cell survival, processes crucial for lifelong viral latency. In transfected neuroblastoma cells, a viral protein expressed during latency (ORF2) interacts with and enhances Akt3 protein kinase activity. These findings provide insight into how cellular factors associated with the Wnt signaling pathway cooperate with LR gene products to regulate the BoHV-1 latency-reactivation cycle.

Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension.

BACKGROUND: Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. METHODS: We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH. RESULTS: Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses. CONCLUSIONS: Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

Estrogen Metabolite 16α-Hydroxyestrone Exacerbates Bone Morphogenetic Protein Receptor Type II-Associated Pulmonary Arterial Hypertension Through MicroRNA-29-Mediated Modulation of Cellular Metabolism.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a proliferative disease of the pulmonary vasculature that preferentially affects women. Estrogens such as the metabolite 16α-hydroxyestrone (16αOHE) may contribute to PAH pathogenesis, and alterations in cellular energy metabolism associate with PAH. We hypothesized that 16αOHE promotes heritable PAH (HPAH) via microRNA-29 (miR-29) family upregulation and that antagonism of miR-29 would attenuate pulmonary hypertension in transgenic mouse models of Bmpr2 mutation. METHODS AND RESULTS: MicroRNA array profiling of human lung tissue found elevation of microRNAs associated with energy metabolism, including the miR-29 family, among HPAH patients. miR-29 expression was 2-fold higher in Bmpr2 mutant mice lungs at baseline compared with controls and 4 to 8-fold higher in Bmpr2 mice exposed to 16αOHE 1.25 µg/h for 4 weeks. Blot analyses of Bmpr2 mouse lung protein showed significant reductions in peroxisome proliferator-activated receptor-γ and CD36 in those mice exposed to 16αOHE and protein derived from HPAH lungs compared with controls. Bmpr2 mice treated with anti-miR-29 (20-mg/kg injections for 6 weeks) had improvements in hemodynamic profile, histology, and markers of dysregulated energy metabolism compared with controls. Pulmonary artery smooth muscle cells derived from Bmpr2 murine lungs demonstrated mitochondrial abnormalities, which improved with anti-miR-29 transfection in vitro; endothelial-like cells derived from HPAH patient induced pluripotent stem cell lines were similar and improved with anti-miR-29 treatment. CONCLUSIONS: 16αOHE promotes the development of HPAH via upregulation of miR-29, which alters molecular and functional indexes of energy metabolism. Antagonism of miR-29 improves in vivo and in vitro features of HPAH and reveals a possible novel therapeutic target.

Endogenous Parathyroid Hormone Promotes Fracture Healing by Increasing Expression of BMPR2 through cAMP/PKA/CREB Pathway in Mice.

BACKGROUND/AIMS: Endogenous parathyroid hormone (PTH) plays an important role in fracture healing. This study investigated whether endogenous PTH regulates fracture healing by bone morphogenetic protein (BMP) and/or the transforming growth factor-ß (TGF-ß) signaling pathway. METHODS: Eight-week-old wild-type (WT) and PTH-knockout (PTH KO) male mice were selected, and models of open right-femoral fracture were constructed. Fracture healing and callus characteristics of mice in the two groups were compared by X-ray, micro-computed tomography, histological, and immunohistochemical examinations. Bone marrow mesenchymal stem cells (BMMSCs) of 8-week-old WT and PTHKO male mice were obtained and induced into osteoblasts and chondrocytes. RESULTS: We found that expression levels of Runt-related transcription factor (RUNX2), bone morphogenetic protein-receptor-type Ⅱ (BMPR2), phosphorylated Smad 1/5/8, and phosphorylated cyclic adenosine monophosphate-responsive element binding protein (CREB) in the callus of PTHKO mice were significantly decreased, whereas no significant difference in expression of SOX9, TGF-ßR2,or pSMAD2/3 was observed between PTHKO and WT mice. Additionally, the activity of osteoblast alkaline phosphatase was low at 7 days post-induction, and was upregulated by addition of PTH or dibutyryl cyclic adenosine monophosphate (dbcAMP) to the cell culture. Furthermore, H89 (protein kinase A inhibitor)eliminated the simulating effects of PTH and dbcAMP, and a low concentration of cyclic adenosine monophosphate (cAMP) was observed in PTHKO mouse BMMSCs. CONCLUSION: These results suggested that endogenous PTH enhanced BMPR2 expression by a cAMP/PKA/CREB pathway in osteoblasts, and increased RUNX2 expression through transduction of the BMP/pSMAD1/5/8 signaling pathway.

Endothelial-to-mesenchymal transition in pulmonary hypertension.

BACKGROUND: The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells. METHODS AND RESULTS: In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2(Δ140Ex1/+) rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. CONCLUSIONS: EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.

The microRNA-342-5p fosters inflammatory macrophage activation through an Akt1- and microRNA-155-dependent pathway during atherosclerosis.

BACKGROUND: Atherosclerosis is a chronic inflammatory vascular disease driven by the subendothelial accumulation of macrophages. The mechanism regulating the inflammatory response in macrophages during atherogenesis remains unclear. Because microRNAs (miRNAs) play a crucial role in cellular signaling by posttranscriptional regulation of gene expression, we studied the miRNA expression profiles during the progression of atherosclerosis. METHODS AND RESULTS: Using an miRNA real-time polymerase chain reaction array, we found that macrophage-derived miR-342-5p and miR-155 are selectively upregulated in early atherosclerotic lesions in Apoe(-/-) mice. miR-342-5p directly targets Akt1 through its 3'-untranslated region. Akt1 suppression by miR-342-5p induces proinflammatory mediators such as Nos2 and II6 in macrophages via the upregulation of miR-155. The local application of an miR-342-5p antagomir inhibits the development of atherosclerosis in partially ligated carotid arteries. In atherosclerotic lesions, the miR-342-5p antagomir upregulated Akt1 expression and suppressed the expression of miR-155 and Nos2. This reduced Nos2 expression was associated with a diminished generation of nitrotyrosine in the plaques. Furthermore, systemic treatment with an inhibitor of miR-342-5p reduced the progression of atherosclerosis in the aorta of Apoe(-/-) mice. CONCLUSIONS: Macrophage-derived miR-342-5p promotes atherosclerosis and enhances the inflammatory stimulation of macrophages by suppressing the Akt1-mediated inhibition of miR-155 expression. Therefore, targeting miR-342-5p may offer a promising strategy to treat atherosclerotic vascular disease.

BMP signaling in rats with TNBS-induced colitis following BMP7 therapy.

Beyond stimulating bone formation, bone morphogenetic proteins (BMPs) are important in development, inflammation, and malignancy of the gut. We have previously shown that BMP7 has a regenerative, anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the BMP signaling pathway we monitored the effect of BMP7 therapy on the BMP signaling components in the rat colon during different stages of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed a significantly decreased BMP7 expression in the acute phase, followed by a significantly increased BMP2 and decreased BMP6 expression during the chronic phase of colitis. BMP7 therapy influenced the expression of several BMPs with the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the acute stage and significantly decreased upon BMP7 therapy. BMP receptor I expression was unchanged, whereas BMP receptor II was decreased at day 2 and increased at days 14 and 30 of TNBS inflammation. However, an opposite pattern of expression following BMP7 therapy has been observed. BMP7 increased the expression of BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy at later stages of the disease. We suggest that BMP signaling was altered during TNBS-induced colitis and was recovered with BMP7 administration, suggesting that IBD is a reversible process.

Plumbagin reverses proliferation and resistance to apoptosis in experimental PAH.

Like cancer, pulmonary arterial hypertension (PAH) is characterised by a pro-proliferative and anti-apoptotic phenotype. In PAH, pulmonary artery smooth muscle cell (PASMC) proliferation is enhanced and apoptosis suppressed. The sustainability of this phenotype requires the activation of pro-survival transcription factors, such as signal transducer and activator of transcription (STAT)3 and nuclear factor of activated T-cells (NFAT). There are no drugs currently available that are able to efficiently and safely inhibit this axis. We hypothesised that plumbagin (PLB), a natural organic compound known to block STAT3 in cancer cells, would reverse experimental pulmonary hypertension. Using human PAH-PASMC, we demonstrated in vitro that PLB inhibits the activation of the STAT3/NFAT axis, increasing the voltage-gated K(+) current bone morphogenetic protein receptor type II (BMPR2), and decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), rho-associated coiled-coil containing protein kinase (ROCK)1 and interleukin (IL)-6, contributing to the inhibition of PAH-PASMC proliferation and resistance to apoptosis (proliferating cell nuclear antigen (PCNA), TUNEL, Ki67 and anexine V). In vivo, PLB oral administration decreases distal pulmonary artery remodelling, mean pulmonary artery pressure and right ventricular hypertrophy without affecting systemic circulation in both monocrotaline- and suden/chronic hypoxia-induced PAH in rats. This study demonstrates that the STAT3/NFAT axis can be therapeutically targeted by PLB in human PAH-PASMC and experimental PAH rat models. Thus, PLB could be considered a specific and attractive future therapeutic strategy for PAH.

The expression and potential function of bone morphogenetic proteins 2 and 4 in bovine trophectoderm.

BACKGROUND: Bone morphogenetic proteins (BMPs) were first described for their roles in bone formation, but they now also are known to possess additional activities, including those relating to embryogenesis. The objectives of this work were to 1) determine if peri-attachment bovine conceptuses and bovine trophoblast cells (CT1) contain transcripts for BMP2 and 4, an innate inhibitor noggin (NOG), and BMP2/4 receptors (BMPRII, ACVR1, BMPR1A, BMPR1B), and 2) determine if BMP2 or 4 supplementation to CT1 cells affects cell proliferation, differentiation or trophoblast-specific gene expression. METHODS: RNA was isolated from day 17 bovine conceptuses and CT1 cells. After RT-PCR, amplified products were cloned and sequenced. In other studies CT1 cells were treated with BMP2 or 4 at various concentrations and effects on cell viability, cell differentiation and abundance of IFNT and CSH1 mRNA were evaluated. RESULTS: Transcripts for BMP2 and 4 were detected in bovine conceptuses and CT1 cells. Also, transcripts for each BMP receptor were detected in conceptuses and CT1 cells. Transcripts for NOG were detected in conceptuses but not CT1 cells. Cell proliferation was reduced by BMP4 but not BMP2 supplementation. Both factors reduced IFNT mRNA abundance but had no effect on CSH1 mRNA abundance in CT1 cells. CONCLUSIONS: The BMP2/4 ligand and receptor system presides within bovine trophectoderm prior to uterine attachment. BMP4 negatively impacts CT1 cell growth and both BMPs affect IFNT mRNA abundance.

Immunoexpression of bone morphogenetic protein-2 (BMP-2), BMP receptor type IA, and BMP receptor type II in metastatic and non-metastatic lower lip squamous cell carcinoma.

BACKGROUND: The aim of this study was to evaluate the expression of bone morphogenetic protein-2 (BMP-2), BMPR-IA, and BMPR-II in metastatic and non-metastatic lower lip squamous cell carcinoma (LLSCC). METHODS: Twenty LLSCC with regional nodal metastasis and 20 LLSCC without metastasis were selected for analysis of the immunoexpression of BMP-2, BMPR-IA, and BMPR-II. The percentage of positive cells was evaluated according to the following parameters: score 1 (staining in 0-50% of cells), score 2 (staining in 51-75% of cells), and score 3 (staining in >75% of cells). Immunostaining intensity was evaluated according to the following scores: score 1 (absent or weak expression) and score 2 (strong expression). RESULTS: Regarding the percentage of positive cells, most tumors with and without metastasis showed score 3 for both BMP-2 (P = 0.655) and BMPR-IA (P = 0.655). For BMPR-II, metastatic tumors exhibited higher percentage of positive cells in comparison with non-metastatic tumors (P = 0.049). With respect to immunostaining intensity, most LLSCCs with and without metastasis showed score 2 for BMP-2 (P = 1.000) and score 1 for BMPR-II (P = 1.000). For BMPR-IA, most metastatic tumors presented score 2, whereas most non-metastatic tumors showed score 1 (P < 0.001). Strong expression of BMPR-IA showed a statistically significant association with advanced clinical staging (P < 0.001) and high score of malignancy (P = 0.028). CONCLUSIONS: The results of this study suggest that disturbances in the BMP-2 signaling pathway may be involved in the development of LLSCC and that the strong expression of BMPR-IA might be indicative of the development of metastasis in these lesions.

Transfection of truncated bone morphogenetic protein receptor-II into oral squamous cell carcinoma cell line Tca8113 and inhibitory effect on proliferation and inductive effect on apoptosis.

Bone morphogenetic proteins (BMPs), one of the crucial regulators in embryonic development and bone formation, have been implicated in epithelium-derived tumors. Previous results showed the involvement of overexpression of BMP 2, 4, 5 in the carcinogenesis of oral epithelia. The ability of BMP receptor-II mutant to modify the malignant phenotype of oral squamous cell carcinoma cell line Tca8113 by blocking the BMP signal transduction pathway has been proposed. In this study, a negative truncated mutant of the BMP receptor-II (tBMPR-II) was transfected into Tca8113 cells. The effects were evaluated though RT-PCR, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, BrdU staining, cell cyclin assay, TdT-mediated dUTP nick end labeling (TUNEL) staining, and cell cycle protein detection. Overexpression of tBMPR-II gene transfection truncates the expression of BMPR-II mRNA expression, but not BMP 2, 4, 5. tBMPR-II resulted in a remarkable inhibition of cell proliferation and viability compared with control Tca8113. The inhibitory effects were partly attributed to the induction of apoptosis and cell cycle arrest in G(0) /G(1) accompanied by downregulation of the intracellular cell cycle proteins of cyclin D1 and cyclin-dependent kinases 4, as well as the upregulation of p27 and p57. Loss of BMP signals correlates tightly with suppression of cell proliferation, induction of apoptosis, and benign transformation of Tca8113 cells phenotype.

Retinoic acid inhibits osteogenic differentiation of rat bone marrow stromal cells.

It has been well established that all-trans-retinoic acid (ATRA) influences bone metabolism when given in the treatment or prevention of cancer. However, the molecular mechanisms underlying this are unknown. In the present study, we investigated the effect of ATRA on differentiation of rat bone marrow stromal cells (BMSCs). BMSCs were harvested from rats and induced to differentiate in the presence or absence of ATRA in either osteogenic (OM) or control medium (CM). BMSCs underwent osteogenic differentiation, showed alkaline phosphatase (ALP) activity, a high level of matrix mineralization, and expressed osteonectin when cultured in OM. Although ATRA induced ALP activity, it failed to induce matrix mineralization and osteonectin, decrease mineralization in OM, and induce lipid accumulation in BMSCs. Moreover, while ATRA induced the expression of BMP-RIA, both BMP-RII and Smad5 mRNA were induced by OM and ATRA. Thus, ATRA inhibited osteogenesis and promoted adipogenesis of BMSCs. BMP signaling cooperated with ATRA in the differentiation of BMSCs.

Emodin accelerates osteoblast differentiation through phosphatidylinositol 3-kinase activation and bone morphogenetic protein-2 gene expression.

Emodin is a naturally occurring anthraquinone present in the roots and bark of numerous plants of the genus Rhamnus. Here, we identified emodin as one of compounds activating the mRNA expression of bone morphogenetic protein (BMP)-2 in the differentiation process of mouse osteoblastic MC3T3-E1 subclone 4 cells. Without any effect on cell growth, the low concentration (up to 5 microM) of emodin highly induced the mRNA expression of BMP-2, the expression of alkaline phosphatase (an early marker of osteoblast differentiation), and the mineralization. Interestingly, emodin induced the activation of phosphatidylinositol 3-kinase (PI3K), Akt and mitogen-activated protein (MAP) kinases, but those inductions by emodin were completely inhibited by the PI3K inhibitor, LY294002, suggesting that the up-regulation of BMP-2 by emodin could be mediated through the activation of both Akt and MAP kinases by activating PI3K. Additionally, emodin-induced activation of NF-kappaB suggested that NF-kappaB might be required for its anabolic activity in part. In conclusion, the use of natural compounds with anabolic activity such as emodin could have a beneficial effect on bone health and this kind of studies further elucidate the pharmacological roles of natural compounds in the prevention of osteoporosis and provide the initiative in the early drug discovery and development for osteoporosis.

Hepatocyte growth factor up-regulates the expression of the bone morphogenetic protein (BMP) receptors, BMPR-IB and BMPR-II, in human prostate cancer cells.

Hepatocyte growth factor (HGF) plays multiple roles in cancer, by acting as a motility, invasion and angiogenesis stimulating factor, which promotes metastasis and tumour growth. Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily. The effects of BMPs are mediated by two subgroups of receptors, type I and type II. Recent studies have shown that some BMPs, via their signaling pathways, affect the growth of prostate cancer cells. BMPR-IB and BMPR-II have been reported to be expressed at low levels in prostate cancer. However, little is known about the crosstalk between HGF and BMP pathways. In this study, prostate cancer cells (PC-3 and DU-145) were exposed to HGF at different concentrations (1-75 ng/ml) for 18 h, or were treated with HGF at 40 ng/ml over various time periods (up to 24 h). The effect of HGF on BMP receptor expression was further investigated in a nude mouse PC-3 xenograft model. Mice were treated with either HGF, the HGF antagonist NK4, or a combination of both. The expression of BMPR-IB and BMPR-II mRNA was up-regulated by HGF, as shown by both conventional PCR and quantitative PCR. An elevation of BMPR-IB and BMPR-II at the protein level was confirmed by both Western blot analysis and immunocytochemical staining. In a murine prostate tumour model, infusion of recombinant HGF resulted in an increase in the levels of both BMPR-IB and BMPR-II transcript in prostate tumours. Concomitant delivery of NK4, an HGF antagonist, prevented this effect. In conclusion, HGF up-regulates the expression of the bone morphogenetic protein receptors, BMPR-IB and BMPR-II, in prostate cancer cells, both in vitro and in vivo. This may have important implications in the development of bone metastasis in prostate cancer.

Bone morphogenetic protein (BMP)-7 but not BMP-2 and BMP-4 improves maintenance of primitive peripheral blood-derived hematopoietic progenitor cells (HPC) cultured in serum-free medium supplemented with early acting cytokines.

BMPs regulate the developmental program of hematopoiesis. We demonstrate an increased expression of the BMP receptors Ia and II on cultured CD34+ cells and examine the impact of BMP-2, -4 and -7 on postnatal HPC cultured with stem cell factor, flt3-ligand and interleukin-3 (SF3). The addition of BMP-2 at 5, 25 and 50 ng/m to serum-free medium with SF3 yielded a 1.4- to 1.2-fold increase of CD34+ cells after seven days, but no effect on CFC or LTC-IC was observed. BMP-4 at 25 ng/ml induced a 2.9-fold expansion of colony-forming cells (CFC) within 1 week followed by a decrease to pre-culture values on day 14. The number of long-term culture initiating cells (LTC-IC) decreased by the factor 40 from day 0 to day 14. BMP-7 at 5-50 ng/ml had not effect on the expansion of CD34+ cells and CFC, but improved at 5 ng/ml the survival of LTC-IC significantly as compared to SF3 alone. In summary, BMP-2, -4 and -7 have no effect on the proliferation of CD34+ cells and CFC cultured with serum-free medium and SF3. However, BMP-7 but not BMP-2 and BMP-4 prevents the loss of primitive hematopoietic progenitor cells cultured in SFM plus SF3.

HIV-1 TAT represses transcription of the bone morphogenic protein receptor-2 in U937 monocytic cells.

The bone morphogenetic protein receptor-2 (BMPR2) is a member of the transforming growth factor-beta receptor family and is expressed on the surface of several cell types including endothelial cells and macrophages. Recently, a cause for familial primary pulmonary hypertension (FPPH) has been identified as mutations in the gene encoding BMPR2. Three forms of pulmonary hypertension (PH) exist, including PPH, FPPH, and PH secondary to other etiologies (sporadic PH) such as drug abuse and human immunodeficiency virus (HIV) infection. It is interesting that these subtypes are histologically indistinguishable. The macrophage is a key target cell for HIV-1, significantly altering macrophage cell function upon infection. HIV-1 trans-activator of transcription (Tat), an immediate-early product of the HIV-1 lifecycle, plays an important role in mediating HIV-induced modulation of host cell function. Our laboratory has previously shown that Tat represses mannose receptor transcription in macrophages. In the current study, we examined activity from the BMPR2 promoter in the macrophage cell line U937 and potential regulation by Tat. Transfection of U937 cells with BMPR2 promoter-reporter constructs revealed dose-dependent repression of BMPR2 promoter activity in the presence of Tat. Experiments using truncations of the BMPR2 promoter localized Tat-mediated repression to the first 208 bases of the promoter. Decreased BMPR2 transcription resulted in altered downstream signaling. Similar to mothers against decapentaplegics (SMAD) phosphorylation and SMAD6 expression, in response to BMP2 treatment, were down-regulated after Tat treatment. Finally, HIV-1 infection and treatment with Tat protein of the U937 human monocytic cell line resulted in a decreased, endogenous BMPR2 transcript copy number.

BMP signaling pathways affect differently migration and invasion of esophageal squamous cancer cells.

Bone morphogenetic proteins (BMPs) are broadly involved in normal embryo development and abnormal pathological process such as cancer. The complexity and diversity of BMPs and their signaling pathways impose quite different or even conflicting effects on clinical traits of tumors. The aim of the present study was to investigate whether different BMPs, including BMP2, BMP4, BMP6 and BMP7, influence esophageal squamous cancer cell (ESCC) growth, invasion and metastasis. BMP6 and type I receptor ALK2 and type II receptor BMPRII, ActRIIA and ActRIIB were expressed in all ESCC cell lines. In addition, adenovirus-mediated BMP overexpression did not affect ECA-109 cell growth. BMP6/7 overexpression increased ECA-109 cell invasion and metastasis, activated SMAD1/5/8 signal pathway and induced downstream gene ID1 expression. While BMP2/4 overexpression reduced ECA-109 cell invasion and metastasis and obviously promoted ERK1/2, P-38 and JNK activation with weak SMAD1/5/8 phosphorylation. When BMP6/7 favorite type I receptor ALK2 or type II receptor BMPRII was interfered with by dominant-negative mutation, BMP6/7-induced invasion and metastasis augmentation disappeared. Further investigation on clinical ESCC samples and non-tumorous adjacent tissue found that tumors with triple-positive BMP6, ALK2 and BMPRII had deeper growth than tumors with only BMP6 expression. These results suggested that different BMPs distinctly affected esophageal squamous cancer cell invasion and metastasis by employing different signal pathways.

Immunoexpression of BMP-2 and BMP-4 and their receptors, BMPR-IA and BMPR-II, in ameloblastomas and adenomatoid odontogenic tumors.

OBJECTIVES: The present study evaluated the immunohistochemical expression of BMP-2 and BMP-4 and of their receptors (BMPR-IA and BMPR-II) in solid ameloblastoma (SA), unicystic ameloblastoma (UA) and adenomatoid odontogenic tumor (AOT) in order to obtain a better understanding of their role in the development and biological behavior of these tumors. DESIGN: This study analyzed these proteins in 30 cases of SA, 10 cases of UA, and 30 cases of AOT. Immunoexpression was evaluated in the parenchyma and stroma by attributing the following scores: 0, no stained cells; 1, ≤10%; 2, >10% and ≤25%; 3, >25% and ≤50%; 4, >50% and ≤75%.; 5, >75% stained cells. RESULTS: In SAs, positive correlations were observed between the stromal and parenchymal expression of BMP-2 (p<0.001) and between the stromal expression of BMP-2 and BMP-4 (p=0.020), as well as between the stromal expression of BMPR-II and BMP-4 (p=0.001) and the stromal and parenchymal expression of BMPR-II (p<0.001). In UAs, correlations were detected between the stromal and parenchymal expression of BMP-4 (p=0.035) and between the stromal expression of BMP-4 and BMPR-IA (p=0.022). In AOTs, analysis of immunoexpression in the parenchyma revealed positive correlations between all proteins. CONCLUSION: BMPs and their receptors play an important role in the differentiation and development of ameloblastomas and AOTs, but may not explain the different biological behaviors of these lesions. The positive correlation observed in AOTs might be related to the formation of mineralized material in this tumor.

[Experiment on induction of fibroblasts on 3-D cell-foam structures to express osteoblastic phenotype and its mechanism].

OBJECTIVE: To study the feasibility of osteogenic phenotype expression by human skin fibroblasts induced in polyglycolic acid (PGA) foams and the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of bone morphogenetic protein (BMP) receptors. METHODS: The fibroblasts were isolated, purified from human skin. (1) Fibroblasts were seeded onto PGA foams. The cell-PGA complexes were cultured in RCCS for 6 weeks, in the media of TNF-alpha (50 U/ml) and BMP-2 (0.1 microg/ml). 1 d, 3 and 6 weeks later, cells and extracellular matrix were investigated by electron microscopic and histochemistry observation respectively. Secretion of osteogenic markers were analyzed by biochemical methods. (2) Fibroblasts were seeded on the glass fragments or culture flasks and treated with TNF-alpha (50 U/ml) in different usage (one-time, all-time). The RT-PCR method and the immunohistochemistry fluorescence staining were used to examine the influence of TNF-alpha on the mRNA expression and the protein expression of the type I BMP receptors at 2, 4, 6, 8 d after treatment. RESULTS: Fibroblasts seeded on the PGA foams formed 3-dimensional matrix 3 weeks after seeding, which was demonstrated as osteo-tissue by tetracycline labeling and ARS staining. Cells secreted much more bone-specific alkaline phosphatase (B-AKP) and osteocalcin (OCN) into supernatant than the cells that were cultured in the media without TNF-a and BMP2. Eight days after all-time usage, the TNF-alpha (50 U/ml) increased the expression of the mRNA and protein of the type IB BMP receptor. CONCLUSIONS: Fibroblasts on 3-D cell-foam structures can express osteoblastic phenotype under certain inducing conditions. The numerous fibroblasts in body would be a promising resource for cell seeds candidate of tissue- engineered bone. TNF-alpha provides the essential condition for BMP2's target effect on fibroblasts, and combined use of TNF-alpha and BMP2 is one of the regulating factors.