Methotrexate inhibits BMP4 and abrogates the hypertrophic chondrocyte phenotype of synovial fibroblasts in juvenile idiopathic arthritis.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) induces growth disturbances in affected joints. Fibroblast-like synoviocytes (FLS) play a crucial role in JIA pathogenesis. FLS overexpress bone morphogenetic protein 4 (BMP4) and have a chondrocyte-like phenotype. FLS contribute directly to joint growth disturbances through endochondral bone formation. We investigated the ability of methotrexate to inhibit BMP4 expression and alter the hypertrophic chondrocyte-like phenotype of JIA FLS. METHODS: We selected primary cells from three subjects with persistent oligoarticular JIA, three subjects who eventually extended to a polyarticular disease course, which we termed extended-to-be (ETB), and three subjects who had polyarticular arthritis at time of diagnosis. We treated cells with methotrexate and two BMP4 inhibitors: noggin and chordin. We measured protein concentration from three chondrocyte cell markers: collagen II, aggrecan, and collagen X as well as BMP4. RESULTS: ColX, marker of chondrocyte hypertrophy, was significantly increased in polyarticular FLS when compared to both persistent FLS and ETB FLS, making polyarticular FLS the most like hypertrophic chondrocytes. Methotrexate caused significant decreases in BMP4 and ColX expression in persistent, ETB, and polyarticular FLS when compared to respective untreated cells. Ligand-binding BMP4 antagonists, noggin and chordin, caused significant decreases in ColX expression in FLS from all three disease courses and significant increases in collagen II protein, an early chondrocyte marker, when compared to respective untreated cells. CONCLUSIONS: Methotrexate, the first-line therapy in the treatment of JIA, mimics BMP4 antagonists by effectively lowering BMP4 and ColX expression in FLS. Inhibiting FLS from undergoing hypertrophy could prevent these cells from contributing to joint growth disturbances via endochondral bone formation.

The Role of Bone Morphogenetic Protein 4 in Lung Diseases.

Bone morphogenetic protein 4 (BMP4) is a multifunctional secretory protein that belongs to the transforming growth factor ß superfamily. BMPs transduce their signaling to the cytoplasm by binding to membrane receptors of the serine/threonine kinase family, including BMP type I and type II receptors. BMP4 participates in various biological processes, such as embryonic development, epithelial-mesenchymal transition, and maintenance of tissue homeostasis. The interaction between BMP4 and the corresponding endogenous antagonists plays a key role in the precise regulation of BMP4 signaling. In this paper, we review the pathogenesis of BMP4-related lung diseases and the foundation on which BMP4 endogenous antagonists have been developed as potential targets.

BMP-4 impedes endothelial cell migration in neointimal hyperplasia via FoXO-3 specific modulation of reactive oxygen species.

BACKGROUND AND AIMS: Endothelial cell injury causes vascular barrier dysfunction and leukocyte recruitment to the underlying tissue. Bone morphogenetic protein 4 (BMP-4) is a transforming growth factor that exerts pro-inflammatory effects on the endothelium. Here, we investigated the effects of BMP-4 on endothelial cell (EC) migration following balloon injury in SD rats. METHODS: An intimal hyperplasia model was established using balloon injury. Hematoxylin-eosin staining (HE) and silver staining were used to detect the alteration of endothelial cells recovery after balloon injury. Serum BMP-4 levels were assessed by ELISA. Human umbilical vein endothelial cells (HUVECs) were cultured. MTT assay was used to measure cell viability. Protein expression was detected by Western blot. Intracellular reactive oxygen species (ROS) was detected by dichloro-dihydro-fluorescein diacetate (DCFH-DA). HUVECs migration was measured via transwell assay and scratch wound assay. RESULTS: The results indicated that BMP-4 inhibition significantly decreased total plasma activity of BMP-4 and reduced neointimal hyperplasia by stimulating endothelial cell migration, but did not affect the medial area following balloon injury. BMP-4 suppressed the formation of ROS via forkhead box O3 (FoXO-3)/superoxide dismutase 1 (SOD-1). In vitro, a high level of ROS induced by BMP-4 impeded HUVECs migration. CONCLUSIONS: The results suggest that BMP-4 inhibition is a potential means of preventing intimal hyperplasia formation after balloon injury.

Overexpression of BMP4 protects retinal ganglion cells in a mouse model of experimental glaucoma.

PURPOSE: Activation of bone morphogenetic protein (BMP) 4 signaling promotes the survival of retinal ganglion cell (RGC) after acute injury. Chordin-like 1 (CHRDL1) is an endogenous BMP antagonist. In this study, we researched whether CHRDL1 was involved in BMP4 signaling and regulation of RGC degeneration in a mouse model of glaucoma. METHODS: Magnetic microbeads were intracameral injected to induce experimental glaucoma in a mouse model. A recombinant adeno-associated virus (rAAV) system was designed for overexpression of BMP4 or CHRDL1 in mouse retina. Immunohistochemistry and hematoxylin-eosin (HE) stains were performed to identify changes in retinal morphology. Electroretinogram (ERG) recordings were used to assess changes in visual function. RESULTS: The mRNA expression levels of Bmp4 and its downstream BMPRIa, small mothers against decapentaplegic 1 (Smad1), were significantly upregulated in retinas with glaucoma. RGC survival was significantly enhanced in the beads + AAV-BMP4 group and significantly reduced in the beads + AAV-CHRDL1 group, compared with the beads + AAV-EGFP group. Similar results were observed in retinal explant culture in vitro. Consistent with these findings, the photopic negative response (PhNR)responses in ERG, which indicate RGC function, were restored in mice overexpressing BMP4, whereas a-wave and b-wave responses were not. Activation of CHRLD1 inhibited Smad1/5/8 phosphorylation and exacerbated RGC damage. The expression of Glial fibrillary acidic protein (GFAP) was decreased significantly in beads + AAV-BMP4 group. CONCLUSIONS: BMP4 promoted RGC survival and visual function in an experimental glaucoma model. Activation of CHRDL1 exaggerated RGC degeneration by inhibiting the BMP4/Smad1/5/8 pathway. The mechanism of BMP4/Smad1/5/8 pathway may be related to the inhibition of glial cell activation. Our studies suggested that BMP4 and CHRLD1 might serve as therapeutic targets in glaucoma.

Conophylline Suppresses Angiotensin II-Induced Myocardial Fibrosis In Vitro via the BMP4/JNK Pathway.

We studied the effects and mechanisms of action of conophylline in different concentrations in the original in vitro model of myocardial fibrosis (treatment of cardiac fibroblasts isolated form the hearts of newborn rats with angiotensin II). Viability, collagen content, and expression of related protein in cardiac fibroblasts were assessed using the MTT-test, Sircol assay, and Western blotting, respectively. Conophylline markedly protected the cultured cells against the development of angiotensin II-induced fibrosis, which was seen from reduced viability of fibroblasts, decreased collagen content, and down-regulation of the expression of α-smooth muscle actin (α-SMA). Conophylline did not affect the TGF-ß pathway altered by angiotensin II, but markedly decreased the level of bone morphogenetic protein-4 (BMP4) enhanced by angiotensin II and BMP4 itself. Conophylline produced no effect on phosphorylation of α-SMA and Smad homologue-1/5/8, the classic BMP4 downstream pathway elements, but reduced the level of c-Jun N-terminal kinase (JNK) elevated by BMP4. Conophylline did not inhibit the development of myocardial fibrosis in the presence of JNK activator anisomycin. Thus, conophylline inhibited angiotensin II-provoked myocardial fibrosis via the BMP4/JNK pathway.

Specific Blockade of Bone Morphogenetic Protein-2/4 Induces Oligodendrogenesis and Remyelination in Demyelinating Disorders.

Oligodendrocyte precursor cells (OPCs) are present in demyelinated lesions of multiple sclerosis (MS) patients. However, their differentiation into functional oligodendrocytes is insufficient, and most lesions evolve into nonfunctional astroglial scars. Blockade of bone morphogenetic protein (BMP) signaling induces differentiation of OPCs into myelin-producing oligodendrocytes. We studied the effect of specific blockade of BMP-2/4 signaling, by intravenous (IV) treatment with anti-BMP-2/4 neutralizing mAb in both the inflammatory model of relapsing experimental autoimmune encephalomyelitis (R-EAE) and the cuprizone-toxic model of demyelination in mice. Administration of anti-BMP-2/4 to R-EAE-induced mice, on day 9 post-immunization (p.i.), ameliorated R-EAE signs, diminished the expression of phospho-SMAD1/5/8, primarily within the astrocytic lineage, increased the numbers of de novo immature and mature oligodendrocytes, and reduced the numbers of newly generated astrocytes within the spinal cord as early as day 18 p.i. This effect was accompanied with elevated remyelination, manifested by increased density of remyelinating axons (0.8 < g-ratios < 1), and reduced fully demyelinated and demyelinating axons, in the anti-BMP-2/4-treated R-EAE mice, studied by electron microscopy. No significant immunosuppressive effect was observed in the CNS and in the periphery, during the peak of the first attack, or at the end of the experiment. Moreover, IV treatment with anti-BMP-2/4 mAb in the cuprizone-challenged mice augmented the numbers of mature oligodendrocytes and remyelination in the corpus callosum during the recovery phase of the disease. Based on our findings, the specific blockade of BMP-2/4 has a therapeutic potential in demyelinating disorders such as MS, by inducing early oligodendrogenesis-mediated remyelination in the affected tissue.

The culture microenvironment of juvenile idiopathic arthritis synovial fibroblasts is favorable for endochondral bone formation through BMP4 and repressed by chondrocytes.

BACKGROUND: We examined influences of conditioned media from chondrocytes (Ch) on juvenile idiopathic arthritis synovial fibroblasts (JFLS) and potential for JFLS to undergo endochondral bone formation (EBF). METHODS: Primary cells from three control fibroblast-like synoviocytes (CFLS) and three JFLS were cultured in Ch-conditioned media and compared with untreated fibroblast-like synoviocytes (FLS). RNA was analyzed by ClariomS microarray. FLS cells cultured in conditioned media were exposed to either TGFBR1 inhibitor LY3200882 or exogenous BMP4 and compared with FLS cultured in conditioned media from Ch (JFLS-Ch). Media supernatants were analyzed by ELISA. RESULTS: In culture, JFLS downregulate BMP2 and its receptor BMPR1a while upregulating BMP antagonists (NOG and CHRD) and express genes (MMP9, PCNA, MMP12) and proteins (COL2, COLX, COMP) associated with chondrocytes. Important TGFß superfamily member gene expression (TGFBI, MMP9, COL1A1, SOX6, and MMP2) is downregulated when JFLS are cultured in Ch-conditioned media. COL2, COLX and COMP protein expression decreases in JFLS-Ch. BMP antagonist protein (NOG, CHRD, GREM, and FST) secretion is significantly increased in JFLS-Ch. Protein phosphorylation increases in JFLS-Ch exposed to exogenous BMP4, and chondrocyte-like phenotype is restored in BMP4 presence, evidenced by increased secretion of COL2 and COLX. Inhibition of TGFBR1 in JFLS-Ch results in overexpression of COL2. CONCLUSIONS: JFLS are chondrocyte-like, and Ch-conditioned media can abrogate this phenotype. The addition of exogenous BMP4 causes JFLS-Ch to restore this chondrocyte-like phenotype, suggesting that JFLS create a microenvironment favorable for endochondral bone formation, thereby contributing to joint growth disturbances in juvenile idiopathic arthritis.

Duloxetine suppresses BMP-4-induced release of osteoprotegerin via inhibition of the SMAD signaling pathway in osteoblasts.

Duloxetine, a selective serotonin-norepinephrine reuptake inhibitor, is currently recommended for the treatment of chronic painful disorders such as fibromyalgia, chronic musculoskeletal pain, and diabetic peripheral neuropathy. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates osteoprotegerin (OPG) production in osteoblast-like MC3T3-E1 cells, and that p70 S6 kinase positively regulates OPG synthesis. The present study aimed to investigate the effect of duloxetine on BMP-4-stimulated OPG synthesis in these cells. Duloxetine dose-dependently suppressed OPG release stimulated by BMP-4. Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), reduced BMP-4-stimulated OPG release, whereas a selective and specific norepinephrine reuptake inhibitor, reboxetine, failed to affect OPG release. In addition, another SSRI sertraline also inhibited BMP-4-stimulated OPG release. On the other hand, siRNA of SMAD1 reduced the OPG release stimulated by BMP-4, indicating the involvement of the SMAD1/5/8 pathway in OPG release. Rapamycin inhibited BMP-4-stimulated p70 S6 kinase phosphorylation, and compound C suppressed the SMAD1/5/8 phosphorylation stimulated by BMP-4. Duloxetine did not affect BMP-4-induced phosphorylation of p70 S6 kinase but suppressed SMAD1/5/8 phosphorylation. Both fluvoxamine and sertraline also inhibited BMP-4-elicited phosphorylation of SMAD1/5/8. These results strongly suggest that duloxetine suppresses BMP-4-stimulated OPG release via inhibition of the Smad1/5/8 signaling pathway in osteoblasts.

RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR), currently considered as a neurovascular disease, has become the major cause of blindness. More and more scholars believe that DR is no longer just a kind of microvascular disease, but accompanied by retinal neurodegenerative changes. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs is a classic treatment for DR; however, anti-VEGF drugs can exacerbate fibrosis and eventually lead to retinal detachment. The aim of this study was to explore the pathogenesis of DR and identified new treatments that can provide dual-target intervention for angiogenesis and fibrosis. METHODS: We explored changes in gene expression in high glucose-induced vascular endothelial cells using RNA sequencing (RNA-seq) technology. We identified bone morphogenetic protein 4 (BMP4) and SMAD family member 9 (SMAD9) among 449 differentially expressed genes from RNA-seq data and confirmed the expression of these two genes in the blood of diabetes patients by RT-PCR and in streptozotocin-induced rat retinas by RT-PCR, immunofluorescence, and western blot. Moreover, considering that DR is a multifactorial and multicellular disease, we used hydrogen peroxide (H2O2), advanced glycation end products (AGEs), CoCl2, 4-hydroxynonenal (4-HNE), and hypoxia to induce three human retinal cell types (Müller, retinal pigment epithelium, and human retinal capillary endothelial cells) to simulate the pathogenesis of DR, and MTT experiment, scratch experiment, Transwell experiment, and lumen formation experiment were used to test whether the model was successfully established. Then, we verified the overexpression of these two genes in the cell models by RT-PCR, immunofluorescence, and western blot. We further tested the effects of BMP4 on retinal cells. We use BMP4 to stimulate retinal cells and observe the effect of BMP4 on retinal cells by MTT experiment, scratch experiment, and RT-PCR. RESULTS: The results demonstrated that BMP4 and SMAD9 were highly expressed in both in vivo and in vitro models, while BMP4 could significantly upregulate the expression of SMAD9 and promote the expression of VEGF and fibrosis factors. CONCLUSIONS: This study is the first to analyze the mechanism by which high glucose levels affect retinal vascular endothelial cells through RNA transcriptome sequencing and indicates that BMP4 may be a potential target for the dual-target treatment (anti-VEGF and anti-fibrosis) of DR. KEY MESSAGES: • High-glucose effect on vascular endothelial cell was analyzed by RNA-seq. • KEGG analysis revealed enrichment of TGF-beta signaling pathway. • SMAD9 and BMP4 expression was upregulated in all samples. • Dual-target therapy of PDR by antagonizing BMP4.

R-spondins are BMP receptor antagonists in Xenopus early embryonic development.

BMP signaling plays key roles in development, stem cells, adult tissue homeostasis, and disease. How BMP receptors are extracellularly modulated and in which physiological context, is therefore of prime importance. R-spondins (RSPOs) are a small family of secreted proteins that co-activate WNT signaling and function as potent stem cell effectors and oncogenes. Evidence is mounting that RSPOs act WNT-independently but how and in which physiological processes remains enigmatic. Here we show that RSPO2 and RSPO3 also act as BMP antagonists. RSPO2 is a high affinity ligand for the type I BMP receptor BMPR1A/ALK3, and it engages ZNRF3 to trigger internalization and degradation of BMPR1A. In early Xenopus embryos, Rspo2 is a negative feedback inhibitor in the BMP4 synexpression group and regulates dorsoventral axis formation. We conclude that R-spondins are bifunctional ligands, which activate WNT- and inhibit BMP signaling via ZNRF3, with implications for development and cancer.

miR-18a Inhibits BMP4 and HIF-1α Normalizing Brain Arteriovenous Malformations.

RATIONALE: Brain arteriovenous malformations (AVMs) are abnormal tangles of vessels where arteries and veins directly connect without intervening capillary nets, increasing the risk of intracerebral hemorrhage and stroke. Current treatments are highly invasive and often not feasible. Thus, effective noninvasive treatments are needed. We previously showed that AVM-brain endothelial cells (BECs) secreted higher VEGF (vascular endothelial growth factor) and lower TSP-1 (thrombospondin-1) levels than control BEC; and that microRNA-18a (miR-18a) normalized AVM-BEC function and phenotype, although its mechanism remained unclear. OBJECTIVE: To elucidate the mechanism of action and potential clinical application of miR-18a as an effective noninvasive treatment to selectively restore the phenotype and functionality of AVM vasculature. METHODS AND RESULTS: The molecular pathways affected by miR-18a in patient-derived BECs and AVM-BECs were determined by Western blot, RT-qPCR (quantitative reverse transcription polymerase chain reaction), ELISA, co-IP, immunostaining, knockdown and overexpression studies, flow cytometry, and luciferase reporter assays. miR-18a was shown to increase TSP-1 and decrease VEGF by reducing PAI-1 (plasminogen activator inhibitor-1/SERPINE1) levels. Furthermore, miR-18a decreased the expression of BMP4 (bone morphogenetic protein 4) and HIF-1α (hypoxia-inducible factor 1α), blocking the BMP4/ALK (activin-like kinase) 2/ALK1/ALK5 and Notch signaling pathways. As determined by Boyden chamber assays, miR-18a also reduced the abnormal AVM-BEC invasiveness, which correlated with a decrease in MMP2 (matrix metalloproteinase 2), MMP9, and ADAM10 (ADAM metallopeptidase domain 10) levels. In vivo pharmacokinetic studies showed that miR-18a reaches the brain following intravenous and intranasal administration. Intranasal co-delivery of miR-18a and NEO100, a good manufacturing practices-quality form of perillyl alcohol, improved the pharmacokinetic profile of miR-18a in the brain without affecting its pharmacological properties. Ultra-high-resolution computed tomography angiography and immunostaining studies in an Mgp-/- AVM mouse model showed that miR-18a decreased abnormal cerebral vasculature and restored the functionality of the bone marrow, lungs, spleen, and liver. CONCLUSIONS: miR-18a may have significant clinical value in preventing, reducing, and potentially reversing AVM.

Inhibition of the BMP Signaling Pathway Ameliorated Established Clinical Symptoms of Experimental Autoimmune Encephalomyelitis.

Bone morphogenetic proteins (BMPs) are secreted growth factors that belong to the transforming growth factor beta superfamily. BMPs have been implicated in physiological processes, but they are also involved in many pathological conditions. Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS); however, its etiology remains elusive. Some evidence points to BMPs as important players in the pathogenesis of inflammatory and autoimmune disorders. In the present work, we studied the expression of BMP2, BMP4, BMP5, BMP6, BMP7, BMP type II receptor, and noggin in the immune system during different phases of experimental autoimmune encephalomyelitis (EAE). Major changes in the expression of BMPs took place in the initial phases of EAE. Indeed, those changes mainly affected BMP6 (whose expression was abrogated), BMP2, and BMP7 (whose expression was increased). In addition, we showed that in vivo inhibition of the BMP signaling pathway with small molecules ameliorated the already established clinical symptoms of EAE, as well as the CNS histopathological features. At the immune level, we observed an expansion of plasmacytoid dendritic cells (pDCs) in mice treated with small molecules that inhibit the BMP signaling pathway. pDCs could play an important role in promoting the expansion of antigen-specific regulatory T cells. Altogether, our data suggest a role for BMPs in early immune events that take place in myelin oligodendrocyte glycoprotein (MOG)-induced EAE. In addition, the clinical outcome of the disease was improved when the BMP signaling pathway was inhibited in mice that presented established EAE symptoms.

Potential Role of Circulating Endoglin in Hypertension via the Upregulated Expression of BMP4.

Endoglin is a membrane glycoprotein primarily expressed by the vascular endothelium and involved in cardiovascular diseases. Upon the proteolytic processing of the membrane-bound protein, a circulating form of endoglin (soluble endoglin, sEng) can be released, and high levels of sEng have been observed in several endothelial-related pathological conditions, where it appears to contribute to endothelial dysfunction. Preeclampsia is a multisystem disorder of high prevalence in pregnant women characterized by the onset of high blood pressure and associated with increased levels of sEng. Although a pathogenic role for sEng involving hypertension has been reported in several animal models of preeclampsia, the exact molecular mechanisms implicated remain to be identified. To search for sEng-induced mediators of hypertension, we analyzed the protein secretome of human endothelial cells in the presence of sEng. We found that sEng induces the expression of BMP4 in endothelial cells, as evidenced by their proteomic signature, gene transcript levels, and BMP4 promoter activity. A mouse model of preeclampsia with high sEng plasma levels (sEng+) showed increased transcript levels of BMP4 in lungs, stomach, and duodenum, and increased circulating levels of BMP4, compared to those of control animals. In addition, after crossing female wild type with male sEng+ mice, hypertension appeared 18 days after mating, coinciding with the appearance of high plasma levels of BMP4. Also, serum levels of sEng and BMP4 were positively correlated in pregnant women with and without preeclampsia. Interestingly, sEng-induced arterial pressure elevation in sEng+ mice was abolished in the presence of the BMP4 inhibitor noggin, suggesting that BMP4 is a downstream mediator of sEng. These results provide a better understanding on the role of sEng in the physiopathology of preeclampsia and other cardiovascular diseases, where sEng levels are increased.

Inhibition of bone morphogenetic protein signaling reduces viability, growth and migratory potential of non-small cell lung carcinoma cells.

PURPOSE: BMP signaling has an oncogenic and tumor-suppressing activity in lung cancer that makes the prospective therapeutic utility of BMP signaling in lung cancer treatment complex. A more in-depth analysis of lung cancer subtypes is needed to identify BMP-related therapeutic targets. We sought to examine the influence of BMP signaling on the viability, growth and migration properties of the cell line LCLC-103H, which originates from a large cell lung carcinoma with giant cells and an extended aneuploidy. METHODS: We used BMP-4 and LDN-214117 as agonist/antagonist system for the BMP receptor type I signaling. Using flow cytometry, wound healing assay, trans-well assay and spheroid culture, we examined the influence of BMP signaling on cell viability, growth and migration. Molecular mechanisms underlying observed changes in cell migration were investigated via gene expression analysis of epithelial-mesenchymal transition (EMT) markers. RESULTS: BMP signaling inhibition resulted in LCLC-103H cell apoptosis and necrosis 72 h after LDN-214117 treatment. Cell growth and proliferation are markedly affected by BMP signaling inhibition. Chemotactic motility and migratory ability of LCLC-103H cells were clearly hampered by LDN-214117 treatment. Cell migration changes after BMP signaling inhibition were shown to be coupled with considerable down-regulation of transcription factors involved in EMT, especially Snail. CONCLUSIONS: BMP signaling inhibition in LCLC-103H cells leads to reduced growth and proliferation, hindered migration and accelerated cell death. The findings contribute to the pool of evidence on BMP signaling in lung cancer with a possibility of introducing BMP signaling inhibition as a novel therapeutic approach for the disease.

Inhibiting Bone Morphogenetic Protein 4 Type I Receptor Signaling Promotes Remyelination by Potentiating Oligodendrocyte Differentiation.

Blocking inhibitory factors within CNS demyelinating lesions is regarded as a promising strategy to promote remyelination. Bone morphogenetic protein 4 (BMP4) is an inhibitory factor present in demyelinating lesions. Noggin, an endogenous antagonist to BMP, has previously been shown to increase the number of oligodendrocytes and promote remyelination in vivo. However, it remains unclear how BMP4 signaling inhibits remyelination. Here we investigated the downstream signaling pathway that mediates the inhibitory effect that BMP4 exerts upon remyelination through pharmacological and transgenic approaches. Using the cuprizone mouse model of central demyelination, we demonstrate that selectively blocking BMP4 signaling via the pharmacological inhibitor LDN-193189 significantly promotes oligodendroglial differentiation and the extent of remyelination in vivo This was accompanied by the downregulation of transcriptional targets that suppress oligodendrocyte differentiation. Further, selective deletion of BMP receptor type IA (BMPRIA) within primary mouse oligodendrocyte progenitor cells (OPCs) significantly enhanced their differentiation and subsequent myelination in vitro Together, the results of this study identify that BMP4 signals via BMPRIA within OPCs to inhibit oligodendroglial differentiation and their capacity to myelinate axons, and suggest that blocking the BMP4/BMPRIA pathway in OPCs is a promising strategy to promote CNS remyelination.

Myelination in Multiple Sclerosis Lesions Is Associated with Regulation of Bone Morphogenetic Protein 4 and Its Antagonist Noggin.

Remyelination is a central aspect of new multiple sclerosis (MS) therapies, in which one aims to alleviate disease symptoms by improving axonal protection. However, a central problem is mediators expressed in MS lesions that prevent effective remyelination. Bone morphogenetic protein4 (BMP4) inhibits the development of mature oligodendrocytes in cell culture and also blocks the expression of myelin proteins. Additionally, numerous studies have shown that Noggin (SYM1)-among other physiological antagonists of BMP4-plays a prominent role in myelin formation in the developing but also the adult central nervous system. Nonetheless, neither BMP4 nor Noggin have been systematically studied in human MS lesions. In this study, we demonstrated by transcript analysis and immunohistochemistry that BMP4 is expressed by astrocytes and microglia/macrophages in association with inflammatory infiltrates in MS lesions, and that astrocytes also express BMP4 in chronic inactive lesions that failed to remyelinate. Furthermore, the demonstration of an increased expression of Noggin in so-called shadow plaques (i.e., remyelinated lesions with thinner myelin sheaths) in comparison to chronically inactive demyelinated lesions implies that antagonizing BMP4 is associated with successful remyelination in MS plaques in humans. However, although BMP4 is strongly overexpressed in inflammatory lesion areas, its levels are also elevated in remyelinated lesion areas, which raises the possibility that BMP4 signaling itself may be required for remyelination. Therefore, remyelination might be influenced by a small number of key factors. Manipulating these molecules, i.e., BMP4 and Noggin, could be a promising therapeutic approach for effective remyelination.

Physical stimulation by REAC and BMP4/WNT-1 inhibitor synergistically enhance cardiogenic commitment in iPSCs.

It is currently known that pluripotent stem cells can be committed in vitro to the cardiac lineage by the modulation of specific signaling pathways, but it is also well known that, despite the significant increase in cardiomyocyte yield provided by the currently available conditioned media, the resulting cardiogenic commitment remains a highly variable process. Previous studies provided evidence that radio electric fields asymmetrically conveyed through the Radio Electric Asymmetric Conveyer (REAC) technology are able to commit R1 embryonic stem cells and human adipose derived stem cells toward a cardiac phenotype. The present study aimed at investigating whether the effect of physical stimulation by REAC in combination with specific chemical inductors enhance the cardiogenic potential in human induced pluripotent stem cells (iPSCs). The appearance of a cardiac-like phenotype in iPSCs cultured in the presence of a cardiogenic medium, based upon BMP4 and a WNT-inhibitor, was consistently increased by REAC treatment used only during the early fate differentiation for the first 72 hours. REAC-exposed iPSCs exhibited an upregulation in the expression of specific cardiogenic transcripts and morphologically in the number of beating clusters, as compared to cells cultured in the cardiogenic medium alone. Our results indicate that physical modulation of cellular dynamics provided by the REAC offers an affordable strategy to mimic iPSC cardiac-like fates in the presence of a cardiogenic milieu.

Liver X receptor activation reduces gastric cancer cell proliferation by suppressing Wnt signalling via LXRß relocalization.

Liver X receptors (LXRs) are involved in various diseases associated with lipid disorders, and in regulating cancer cell proliferation. However, the underlying molecular mechanisms, especially those in gastric cancer (GC) remain to be clarified. In this study, immunohistochemistry analysis revealed that LXRß was mainly expressed in GC tissue, with less expression in adjacent normal tissues. The LXRß agonist T0901317 efficiently suppressed the proliferation and colony formation of various GC cell lines. We further showed that LXRß translocated from the cytoplasm to the nucleus when activated by T0901317. LXRß nuclear localization suppressed the activation of Wnt signalling and decreased the expression of target genes such as MYC, BMP4, and MMP7 through binding to their promoters. Moreover, we demonstrated that the LXR agonist efficiently suppressed GC tumour growth in a nude mouse xenograft model. Taken together, these results revealed that LXRß agonist inhibited GC cells proliferation by suppressing Wnt signalling via LXRß relocalization. The results strongly suggest that LXRß could be a promising target in GC therapy.

E4BP4 promotes thyroid cancer proliferation by modulating iron homeostasis through repression of hepcidin.

Iron homeostasis is critical to mammals, and dysregulation in iron homeostasis usually leads to severe disorders including various cancers. Massive hepcidin secretion is an indicator of thyroid cancer, but the molecular mechanisms responsible for this dysregulation are unknown. Hepcidin secretion from thyroid cancer cells also leads to decreased expression of the iron exporter, ferroportin (FPN), and increased intracellular iron retention, which promote cancer proliferation. In this study, we examined the role of hepcidin in thyroid cancer (TC) and the molecular bases of its signaling. Synthesis of hepcidin is regulated by the BMP4/7 agonist SOSTDC1, which was downregulated in TC; SOSTDC1 downregulation was correlated with G9a-mediated hypermethylation in its promoter. The binding of G9a to the SOSTDC1 promoter requires E4BP4, which interacts with G9a to form a multi-molecular complex that contributes to SOSTDC1 silencing. Silencing of E4BP4 or G9a has similar effects to SOSTDC1 overexpression, which suppresses secretion of hepcidin and inhibits TC cell proliferation. Furthermore, our in vivo xenograft data indicated that depletion of E4BP4 also inhibits cancer growth, reduces hepcidin secretion, and reduces G9a nuclear transportation. Iron homeostasis and tumor growth in TC may be regulated by an E4BP4-dependent epigenetic mechanism. These findings suggest a new mechanism of cellular iron dysfunction through the E4BP4/G9a/SOSTDC1/hepcidin pathway, which is an essential link in TC.

Chronic myeloid leukaemia cells require the bone morphogenic protein pathway for cell cycle progression and self-renewal.

Leukaemic stem cell (LSC) persistence remains a major obstacle to curing chronic myeloid leukaemia (CML). The bone morphogenic protein (BMP) pathway is deregulated in CML, with altered expression and response to the BMP ligands shown to impact on LSC expansion and behaviour. In this study, we determined whether alterations in the BMP pathway gene signature had any predictive value for therapeutic response by profiling 60 CML samples at diagnosis from the UK SPIRIT2 trial and correlating the data to treatment response using the 18-month follow-up data. There was significant deregulation of several genes involved in the BMP pathway with ACV1C, INHBA, SMAD7, SNAIL1 and SMURF2 showing differential expression in relation to response. Therapeutic targeting of CML cells using BMP receptor inhibitors, in combination with tyrosine kinase inhibitor (TKI), indicate a synergistic mode of action. Furthermore, dual treatment resulted in altered cell cycle gene transcription and irreversible cell cycle arrest, along with increased apoptosis compared to single agents. Targeting CML CD34+ cells with BMP receptor inhibitors resulted in fewer cell divisions, reduced numbers of CD34+ cells and colony formation when compared to normal donor CD34+ cells, both in the presence and absence of BMP4. In an induced pluripotent stem cell (iPSC) model generated from CD34+ hematopoietic cells, we demonstrate altered cell cycle profiles and dynamics of ALK expression in CML-iPSCs in the presence and absence of BMP4 stimulation, when compared to normal iPSC. Moreover, dual targeting with TKI and BMP inhibitor prevented the self-renewal of CML-iPSC and increased meso-endodermal differentiation. These findings indicate that transformed stem cells may be more reliant on BMP signalling than normal stem cells. These changes offer a therapeutic window in CML, with intervention using BMP inhibitors in combination with TKI having the potential to target LSC self-renewal and improve long-term outcome for patients.