Heterodimer-heterotetramer formation mediates enhanced sensor activity in a biophysical model for BMP signaling

Numerous stages of organismal development rely on the cellular interpretation of gradients of secreted morphogens including members of the Bone Morphogenetic Protein (BMP) family through transmembrane receptors. Early gradients of BMPs drive dorsal/ventral patterning throughout the animal kingdom in both vertebrates and invertebrates. Growing evidence in Drosophila, zebrafish, murine and other systems suggests that BMP ligand heterodimers are the primary BMP signaling ligand, even in systems in which mixtures of BMP homodimers and heterodimers are present. Signaling by heterodimers occurs through a hetero-tetrameric receptor complex comprising of two distinct type one BMP receptors and two type II receptors. To understand the system dynamics and determine whether kinetic assembly of heterodimer-heterotetramer BMP complexes is favored, as compared to other plausible BMP ligand-receptor configurations, we developed a kinetic model for BMP tetramer formation based on current measurements for binding rates and affinities. We find that contrary to a common hypothesis, heterodimer-heterotetramer formation is not kinetically favored over the formation of homodimer-tetramer complexes under physiological conditions of receptor and ligand concentrations and therefore other mechanisms, potentially including differential kinase activities of the formed heterotetramer complexes, must be the cause of heterodimer-heterotetramer signaling primacy. Further, although BMP complex assembly favors homodimer and homomeric complex formation over a wide range of parameters, ignoring these signals and instead relying on the heterodimer improves the range of morphogen interpretation in a broad set of conditions, suggesting a performance advantage for heterodimer signaling in patterning multiple cell types in a gradient.

Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization

Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

O29 Aberrant expression of BMP8A and the BMPRs involves in the progression of breast cancer

Introduction Role of Bone morphogenetic protein 8A (BMP8A) and BMP receptors (BMPRs) in the tumourigenesis and progression of breast cancer remains elusive. Present study aims to investigate the expression of BMP8A and related BMPRs in breast cancer and their clinical implication. Method Expression of BMP8A and BMPRs was analysed using the RNA sequencing data of the TCGA breast cancer cohort. Findings were further validated in a meta gene array dataset (E-MDTA6703, n = 2302). STRING dataset was applied to explore the predicted receptors of BMP8A. Clinical relevance of deregulated BMP8A and BMPRs in breast cancer was assessed using both ANOVA and Kaplan-Meier tests. Correlation with markers of proliferation and invasion was evaluated using Spearman test. Result Analysis of datasets revealed that BMP8A and BMPR1B were highly expressed in breast cancer while ACVRL1, ACVR1, BMPR1A, ACVR1C, TGFBR2, TGFBR3, BMPR2 and ACVR2A were lower-expressed compared with normal controls. Expressions of BMPR1B, BMPR1A, BMPR2, ACVR2A and ACVR2B were highly correlated with BMP8A in the breast cancers. Overall survival in the group with higher BMP8A expression was shorter(median= 122.3 months), P = 0.012 compared with lower-expressed group(median = 215.2 months). No significant difference was observed in BMP8A and BMPRs in tumours according to their staging and lymph node involvement. Positive correlations were found between BMP8A and tumour proliferation, EMT, angiogenic markers. Conclusion BMP8A is increased in breast cancer and correlates with poor prognosis. The highly correlated BMPRs might be involved in the signal transduction of BMP8A to co-regulate BMP responsive genes and cellular functions which is yet to be investigated. Take-home Message BMP8A is increased in breast cancer and correlates with poor prognosis.

Iron overload inhibits BMP/SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes

Hepcidin is a peptide hormone that targets the iron exporter ferroportin, thereby limiting iron entry into the bloodstream. It is generated in hepatocytes mainly in response to increased body iron stores or inflammatory cues. Iron stimulates expression of bone morphogenetic protein 6 (BMP6) from liver sinusoidal endothelial cells, which in turn binds to BMP receptors on hepatocytes and induces the SMAD signaling cascade for transcriptional activation of the hepcidin-encoding HAMP mRNA. SMAD signaling is also essential for inflammatory HAMP mRNA induction by the IL-6/STAT3 pathway. Herein, we utilized human Huh7 hepatoma cells and primary murine hepatocytes to assess the effects of iron perturbations on signaling to hepcidin. Iron chelation appeared to slightly impair signaling to hepcidin. Subsequent iron supplementation not only failed to reverse these effects, but drastically reduced basal HAMP mRNA and inhibited HAMP mRNA induction by BMP6 and/or IL-6. Thus, treatment of cells with excess iron inhibited basal and BMP6-mediated SMAD5 phosphorylation and induction of HAMP, ID1 and SMAD7 mRNAs in a dose-dependent manner. Iron also inhibited IL-6-mediated STAT3 phosphorylation and induction of HAMP and SOCS3 mRNAs. These responses were accompanied by induction of GCLC and HMOX1 mRNAs, known markers of oxidative stress. We conclude that hepatocellular iron overload suppresses hepcidin by inhibiting the SMAD and STAT3 signaling pathways downstream of their respective ligands.

Abnormal Expression and Prognostic Significance of Bone Morphogenetic Proteins and Their Receptors in Lung Adenocarcinoma

Background. Lung adenocarcinoma (LUAD) is one of the most life-threatening malignancies. The crucial role of bone morphogenetic protein (BMP)/BMP receptors reveals the significance of exploring BMP protein-related prognostic predictors in LUAD. Methods. The mRNA expression of BMPs/BMP receptors was investigated in LUAD and normal lung tissues. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed, and the prognostic values were assessed by Kaplan-Meier Plotter. Univariate and multivariate Cox regression analyses were executed to ascertain the correlation between overall survival (OS) and the mRNA expression of BMPs/BMP receptors. The receiver operating characteristic (ROC) curves were implemented to evaluate the predictive power of the prognostic model. Then, the prognostic model was validated in the GEO cohort. Furthermore, a nomogram comprising the prognostic model was established. Results. The mRNA expression of BMP2/5/6/R2, ACVRL1, and TGFBR2/3 was lower in LUAD tissues than in normal lung tissues. High expression of BMP2/4/5/R1A/R2, ACVR1/2A/L1, and TGFBR1/3 was associated with better OS, while BMP7 and ACVR1C/2B were associated with poorer OS. Three genes (BMP5, BMP7, and ACVR2A) were screened by univariate and multivariate Cox regression analyses to develop the prognostic model in TCGA. Significantly better survival was observed in LUAD patients with a low-risk score than those with a high-risk score. The ROC curves confirmed the good performance of the prognostic model, then, the prognostic model was validated in the GSE31210 dataset. A nomogram was constructed (AUCs>0.7). And hub genes were further evaluated, including gene set enrichment analysis and immune cell infiltration. Conclusions. BMP5, BMP7, and ACVR2A are potential therapeutic targets in LUAD. The three-gene prognostic model and the nomogram are reliable tools for predicting the OS of LUAD patients.

Involvement of BMP-15 in Glucocorticoid Actions on Ovarian Steroidogenesis by Rat Granulosa Cells

Glucocorticoid receptor (GR) are known to be expressed in the ovary and glucocorticoids are shown to exert direct effects on granulosa cell functions. In the clinical setting, menstrual abnormality, amenorrhea and hypermenorrhea can be shown in patients with glucocorticoid excess. On the other hand, glucocorticoids can also be used for the treatment of PCOS with hyperandrogenism. However, the effects of glucocorticoids on the reproductive system have not been fully elucidated. In the present study, we investigated the influence of glucocorticoids on follicular steroidogenesis using primary culture of rat granulosa cells, by focusing on the ovarian bone morphogenetic proteins (BMPs) acting as a luteinizing inhibitor. Granulosa cells isolated from female immature rats were treated with follicle-stimulating hormone (FSH) in the presence of dexamethasone (Dex) in serum-free conditions. After treatment with Dex for 48 h, the changes of estradiol (E2) and progesterone (P4) production and cAMP synthesis induced by FSH treatments were measured by ELISA. Total RNAs of granulosa cells treated with FSH, Dex and BMPs were extracted and mRNA levels of steroidogenetic factors and enzymes, BMP receptors and Id-1 were quantified by real-time RT-PCR. Phosphorylation of Smad1/5/9 induced by BMPs was evaluated by Western blotting using cell lysates in the presence or absence of Dex. As a result, it was revealed that Dex treatment decreased FSH-induced E2 production by granulosa cells. In accordance with the steroid results, Dex suppressed FSH-induced P450arom mRNA expression as well as FSH-induced cAMP synthesis by granulosa cells. By contrast, Dex treatment augmented FSH-induced P4 production by granulosa cells in a concentration-dependent manner. Dex treatment was found to enhance basal and FSH-induced mRNA levels of P4-synthetic enzymes including P450scc and 3βHSD. Of note, Dex treatment activated the BMP target gene Id-1 transcription and Smad1/5/9 phosphorylation, in particular, induced by BMP-15 among various BMP ligands including BMP-2, -4, -6, -7, -9 and -15. It was also revealed that Dex treatment increased mRNA levels of ALK-6, a type-I receptor for BMP-15, and that BMP-15 treatment in turn upregulated GR mRNA levels expressed by granulosa cells. Given that BMP-15 acts as an inhibitor for P4 production by suppressing FSH-receptor actions, it was suggested that glucocorticoid is functionally linked to the enhancement of endogenous BMP-15, leading to the negative feedback toward the P4 overproduction induced by FSH and Dex in granulosa cells. Collectively, it was revealed that glucocorticoids elicit differential effects on the ovarian steroidogenesis of E2 and P4, in which GR and BMP-15 actions are mutually enhanced in granulosa cells.

Orexin A Enhances Pro-Opiomelanocortin Transcription Regulated by BMP-4 in Mouse Corticotrope AtT20 Cells

Orexin is expressed mainly in the hypothalamus and is known to activate the hypothalamic–pituitary–adrenal (HPA) axis that is involved in various stress responses and its resilience. However, the effects of orexin on the endocrine function of pituitary corticotrope cells remain unclear. In this study, we investigated the roles of orexin A in pro-opiomelanocortin (POMC) transcription using mouse corticotrope AtT20 cells, focusing on the bone morphogenetic protein (BMP) system expressed in the pituitary. Regarding the receptors for orexin, type 2 (OXR2) rather than type 1 (OX1R) receptor mRNA was predominantly expressed in AtT20 cells. It was found that orexin A treatment enhanced POMC expression, induced by corticotropin-releasing hormone (CRH) stimulation through upregulation of CRH receptor type-1 (CRHR1). Orexin A had no direct effect on the POMC transcription suppressed by BMP-4 treatment, whereas it suppressed Smad1/5/9 phosphorylation and Id-1 mRNA expression induced by BMP-4. It was further revealed that orexin A had no significant effect on the expression levels of type I and II BMP receptors but upregulated inhibitory Smad6/7 mRNA and protein levels in AtT20 cells. The results demonstrated that orexin A upregulated CRHR signaling and downregulated BMP-Smad signaling, leading to an enhancement of POMC transcription by corticotrope cells.

Differential bioactivity of four BMP-family members as function of biomaterial stiffness

ABSTRACTWhereas soft biomaterial is not able to induce cell spreading, BMP-2 presented by a soft film has been described to be sufficient to trigger cell spreading, migration and downstream BMP-2 signaling. Based on thin polyelectrolyte films of controlled stiffness, we investigated whether the presentation of four BMP members (2, 4, 7, 9) in a matrix-bound manner may differentially impact cell adhesion and bone differentiation of skeletal progenitors. We performed high content and automated screening of cellular responses, including cell number, cell spreading area, SMAD phosphorylation and alkaline phosphatase activity. The basolateral presentation of the different BMPs allowed us to discriminate the specificity of cellular response and the role of BMP receptors type I, type II, as well as three β integrins, in a BMP type and stiffness-dependent manner.

Bone Morphogenetic Protein-2 Induces Non-Canonical Inflammatory and Oxidative Pathways in Human Retinal Endothelial Cells

The mechanisms of diabetic retinopathy (DR), are not yet fully understood. We previously demonstrated an upregulation of retinal bone morphogenetic protein-2 (BMP2) in experimental diabetes and in retinas of diabetic human subjects. The purpose of current study was to investigate the role of non-canonical inflammatory pathway in BMP2-induced retinal endothelial cell (REC) barrier dysfunction. For this purpose, we used RT-PCR and western blotting to evaluate the levels of BMP2 signaling components (BMP2, BMP4, BMP receptors), VEGF, phosphorylated p38 MAPK and NFκB, and oxidative stress markers in cultured human retinal endothelial cells (HRECs) subjected to BMP2 (50ng/ml) for up to 24 h. Also, effect of high glucose (HG, 30mM D-glucose) on the expression of BMP2 and its downstream genes was examined in HRECs. H2-DCF is a fluorogenic dye that measures the levels of cellular reactive oxygen species (ROS) was used to measure the pro-oxidative effect of BMP2. Moreover, we evaluated the effect of inhibiting p38 and VEGF signaling on BMP2-induced HRECs barrier dysfunction by measuring the trans-endothelial cell electrical resistance (TER) using electric cell-substrate impedance sensing (ECIS). We also tested the effect of HG on the integrity of HRECs barrier in the presence or absence of inhibitors of BMP2 signaling. Our data reveals that BMP2 and high glucose upregulates BMP components of the BMP signaling pathway (SMAD effectors, BMP receptors, and TGFβ ligand itself) and induces phosphorylation of p38 MAPK and NFκB with nuclear translocation of NFκB. Inhibition of p38 or NFκB attenuated BMP2-induced VEGF expression and barrier dysfunction in HRECs. Also, inhibition of VEGFR2 attenuated BMP2-induced barrier dysfunction. Moreover, BMP2 induces generation of ROS and endothelial nitric oxide synthase (eNOS) expression and activity in HRECs. Finally, HG upregulated BMP2 and its downstream genes (SMAD, BMP4, ALKs, and TGF-β) in HRECs and BMP2 inhibitors attenuated HG-induced HRECs barrier dysfunction. Our results suggest that in addition to the regular canonical SMAD signaling BMP2 induces non-canonical inflammatory pathway in HRECs via activation of p38/NFκB pathway that causes the upregulation of VEGF and the disruption of HRECs. Inhibition of BMP2 signaling is a potential therapeutic intervention to preserve endothelial cell barrier function in DR.

ETMR-05. SINGLE-CELL RNA-SEQ OF ETMR REVEALS CELL PROGRAMS OF DEVELOPMENTAL HIERARCHY AND CELLULAR DIVERSITY IN THE TUMOR MICROENVIRONMENT

Embryonal tumors with multilayered rosettes (ETMR) are deadly brain malignancies affecting young children. No standard treatment is available and the median survival is less than 12 months. Molecularly, the disease is characterized by the miRNA C19MC cluster amplification, with the expression of multiples miRNAs related to a stem cell program. The discoveries on the purely molecular mechanisms of the disease did not help to create a bridge for new treatment strategies so far and the cellular diversity of ETMR remains poorly understood. In this study, we used single-cell RNA sequencing of murine and human tumors to describe ETMR cellular heterogeneity. Our findings support that intra-tumoral heterogeneity is mainly characterized by 4 cellular programs defining a developmental hierarchy related to different metabolic states: 1) Early quiescent NSC-like cells supported by fatty-acid oxidation 2) Late NSC and NP-like proliferative cells fueled by glycolytic metabolism; 3) Post-mitotic neuroblast-like cells, relying on oxidative-phosphorylation; 4) NSC-like proliferative cells, with metabolic plasticity and capable of performing the three types of metabolism. Tumor-specific ligand-receptor interaction analysis revealed that ETMR exchange with microglia and vascular mural cells (MC) signals related to extracellular matrix (ECM) organization (Cxcl12-CxCr4), stem cell signaling (BMPs-BMP receptors), anti-apoptosis and survival (Ntf3-Ntrk), not seen in the control brain. In addition, the vascular MC showed a cancer-associated fibroblast (CAF) phenotype, with potential prognostic implications, as previously demonstrated for other tumors. This study provides new findings to build up a more robust understanding of ETMR biology and opens space for further studies in the field.