Bone Morphogenetic Protein Receptor Inhibitor Research Articles

Mesenchyme governs hair follicle induction.

Tissue interactions are essential for guiding organ development and regeneration. Hair follicle formation relies on inductive signalling between two tissues, the embryonic surface epithelium and the adjacent mesenchyme. Although previous research has highlighted the hair-inducing potential of the mesenchymal component of the hair follicle - the dermal papilla and its precursor, the dermal condensate - the source and nature of the primary inductive signal before dermal condensate formation have remained elusive. Here, we performed epithelial-mesenchymal tissue recombination experiments using hair-forming back skin and glabrous plantar skin from mouse embryos to unveil that the back skin mesenchyme is inductive even before dermal condensate formation. Moreover, the naïve, unpatterned mesenchyme was sufficient to trigger hair follicle formation even in the oral epithelium. Building on previous knowledge, we explored the hair-inductive ability of the Wnt agonist R-spondin 1 and a Bmp receptor inhibitor in embryonic skin explants. Although R-spondin 1 instigated precocious placode-specific transcriptional responses, it was insufficient for hair follicle induction, either alone or in combination with Bmp receptor inhibition. Our findings pave the way for identifying the hair follicle-inducing cue.

SP600125 Enhances Temperature-Controlled Repeated Thermal Stimulation-Induced Neurite Outgrowth in PC12-P1F1 Cells.

This study evaluated the mechanism of temperature-controlled repeated thermal stimulation (TRTS)-mediated neuronal differentiation. We assessed the effect of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, on neuronal differentiation of rat PC12-P1F1 cells, which can differentiate into neuron-like cells by exposure to TRTS or neurotrophic factors, including bone morphogenetic protein (BMP) 4. We evaluated neuritogenesis by incubating the cells under conditions of TRTS and/or SP600125. Cotreatment with SP600125 significantly enhanced TRTS-mediated neuritogenesis, whereas that with other selective mitogen-activated protein kinase (MAPK) inhibitors did not-e.g., extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126, and p38 MAPK inhibitor SB203580. We tried to clarify the mechanism of SP600125 action by testing the effect of U0126 and the BMP receptor inhibitor LDN193189 on the SP600125-mediated enhancement of intracellular signaling. SP600125-enhanced TRTS-induced neuritogenesis was significantly inhibited by U0126 or LDN193189. Gene expression analysis revealed that TRTS significantly increased β3-Tubulin, MKK3, and Smad7 gene expressions. Additionally, Smad6 and Smad7 gene expressions were substantially attenuated through SP600125 co-treatment during TRTS. Therefore, SP600125 may partly enhance TRTS-induced neuritogenesis by attenuating the negative feedback loop of BMP signaling. Further investigation of the mechanisms underlying the effect of SP600125 during TRTS-mediated neuritogenesis may contribute to the future development of regenerative neuromedicine.

Bone morphogenetic protein receptor inhibitors suppress the growth of glioblastoma cells.

Glioblastomas (GBMs) are aggressive brain tumors that are resistant to chemotherapy and radiation. Bone morphogenetic protein (BMP) ligand BMP4 is being examined as a potential therapeutic for GBMs because it induces differentiation of cancer stem cells (CSCs) to an astrocyte phenotype. ID1 is reported to promote self-renewal and inhibit CSC differentiation. In most cancers, ID1 is transcriptionally upregulated by BMP4 promoting invasion and stemness. This conflicting data bring into question whether BMP signaling is growth suppressive or growth promoting in GBMs. We utilized BMP inhibitors DMH1, JL5, and Ym155 to examine the role of BMP signaling on the growth of GBMs. DMH1 targets BMP type 1 receptors whereas JL5 inhibits both the type 1 and type 2 BMP receptors. Ym155 does not bind the BMP receptors but rather inhibits BMP signaling by inducing the degradation of BMPR2. We show that JL5, DMH1, and Ym155 decreased the expression of ID1 in SD2 and U87 cells. JL5 and Ym155 also decreased the expression of BMPR2 and its downstream target inhibitor of apoptosis protein XIAP. JL5 treatment resulted in significant cell death and suppressed self-renewal to a greater extent than that induced by BMP4 ligand. The lysosome inhibitor chloroquine increases the localization of BMPR2 to the plasma membrane enhancing JL5-induced downregulation of ID1 and cell death in SD2 cells. We show that BMP signaling is growth promoting in GBMs. These studies suggest the need for development of BMP inhibitors and evaluation as potential therapeutic for GBMs.

BMP9-ID1 Signaling Activates HIF-1α and VEGFA Expression to Promote Tumor Angiogenesis in Hepatocellular Carcinoma.

Since hepatocellular carcinoma (HCC) is a typical hypervascular malignant tumor with poor prognosis, targeting angiogenesis is an important therapeutic strategy for advanced HCC. Involvement of bone morphologic protein 9 (BMP9), a transforming growth factor-beta superfamily member, has recently been reported in the development of liver diseases and angiogenesis. Here, we aimed to elucidate the role of BMP9 signaling in promoting HCC angiogenesis and to assess the antiangiogenic effect of BMP receptor inhibitors in HCC. By analyzing HCC tissue gene expression profiles, we found that BMP9 expression was significantly correlated with angiogenesis-associated genes, including HIF-1α and VEGFR2. In vitro, BMP9 induced HCC cell HIF-1α/VEGFA expression and VEGFA secretion. Silencing of the inhibitor of DNA-binding protein 1 (ID1), a transcription factor targeted by BMP9 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression and VEGFA secretion, resulting in decreased human umbilical vein endothelial cell (HUVEC) lumen formation. BMP receptor inhibitors, which inhibit BMP9-ID1 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression, VEGFA secretion, and HUVEC lumen formation. In vivo, the BMP receptor inhibitor LDN-212854 successfully inhibited HCC tumor growth and angiogenesis by inhibiting BMP9-ID1 signaling. In summary, BMP9-ID1 signaling promotes HCC angiogenesis by activating HIF-1α/VEGFA expression. Thus, targeting BMP9-ID1 signaling could be a pivotal therapeutic option for advanced HCC.

Natural Herb Mixture Extract Accelerates Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells by Activating the SMAD Pathway.

We aimed to analyze the effects and explore the molecular mechanisms of a natural herb mixture extract (NME) on osteoblasts during differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs). We tried to confirm the regulation of osteogenic differentiation during NME treatment. Alkaline phosphatase assay and Alizarin red S staining were performed to evaluate the regulation of osteogenic differentiation. Real-time polymerase chain reaction was performed to analyze the expression of osteoblast maker genes, and Western blot was used to verify the signaling pathway. Signaling pathway conformation, selective bone morphogenetic protein receptor inhibitor, and dorsomorphin homolog 1 were used as pretreatments before inducing osteogenic differentiation. We determined that MME (natural herb mixture extract) was a safe material and significantly increased osteoblast differentiation and that SMAD phosphorylation is a key signaling pathway that regulates osteogenic differentiation in hBMSCs.

Adipocytes and Stromal Cells Regulate Brown Adipogenesis Through Secretory Factors During the Postnatal White-to-Brown Conversion of Adipose Tissue in Syrian Hamsters.

Brown adipose tissue (BAT) is a specialized tissue that regulates non-shivering thermogenesis. In Syrian hamsters, interscapular adipose tissue is composed primarily of white adipocytes at birth, which is converted to BAT through the proliferation and differentiation of brown adipocyte progenitors and the simultaneous disappearance of white adipocytes. In this study, we investigated the regulatory mechanism of brown adipogenesis during postnatal BAT formation in hamsters. Interscapular adipose tissue of a 10-day-old hamster, which primarily consists of brown adipocyte progenitors and white adipocytes, was digested with collagenase and fractioned into stromal–vascular (SV) cells and white adipocytes. SV cells spontaneously differentiated into brown adipocytes that contained multilocular lipid droplets and expressed uncoupling protein 1 (Ucp1), a marker of brown adipocytes, without treatment of adipogenic cocktail such as dexamethasone and insulin. The spontaneous differentiation of SV cells was suppressed by co-culture with adipocytes or by the addition of white adipocyte-conditioned medium. Conversely, the addition of SV cell-conditioned medium increased the expression of Ucp1. These results indicate that adipocytes secrete factors that suppress brown adipogenesis, whereas SV cells secrete factors that promote brown adipogenesis. Transcriptome analysis was conducted; however, no candidate suppressing factors secreted from adipocytes were identified. In contrast, 19 genes that encode secretory factors, including bone morphogenetic protein (BMP) family members, BMP3B, BMP5, and BMP7, were highly expressed in SV cells compared with adipocytes. Furthermore, the SMAD and MAPK signaling pathways, which represent the major BMP signaling pathways, were activated in SV cells, suggesting that BMPs secreted from SV cells induce brown adipogenesis in an autocrine manner through the SMAD/MAPK signaling pathways. Treatment of 5-day-old hamsters with type I BMP receptor inhibitor, LDN-193189, for 5 days reduced p38 MAPK phosphorylation and drastically suppressed BAT formation of interscapular adipose tissue. In conclusion, adipocytes and stromal cells regulate brown adipogenesis through secretory factors during the postnatal white-to-brown conversion of adipose tissue in Syrian hamsters.

Macrophages Inhibit Ciliary Protein Levels by Secreting BMP-2 Leading to Airway Epithelial Remodeling Under Cigarette Smoke Exposure.

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease with high morbidity and mortality worldwide. So far, smoking is still its leading cause. The characteristics of COPD are emphysema and airway remodeling, as well as chronic inflammation, which were predominated by macrophages. Some studies have reported that macrophages were involved in emphysema and chronic inflammation, but whether there is a link between airway remodeling and macrophages remains unclear. In this study, we found that both acute and chronic cigarette smoke exposure led to an increase of macrophages in the lung and a decrease of ciliated cells in the airway epithelium of a mouse model. The results of in vitro experiments showed that the ciliary protein (β-tubulin-IV) levels of BEAS-2B cells could be inhibited when co-cultured with human macrophage line THP-1, and the inhibitory effect was augmented with the stimulation of cigarette smoke extract (CSE). Based on the results of transcriptome sequencing, we focused on the protein, bone morphogenetic protein-2 (BMP-2), secreted by the macrophage, which might mediate this inhibitory effect. Further studies confirmed that BMP-2 protein inhibited β-tubulin-IV protein levels of BEAS-2B cells under the stimulation of CSE. Coincidentally, this inhibitory effect could be nearly blocked by the BMP receptor inhibitor, LDN, or could be interfered with BMP-2 siRNA. This study suggests that activation and infiltration of macrophages in the lung induced by smoke exposure lead to a high expression of BMP-2, which in turn inhibits the ciliary protein levels of the bronchial epithelial cells, contributing to the remodeling of airway epithelium, and aggravates the development of COPD.

BMP signaling suppresses Gemc1 expression and ependymal differentiation of mouse telencephalic progenitors

The lateral ventricles of the adult mammalian brain are lined by a single layer of multiciliated ependymal cells, which generate a flow of cerebrospinal fluid through directional beating of their cilia as well as regulate neurogenesis through interaction with adult neural stem cells. Ependymal cells are derived from a subset of embryonic neural stem-progenitor cells (NPCs, also known as radial glial cells) that becomes postmitotic during the late embryonic stage of development. Members of the Geminin family of transcriptional regulators including GemC1 and Mcidas play key roles in the differentiation of ependymal cells, but it remains largely unclear what extracellular signals regulate these factors and ependymal differentiation during embryonic and early-postnatal development. We now show that the levels of Smad1/5/8 phosphorylation and Id1/4 protein expression—both of which are downstream events of bone morphogenetic protein (BMP) signaling—decline in cells of the ventricular-subventricular zone in the mouse lateral ganglionic eminence in association with ependymal differentiation. Exposure of postnatal NPC cultures to BMP ligands or to a BMP receptor inhibitor suppressed and promoted the emergence of multiciliated ependymal cells, respectively. Moreover, treatment of embryonic NPC cultures with BMP ligands reduced the expression level of the ependymal marker Foxj1 and suppressed the emergence of ependymal-like cells. Finally, BMP ligands reduced the expression levels of Gemc1 and Mcidas in postnatal NPC cultures, whereas the BMP receptor inhibitor increased them. Our results thus implicate BMP signaling in suppression of ependymal differentiation from NPCs through regulation of Gemc1 and Mcidas expression during embryonic and early-postnatal stages of mouse telencephalic development.

Ablation of Fat Cells in Adult Mice Induces Massive Bone Gain

Adipocytes control bone mass, but the mechanism is unclear. To explore the effect of postnatal adipocyte elimination on bone cells, we mated mice expressing an inducible primate diphtheria toxin receptor (DTR) to those bearing adiponectin (ADQ)-Cre. DTR activation eliminates peripheral and marrow adipocytes in these DTRADQ mice. Within 4days of DTR activation, the systemic bone mass of DTRADQ mice began to increase due to stimulated osteogenesis, with a 1,000% expansion by 10-14days post-DTR treatment. This adipocyte ablation-mediated enhancement of skeletal mass reflected bone morphogenetic protein (BMP) receptor activation following the elimination of its inhibitors, associated with simultaneous epidermal growth factor (EGF) receptor signaling. DTRADQ-induced osteosclerosis is not due to ablation of peripheral adipocytes but likely reflects the elimination of marrow ADQ-expressing cells. Thus, anabolic drugs targeting BMP receptor inhibitors with short-term EGF receptor activation may be a means of profoundly increasing skeletal mass to prevent or reverse pathological bone loss.

Autocrine BMP4 Signaling Enhances Tumor Aggressiveness via Promoting Wnt/β-Catenin Signaling in IDH1-mutant Gliomas

The isocitrate dehydrogenase (IDH1/2) mutations are frequent genetic abnormalities in the majority of WHO grade II/III glioma and secondary GBM. IDH1-mutated (IDH1Mut) glioma exhibits distinctive patterns in cancer biology and metabolism. In the present study, we showed that bone morphogenetic proteins (BMP4) are significantly upregulated in IDH1Mut glioma. Further, we demonstrated that cancer-associated BMP4 is secreted to tumor microenvironment, which enhances the tumor migration and invasion through an autocrine manner. Mechanistically, BMP4 activates its receptor and concomitant SMAD1/5/8 signaling, which potentiates Wnt/β-catenin signaling by enhancing Frizzled receptor expression. LDN-193189, a selective BMP receptor inhibitor, prolonged the overall survival of mice bearing IDH1-mutated intracranial xenografts by limiting BMP/catenin signaling. These findings demonstrate the pivotal role of BMP4 on tumor aggressiveness in IDH1Mut gliomas, suggesting a possible therapeutic strategy for this type of malignancy.

The Role of the BMP Pathway in Sustaining CML Stem Cells in the Bone Marrow Niche

Despite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) patients require life-long treatment due to leukemic stem cell (LSC) persistence. LSCs reside together with mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) in the bone marrow (BM) niche, which they modify to their advantage whilst impairing normal hematopoiesis. To date it has proved difficult to understand how LSCs both dominate and alter the niche, and to effectively target LSCs with current therapies. Recent studies have shown intrinsic and extrinsic deregulation of the bone morphogenetic protein (BMP) pathway in CML. These cells show altered stem cell fate, persistence, and response to BMP receptor (BMPR) antagonists, which affect cell behavior including cell cycle, apoptosis, and expansion (Laperrousaz et al. 2013; Grockowiak et al. 2017; Zylbersztejn et al. 2018; Toofan et al. 2018). We compared TKIs alone and in combination with a BMPR inhibitor to gain improved insights into BCR-ABL1 dependent and independent regulatory mechanisms within the niche environment using this therapeutic approach. K562 CML cell line and CML CD34+ primary cells were used in this study in combination with HS5 stromal cell co-culture. CML cells were treated with single or combination treatments of imatinib, the dual SRC-ABL1 TKI saracatinib, and the BMPR inhibitor dorsomorphin for 4h or 72h with and without BMP4 stimulation/co-culture. HSC and LSC interactions were also investigated using our artificial 3D BM niche model which comprises of magnetically levitated MSC spheroids embedded in medical-grade collagen type I, mimicking the BM biological and mechanical microenvironment, along with a high throughput microfluidic MSC spheroid formation system for drug testing. Kinase screens were performed on CML CD34+ cells using a chip-based microarray assay (PamGene) following 4h drug treatments. Phosphorylation data were then utilized for upstream kinase and pathway analysis using the metacore platform. This analysis facilitated identification of proteins showing a change in expression of ≥0.5-fold across all (n=3) patient samples, which was validated by qPCR and immunoblotting. Fluidigm multiplex qPCR was utilized to assess changes in expression of early response, self-renewal and differentiation genes. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays of primary CD34+CP-CML samples (n=3) following treatment. We demonstrated a synergistic mode of action upon inhibition of the BMP pathway in combination with TKI treatment, resulting in increased apoptosis (p< 0.001), altered cell cycle (G2-M, p< 0.01), fewer cell divisions, and a reduction in CD34+cells. Primary patient samples displayed differential gene expression in relation to response for genes involved in cell cycle (CDKN1A, CDKN2B, RB1; p< 0.05), self-renewal (PBX1; p< 0.01) and cell survival (GATA1, CKIT, p< 0.05). Kinase prediction analysis identified kinases involved in cell growth, development, differentiation, apoptosis, and cell-cell adhesion in treated sample lysates, with consequent pathway analysis highlighting transcription factors, ETS1, TP53 and C-MYC as main regulators across all common pathways. QPCR identified significant changes in expression of ETS1 (p< 0.001) and C-MYC (p< 0.01) following treatment. Within the top 10 deregulated pathways based on PamGene TK profiling we also identified GAB1 and GAB2 as common effectors, known to play an important role in growth and differentiation of myeloid cells, and which were demonstrated by immunoblotting to be considerably downregulated following 72h single and dual treatments in K562. Co-culture of CD34+ cells on stroma was chemo-protective, however dual treatment was still able to elicit strong anti-proliferative effects. We are currently investigating these promising findings in more detail using our 3D niche model and microfluidic spheroid platform. Taken together, these results provide vital insights into the mechanisms by which CML cells respond to current treatments, which is critical for improving therapeutic approaches and avoiding patient resistance or relapse. A combinatorial approach targeting the BMP pathway with BMP antagonists or small molecule inhibitors together with second-generation TKIs could open up new therapeutic possibilities. Disclosures No relevant conflicts of interest to declare.

Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling.

Parkinson’s disease is characterized by the intracellular accumulation of α-synuclein which has been linked to early dopaminergic axonal degeneration. Identifying druggable targets that can promote axonal growth in cells overexpressing α-synuclein is important in order to develop strategies for early intervention. Class-IIa histone deacetylases (HDACs) have previously emerged as druggable targets, however, it is not known which specific class-IIa HDACs should be targeted to promote neurite growth in dopaminergic neurons. To provide insight into this, we used gene co-expression analysis to identify which, if any, of the class-IIa HDACs had a positive correlation with markers of dopaminergic neurons in the human substantia nigra. This revealed that two histone deacetylases, HDAC5 and HDAC9, are co-expressed with TH, GIRK2 and ALDH1A1 in the human SN. We further found that HDAC5 and HDAC9 are expressed in dopaminergic neurons in the adult mouse substantia nigra. We show that siRNAs targeting HDAC5 or HDAC9 can promote neurite growth in SH-SY5Y cells, and that their pharmacological inhibition, using the drug MC1568, promoted neurite growth in cultured rat dopaminergic neurons. Moreover, MC1568 treatment upregulated the expression of the neurotrophic factor, BMP2, and its downstream transcription factor, SMAD1. In addition, MC1568 or siRNAs targeting HDAC5 or HDAC9 led to an increase in Smad-dependent GFP expression in a reporter assay. Furthermore, MC1568 treatment of cultured rat dopaminergic neurons increased cellular levels of phosphorylated Smad1, which was prevented by the BMP receptor inhibitor, dorsomorphin. Dorsomorphin treatment prevented the neurite growth-promoting effects of siRNAs targeting HDAC5, as did overexpression of dominant-negative Smad4 or of the inhibitory Smad7, demonstrating a functional link to BMP signaling. Supplementation with BMP2 prevented the neurite growth-inhibitory effects of nuclear-restricted HDAC5. Finally, we report that siRNAs targeting HDAC5 or HDAC9 promoted neurite growth in cells overexpressing wild-type or A53T-α-synuclein and that MC1568 protected cultured rat dopaminergic neurons against the neurotoxin, MPP+. These findings establish HDAC5 and HDAC9 as novel regulators of BMP-Smad signaling, that additionally may be therapeutic targets worthy of further exploration in iPSC-derived human DA neurons and in vivo models of Parkinson’s disease.

Developing a Novel and Convenient Model for Investigating Sweat Gland Morphogenesis from Epidermal Stem Cells.

Sweat glands developed from the embryonic epidermis. To elucidate the underlying mechanisms of morphogenesis, a reliable in vitro test system for bioactive screening must be developed. Here, we described a novel and convenient model by coculturing embryonic tissue and epidermal stem cells (ESCs) using Transwell insert for evaluating the effects of soluble morphogens on sweat gland morphogenesis in vitro. Using this coculture system, morphological alteration, histological features, and specific markers were observed. Initial experiments revealed that ESCs cocultured with embryonic paw pad (EPP) tissue demonstrated glandular structure and cytokeratin 8 (K8) and cytokeratin 18 (K18) positive, while ESCs cocultured with embryonic dorsal skin demonstrated “sea snail” structure and K8, K18 negative. Moreover, bone morphogenetic protein 4 (BMP4) and epidermal growth factor (EGF) concentrations were detected in the medium of the EPP group. BMP receptor inhibitor could effectively block the ESC differentiation to sweat glands, while EGF receptor blocker did not show the effect. Our results showed clear benefits of this novel and convenient model in terms of in vitro-in vivo correlation. It was an appropriate alternative for screening of potential bioactives regulating the sweat gland morphogenesis mechanism.

Exaggerated BMP4 signalling alters human airway basal progenitor celldifferentiation to cigarette smoking-related phenotypes.

Airway remodelling in chronic obstructive pulmonary disease (COPD) originates, in part, from smoking-induced changes in airway basal stem/progenitor cells (BCs). Based on the knowledge that bone morphogenetic protein 4 (BMP4) influences epithelial progenitor function in the developing and adult mouse lung, we hypothesised that BMP4 signalling may regulate the biology of adult human airway BCs relevant to COPD.BMP4 signalling components in human airway epithelium were analysed at the mRNA and protein levels, and the differentiation of BCs was assessed using the BC expansion and air-liquid interface models in the absence/presence of BMP4, BMP receptor inhibitor and/or small interfering RNAs against BMP receptors and downstream signalling.The data demonstrate that in cigarette smokers, BMP4 is upregulated in ciliated and intermediate undifferentiated cells, and expression of the BMP4 receptor BMPR1A is enriched in BCs. BMP4 induced BCs to acquire a smoking-related abnormal phenotype in vitro mediated by BMPR1A/Smad signalling, characterised by decreased capacity to differentiate into normal mucociliary epithelium, while generating squamous metaplasia.Exaggerated BMP4 signalling promotes cigarette smoking-relevant airway epithelial remodelling by inducing abnormal phenotypes in human airway BCs. Targeting of BMP4 signalling in airway BCs may represent a novel target to prevent/treat COPD-associated airway disease.

Double-Humanized Mouse Model to Study Bone Morphogenetic Protein (BMP) Signaling in Tumor Xenografts.

The activation of the bone morphogenic protein (BMP) signaling pathway in cancer cells has been shown to enhance migration and tumor angiogenesis and promote survival. The BMP signaling pathway regulates benign cells in the tumor microenvironment and is a known regulator of immune cells. The development of BMP receptor inhibitors has allowed the study of tumor xenografts in mice. We describe a double-humanized mouse model with adoptively transferred human immune and human tumor cells that can be used to assess the effects of BMP inhibitors on these human cells in vivo.

BMP-2 induces human mononuclear cell chemotaxis and adhesion and modulates monocyte-to-macrophage differentiation.

Type 2 diabetes mellitus (T2DM) is a cardiovascular risk factor which leads to atherosclerosis, an inflammatory disease characterized by the infiltration of mononuclear cells in the vessel. Bone morphogenetic protein (BMP)‐2 is a cytokine which has been recently shown to be elevated in atherosclerosis and T2DM and to contribute to vascular inflammation. However, the role of BMP‐2 in the regulation of mononuclear cell function remains to be established. Herein, we demonstrate that BMP‐2 induced human monocyte chemotaxis via phosphoinositide 3 kinase and mitogen‐activated protein kinases. Inhibition of endogenous BMP‐2 signalling, by Noggin or a BMP receptor inhibitor, interfered with monocyte migration. Although BMP‐2 expression was increased in monocytes from T2DM patients, it could still stimulate their migration. Furthermore, BMP‐2 interfered with their differentiation into M2 macrophages. Finally, BMP‐2 both induced the adhesion of monocytes to fibronectin and endothelial cells (ECs), and promoted the adhesive properties of ECs, by increasing expression of adhesion and pro‐inflammatory molecules. Our data demonstrate that BMP‐2 could exert its pro‐inflammatory effects by inducing monocyte migration and adhesiveness to ECs and by interfering with the monocyte differentiation into M2 macrophages. Our findings provide novel insights into the mechanisms by which BMP‐2 may contribute to the development of atherosclerosis.

Abstract LB-189: Novel bone morphogenetic protein receptor inhibitor JL5 suppresses tumor cell survival signaling and induces regression of human lung cancer

Abstract Purpose: Studies have shown that bone morphogenetic protein (BMP) signaling is aberrantly expressed in lung and other carcinoma leading to pro-oncogenic effects on tumor growth. The BMP receptor inhibitor DMH2 induces death of lung cancer cells through the downregulation of anti-apoptotic proteins XIAP, TAK1, and Id1-Id3. Since DMH2 does not downregulate BMP signaling in vivo because of metabolic instability and poor pharmacokinetics better BMP inhibitors are needed. Experimental Design: Here, we identified a site of metabolic instability of DMH2 and designed a novel BMP receptor inhibitor, JL5. We examined the effects of JL5 to downregulate BMP signaling and induce cell death of lung cancer cells in vitro and in vivo. We also queried the The Cancer Genome Atlas (TCGA) to assess if genetic alterations in lung cancer would affect drug targetability of the BMP receptors. Results: We show that JL5 has improved pharmacokinetic profile compared to DMH2. JL5 suppresses BMP signaling in lung cancer cells in vitro and in tumor xenografts. Moreover, we demonstrate JL5-induced tumor cell death and tumor regression in xenograft mouse models without immune cells and humanized with adoptively transferred human immune cells. In humanized mice, JL5 additionally induces the infiltration of immune cells within the tumor microenvironment. The TCBA database analysis suggests that genetic alterations in the BMP signaling cascade will not limit targeting with small molecule inhibitors. Conclusion: Our studies show that the BMP signaling pathway is targetable and BMP receptor inhibitors should be developed as a therapeutic to treat lung and other cancer patients. Citation Format: Jenna H. Newman, Rachel E. NeMoyer, David Augeri, Jyoti Malhotra, Elaine Langenfeld, Charles B. Chesson, Micheal J. Lee, Saeed Tarabichi, Sachin R. Jhawar, Praveen K. Bommareddy, Sh'rae Marshall, Evita T. Sadimin, John E. Kerrigan, Michael Goedken, Christine Minerowicz, Salma K. Jabbour, Shengguo Li, Natalie Dobias, Mary O. Carayannopolous, Andrew Zloza, John Langenfeld. Novel bone morphogenetic protein receptor inhibitor JL5 suppresses tumor cell survival signaling and induces regression of human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-189.

TGF-β and BMP signals regulate insect diapause through Smad1-POU-TFAM pathway

The transforming growth factor-β (TGF-β) superfamily signaling pathway contains two general branches, known as TGF-β and bone morphogenetic protein (BMP), that regulate development in animals. It is well known that TGF-β superfamily signaling participates in the regulation of dauer (lifespan extension) in Caenorhabditis elegans, but little is known about the molecular mechanisms of lifespan extension in the pathway. Diapause, a programmed developmental arrest in insects, is similar to dauer in C. elegans. In this study, we find that TGF-β superfamily signaling regulates Helicoverpa armigera diapause via a novel mechanism. Both TGF-β and BMP signals are weaker in the brains of diapause-destined pupae than in nondiapause-destined pupae, and the levels of p-Smad1, POU, TFAM, and mitochondrial activity are decreased in diapause pupae. Development in nondiapause pupae is delayed by an injection of TGF-β or BMP receptor inhibitors. Both TGF-β and BMP signals can activate a common target, Smad1. ChIP and EMSA assays indicate that Smad1 can bind to the POU promoter to regulate its expression. POU can improve the transcription of TFAM, which regulates mitochondrial activity. This is the first report showing that both TGF-β and BMP signals regulate development or diapause through the Smad1-POU-TFAM-mitochondrial activity in insects.

Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors

Human induced pluripotent stem cells (hiPSCs) are a potential source for cell therapy of Duchenne muscular dystrophy. To reliably obtain skeletal muscle progenitors from hiPSCs, we treated hiPS cells with a Wnt activator, CHIR-99021 and a BMP receptor inhibitor, LDN-193189, and then induced skeletal muscle cells using a previously reported sphere-based culture. This protocol greatly improved sphere formation efficiency and stably induced the differentiation of myogenic cells from hiPS cells generated from both healthy donors and a patient with congenital myasthenic syndrome. hiPSC-derived myogenic progenitors were enriched in the CD57(−) CD108(−) CD271(+) ERBB3(+) cell fraction, and their differentiation was greatly promoted by TGF-β inhibitors. TGF-β inhibitors down-regulated the NFIX transcription factor, and NFIX short hairpin RNA (shRNA) improved the differentiation of iPS cell-derived myogenic progenitors. These results suggest that NFIX inhibited differentiation of myogenic progenitors. hiPSC-derived myogenic cells differentiated into myofibers in muscles of NSG-mdx4Cv mice after direct transplantation. Our results indicate that our new muscle induction protocol is useful for cell therapy of muscular dystrophies.

Novel bone morphogenetic protein receptor inhibitor JL5 suppresses tumor cell survival signaling and induces regression of human lung cancer.

BMP receptor inhibitors induce death of cancer cells through the downregulation of antiapoptotic proteins XIAP, pTAK1, and Id1-Id3. However, the current most potent BMP receptor inhibitor, DMH2, does not downregulate BMP signaling in vivo because of metabolic instability and poor pharmacokinetics. Here we identified the site of metabolic instability of DMH2 and designed a novel BMP receptor inhibitor, JL5. We show that JL5 has a greater volume of distribution and suppresses the expression of Id1 and pTak1 in tumor xenografts. Moreover, we demonstrate JL5-induced tumor cell death and tumor regression in xenograft mouse models without immune cells and humanized with adoptively transferred human immune cells. In humanized mice, JL5 additionally induces the infiltration of immune cells within the tumor microenvironment. Our studies show that the BMP signaling pathway is targetable in vivo and BMP receptor inhibitors can be developed as a therapeutic to treat cancer patients.