Bone Morphogenetic Protein Signaling Research Articles

KRAS mutation-induced EndMT of brain arteriovenous malformation is mediated through the TGF-β/BMP-SMAD4 pathway

ObjectiveSomatic KRAS mutations have been identified in the majority of brain arteriovenous malformations (bAVMs), and subsequent in vivo experiments have confirmed that KRAS mutation in endothelial cells (ECs) causes AVMs...

E3 ligase Cul2 mediates Drosophila early germ cell differentiation through targeting Bam

Drosophila ovary has been one of the most mature and excellent systems for studying the in vivo regulatory mechanisms of stem cell fate determination. It has been well-known that the bone morphogenetic protein (BMP) signaling released by the niche cells promotes the maintenance of germline stem cells (GSCs) through inhibiting the transcription of the bag-of-marbles (bam) gene, which encodes a key factor for GSC differentiation. However, whether Bam is regulated at the post-translational level remains largely unknown. Here we show that the E3 ligase Cullin-2 (Cul2) is involved in modulating Bam ubiquitination, which occurs probably at multiple lysine residues of Bam's C-terminal region. Genetic evidence further supports the notion that Cul2-mediated Bam ubiquitination and turnover are essential for GSC maintenance and proper germline development. Collectively, our data not only uncovers a novel regulatory mechanism by which Bam is controlled at the post-translational level, but also provides new insights into how Cullin family protein determines the differentiation fate of early germ cells.

183 Reduced FLG expression in atopic eczema reduces expression of key barrier genes and increases BMP signalling

Filaggrin (FLG) is a key cornified envelope and epidermal barrier protein. FLG mutations are the most strongly implicated genetic risk factor for atopic eczema (AE). FLG is part of a gene complex, called the epidermal differentiation complex (EDC), the genes of which contribute to various aspects of epidermal barrier function. FLG siRNA knockdown in keratinocytes caused a global reduction in EDC and Keratin (KRT) gene expression concomitant with up-regulation of bone morphogenetic protein (BMP) signalling.

Novel roles for RNA binding proteins squid, hephaesteus, and Hrb27C in Drosophila oogenesis.

Reproductive capacity in many organisms is maintained by germline stem cells (GSCs). A complex regulatory network influences stem cell fate, including intrinsic factors, local signals, and hormonal and nutritional cues. Posttranscriptional regulatory mechanisms ensure proper cell fate transitions, promoting germ cell differentiation to oocytes. As essential RNA binding proteins with constitutive functions in RNA metabolism, heterogeneous nuclear ribonucleoproteins (hnRNPs) have been implicated in GSC function and axis specification during oocyte development. HnRNPs support biogenesis, localization, maturation, and translation of nascent transcripts. Whether and individual hnRNPs specifically regulate GSC function has yet to be explored. We demonstrate that hnRNPs are expressed in distinct patterns in the Drosophila germarium. We show that three hnRNPs, squid, hephaestus, and Hrb27C are cell-autonomously required in GSCs for their maintenance. Although these hnRNPs do not impact adhesion of GSCs to adjacent cap cells, squid and hephaestus (but not Hrb27C) are necessary for proper bone morphogenetic protein signaling in GSCs. Moreover, Hrb27C promotes proper GSC proliferation, whereas hephaestus promotes cyst division. We find that hnRNPs are independently and intrinsically required in GSCs for their maintenance in adults. Our results support the model that hnRNPs play unique roles in stem cells essential for their self-renewal and proliferation.

DISC-0974, an Anti-Hemojuvelin (HJV) Monoclonal Antibody, Reduced Hepcidin and Improved Anemia in a Rat Model of Chronic Kidney Disease

Introduction DISC-0974 is a humanized monoclonal antibody against hemojuvelin (HJV) and is currently in Phase I clinical studies. HJV is a pathway-specific coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. Loss of function mutations of the HFE2 gene encoding HJV in humans cause profound reductions in hepcidin synthesis and severe iron overload. Consequently, it is hypothesized that pharmacological reduction of HJV activity will lead to reduction of hepcidin synthesis and increased iron availability. This hypothesis has been confirmed in studies of DISC-0974 in rodents and non-human primates and in healthy human volunteers. We have conducted studies to evaluate the effect of DISC-0974 in an animal model of CKD anemia. Anemia is a common complication in patients with CKD and has been associated with multiple adverse outcomes in this population. In CKD hepcidin is increased because of both reduced renal clearance and increased synthesis. This increase is believed to be a central contributor to the development of anemia by reducing the availability of iron from systemic iron stores and by reducing dietary iron absorption. Supporting this hypothesis, treatment with hepcidin lowering drugs has been demonstrated to increase serum iron and to increase hemoglobin (Sheetz et al, Br J Clin Pharmacol. 2019;85:935-948). We hypothesize that by downregulating hepcidin, DISC-0974 will have beneficial effects in treating anemia in the CKD patients because it will make iron available for improved erythropoiesis and increased hemoglobin synthesis as well as reducing erythropoietin resistance. Material and Methods The effect of DISC-0974 on improving anemia was evaluated in an adenine-induced rat model of CKD. In this study, renal impairment was induced by giving Sprague Dawley rats a diet containing 0.75% adenine for 3 weeks to induce kidney injury and then switched to a control diet for the remainder of the study (i.e., 3 more weeks). DISC-0974 (20 mg/kg) or vehicle (n-5/group) was administered intravenously once per week starting on Day 7 and continuing to Day 35. An additional group of rats (n=5/group) was fed with the control diet for the entire study and dosed intravenously with vehicle. Rats were euthanized on Day 42. Blood was collected on Days 0, 7, 21, 35, and 42 for evaluation of hematology parameters, as well as serum iron and hepcidin. Livers were also collected at study end for evaluation of hepcidin gene (HAMP) mRNA expression level. Results Consistent with a previous report (Sun et al, Nephrol Dial Transplant. 2013;28:1733-1743), the 0.75% adenine diet induced kidney dysfunction, as evidenced by marked increase in both serum creatinine and blood urea nitrogen. Rats on adenine diet developed reduced serum iron, reduced red cell production, and lowered hemoglobin with a hypochromic microcytic profile reflecting reduced iron availability. CKD rats appeared to tolerate DISC-0974 treatment well, as DISC-0974 @ 20mg/kg did not affect body weight. Consistent with the mechanism of action of blocking the formation of the BMP/BMPR/HJV complex, DISC-0974 reduced HAMP gene expression in the liver as measured at the end the study, which led to reduced serum hepcidin and increased serum iron concentration. Cellular hemoglobinization measured as reticulocyte Hemoglobin was increased. The maximum improvement in hemoglobin by DISC-0974 compared to the vehicle group was 17g/L in the rat CKD model. Conclusions We conclude that treatment with DISC-0974 at a dose that reduces hepcidin gene expression is able to increase iron availability and substantially prevents the reduction in hemoglobin that is seen in animals that develop adenine-induced renal impairment. This study supports the development of DISC-0974 for the treatment of patients with CKD anemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Activin-like Kinase 2 (ALK2/ACVR1) Is a Resistance Factor and Therapeutic Vulnerability to FLT3 Inhibition in Acute Myeloid Leukemia

Background: FMS-like tyrosine kinase-3 (FLT3), a transmembrane receptor, is frequently mutated in acute myeloid leukemia (AML), and this is associated with poor prognosis. Although FLT3 inhibitors (FLT3i) have significantly improved overall survival, their clinical effectiveness is limited due to treatment resistance, and the molecular mechanism remains unknown. We previously reported that the bone morphogenetic protein (BMP) signaling pathway supports AML cell growth via osteogenic differentiation in mesenchymal stromal cells. However, the role of BMP in FLT3i resistance has not been studied. We hypothesized that BMP type-1 receptor (ALK2/ACVR1) is a resistance factor for FLT3i, and therefore targeting FLT3 and ACVR1 using a novel dual inhibitor, TP-0184, would overcome FLT3i resistance and sensitize AML cells to standard therapy. Methods: We assessed the association of ACVR1 expression with molecular mutations in AML by analyzing gene expression datasets (OHSU and TCGA). We also measured ACVR1 expression using qRT-PCR in FLT3-mutant AML cell lines (molm-13, molm-14, and MV4-11) treated with different doses of FLT3i (gilteritinib or midostaurin), as well as in peripheral blood mononuclear cells isolated from patients with FLT3-mutant AML treated with FLT3i. To determine the role of ACVR1 in FLT3i resistance, we knocked down ACVR1 in FLT3-mutant cells, treated them with various doses of FLT3i, and measured apoptosis using IncuCyte. We also measured the effect of TP-0184 on cell proliferation and the cell cycle in FLT3-wild-type (WT) and FLT3-mutant cells. To determine the mechanism of TP-0184-mediated cell cycle arrest, we measured the effect of TP-0184 on cell signaling pathways using Western blot analysis and RNA sequencing. We also performed human RTK kinase binding assay to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. MTT assay was used to determine the synergistic effect of TP-0184 with cytarabine or venetoclax (BCL2 inhibitor). We investigated the effect of TP-0184 on AML growth in vivo using FLT3-mutant and FLT3-WT AML xenograft and patient-derived xenograft models. Results: Analysis of AML datasets showed that ACVR1 expression is upregulated in FLT3-mutant compared with FLT3-WT AML patient samples (p<0.01) and predicts poor overall survival (p=0.05). ACVR1 expression was also higher in FLT3-mutant than in FLT3-WT cell lines (p=0.039). Interestingly, ACVR1 expression was upregulated in patient samples of FLT3-mutant AML treated with gilteritinib (p=0.02) and FLT3-mutant cell lines treated with FLT3i in a dose- and time-dependent manner. Moreover, ACVR1 knockdown in FLT3-mutant cell lines sensitized AML cells to FLT3i 2- to 3-fold compared with scrambled control cells, suggesting that ACVR1 is a resistance factor in AML cells treated with FLT3i, and that targeting ACVR1 could sensitize AML cells to FLT3i. In FLT3-WT and FLT3-mutant AML cell lines treated with TP-0184, cell growth was significantly reduced in FLT3-mutant cells (IC50<25nM) but not in FLT3-WT cells. TP-0184 induced G1/G0 arrest in FLT3-mutant cells but had minimal or no effect in FLT3-WT cells, suggesting that TP-0184 overcomes ACVR1-mediated resistance in FLT3-mutant cells. Treatment with TP-0184 significantly inhibited multiple proteins downstream of FLT3, including STAT-5, ERK, PI3K, and mTOR pathways, and decreased in pSmad1/5 proteins downstream of ACVR1 in AML cells. These findings confirmed that TP-0184 inhibits both mutant FLT3 and ACVR1 in AML cells. Gene expression analysis revealed that TP-0184 significantly downregulated the serine biosynthesis pathway in FLT3-mutant cell lines. Moreover, molecular docking and kinase-binding studies confirmed that TP-0184 binds to FLT3 mutants with high affinity (KD=<5nM). Interestingly,TP-0184 with venetoclax or cytarabine showed a synergistic effect in FLT3-mutant cells (combination index 0.01; p<0.01). In vivo data also showed that TP-0184 inhibited AML growth and significantly prolonged survival in FLT3-mutant AML xenograft and patient-derived xenograft models (p<0.01). Conclusion: Our data indicate that ACVR1 causes resistance to FLT3 inhibitors. TP-0184, a dual inhibitor, targets mutant FLT3 and ACVR1 in AML cell lines. TP-0184 sensitizes AML cells with chemotherapy and venetoclax and inhibits AML growth in vivo.

Low intensity near-infrared light promotes bone regeneration via circadian clock protein cryptochrome 1

Bone regeneration remains a great clinical challenge. Low intensity near-infrared (NIR) light showed strong potential to promote tissue regeneration, offering a promising strategy for bone defect regeneration. However, the effect and underlying mechanism of NIR on bone regeneration remain unclear. We demonstrated that bone regeneration in the rat skull defect model was significantly accelerated with low-intensity NIR stimulation. In vitro studies showed that NIR stimulation could promote the osteoblast differentiation in bone mesenchymal stem cells (BMSCs) and MC3T3-E1 cells, which was associated with increased ubiquitination of the core circadian clock protein Cryptochrome 1 (CRY1) in the nucleus. We found that the reduction of CRY1 induced by NIR light activated the bone morphogenetic protein (BMP) signaling pathways, promoting SMAD1/5/9 phosphorylation and increasing the expression levels of Runx2 and Osterix. NIR light treatment may act through sodium voltage-gated channel Scn4a, which may be a potential responder of NIR light to accelerate bone regeneration. Together, these findings suggest that low-intensity NIR light may promote in situ bone regeneration in a CRY1-dependent manner, providing a novel, efficient and non-invasive strategy to promote bone regeneration for clinical bone defects.

Transmembrane anterior posterior transformation 1 regulates BMP signaling and modulates the protein stability of SMAD1/5

The bone morphogenetic protein (BMP) signaling pathway plays pivotal roles in various biological processes during embryogenesis and adult homeostasis. Transmembrane anterior posterior transformation 1 (TAPT1) is an evolutionarily conserved protein involved in murine axial skeletal patterning. Genetic defects in TAPT1 result in complex lethal osteochondrodysplasia. However, the specific cellular activity of TAPT1 is not clear. Herein, we report that TAPT1 inhibits BMP signaling and destabilizes the SMAD1/5 protein by facilitating its interaction with SMURF1 E3 ubiquitin ligase, which leads to SMAD1/5 proteasomal degradation. In addition, we found that the activation of BMP signaling facilitates the redistribution of TAPT1 and promotes its association with SMAD1. TAPT1-deficient murine C2C12 myoblasts or C3H/10T1/2 mesenchymal stem cells exhibit elevated SMAD1/5/9 protein levels, which amplifies BMP activation, in turn leading to a boost in the transdifferentiation or differentiation processing of these distinct TAPT1-deficient cell lines changing into mature osteoblasts. Furthermore, the enhancing effect of TAPT1 deficiency on osteogenic differentiation of C3H/10T1/2cells was observed in an invivo ectopic bone formation model. Importantly, a subset of TAPT1 mutations identified in humans with lethal skeletal dysplasia exhibited gain-of-function activity on SMAD1 protein levels. Thus, this finding elucidates the role of TAPT1 in the regulation of SMAD1/5 protein stability for controlling BMP signaling.

Aneurysmal Bone Cyst (ABC), A Rare Diagnosis in Oral Pathology Based on Our 15-Year Institutional Experience

Abstract Introduction/Objective Aneurysmal bone cyst (ABC) is a rare, benign bone lesion that was first described by Jaffe and Lichtenstein in 1942. Primary ABC usually affects vertebrae and metaphyseal regions of long bones, particularly femur and tibia. Only 2-3% of ABCs occur in the head and neck region, with the mandible and maxilla being the most common. It arises mostly in children and young adults, mean age 20 yo, with no gender predilection. ABC can manifest with pain, swelling, and pathological fractures. Radiographically, it appears as a metaphyseal expansile lesion that thins the cortex and has a sclerotic rim. Lesions containing “fluid-fluid” levels are best appreciated on MRI. Approximately 70% of primary ABCs have rearrangements of the USP6 gene. The most common translocation seen in primary ABCs is t(16;17) (q22;p13) leading to the fusion of the cadherin 11 gene with USP6 gene. The USP6 gene product inhibits the differentiation of osteoblasts and dysregulates bone morphogenetic protein signaling pathway. Methods/Case Report A retrospective chart review of all oral pathology cases referred to our institution between 1996 and 2022 was performed; three patients diagnosed with primary ABC were identified. Patient demographic data, clinical presentation, treatment, slides, and pathologic features were reviewed. Results (if a Case Study Enter NA) We report of three primary ABCs of the mandible and maxilla. The mean age was 39 yo (range, 26-59 years). The mandible was affected in two cases, and the maxilla involved in one. Female: Male ratio was 2:1. All lesions were primary ABC, with no other associated lesion - they showed pseudocystic structures with fibrous stroma and a variable numbers of multinucleated giant cells in relation to sinusoidal blood spaces, as well as irregular trabeculae of osteoid and reactive woven bone. Conclusion Primary ABC is a rare tumor in oral pathology. The importance of this study is to increase awareness of this neoplasm in the head and neck region, as well as delineate features that help differentiate ABCs from other fibro- osseous lesions of the mandible and maxilla.

Abstract 11735: Single-Nucleus RNA Sequencing Analysis Reveals Subpopulation of Endothelial Cells Activation in Hearts of Porcine Double Injury Model

Pigs underwent a double injury model: neonatal day 1 apical resection (AR P1 ) and postnatal day 28 LAD ligation (MI P28 ) can remuscularize the AMI by postnatal day 56 (P56) with little evidence of myocardial scarring, which is accompanied by increased cardiomyocytes (CMs) cell-cycle activity. The vascularization that occurred to support remuscularization has not been studied. Therefore, we analyze the single-nucleus RNA sequencing (snRNA-seq) data collected from the MI-border zone of AR P1 -MI P28 , MI P28 (without AR P1 ), naïve, and fetal pig endothelial cells (ECs) using the Artificial Intelligence autoencoder and cluster analyses. Three clusters of EC were identified. Among them, vascular and lymphatic EC clusters (EC1 and EC2) were presented in all postnatal hearts. Besides expressing vascular EC gene signatures, the remaining cluster, named ‘EC3’, also upregulated Transforming grow factor-beta (TGFβ) and Bone Morphogenetic Proteins (BMP) signaling markers. The signaling indicated EC3 was still under development. EC3 strongly presented in the fetal heart (31% of ECs); then, EC3 proportions decreased as the pig matured and virtually dismissed (&lt;2%) in the naïve heart from P28 to later. Up to 1-week following MI P28 injury, EC3 proportions remained low (&lt;2%) in MI P28 -only hearts; meanwhile, EC3 proportions significantly increased (&gt;7%) in the regenerative AR P1 -MI P28 hearts. Thus, in the AR P1 -MI P28 injured hearts, the subpopulation of EC3 is activated to support remuscularization. Figure. SnRNA-seq analysis identifies a cluster of under-development endothelial cells (EC3). A) UMAP: three EC clusters. B) UMAP: vascular EC marker PLVAP expressesed (calculated using Seurat) in EC1 and EC3. C) UMAP: lymphatic EC marker CCL21 expressesed in EC2. D) Heatmap: TGFβ and BMP signaling upregulated in EC3. C) Bargraph: proportions of EC3 in naïve and injured hearts on postnatal days P28, P30, and P35; P-value and Fold-change (FC) were calculated using Fisher’s exact test.

Abstract 9832: Liver-Heart Crosstalk Mediated by Coagulation Factor XI Protects Against Heart Failure

Introduction: Tissue-tissue crosstalk by endocrine factors is a vital mechanism to maintain proper physiologic homeostasis. The heart and the liver display multifaceted interactions and it is common to observe heart diseases affecting the liver and vice versa. Hypothesis: We hypothesized that the liver-heart interactions may be mediated by novel endocrine factors. Methods: Global transcriptomic data from the heart and the liver were generated across all 100 diverse inbred strains of mice, the Hybrid Mouse Diversity Panel (HMDP), and used to detect the correlation structure between secreted proteins from the liver and their downstream effects on the heart. Candidates were validated in a “two-hit” model of heart failure with preserved ejection fraction (HFpEF) and plasma samples from HFpEF patients and controls were used to examine the clinical relevance. Results: Using the bioinformatics framework, we predicted that coagulation factor XI (FXI) is a liver-derived protein that influences heart gene expression and diastolic dysfunction, a key trait of HFpEF. In a mouse model of HFpEF, mice overexpressing FXI in the liver showed improved diastolic function and reduced inflammation and fibrosis, whereas FXI knockout mice were sensitized for diastolic dysfunction. FXI overexpression activated the bone morphogenetic protein (BMP)-Smad1/5 pathway in the heart, thus inhibiting genes involved in inflammation and fibrosis. The action of FXI on heart requires proteolytic activity, as point mutations in its catalytic domain eliminated effects on BMP signaling and heart function. BMP7 is secreted as an inactive precursor and our results indicate that it is cleaved by FXI, releasing the active dimer and monomer growth factor from the prodomain that binds to the extracellular matrix. Further, plasma FXI was inversely correlated with E/e’ ratio in all human participants and HFpEF patients. Conclusions: Our studies indicate that liver-derived FXI specifically regulates cardiomyocytes through the BMP-Smad1/5 pathway, resulting in attenuation of fibrosis, inflammation, and diastolic dysfunction in the context of a HFpEF model. Our study reveals a protective role of FXI on heart injury, distinct from its role in coagulation.

Phenotypic Discovery of Triazolo[1,5-c]quinazolines as a First-In-Class Bone Morphogenetic Protein Amplifier Chemotype.

Phenotypic drug discovery (PDD) continues to fuel the research and development pipelines with first-in-class therapeutic modalities, but success rates critically depend on the quality of the underlying model system. Here, we employed a stem cell-based approach for the target-agnostic, yet pathway-centric discovery of small-molecule cytokine signaling activators to act as morphogens during development and regeneration. Unbiased screening identified triazolo[1,5-c]quinazolines as a new-in-class in vitro and in vivo active amplifier of the bone morphogenetic protein (BMP) pathway. Cellular BMP outputs were stimulated via enhanced and sustained availability of BMP-Smad proteins, strictly dependent on a minimal BMP input. Holistic target deconvolution unveiled a unique mechanism of dual targeting of casein kinase 1 and phosphatidyl inositol 3-kinase isoforms as key effectors for efficient amplification of osteogenic BMP signaling. This work underscores the asset of PDD to discover unrecognized polypharmacology signatures, in this case significantly expanding the chemical and druggable space of BMP modulators.

Iron metabolism disorder regulated by BMP signaling in hypoxic pulmonary hypertension

Backgrounds and aimsUnexplained iron deficiency is associated with poorer survival in patients with pulmonary hypertension (PH). Bone morphogenetic protein (BMP) signaling and BMP protein type II receptor (BMPR2) expression are important in the pathogenesis of PH. BMP6 in hepatocytes is a central transcriptional regulator of the iron hormone hepcidin that controls systemic iron balance. This study aimed to investigate the effects of BMP signaling on iron metabolism and its implication in hypoxia-induced PH. Methods and resultsPH was induced in Sprague–Dawley Rats under hypoxia for 4 weeks. Compared with the control group, right ventricular systolic pressure and right ventricle hypertrophy index were both markedly increased, and serum iron level was significantly decreased with iron metabolic disorder in the hypoxia group. In cultured human pulmonary artery endothelial cells (HPAECs), hypoxia increased oxidative stress and apoptosis, which were reversed by supplementation with Fe agent. Meanwhile, iron chelator deferoxamine triggered oxidative stress and apoptosis in HPAECs, and treatment with antioxidant alleviated iron-deficiency-induced apoptosis by reducing reactive oxygen species production. Expression of hepcidin, BMP6 and hypoxia-inducible factor (HIF)-1α were significantly upregulated, while expression of BMPR2 was downregulated in hepatocytes in the hypoxia group, both in vivo and in vitro. Expression of hepcidin and HIF-1α were significantly increased by BMP6, while pretreatment with siRNA-BMPR2 augmented the enhanced expression of hepcidin and HIF-1α induced by BMP6. ConclusionsIron deficiency promoted oxidative stress and apoptosis in HPAECs in hypoxia-induced PH, and enhanced expression of hepcidin regulated by BMP6/BMPR2 signaling may contribute to iron metabolic disorder.

Loss of bone morphogenetic protein signaling in fibroblasts results in CXCL12-driven serrated polyp development

Mutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The established role of stromal cells in regulating BMP activity in the intestine implies a role for stromal cells in polyp development. We used conditional Cre-LoxP mice to investigate how specific loss of BMPR1A in endothelial cells, fibroblasts, or myofibroblasts/smooth muscle cells affects intestinal homeostasis. Selective loss of BMPR1A in fibroblasts causes severe histological changes in the intestines with a significant increase in stromal cell content and epithelial cell hyperproliferation, leading to numerous serrated polyps. This phenotype suggests that crucial changes occur in the fibroblast secretome that influences polyp development. Analyses of publicly available RNA expression databases identified CXCL12 as a potential candidate. RNAscope in situ hybridization showed an evident increase of Cxcl12-expressing fibroblasts. In vitro, stimulation of fibroblasts with BMPs resulted in downregulation of CXCL12, while inhibition of the BMP pathway resulted in gradual upregulation of CXCL12 over time. Moreover, neutralization of CXCL12 in vivo in the fibroblast-specific BMPR1A KO mice resulted in a significant decrease in polyp formation. Finally, in CRC patient specimens, mRNA-expression data showed that patients with high GREMLIN1 and CXCL12 expression had a significantly poorer overall survival. Significantly higher GREMLIN1, NOGGIN, and CXCL12 expression were detected in the Consensus Molecular Subtype 4 (CMS4) colorectal cancers, which are thought to arise from serrated polyps. Taken together, these data imply that fibroblast-specific BMP signaling–CXCL12 interaction could have a role in the etiology of serrated polyp formation.

Brasenia-inspired hydrogel with sustained and sequential release of BMP and WNT activators for improved bone regeneration

Although bone morphogenetic protein (BMP) and WNT signaling play pivotal roles in bone development, homeostasis, and regeneration, the applications of proteins to stimulate corresponding signaling pathways showed limited outcomes in the repair and regeneration of bone defects that might be attributed to the reciprocal interventions of these pathways. In order to satisfy the combinational and sequential activation of BMP and WNT pathways, inspired by the heterogeneous hydrogel-liked structures of Brasenia, heterogeneous alginate/chitosan hydrogels were fabricated and spatially loaded with FK506 and BIO to achieve sustained and sequential release of the activators. Alkaline phosphatase staining, alizarin red staining and qRT-PCR results suggested that FK506 and BIO enhanced osteoblastic differentiation in vitro when used separately. Besides, by mixing and matching the activators and the hydrogel layers, a superior releasing mode that a combination of early FK506 release and following BIO release was identified via both in vitro and in vivo explorations for most efficient bone regeneration. These results suggested that drug-loaded heterogeneous hydrogels possess great potentials in treating bone loss defects for future clinical practice.

ODP335 Mutual Effects of Orexin and BMPs on Gonadotropin Expression by Mouse Gonadotrope LβT2 Cells

Abstract Orexins are neuropeptides that express primarily in the hypothalamus and are produced in two isoforms, orexin A and orexin B. There are two kinds of G protein coupled receptors, orexin type 1 (OX1R) and type 2 (OX2R) receptors. Orexin has been reported to have key roles on sleep-wake regulation and feeding behavior in the central nervous system, whereas its receptors are also expressed in peripheral tissues including the endocrine organs, and orexin affects the regulation of the endocrine system. In our previous experiments, we have revealed the impact of orexins on anterior pituitary functions. For instance, we reported that orexin A plays an inhibitory role in prolactin production through the suppression of endogenous bone morphogenetic protein (BMP) activity in rat pituitary lactotrope GH3 cells. It was also reported that orexin A enhances pro-opiomelanocortin (POMC) transcription by upregulating corticotropin-releasing hormone (CRH) receptor signaling and by downregulating BMP-Smad signaling in mouse corticotope AtT20 cells. However, the effects of orexin on the endocrine function regarding gonadotrope cells remain unclear. We have recently uncovered that core clock genes and BMPs have mutual effects on luteinizing hormone (LH) expression in a phase-dependent manner by mouse gonadotrope LβT2 cells. In the present study, we investigated the effect of orexin on pituitary gonadotropin expression using LβT2 cells, which express OX1R and OX2R, by focusing on the functional involvement of BMP system and clock genes. It was revealed that orexin A stimulation increased LHβ and FSHβ mRNA expression in a concentration-responsive manner in the absence of GnRH, and interestingly, GnRH co-treatment further upregulated LHβ mRNA expression in LβT2 cells. Regarding the interrelationships between the signalings of orexin and BMPs, it was also revealed that orexin A pretreatment enhanced the BMP receptor signaling detected as the Smad1/5/9 phosphorylation, indicating that orexin enables to upregulate the BMP actions in LβT2 cells. In our previous studies, we reported that several BMP ligands such as BMP-6, -7 and 15 expressed in LβT2 cells can promote gonadotropin transcription, in which BMP-6 regulates GnRH-stimulated LH expression by modulating the sensitivity to somatostatin analogs. Based on the present results, it was implied that endogenous orexin can be functionally involved in the underlying mechanisms of gonadotropin expression. Collectively, orexin enhances gonadotropin expression by regulating BMP signaling in gonadotrope cells. Here, we will also discuss functional involvement of clock genes in the regulatory system of gonadotropin secretion induced by orexin and BMPs. Presentation: No date and time listed

ODP407 Effects of Leptin and Ghrelin on Ovarian Steroidogenesis and Involvement of BMP Action in Rat Granulosa Cells.

Abstract Leptin and ghrelin are the two major hormones which regulate food intake and energy balance. They have also been reported to have pleiotropic functions, including the modulation of ovarian function via the hypothalamic-pituitary-ovarian system or in a direct manner to the ovaries. Blood leptin levels increase over the menstrual cycle and peak at the time of the luteinizing hormone (LH) surge. Leptin secreted from adipocytes acts on the hypothalamus and pituitary gland to stimulate the secretion of GnRH and gonadotropins. In the ovaries, high levels of leptin inhibit follicle-stimulating hormone (FSH)-induced steroid secretion and follicular development. Ghrelin, secreted by the stomach, has been reported to modulate GnRH secretion in the hypothalamus and to affect FSH and LH secretion, as well as steroidogenesis in the ovaries. Knockout of these hormones leads to impaired fertility. However, the mechanism of the direct effects of leptin and ghrelin on the ovaries has not been well elucidated. In the present study, we investigated the effects of leptin and ghrelin on the ovarian steroidogenesis using primary culture of rat granulosa cells, by focusing on the ovarian bone morphogenetic protein (BMP) system, which plays an important role in follicular development and steroidogenesis and acts as a luteinizing inhibitor. The results showed that treatment with leptin (1 to 300 ng/mL) did not affect either estradiol (E2) or progesterone (P4) production, but enhanced FSH-induced P4 production at low concentrations (10 ng/mL). On the other hand, treatment with ghrelin (1 to 300 nM) did not affect E2 and P4 production, but enhanced FSH-induced P4 production in a concentration-responsive manner. The mRNA levels of the follicular steroid synthases (StAR, P450scc, 3βHSD, 20αHSD) were examined. Leptin treatment enhanced 3βHSD mRNA levels. Both leptin treatment and ghrelin treatment decreased FSH-induced 20αHSD mRNA levels. However, both treatments decreased FSH-induced cAMP production. In addition, leptin treatment tended to suppress the transcriptional activity of Id-1 induced by BMP-6. Collectively, these results indicate that both leptin and ghrelin enhance FSH-induced P4 production by suppressing BMP signaling in rat granulosa cells. It was thus suggested that leptin and ghrelin which regulate food intake and energy balance can also act as endogenous regulators for the modulation of P4 synthesis and luteinizing process by ovarian follicles. Presentation: No date and time listed

Glioma stem cells and neural stem cells respond differently to BMP4 signaling

Malignant glioma is a highly heterogeneous and invasive primary brain tumor characterized by high recurrence rates, resistance to combined therapy, and dismal prognosis. Glioma stem cells (GSCs) are likely responsible for tumor progression, resistance to therapy, recurrence, and poor prognosis owing to their high self-renewal and tumorigenic potential. As a family member of BMP signaling, bone morphogenetic protein4 (BMP4) has been reported to induce the differentiation of GSCs and neural stem cells (NSCs). However, the molecular mechanisms underlying the BMP4-mediated effects in these two cell types are unclear. In this study, we treated hGSCs and hNSCs with BMP4 and compared the phenotypic and transcriptional changes between these two cell types. Phenotypically, we found that the growth of hGSCs was greatly inhibited by BMP4, but the same treatment only increased the cell size of hNSCs. While the RNA sequencing results showed that BMP4 treatment evoked significantly transcriptional changes in both hGSCs and hNSCs, the profiles of differentially expressed genes were distinct between the two groups. A gene set that specifically targeted the proliferation and differentiation of hGSCs but not hNSCs was enriched and then validated in hGSC culture. Our results suggested that hGSCs and hNSCs responded differently to BMP4 stimulation. Understanding and investigating different responses between hGSCs and hNSCs will benefit finding partner factors working together with BMP4 to further suppress GSCs proliferation and stemness without disturbing NSCs.

Mechanical strain drives exosome production, function, and miRNA cargo in C2C12 muscle progenitor cells.

Mesenchymal stem cells (MSCs) have been proven to promote tissue repair. However, concerns related to their clinical application and regulatory hurdles remain. Recent data has demonstrated the proregenerative secretome of MSCs can result in similar effects in the absence of the cells themselves. Within the secretome, exosomes have emerged as a promising regenerative component. Exosomes, which are nanosized lipid vesicles secreted by cells, encapsulate micro-RNA(miRNA), RNA, and proteins that drive MSCs regenerative potential with cell specific content. As such, there is an opportunity to optimize the regenerative potential of MSCs, and thus their secreted exosome fraction, to improve clinical efficacy. Exercise is one factor that has been shown to improve muscle progenitor cell function and regenerative potential. However, the effect of exercise on MSC exosome content and function is still unclear. To address this, we used an in vitro culture system to evaluate the effects of mechanical strain, an exercise mimetic, on C2C12 (muscle progenitor cell) exosome production and proregenerative function. Our results indicate that the total exosome production is increased by mechanical strain and can be regulated with different tensile loading regimens. Furthermore, we found that exosomes from mechanically stimulated cells increase proliferation and myogenic differentiation of naïve C2C12 cells. Lastly, we show that exosomal miRNA cargo is differentially expressed following strain. Gene ontology mapping suggests positive regulation of bone morphogenetic protein signaling, regulation of actin-filament-based processes, and muscle cell apoptosis may be at least partially responsible for the proregenerative effects of exosomes from mechanically stimulated C2C12 muscle progenitor cells.

Multifaceted Functions of TWSG1: From Embryogenesis to Cancer Development.

Bone morphogenetic proteins (BMPs) play an important role in development. Twisted gastrulation BMP signaling modulator 1 (TWSG1) was initially identified as a regulator of the dorsoventral axis formation in Drosophila. The mechanism of BMP signaling modulation by TWSG1 is complex. TWSG1 inhibits BMP signaling by binding to BMP ligands including BMP4, whereas it enhances signaling by interacting with Chordin, a BMP antagonist. Therefore, TWSG1 can act as both a BMP agonist and antagonist. TWSG1 has various functions ranging from embryogenesis to cancer progression. TWSG1 knockout mice showed neural, craniofacial, and mammary defects. TWSG1 also regulated erythropoiesis and thymocyte development. Furthermore, the relationship between TWSG1 and cancer has been elucidated. Allelic loss of TWSG1 was detected in colorectal cancer. TWSG1 expression was upregulated in papillary thyroid carcinoma and glioblastoma but downregulated in gastric and endometrial cancers. TWSG1 suppressed BMP7-enhanced sphere formation and migration in endometrial cancer cells, indicating its tumor-suppressive role. Further studies are required to clarify the TWSG1 function and its association with BMP signaling in cancer development. Finally, TWSG1 is abundantly expressed in human and mouse ovaries and sustains follicular growth in rodent ovaries. Thus, TWSG1 has various functions ranging from fertility to cancer. Therefore, TWSG1 signaling modulation may be beneficial in treating specific diseases such as cancer.