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Extracellular matrix proteins play important roles in the development of pulmonary hypertension(pH). However, the role of Cartilage oligomeric matrix protein (COMP) in the development of hypoxia-induced pH is largely unknown. We tested the hypothesis that COMP deficiency induced by hypoxia leads to the phenotype switching of pulmonary arterial smooth muscle cells (PASMCs). The expression of COMP decreased in a chronic hypoxia rat pH model (P<0.05) and in PASMCs under hypoxia (3%O 2 ) (P<0.05). The expressions of differentiated marker proteins reduced in the pulmonary arteries from 5 month old COMP -/- mice and in PASMCs under hypoxia or with the siRNA of COMP treatment under normoxia, but increased in PASMCs with adenovirus-increased COMP under hypoxia. The absorbance of cell counting kit-8 at 450nm and the expressions of proliferating cell nuclear antigen (PCNA) and osteopontin increased in PASMCs with the siRNA of COMP under normoxia (P<0.05). PCNA and osteopontin decreased in PASMCs with adenovirus-increased COMP under hypoxia (P<0.05). Additionally, the expression of bone morphogenetic protein receptor 2 (BMPR2) was reduced in COMP -/- mice (P<0.01). Both mRNA and protein levels of bone morphogenetic protein 2 (BMP2) were lower in PASMCs with the siRNA of COMP (P<0.05). The protein level of BMP2 could be reversed by adenovirus-increased COMP under hypoxia (P<0.05). These data suggest that COMP could normally have a protective role against PASMC phenotype switching and maintain BMP2/BMPR2 signaling, and these protective actions could be lost as a result of hypoxia promoting a depletion of COMP., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disease which is associated with pulmonary arterial endothelial cells (PAEC) dysfunction and pulmonary arterial smooth muscle cells proliferation. Moreover, inflammation is contributing a critical role in EC dysfunction and remains elusive. Nuclear factor-kappa B (NF-κB) is a master transcriptional regulator in various cardiovascular pathologies; but, NF-κB's role in EC dysfunction in unknown. Our previous study using cardiac and lung specific IκBα mutant mice (3M and IKBM) showed that PAH induced right ventricular hypertrophy (RVH) was prevented in monocrotaline (MCT) treated 3M and IKBM mice, compared to the wild-type mice. Recently, microRNAs (miRNAs) have emerged as a new class of post-transcriptional regulators in vascular remodeling; but, NF-κB regulated miRNA modulation in EC dysfunction is unknown. Using miRNA array analysis, we identified miR-130a which is upregulated in MCT-induced PAH mouse model, as a possible candidate to study. We showed that overexpressing miR-130a in lung microvascular endothelial cells (MVEC) promoted activation of α-smooth muscle actin, a critical component in endothelial-to-mesenchymal transition in EC remodeling. In this study, we demonstrated that bone morphogenetic protein receptor 2 (BMPR2) was a target for miR-130a and miR-130a was regulated by NF-κB which controlled apoptosis and vascular genes in MVEC. The findings reveal that NF-κB-mediated miR-130a modulation is critical in lung vascular remodeling., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pulmonary arterial hypertension (PAH) is a devastating disease characterised by occlusive pulmonary vasculopathy. Activation of bone morphogenetic protein receptor 2 (BMPR2) signalling by FK506 (tacrolimus) reverses occlusive vasculopathy in rodent PAH models. Here, we determined the safety and tolerability of low-level FK506 therapy in stable PAH patients.We performed a randomised, double-blind, placebo-controlled, 16-week, single-centre, phase IIa trial in PAH patients with New York Heart Association functional class II/III symptoms using three FK506 target levels (<2, 2-3 and 3-5 ng·mL -1 ). 23 patients were randomised and 20 patients completed the trial.FK506 was generally well tolerated, with nausea/diarrhoea being the most commonly reported adverse event and no observation of line infections in patients on intravenous prostacyclin therapy. PAH patients had significantly lower BMPR2 expression in peripheral blood mononuclear cells versus healthy controls (n=13; p=0.005), which improved after FK506 treatment. While we observed that some patients responded with a pronounced increase in BMPR2 expression as well as improvement in 6-min walk distance, and serological and echocardiographic parameters of heart failure, these changes were not significant.Low-level FK506 is well tolerated and increases BMPR2 in subsets of PAH patients. These results support the study of FK506 in a phase IIb efficacy trial., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

We earlier demonstrated synergistic increase in the proliferation of pulmonary smooth muscle cells on exposure to HIV-proteins and/or cocaine due to severe down-modulation of bone morphogenetic protein receptor (BMPR) axis: the anti-proliferative arm of TGF-β super family of receptors. Here, now we demonstrate the effect of HIV-Tat and cocaine on the proliferative TGF-β signaling cascade. We observed a significant increase in the secretion of TGF-β1 ligand along with enhanced protein expression of TGFβ Receptor (TGFβR)-1, TGFβR-2 and phosphorylated SMAD2/3 in human pulmonary arterial smooth muscle cells on treatment with cocaine and Tat. Further, we noticed an increase in the levels of p-TAK1 complexed with TGFβR-2. Concomitant to this a significant increase in the activation of TAK1-mediated, SMAD-independent downstream signaling molecules: p-MKK4 and p-JNK was observed. However, activation of MKK3/6-p38MAPK, another axis downstream of TAK1 was found to be reduced due to attenuation in the protein levels of BMPR2. Both SMAD and non-SMAD dependent TGFβR cascades were found to contribute to hyper-proliferation. Finally the increase in the levels of phosphorylated TGFβR1 and TGFβR2 on exposure to HIV-proteins and cocaine was confirmed in pulmonary smooth muscle cells from cocaine injected HIV-transgenic rats and in total lung extracts from HIV infected cocaine and/or opioid users.

Elaborate regulatory networks of the Bone Morphogenetic Protein (BMP) pathways ensure precise signalling outcome during cell differentiation and tissue homeostasis. Here, we identified IRS4 as a novel regulator of BMP signal transduction and provide molecular insights how it integrates into the signalling pathway. We found that IRS4 interacts with the BMP receptor BMPRII and specifically targets Smad1 for proteasomal degradation consequently leading to repressed BMP/Smad signalling in C2C12 myoblasts while concomitantly activating the PI3K/Akt axis. IRS4 is present in human and primary mouse myoblasts, the expression increases during myogenic differentiation but is downregulated upon final commitment coinciding with Myogenin expression. Functionally, IRS4 promotes myogenesis in C2C12 cells, while IRS4 knockdown inhibits differentiation of myoblasts. We propose that IRS4 is particularly critical in the myoblast stage to serve as a molecular switch between BMP/Smad and Akt signalling and to thereby control cell commitment. These findings provide profound understanding of the role of BMP signalling in early myogenic differentiation and open new ways for targeting the BMP pathway in muscle regeneration.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by a progressive elevation in mean pulmonary arterial pressure. This occurs due to abnormal remodeling of small peripheral lung vasculature resulting in progressive occlusion of the artery lumen that eventually causes right heart failure and death. The most common cause of PAH is inactivating mutations in the gene encoding a bone morphogenetic protein type II receptor (BMPRII). Current therapeutic options for PAH are limited and focused mainly on reversal of pulmonary vasoconstriction and proliferation of vascular cells. Although these treatments can relieve disease symptoms, PAH remains a progressive lethal disease. Emerging data suggest that restoration of BMPRII signaling in PAH is a promising alternative that could prevent and reverse pulmonary vascular remodeling. Here we will focus on recent advances in rescuing BMPRII expression, function or signaling to prevent and reverse pulmonary vascular remodeling in PAH and its feasibility for clinical translation. Furthermore, we summarize the role of described miRNAs that directly target the BMPR2 gene in blood vessels. We discuss the therapeutic potential and the limitations of promising new approaches to restore BMPRII signaling in PAH patients. Different mutations in BMPR2 and environmental/genetic factors make PAH a heterogeneous disease and it is thus likely that the best approach will be patient-tailored therapies.

Objective: We determined in patients with pulmonary arterial (PA) hypertension (PAH) whether in addition to increased production of elastase by PA smooth muscle cells previously reported, PA elastic fibers are susceptible to degradation because of their abnormal assembly., Approach and Results: Fibrillin-1 and elastin are the major components of elastic fibers, and fibrillin-1 binds bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β1 (TGFβ1). Thus, we considered whether BMPs like TGFβ1 contribute to elastic fiber assembly and whether this process is perturbed in PAH particularly when the BMP receptor, BMPR2, is mutant. We also assessed whether in mice with Bmpr2/1a compound heterozygosity, elastic fibers are susceptible to degradation. In PA smooth muscle cells and adventitial fibroblasts, TGFβ1 increased elastin mRNA, but the elevation in elastin protein was dependent on BMPR2; TGFβ1 and BMP4, via BMPR2, increased extracellular accumulation of fibrillin-1. Both BMP4- and TGFβ1-stimulated elastic fiber assembly was impaired in idiopathic (I) PAH-PA adventitial fibroblast versus control cells, particularly those with hereditary (H) PAH and a BMPR2 mutation. This was related to profound reductions in elastin and fibrillin-1 mRNA. Elastin protein was increased in IPAH PA adventitial fibroblast by TGFβ1 but only minimally so in BMPR2 mutant cells. Fibrillin-1 protein increased only modestly in IPAH or HPAH PA adventitial fibroblasts stimulated with BMP4 or TGFβ1. In Bmpr2/1a heterozygote mice, reduced PA fibrillin-1 was associated with elastic fiber susceptibility to degradation and more severe pulmonary hypertension., Conclusions: Disrupting BMPR2 impairs TGFβ1- and BMP4-mediated elastic fiber assembly and is of pathophysiologic significance in PAH., (© 2017 American Heart Association, Inc.)

Lysine methylation of chromosomal and nuclear proteins is a well-known mechanism of epigenetic regulation, but relatively little is known about the role of this protein modification in signal transduction. Using an RNAi-based functional screening of the SMYD family of lysine methyltransferases (KMTs), we identified SMYD2 as a KMT essential for robust bone morphogenic protein (BMP)- but not TGFβ-induced target gene expression in HaCaT keratinocyte cells. A role for SMYD2 in BMP-induced gene expression was confirmed by shRNA knockdown and CRISPR/Cas9-mediated knock-out of SMYD2 We further demonstrate that SMYD2 knockdown or knock-out impairs BMP-induced phosphorylation of the signal-transducing protein SMAD1/5 and SMAD1/5 nuclear localization and interaction with SMAD4. The SMYD2 KMT activity was required to facilitate BMP-mediated signal transduction, as treatment with the SMYD2 inhibitor AZ505 suppressed BMP2-induced SMAD1/5 phosphorylation. Furthermore, we present evidence that SMYD2 likely modulates the BMP response through its function in the cytosol. We show that, although SMYD2 interacted with multiple components in the BMP pathway, it specifically methylated the kinase domain of BMP type II receptor BMPR2. Taken together, our findings suggest that SMYD2 may promote BMP signaling by directly methylating BMPR2, which, in turn, stimulates BMPR2 kinase activity and activation of the BMP pathway., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2+) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/β-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2+ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/β-catenin signaling in ABCG2+ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2+ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.

Background: Pulmonary arterial hypertension (PAH) is a deadly disease, and the molecular mechanism of PAH has not been clarified clearly. The objective of this study was to identify possible biomarkers and explore the potential mechanisms of Schistosoma-induced PAH., Methods: GSE49114 RNA-Seq data developed from mouse whole lung tissues were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between control samples and schistosomiasis-induced PAH samples were identified by the edgeR software. Gene Ontology (GO) and pathway enrichment analysis of DEGs were performed, followed by metabolic pathway network construction. Moreover, pathways with higher connectivity degrees in the metabolic pathway network were identified., Results: Totally, 877 up- and 520 downregulated DEGs were screened. The upregulated DEGs such as IL-4 (Interleukin-4) were significantly related with immune system process, transmembrane signaling receptor activity, and signal transducer activity. Downregulated DEGs (i.e., Smad9 (SMAD family member 9), BMPR2 (bone morphogenetic protein type 2 receptor), and Eng (endoglin)) were significantly enriched in signal transducer activity, growth factor binding, and signal transduction. The top 10 metabolic pathways with highest connectivity degree were screened, including leishmaniasis (degree = 26), antigen processing and presentation (degree = 20), hematopoietic cell lineage (degree = 20), chemokine signaling pathway (degree = 18), and JAK-STAT signaling pathway (degree = 18)., Conclusions: Smad9, BMPR2, Eng and IL4, and their relative functions such as signal transduction, signal transducer activity, and immune system process might play important roles in schistosomiasis-induced PAH. Moreover, the interaction of metabolic pathways was critical in the development of schistosomiasis-PAH.

Primary ovarian insufficiency (POI) affects ~1% of women in the general population. Despite numerous attempts at identifying POI genetic aetiology, coding mutations in only a few genes have been functionally related to POI pathogenesis. It has been suggested that mutant BMPR2 might contribute towards the phenotype. Several BMP15 (a BMPR2 ligand) coding mutations in human species have been related to POI pathogenesis. The BMPR2 p.Ser987Phe mutation, previously identified in a woman with POI, might therefore lead to cellular dysfunction contributing to the phenotype. To explore such an assumption, the present study assessed potential pathogenic subcellular localization/aggregation patterns associated with the p.Ser987Phe mutant form of BMPR2 in a relevant model for studying ovarian function. A significant increase in protein-like aggregation patterns was identified at the endoplasmic reticulum (ER) which permitted us to establish, for the first time, a potential functional association between mutant BMPR2 and POI aetiology. Since BMPR2 mutant forms were previously related to idiopathic pulmonary arterial hypertension, BMPR2 mutations may be related to an as-yet-to-be described syndromic form of POI involving pulmonary dysfunction. Additional assays are necessary to confirm that BMPR2 abnormal subcellular patterns are composed by aggregates., Abbreviations: POI: primary ovarian insufficiency; ER: endoplasmic reticulum; NGS: next generation sequencing.

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.)

Bone morphogenetic protein type 2 receptor (BMPR2) is one of the transforming growth factor-β (TGF-β) superfamily receptors, performing diverse roles during embryonic development, vasculogenesis, and osteogenesis. Human BMPR2 consists of 1,038 amino acids, and contains functionally conserved extracellular, transmembrane, kinase, and C-terminal cytoplasmic domains. Bone morphogenetic proteins (BMPs) engage the tetrameric complex, composed of BMPR2 and its corresponding type 1 receptors, which initiates SMAD proteins-mediated signal transduction leading to the expression of target genes implicated in the development or differentiation of the embryo, organs and bones. In particular, genetic alterations of BMPR2 gene are associated with several clinical disorders, including representative pulmonary arterial hypertension, cancers, and metabolic diseases, thus demonstrating the physiological importance of BMPR2. In this mini review, we summarize recent findings regarding the molecular basis of BMPR2 functions in BMP signaling, and the versatile roles of BMPR2. In addition, various aspects of experimentally validated pathogenic mutations of BMPR2 and the linked human diseases will also be discussed, which are important in clinical settings for diagnostics and treatment. [BMB Reports 2017; 50(6): 308-317].

Pulmonary arterial hypertension is a progressive disease that causes the obstruction of precapillary pulmonary arteries and a sustained increase in pulmonary vascular resistance. The aim was to analyze functionally the variants found in the BMPR2 gene and to establish a genotype-phenotype correlation. mRNA expression studies were performed using pSPL3 vector, studies of subcellular localization were performed using pEGFP-N1 vector and luciferase assays were performed using pGL3-Basic vector. We have identified 30 variants in the BMPR2 gene in 27 of 55 patients. In 16 patients we detected pathogenic mutations. Minigene assays revealed that 6 variants (synonymous, missense) result in splicing defect. By immunofluorescence assay, we observed that 4 mutations affect the protein localization. Finally, 4 mutations located in the 5'UTR region showed a decreased transcriptional activity in luciferase assays. Genotype-phenotype correlation, revealed that patients with pathogenic mutations have a more severe phenotype (sPaP p = 0.042, 6MWT p = 0.041), a lower age at diagnosis (p = 0.040) and seemed to have worse response to phosphodiesterase-5-inhibitors (p = 0.010). Our study confirms that in vitro expression analysis is a suitable approach in order to investigate the phenotypic consequences of the nucleotide variants, especially in cases where the involved genes have a pattern of expression in tissues of difficult access.

Fragile X syndrome (FXS) is the most common cause of heritable intellectual disability and autism and affects ~1 in 4000 males and 1 in 8000 females. The discovery of effective treatments for FXS has been hampered by the lack of effective animal models and phenotypic readouts for drug screening. FXS ensues from the epigenetic silencing or loss-of-function mutation of the fragile X mental retardation 1 ( FMR1 ) gene, which encodes an RNA binding protein that associates with and represses the translation of target mRNAs. We previously found that the activation of LIM kinase 1 (LIMK1) downstream of augmented synthesis of bone morphogenetic protein (BMP) type 2 receptor (BMPR2) promotes aberrant synaptic development in mouse and Drosophila models of FXS and that these molecular and cellular markers were correlated in patients with FXS. We report that larval locomotion is augmented in a Drosophila FXS model. Genetic or pharmacological intervention on the BMPR2-LIMK pathway ameliorated the synaptic abnormality and locomotion phenotypes of FXS larvae, as well as hyperactivity in an FXS mouse model. Our study demonstrates that (i) the BMPR2-LIMK pathway is a promising therapeutic target for FXS and (ii) the locomotion phenotype of FXS larvae is a quantitative functional readout for the neuromorphological phenotype associated with FXS and is amenable to the screening novel FXS therapeutics., (Copyright © 2017, American Association for the Advancement of Science.)

Endothelial cell (EC) activation and vascular injury are hallmark features of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Caveolin-1 (Cav-1) is highly expressed in pulmonary microvascular ECs and plays a key role in maintaining vascular homeostasis. The aim of this study was to determine if the lung inflammatory response to Escherichia coli lipopolysaccharide (LPS) promotes priming of ECs via Cav-1 depletion and if this contributes to the onset of pulmonary vascular remodeling. To test the hypothesis that depletion of Cav-1 primes ECs to respond to profibrotic signals, C57BL6 wild-type (WT) mice ( Tie2.Cre - ;Cav1 fl/fl ) were exposed to nebulized LPS (10 mg; 1 h daily for 4 days) and compared with EC-specific Cav1 -/- ( Tie2.Cre + ;Cav1 fl/fl ). After 96 h of LPS exposure, total lung Cav-1 and bone morphogenetic protein receptor type II (BMPRII) expression were reduced in WT mice. Moreover, plasma albumin leakage, infiltration of immune cells, and levels of IL-6/IL-6R and transforming growth factor-β (TGF-β) were elevated in both LPS-treated WT and EC-Cav1 -/- mice. Finally, EC-Cav1 -/- mice exhibited a modest increase in microvascular thickness basally and even more so on exposure to LPS (96 h). EC-Cav1 -/- mice and LPS-treated WT mice exhibited reduced BMPRII expression and endothelial nitric oxide synthase uncoupling, which along with increased TGF-β promoted TGFβRI-dependent SMAD-2/3 phosphorylation. Finally, human lung sections from patients with ARDS displayed reduced EC Cav-1 expression, elevated TGF-β levels, and severe pulmonary vascular remodeling. Thus EC Cav-1 depletion, oxidative stress-mediated reduction in BMPRII expression, and enhanced TGF-β-driven SMAD-2/3 signaling promote pulmonary vascular remodeling in inflamed lungs., (Copyright © 2017 the American Physiological Society.)

Background: Cell-based gene therapy has become a subject of interest for the treatment of pulmonary arterial hypertension (PAH), a devastating disease characterized by pulmonary artery smooth muscle cell (PASMC) hyperplasia. Mesenchymal stem cells (MSCs) have been recently acknowledged as a potential cell vector for gene therapy. Here, we investigated the effect of MSC-based let-7a for PAH., Methods: After isolation and identification of MSCs from rat bone marrow, cells were infected with recombinant adenovirus vector Ad-let-7a. Lewis rats were subcutaneously injected with monocrotaline (MCT) to induce PAH, followed by the administration of MSCs, MSCs-NC (miR-control), or MSC-let-7a, respectively. Then, right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary vascular remodeling were evaluated. Rat pulmonary artery smooth muscle cells (rPASMCs) under hypoxia were co-cultured with MSCs or MSC-let-7a. Cell proliferation and apoptosis were separately determined by 3 H thymidine incorporation and flow cytometry analysis. The underlying mechanism was also investigated., Results: MSC transplantation enhanced let-7a levels in MCT-induced PAH rats. After injection with MSC-let-7a, RVSP, right ventricular hypertrophy, and pulmonary vascular remodeling were notably ameliorated, indicating a protective effect of MSC-let-7a against PAH. When co-cultured with MSC-let-7a, hypoxia-triggered PASMC proliferation was obviously attenuated, concomitant with the decrease in cell proliferation-associated proteins. Simultaneously, the resistance of PASMCs to apoptosis was remarkably abrogated by MSC-let-7a administration. A mechanism assay revealed that MSC-let-7a restrained the activation of signal transducers and activators of transcription 3 (STAT3) and increased its downstream bone morphogenetic protein receptor 2 (BMPR2) expression. Importantly, preconditioning with BMPR2 siRNA dramatically abated the suppressive effects of MSC-let-7a on PASMC proliferation and apoptosis resistance., Conclusions: Collectively, this study suggests that MSCs modified with let-7a may ameliorate the progression of PAH by inhibiting PASMC growth through the STAT3-BMPR2 signaling, supporting a promising therapeutic strategy for PAH patients.

Although forkhead-box n1 (Foxn1) is a critical thymic epithelial cell regulator in thymus organogenesis, its association with epithelial differentiation and homeostasis in the postnatal and aged thymic microenvironment remains conflicting. Consequently, we have generated a Foxn1 eGFP/+ knock-in mouse model that allows for refined investigation of the aging thymic epithelium. This reporter line differs from those previously published in that concomitant expression of enhanced green fluorescent protein enables live cell sorting of Foxn1 + cell populations. Our heterozygotes did not exhibit haploinsufficiency, with Foxn1 expression resembling that of wild-type mice. Comparative analysis between Foxn1 and enhanced green fluorescent protein at both the transcriptional and translational levels revealed co-localization, with progressive down-regulation observed predominantly in the aging cortical epithelium. Supplementation with bone morphogenetic protein (Bmp)-4 enhanced Foxn1 expression and colony forming efficiency in both embryonic and adult progenitor 3D cultures. Strikingly, selective maintenance of immature cortical and medullary epithelial cells was observed which is consistent with the higher Bmp receptor 2 expression levels seen in these progenitor populations. This study demonstrates the significance of our mouse model in unraveling the role of this master regulator in thymus development, homeostasis and aging, providing a faithful reporter system for phenotypic and functional investigations., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder in which mechanical obstruction of the pulmonary vascular bed is largely responsible for the rise in pulmonary arterial pressures. The discovery of heterozygous BMPR2 germline mutations as critical predisposing factors together with a remarkable progress in our understanding of the pathogenic mechanisms have helped identify the significant and complex roles of the BMPRII axis in PAH. However, their precise contributions to the condition are still incompletely understood. Areas covered: This review aims to assemble and discuss the cellular actions of BMPs together with the possible clinical applications and prospects for their use in the near future. Expert opinion: Although major advances have been made, several questions remain unanswered regarding development of efficacious therapies targeting the BMPRII axis in PAH. Specifically, the reasons why BMPRII signaling is reduced in PAH and how the alterations influence or even drive the pathogenesis need to be understood. Because the BMPRII axis is ubiquitously expressed and exhibits a wide variety of functions in organ regeneration and homeostasis, a better understanding of the overall risk-benefit ratio of these strategies with long-term follow-up is needed. This knowledge will lay the foundation for discovery of innovative therapeutics for PAH.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In this study, we examined the expression of bone morphogenetic protein receptor 2 (BMPR2) in primary NB and adjacent non-tumor samples (adrenal gland). BMPR2 expression was significantly downregulated in NB tissues, particularly in high-grade NB, and was inversely related to the expression of the NB differentiation markers ferritin and enolase. The significance of the downregulation was further explored in cultured NB cells. While enforced expression of BMPR2 decreased cell proliferation and colony-forming activity, shRNA-mediated knockdown of BMPR2 led to increased cell growth and clonogenicity. In mice, NB cells harboring BMPR2 shRNA showed significantly increased tumorigenicity compared with control cells. We also performed a retrospective analysis of NB patients and identified a significant positive correlation between tumor BMPR2 expression and overall survival. These findings suggest that BMPR2 may play an important role in the development of NB., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Heterozygous germ-line mutations in the bone morphogenetic protein type-II receptor (BMPR-II) gene underlie heritable pulmonary arterial hypertension (HPAH). Although inflammation promotes PAH, the mechanisms by which inflammation and BMPR-II dysfunction conspire to cause disease remain unknown. Here we identify that tumour necrosis factor-α (TNFα) selectively reduces BMPR-II transcription and mediates post-translational BMPR-II cleavage via the sheddases, ADAM10 and ADAM17 in pulmonary artery smooth muscle cells (PASMCs). TNFα-mediated suppression of BMPR-II subverts BMP signalling, leading to BMP6-mediated PASMC proliferation via preferential activation of an ALK2/ACTR-IIA signalling axis. Furthermore, TNFα, via SRC family kinases, increases pro-proliferative NOTCH2 signalling in HPAH PASMCs with reduced BMPR-II expression. We confirm this signalling switch in rodent models of PAH and demonstrate that anti-TNFα immunotherapy reverses disease progression, restoring normal BMP/NOTCH signalling. Collectively, these findings identify mechanisms by which BMP and TNFα signalling contribute to disease, and suggest a tractable approach for therapeutic intervention in PAH.

Bone morphogenetic protein 2 (BMP2) and BMP4 are key regulators of the fate and differentiation of human mammary epithelial stem cells (SCs), as well as of their niches, and are involved in breast cancer development. We established that MCF10A immature mammary epithelial cells reliably reproduce the BMP response that we previously identified in human primary epithelial SCs. In this model, we observed that BMP2 promotes luminal progenitor commitment and expansion, whereas BMP4 prevents lineage differentiation. Environmental pollutants are known to promote cancer development, possibly by providing cells with stem-like features and by modifying their niches. Bisphenols, in particular, were shown to increase the risk of developing breast cancer. Here, we demonstrate that chronic exposure to low doses of bisphenol A (BPA) or benzo(a)pyrene (B(a)P) alone has little effect on SCs properties of MCF10A cells. Conversely, we show that this exposure affects the response of immature epithelial cells to BMP2 and BMP4. Furthermore, the modifications triggered in MCF10A cells on exposure to pollutants appeared to be predominantly mediated by altering the expression and localization of type-1 receptors and by pre-activating BMP signaling, through the phosphorylation of small mothers against decapentaplegic 1/5/8 (SMAD1/5/8). By analyzing stem and progenitor properties, we reveal that BPA prevents the maintenance of SC features prompted by BMP4, whereas promoting cell differentiation towards a myoepithelial phenotype. Inversely, B(a)P prevents BMP2-mediated luminal progenitor commitment and expansion, leading to the retention of stem-like properties. Overall, our data indicate that BPA and B(a)P distinctly alter the fate and differentiation potential of mammary epithelial SCs by modulating BMP signaling.

Human telomerase reverse transcriptase (hTERT) plays a central role in telomere lengthening for continuous cell proliferation, but it remains unclear how extracellular cues regulate telomerase lengthening of telomeres. Here we report that the cytokine bone morphogenetic protein-7 (BMP7) induces the hTERT gene repression in a BMPRII receptor- and Smad3-dependent manner in human breast cancer cells. Chonic exposure of human breast cancer cells to BMP7 results in short telomeres, cell senescence and apoptosis. Mutation of the BMPRII receptor, but not TGFbRII, ACTRIIA or ACTRIIB receptor, inhibits BMP7-induced repression of the hTERT gene promoter activity, leading to increased telomerase activity, lengthened telomeres and continued cell proliferation. Expression of hTERT prevents BMP7-induced breast cancer cell senescence and apoptosis. Thus, our data suggest that BMP7 induces breast cancer cell aging by a mechanism involving BMPRII receptor- and Smad3-mediated repression of the hTERT gene.

Osteoporosis is a major complication in patients with diabetes mellitus. Thus, it is crucial to study the signal mechanisms responsible for enhancement of bone mass in diabetes. Administration of human parathyroid hormone (hPTH) has been reported to prevent osteoblast apoptosis and have anabolic effects on bone in animals and humans. In the present study, we examined the effects of hPTH on expression of bone morphogenetic protein type 2 (BMP-2) and its receptor BMPR2 in diabetic rats following spinal fusion. Our data show that hPTH amplified BMP-2 and BMPR2 in bone tissues of non-diabetic rats, but not in diabetic rats. Our data further demonstrate that hPTH plays a role in regulating BMP-2 and BMPR2 via mTOR-PI3K signal pathway. We suggest specific signaling pathways by which hPTH regulates BMP-2 via mTOR-PI3K mechanism in bone formation following spinal fusion. Notably, our data indicate under diabetic conditions this signal pathway is impaired, thereby likely affecting bone formation after spinal fusion. The subsequent induction of BMP-2 and BMPR2 are likely a part of the protective effects aimed at attenuating pathological bone damage as a result of diabetes.

Pulmonary arterial hypertension (PAH) describes a group of conditions with a common hemodynamic phenotype of increased pulmonary artery pressure, driven by progressive remodeling of small pulmonary arteries, leading to right heart failure and death. Vascular remodeling is the key pathological feature of PAH, but treatments targeting this process are lacking. In this review, we summarize important advances in our understanding of PAH pathogenesis from novel genetic and epigenetic factors, to cell metabolism and DNA damage. We show how these processes may integrate and highlight exploitable targets that could alter the relentless vascular remodeling in PAH., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are members of the transforming growth factor beta (TGF-β) superfamily. Through autocrine and paracrine mechanisms, these two factors can regulate cell differentiation, proliferation, and other functions in the ovary locally. Furthermore, GDF9 and BMP15 play vital roles in follicular growth, atresia, ovulation, fertilization, reproduction, and maintenance. Numerous studies have demonstrated a synergy between BMP15 and GDF9. Studies in humans and mice have indicated that the synergy between BMP15 and GDF9 is primarily mediated by the bone morphogenetic protein type II receptor (BMPR2). The BMP15/GDF9 heterodimer needs to bind to the BMPR2-ALK4/5/7-ALK6 receptor complex to activate the SMAD2/3 signaling pathway. However, it is not clear which genes mediate and regulate the effects of the BMP15/GDF9 proteins on bovine cumulus cells (CCs)., Methods: Our earlier study showed that BMPR2 is a gene that is directly targeted and regulated by miR-375. Therefore, we designed and synthesized an miR-375 mimics/inhibitor and regulated BMPR2 expression in bovine CCs by the overexpression or inhibition of miR-375. After the overexpression or inhibition of miR-375, the apoptosis rate of bovine CCs was measured by flow cytometry; changes in critical gene expression were measured by RT-qPCR and western blot assays; and the proliferation of bovine CCs was measured by CCK-8 assay., Results: In bovine CCs, the overexpression of miR-375 resulted in decreased BMPR2 and ALK7 expression, whereas the inhibition of miR-375 caused increased BMPR2 and ALK7 expression. The overexpression of miR-375 attenuated the proliferation ability and significantly increased the apoptosis rate of bovine CCs, whereas the inhibition of miR-375 did not significantly change the proliferation ability or apoptosis rate., Conclusions: BMPR2, a target of miR-375, is regulated by this molecule, thereby affecting expression of BMP15/GDF9 receptors, and the proliferation and apoptosis of bovine CCs., (© 2017 The Author(s)Published by S. Karger AG, Basel.)

Group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH) is a rare disease characterized by proliferation and occlusion of small pulmonary arterioles, leading to progressive elevation of pulmonary artery pressure and pulmonary vascular resistance, and right ventricular failure. Historically, it has been associated with a high mortality rate, although, over the last decade, treatment has improved survival. PAH includes idiopathic PAH (IPAH), heritable PAH (HPAH), and PAH associated with certain medical conditions. The aetiology of PAH is heterogeneous, and genetics play an important role in some cases. Mutations in BMPR2, encoding bone morphogenetic protein receptor 2, a member of the transforming growth factor-β superfamily of receptors, have been identified in 70% of cases of HPAH, and in 10-40% of cases of IPAH. Other genetic causes of PAH include mutations in the genes encoding activin receptor-like type 1, endoglin, SMAD9, caveolin 1, and potassium two-pore-domain channel subfamily K member 3. Mutations in the gene encoding T-box 4 have been identified in 10-30% of paediatric PAH patients, but rarely in adults with PAH. PAH in children is much more heterogeneous than in adults, and can be associated with several genetic syndromes, congenital heart disease, pulmonary disease, and vascular disease. In addition to rare mutations as a monogenic cause of HPAH, common variants in the gene encoding cerebellin 2 increase the risk of PAH by approximately two-fold. A PAH panel of genes is available for clinical testing, and should be considered for use in clinical management, especially for patients with a family history of PAH. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: Gliomas result in the highest morbidity and mortality rates of intracranial primary central nervous system tumors because of their aggressive growth characteristics and high postoperative recurrence. They are characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior in patients. Proliferation is a key aspect of the clinical progression of malignant gliomas, complicating complete surgical resection and enabling tumor regrowth and further proliferation of the surviving tumor cells., Methods: The expression of Fstl1 was detected by western blotting and qRT-PCR. We used cell proliferation and colony formation assays to measure proliferation. Then, flow cytometry was used to analyze cell cycle progression. The expression of Fstl1, p-Smad1/5/8 and p21 in GBM tissue sections was evaluated using immunohistochemical staining. Furthermore, we used coimmunoprecipitation (Co-IP) and immunoprecipitation to validate the relationship between Fstl1, BMP4 and BMPR2. Finally, we used orthotopic xenograft studies to measure the growth of tumors in vivo., Results: We found that follistatin-like 1 (Fstl1) was upregulated in high-grade glioma specimens and that its levels correlated with poor prognosis. Fstl1 upregulation increased cell proliferation, colony formation and cell cycle progression, while its knockdown inhibited these processes. Moreover, Fstl1 interacted with bone morphogenetic protein (BMP) 4, but not BMP receptor (BMPR) 2, and competitively inhibited their association. Furthermore, Fstl1 overexpression suppressed the activation of the BMP4/Smad1/5/8 signaling pathway, while BMP4 overexpression reversed this effect., Conclusion: Our study demonstrated that Fstl1 promoted glioma growth through the BMP4/Smad1/5/8 signaling pathway, and these findings suggest potential new glioblastoma treatment strategies., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Background and Aims: The impairment of intestinal epithelial barrier is the main etiologic factor of inflammatory bowel disease. The proper intestinal epithelial proliferation and differentiation is crucial for maintaining intestinal integrity. Metformin is a common anti-diabetic drug. The objective is to evaluate the protective effects of metformin on ileal epithelial barrier integrity using interleukin-10 deficient (IL10KO) mice., Methods: Wild-type and IL10KO mice were fed with/without metformin for 6 weeks and then ileum was collected for analyses. The mediatory role of AMP-activated protein kinase (AMPK) was further examined by gain and loss of function study in vitro., Results: Compared to wild-type mice, IL10KO mice had increased proliferation, reduced goblet cell and Paneth cell lineage differentiation in the ileum tissue, which was accompanied with increased crypt expansion. Metformin supplementation mitigated intestinal cell proliferation, restored villus/crypt ratio, increased goblet cell and Paneth cell differentiation and improved barrier function. In addition, metformin supplementation in IL10KO mice suppressed macrophage pro-inflammatory activity as indicated by reduced M1 macrophage abundance and decreased pro-inflammatory cytokine IL-1β, TNF-α and IFN-γ expressions. As a target of metformin, AMPK phosphorylation was enhanced in mice treated with metformin, regardless of mouse genotypes. In correlation, the mRNA level of differentiation regulator including bmp4, bmpr2 and math1 were also increased in IL10KO mice supplemented with metformin, which likely explains the enhanced epithelial differentiation in IL10KO mice with metformin. Consistently, in Caco-2 cells, metformin promoted claudin-3 and E-cadherin assembly and mitigated TNF-α-induced fragmentation of tight junction proteins. Gain and loss of function assay also demonstrated AMPK was correlated with epithelial differentiation and proliferation., Conclusions: Metformin supplementation promotes secretory cell lineage differentiation, suppresses inflammation and improves epithelial barrier function in IL10KO mice likely through activation of AMPK, showing its beneficial effects on gut epithelial., Competing Interests: The authors have declared that no competing interests exist.

Pulsed electromagnetic fields (PEMFs) have been considered as a potential candidate for the prevention and treatment of osteoporosis, however, the mechanism of its action is still elusive. We have previously reported that 50Hz 0.6mT PEMFs stimulate osteoblastic differentiation and mineralization in a primary cilium- dependent manner, but did not know the reason. In the current study, we found that the PEMFs promoted osteogenic differentiation and maturation of rat calvarial osteoblasts (ROBs) by activating bone morphogenetic protein BMP-Smad1/5/8 signaling on the condition that primary cilia were normal. Further studies revealed that BMPRII, the primary binding receptor of BMP ligand, was readily and strongly upregulated by PEMF treatment and localized at the bases of primary cilia. Abrogation of primary cilia with small interfering RNA sequence targeting IFT88 abolished the PEMF-induced upregulation of BMPRII and its ciliary localization. Knockdown of BMPRII expression level with RNA interference had no effects on primary cilia but significantly decreased the promoting effect of PEMFs on osteoblastic differentiation and maturation. These results indicated that PEMFs stimulate osteogenic differentiation and maturation of osteoblast by primary cilium-mediated upregulation of BMPRII expression and subsequently activation of BMP-Smad1/5/8 signaling, and that BMPRII is the key component linking primary cilium and BMP-Smad1/5/8 pathway. This study has thus revealed the molecular mechanism for the osteogenic effect of PEMFs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In the present study we developed a new specific gene panel for pulmonary arterial hypertension (PAH) including major disease genes and further candidates. We assessed 37 patients with invasively confirmed PAH and five relatives of affected patients for genetic testing. A new PAH-specific gene panel was designed using next generation sequencing (NGS) including 12 known disease genes and 17 further candidates. Any potential pathogenic variants were reassessed by Sanger sequencing. Twenty-two of the 37 patients (59%) had a mutation in BMPR2, ALK1, ENG or EIF2AK4 genes identified by panel and Sanger sequencing. In addition, 12 unclassified variants were identified in seven genes (known and candidate genes). A sensitivity of 100% was met after quality parameters were adjusted. Specificity increased to 100% when Sanger technique was added as a routine validation. The new PAH-specific gene panel developed in the present study allowed for the first time the assessment of all known PAH genes and further candidates at once and markedly reduced overall sequencing time and costs. Sensitivity and specificity reached 100% when Sanger sequencing was additionally applied. Thus, this technique will potentially change the routine diagnostic genetic testing in PAH patients., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Mutations in the bone morphogenetic protein receptor (BMPR2) gene have been observed in 70 % of patients with heritable pulmonary arterial hypertension (HPAH) and in 11-40 % with idiopathic PAH (IPAH). However, carriers of a BMPR2 mutation have only 20 % risk of developing PAH. Since inflammatory mediators are increased and predict survival in PAH, they could act as a second hit inducing the development of pulmonary hypertension in BMPR2 mutation carriers. Our specific aim was to determine whether inflammatory mediators could contribute to pulmonary vascular cell dysfunction in PAH patients with and without a BMPR2 mutation. Pulmonary microvascular endothelial cells (PMEC) and arterial smooth muscle cells (PASMC) were isolated from lung parenchyma of transplanted PAH patients, carriers of a BMPR2 mutation or not, and from lobectomy patients or lung donors. The effects of CRP and TNFα on mitogenic activity, adhesiveness capacity, and expression of adhesion molecules were investigated in PMECs and PASMCs. PMECs from BMPR2 mutation carriers induced an increase in PASMC mitogenic activity; moreover, endothelin-1 secretion by PMECs from carriers was higher than by PMECs from non-carriers. Recruitment of monocytes by PMECs isolated from carriers was higher compared to PMECs from non-carriers and from controls, with an elevated ICAM-1 expression. CRP increased adhesion of monocytes to PMECs in carriers and non-carriers, and TNFα only in carriers. PMEC from BMPR2 mutation carriers have enhanced adhesiveness for monocytes in response to inflammatory mediators, suggesting that BMPR2 mutation could generate susceptibility to an inflammatory insult in PAH.

Background: Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction, vascular remodelling, and microthrombotic events. Inflammatory cytokine interleukin (IL-6) may be a key factor in the development of PAH, and glycoprotein 130 (Gp130) is an important signal-transducing subunit of IL-6. The aim of our study was to evaluate the effectiveness of Gp130 inhibitor in reducing inflammation and ameliorating PAH-related vascular remodelling in monocrotaline (MCT)-exposed rats., Methods: Sprague-Dawley rats (n = 96; weight, 240-250 g) were randomly divided into 3 groups: control, MCT-exposed (MCT), and MCT-exposed plus Gp130 inhibitor (MCT-Gp) administered daily (5 mg/kg) from days 14-28. Eight rats were killed in each group at weeks 1 through 4, with the following measured variables compared across groups on day 28: hemodynamics, right ventricular hypertrophy, morphometric measurements, immunohistochemical results, levels of IL-6, phosphorylated signal transducer and activator of transcription 3, proliferating cell nuclear antigen (PCNA), bone morphogenetic protein receptor-2 (BMPR2), proangiogenic factor, vascular endothelial growth factor (VEGF), proproliferative kinase extracellular signal-regulated kinase (ERK), survivin, Bcl-2, and Bax., Results: Compared with the MCT group, Gp130 inhibitor, after MCT exposure, improved hemodynamics and significantly reduced the severity of inflammation, as estimated by levels of IL-6 (P < 0.0001), and reversed pulmonary arterial remodelling, as assessed by medial wall thickness (P < 0.0001). Gp130 inhibitor upregulated BMPR2 expression in MCT-exposed lungs (P = 0.040) and decreased the expression of PCNA, VEGF, ERK, and survivin (all P < 0.05)., Conclusions: Gp130 inhibitor upregulated BMPR2 expression in MCT-exposed lungs, restored the BMPR2/IL-6 balance, reduced IL-6-associated inflammation, inhibited pulmonary artery smooth muscle cell proliferation, and ameliorated pulmonary vascular remodelling in MCT-induced PH in rats., (Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

In vertebrates, bone morphogenetic proteins (BMPs) play an important role in various biological processes. However, the function of BMPs in crustaceans is still unknown. In our study, a ligand (BMP7) and two receptors (Sp-BMPRIB and Sp-BMPRII) are cloned firstly in the mud crab, Scylla paramamosain. The qRT-PCR demonstrated that both ligand and receptors were expressed in various tissues, especially in ovary. The expression of BMPRs mRNA increased along the ovarian development, while BMP7 had an opposite tendency. In-situ hybridization revealed that Sp-BMPRIB and Sp-BMPRII were expressed in both oocytes and follicle cells, whereas Sp-BMP7 was exclusively localized in follicle cells. RNAi experiments showed that the expression levels of Smad1 and vitellogenin receptor declined rapidly after BMPRs were silenced. Based on these data, we hypothesized that in S. paramamosain, BMP7 and BMPRs had impact on the ovarian development, presumably via the autocrine/paracrine way., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Granulosa cell proliferation and differentiation are key developmental steps involved in the formation of the dominant follicle eligible for ovulation. This process is, in turn, regulated by spatiotemporally emerging molecular events. MicroRNAs (miRNAs) are one of the molecular signatures believed to regulate granulosa cell function by fine-tuning gene expression. Previously, we showed that the miR-17-92 cluster was differentially expressed in granulosa cells from subordinate and dominant follicles at day 19 of the estrous cycle. However, the role of this miRNA cluster in bovine follicular cell function is not known. Therefore, in the present study, we investigate the role of the miR-17-92 cluster in granulosa cell function by using an in vitro model. Target prediction and luciferase assay analysis revealed that the miR-17-92 cluster coordinately regulated the PTEN and BMPR2 genes. Overexpression of the miR-17-92 cluster by using a mimic promoted granulosa cell proliferation and reduced the proportion of differentiated cells. However, cluster inhibition resulted in decreased proliferation and increased differentiation in granulosa cells. This was further supported by expression analysis of marker genes of proliferation and differentiation. The role of the miR-17-92 cluster was cross-validated by selective knockdown of its target genes by the short interfering RNA technique. Suppression of the PTEN and BMPR2 genes revealed similar phenotypic and molecular alterations as observed when the granulosa cells were transfected with the miR-17-92 cluster mimic. Thus, the miR-17-92 cluster is involved in granulosa cell proliferation and differentiation by coordinately targeting the PTEN and BMPR2 genes.

Objective To observe the effects of Bushen Tiaojing Recipe (BTR) on the counts of survival preantral follicles and the bone morphogenetic protein receptor II (BMPR II )/activin receptor- like kinase 6-drosophila mothers against decapentaplegic proteins (ALK6-Smads) signal pathway in oocytes cultured in vitro, and to study its mechanism for improving the quality of oocytes. Methods Prean- tral follicles were mechanically isolated from 65 female 12-day old healthy Kunming mice, which were inoculated by normal rats' serum (as the control group) , high, medium, low dose BTR containing serums (as Shen-supplementing groups) , high dose BTR containing serum + K02288 (as the inhibitor group) , respectively. All were cultured by common method in vitro. On the 6th day the counts of survival preantral follicles were compared between each Shen-supplementing group and the control group respectively. mR- NA expressions of BMPR II, ALK6, Smad1 , Smad5, and Smad8 were detected by Real-time fluorescence quantitative PCR. The protein expressions of indices mentioned above and phospho-Smadl/5/8 (p- Smadl/5/8) were detected by cellular immunofluorescence test. Results Compared with the control group, the quantity of survival preantral follicles increased in the high dose BTR containing serum group; mRNA expressions of BMPR II, ALK6, Smad5, and Smad8 were elevated, protein expressions of indi- ces mentioned above and p-Smadl/5/8 were increased in the 3 Shen-supplementing groups (P <0. 05) ; mRNA and protein expressions of Smad1 were increased in high and medium dose BTR containing serum groups (P<0.05). Compared with the high dose BTR containing serum group, protein expressions of Smad1/5/8 were reduced in the inhibitor group (P <0.05). Conclusion BTR could elevate the quantity of survival preantral follicles cultured in vitroand improve the quality of oocytes, which might be possibly as- sociated to regulating the BMPR II/ALK6-Smads signal pathway in oocytes.

Ethnopharmacological Relevance: Sambucus Williamsii Hance (SWH) is a plant from a family of Caprifoliaceae, which has a long medical history of use as an effective folk treatment for fracture bruises., Aim of the Study: To evaluate the effects of 50% ethanol extracts of root-bark of Sambucus Williamsii Hance(EE-rbSWH) on fracture healing of rats and explore its mechanism of actions related to the BMP-2 signaling pathway., Materials and Methods: EE-rbSWH was orally administered at the doses of 340 and 680mg/kg to adult Sprague-Dawley rats with operation of open femur fracture completely for 2, 4 and 8 weeks. And the rats of sham operation and Model groups were administered Vehicle (distilled water 0.8mL/200g/day). Firstly, the bone X-ray morphology and bone mineral density(BMD) of the fracture site were observed and measured after anesthesia the rats at weeks 2, 4, and 8 after surgery, then the serum levels of alkaline phosphatase(ALP) and osteocalcin (BGP) were measured; Secondly, the tissue morphology of the fracture site was observed after sacrificed the rats; Thirdly, the formation of mineralized nodules in bone marrow stromal cells(BMSC) were evaluated at week 2; Lastly, the genes levels of BMP-2 and Runx2 in the femur were detected at week 2 and 4, and the proteins expression of BMP-2 signaling pathway (BMP-2, BMPRIB, BMPRII and Runx2) in the femur also were detected at week 2., Results: EE-rbSWH remarkably accelerated fracture healing by promoting bone formation at all the time points of fracture healing. Mainly by increasing the BMD level at the fracture site, the levels of serum ALP and BGP, and also the numbers increasing of calcified nodules in BMSC. The mechanism studies, EE-rbSWH can promote fracture healing by enhancing the expressions of BMP-2 and Runx2 mRNA, and also the proteins of BMP-2, BMPRIB, BMPRII and Runx2 at the fracture site of rats., Conclusions: Our results suggested that 50% ethanol extracts of root-bark of Sambucus Williamsii Hance can accelerate fracture healing by recruitment of osteoblasts at the fracture site and through up-regulation of the BMP-2 signaling pathway., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Context: Few potential candidate genes coding for type I and II receptors of transforming growth factor beta signaling pathway and the serotonin transporter have been associated with pulmonary hypertension (PH). The latter being a phenotype for high altitude pulmonary edema (HAPE), these genes are hypothesized to be crucial markers to investigate under the hypobaric hypoxic environment of high altitude., Aims: We hence aimed to investigate bone-morphogenetic protein-2 (BMP2), bone morphogenetic protein receptor type-II (BMPR-2), activin receptor-like kinase-1 (ALK-1), serotonin transporter (5-HTT) and serotonin (5-HT) for their contribution, individually/epistatically, to clinical endpoints by altering downstream signaling molecules., Methods and Materials: In a case-control design, interactions between/among polymorphisms of BMPR-2, ALK-1 and 5-HTT were screened in 200 HAPE-patients (HAPE-p) and 200 HAPE-free sojourners (HAPE-f). Plasma biomarker BMP-2 and 5-HT were estimated. The relative gene expression was also witnessed in 20 humans/10 rats followed by correlation analyses., Results: The genotype/allele models revealed the prevalence of BMPR-2 rs6717924A-rs4303700A-rs1048829A; ALK-1 rs11169953T-rs3759178C-rs706816C and 5-HTT rs6354C in HAPE (P≤0.05). Multifactor dimensionality reduction for interactions among genes revealed a 4-locus model of BMPR-2 rs6717924G/A; ALK-1 rs11169953C/T-rs706816T/C and 5-HTT rs6354A/C as the best disease predicting (P≤0.001); whereas HapEvolution analysis confirmed the alleles rs6717924A, rs4303700A and rs6354C as the best interacting (P≤0.01). Plasma levels of BMP-2 and 5-HT were elevated in HAPE (P≤0.0001). The expression of BMP-2, ALK-1, 5-HT, 5-HTT was elevated and of BMPR-2 decreased in humans and rats (P≤0.05). The risk alleles BMPR-2 rs6717924A-rs4303700G-rs1048829A; ALK-1 rs11169953T-rs706816C and 5-HTT rs6354C correlated inversely with arterial oxygen saturation (SaO2) and positively with mean arterial pressure (MAP), BMP-2 and 5-HT in HAPE. Likewise, haplotypes BMPR-2 GGGCGAAAA, AAATAGGGA and ALK-1 CCTCAAAG, CCTTAAAG correlated with clinical markers and biomarkers (P≤0.01). BMP-2 and 5-HT correlated positively with MAP and negatively with SaO2 (P≤0.01)., Conclusions: The genetic-interactions among BMPR-2, ALK-1, and 5-HTT polymorphisms, elevated BMP-2 and 5-HT levels and differential gene expression substantiated the strong genetic contribution in HAPE pathophysiology., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology. The development of pulmonary hypertension (PH) is considered the single most significant predictor of mortality in patients with chronic lung diseases. The processes that govern the progression and development of fibroproliferative and vascular lesions in IPF are not fully understood. Using human lung explant samples from patients with IPF with or without a diagnosis of PH as well as normal control tissue, we report reduced BMPR2 expression in patients with IPF or IPF+PH. These changes were consistent with dampened P-SMAD 1/5/8 and elevated P-SMAD 2/3, demonstrating reduced BMPR2 signaling and elevated TGF-β activity in IPF. In the bleomycin (BLM) model of lung fibrosis and PH, we also report decreased BMPR2 expression compared with control animals that correlated with vascular remodeling and PH. We show that genetic abrogation or pharmacological inhibition of interleukin-6 leads to diminished markers of fibrosis and PH consistent with elevated levels of BMPR2 and reduced levels of a collection of microRNAs (miRs) that are able to degrade BMPR2. We also demonstrate that isolated bone marrow-derived macrophages from BLM-exposed mice show reduced BMPR2 levels upon exposure with IL6 or the IL6+IL6R complex that are consistent with immunohistochemistry showing reduced BMPR2 in CD206 expressing macrophages from lung sections from IPF and IPF+PH patients. In conclusion, our data suggest that depletion of BMPR2 mediated by a collection of miRs induced by IL6 and subsequent STAT3 phosphorylation as a novel mechanism participating to fibroproliferative and vascular injuries in IPF., (Copyright © 2016 the American Physiological Society.)

Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: To observe the effect of bone morphogenetic protein 4 (BMP4) on the Smad signaling pathway in rat distal pulmonary artery smooth muscle cells (PASMCs), and to explore the role of the transient receptor potential ion channel (TRPC) protein 1 and 6(TRPC1, 6) in rat distal PASMCs., Methods: Distal pulmonary arteries were isolated from adult male Wistar rats(n=6, 280-300 g). The endothelium-denuded pulmonary artery tissue was digested using Collagenase and PASMCs were cultured. Activation of BMP4 signaling pathway in Smad was detected by Western blotting. Western blotting was used for the measurement of protein to determine the involvement of BMP4/BMPRⅡ signaling in BMP4-inducd TRPC1 and TRPC6 protein expression, and Smad signaling was inhibited by the specific BMPRⅡ small interfering RNA (BMPRⅡSiRNA)., Results: Rapid phosphorylation of Smad1/5/8 was seen after 15 min of stimulation with BMP4, which was reduced with time. The BMPR Ⅱ proteins was effectively down-regulated in the PASMCs after transfection with BMPRⅡ SiRNA, and the phosphorylation of Smad1/5/8 BMP4-induced was decreased in the PASMCs transfected with BMPR Ⅱ SiRNA compared to the normal PASMCs. In addition, transfection of BMPRⅡsiRNA also attenuated BMP4-induced TRPC1 and TRPC6 protein expression compared with transfection of NT-target siRNA in distal PASMCs. PASMCs transfected with BMPRⅡsiRNA showed a markedly decreased ability for BMP4 induction as assessed by gray value ratio of Western blotting, 2.7 times and 2.5 times by TRPC1/β-actin and TRPC6/β-actin respectively, compared with NT siRNA-treated cells(P<0.05)., Conclusion: TRPC1 and TRPC6 protein expression can be up-regulated through Smad1/5/8 signaling activation by combination of BMP4 and BMPRⅡ in PASMCs.

Epigenetic silencing of fragile X mental retardation 1 (FMR1) causes fragile X syndrome (FXS), a common inherited form of intellectual disability and autism. FXS correlates with abnormal synapse and dendritic spine development, but the molecular link between the absence of the FMR1 product FMRP, an RNA binding protein, and the neuropathology is unclear. We found that the messenger RNA encoding bone morphogenetic protein type II receptor (BMPR2) is a target of FMRP. Depletion of FMRP increased BMPR2 abundance, especially that of the full-length isoform that bound and activated LIM domain kinase 1 (LIMK1), a component of the noncanonical BMP signal transduction pathway that stimulates actin reorganization to promote neurite outgrowth and synapse formation. Heterozygosity for BMPR2 rescued the morphological abnormalities in neurons both in Drosophila and in mouse models of FXS, as did the postnatal pharmacological inhibition of LIMK1 activity. Compared with postmortem prefrontal cortex tissue from healthy subjects, the amount of full-length BMPR2 and of a marker of LIMK1 activity was increased in this brain region from FXS patients. These findings suggest that increased BMPR2 signal transduction is linked to FXS and that the BMPR2-LIMK1 pathway is a putative therapeutic target in patients with FXS and possibly other forms of autism., (Copyright © 2016, American Association for the Advancement of Science.)

Transforming growth factor-β (TGF-β) family ligands are pleiotropic cytokines. Their physiological activities are not determined by a simple coupling of stimulus and response, but depend critically on context, i.e. the interplay of receptors, ligands, and regulators that form the TGF-β signal transduction system of a cell or tissue. How these different components combine to regulate signaling activities remains poorly understood. Here, we describe a ligand-mediated mechanism of signaling regulation. Based on the observation that the type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII interact with a large group of overlapping ligands at overlapping epitopes, we hypothesized high affinity ligands compete with low affinity ligands for receptor binding and signaling. We show activin A and other high affinity ligands directly inhibited signaling by the low affinity ligands BMP-2, BMP-7, and BMP-9. We demonstrate activin A functions as a competitive inhibitor that blocks the ligand binding epitope on type II receptors. We propose binding competition and signaling antagonism are integral functions of the TGF-β signal transduction system. These functions could help explain how activin A modulates physiological signaling during extraordinary cellular responses, such as injury and wound healing, and how activin A could elicit disease phenotypes such as cancer-related muscle wasting and fibrosis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background and Objective: Idiopathic, familial and secondary pulmonary arterial hypertension (PAH) are associated with reduced bone morphogenetic protein receptor type 2 (BMPR2) expression, and in some contexts, TGF-β upregulation. Our aims were to assess BMPR2 gene therapy in a PAH mouse model and to assess the impact on TGF-β signalling., Methods: Using a targeted in vivo gene delivery approach, we assessed the impact of BMPR2 gene delivery in a transgenic mouse model in which PAH was first induced by doxycycline driven expression of a dominant negative BMPR2 mutant (R899X). We also assessed the impact of BMPR2 gene delivery on TGF-β-induced changes in cell signalling in human pulmonary vascular endothelial and smooth muscle cells., Results: In the mouse model, changes in TGF-β levels were not detected, but BMPR2 gene delivery reversed the increase in right ventricle systolic pressure (RVSP) and Fulton Index (FI), associated with a trend to increased pulmonary endothelial nitric oxide synthase (eNOS) gene expression. In vitro, BMPR2 gene transfer reduced TGF-β effects on Smad2, Smad1/5/8 and Erk1/2 phosphorylation in human pulmonary arterial smooth muscle cells (HPASMC). BMPR2 was also found to upregulate nitric oxide (NO) production in lung derived human microvascular endothelial cells (HMVEC-L)., Conclusion: This study provides further evidence that BMPR2 modulation may have therapeutic potential. See Editorial, page 406., (© 2015 Asian Pacific Society of Respirology.)

The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and -SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3' terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane., (© 2016 Amsalem et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Inflammation plays a fundamental role in the inception and development of atherosclerosis (ATH). Mechanisms of inflammation include the infiltration of monocytes into the injured area and subsequent differentiation into either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. We have previously published data suggesting bone morphogenetic protein-7 (BMP-7) enhances M2 macrophage differentiation and anti-inflammatory cytokine secretion in vitro. In this regard, we hypothesized BMP-7 would inhibit plaque formation in an animal model of ATH through monocytic plasticity mediation. ATH was generated in male and female Apo E(-/-) mice via partial left carotid artery (PLCA) ligation and mice were divided into 3 groups: Sham, PLCA, and PLCA+BMP-7 (200 ug/kg; i.v.). Our data suggest that BMP-7 inhibits plaque formation and increases arterial systolic velocity. Furthermore, we report inhibition of monocyte infiltration and a decrease in associated pro-inflammatory cytokines (MCP-1, TNF-α, and IL-6) in the PLCA+BMP-7 mice. In contrast, our data suggest a significant (p<0.05) increase in M2 macrophage populations with consequential enhanced anti-inflammatory cytokine (IL-1RA, IL-10, and Arginase 1) expression following BMP-7 treatment. We have also observed that mechanisms promoting monocyte into M2 macrophage differentiation by BMP-7 involve the upregulation and activation of the BMP-7 receptor (BMP-7RII). In conclusion, we report that BMP-7 has the potential to mediate cellular plasticity and mitigate the inflammatory immune response, which results in decreased plaque formation and improved blood velocity.

Background: Bone morphogenetic proteins regulate multiple processes in embryonic development, including early dorso-ventral patterning and neural crest development. BMPs activate heteromeric receptor complexes consisting of type I and type II receptor-serine/threonine kinases. BMP receptors Ia and Ib, also known as ALK3 and ALK6 respectively, are the most common type I receptors that likely mediate most BMP signaling events. Since early expression patterns and functions in Xenopus laevis development have not been described, we have addressed these questions in the present study., Results: Here we have analyzed the temporal and spatial expression patterns of ALK3 and ALK6; we have also carried out loss-of-function studies to define the function of these receptors in early Xenopus development. We detected both redundant and non-redundant roles of ALK3 and ALK6 in dorso-ventral patterning. From late gastrula stages onwards, their expression patterns diverged, which correlated with a specific, non-redundant requirement of ALK6 in post-gastrula neural crest cells. ALK6 was essential for induction of neural crest cell fate and further development of the neural crest and its derivatives., Conclusions: ALK3 and ALK6 both contribute to the gene regulatory network that regulates dorso-ventral patterning; they play partially overlapping and partially non-redundant roles in this process. ALK3 and ALK6 are independently required for the spatially restricted activation of BMP signaling and msx2 upregulation at the neural plate border, whereas in post-gastrula development ALK6 exerts a highly specific, conserved function in neural crest development.

A proliferative endothelial cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Bone morphogenetic protein type II receptor (BMPR2) loss-of-function is the most common cause of heritable PAH and has been closely linked to the formation of pathological plexiform lesions. Although some BMPR2 mutations leave ligand-dependent responses intact, the disruption of ligand-independent, noncanonical functions are universal among PAH-associated BMPR2 genotypes, but incompletely understood. This study examined the noncanonical signaling consequences of BMPR2 silencing in human pulmonary artery endothelial cells to identify potential therapeutic targets. BMPR2 siRNA silencing resulted in a proliferative, promigratory pulmonary artery endothelial cell phenotype and disruption of cytoskeletal architecture. Expression profiling closely reflected these phenotypic changes. Gene set enrichment and promoter analyses, as well as the differential expression of pathway components identified Ras/Raf/ERK signaling as an important consequence of BMPR2 silencing. Raf family members and ERK1/2 were constitutively activated after BMPR2 knockdown. Two Raf inhibitors, sorafenib and AZ628, and low-dose nintedanib, a triple receptor tyrosine kinase inhibitor upstream from Ras, reversed the abnormal proliferation and hypermotility of BMPR2 deficiency. Inhibition of dysregulated Ras/Raf/ERK signaling may be useful in reversing vascular remodeling in PAH.