SMAD9-MYCN positive feedback loop represents a unique dependency for MYCN-amplified neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor occurring during childhood and high-risk NB patients have a poor prognosis. The amplified MYCN gene serves as an important determinant of a high risk of NB. METHODS: We performed an integrative screen using public NB tissue and cell line data, and identified that SMAD9 played an important role in high-risk NB. An investigation of the super-enhancers database (SEdb) and chromatin immunoprecipitation sequencing (ChIP-seq) dataset along with biological experiments of incorporating gene knockdown and CRISPR interference (CRISPRi) were performed to identify upstream regulatory mechanism of SMAD9. Gene knockdown and rescue, quantitative real-time PCR (Q-RT-PCR), cell titer Glo assays, colony formation assays, a subcutaneous xenograft model and immunohistochemistry were used to determine the functional role of SMAD9 in NB. An integrative analysis of ChIP-seq data with the validation of CRISPRi and dual-luciferase reporter assays and RNA sequencing (RNA-seq) data with Q-RT-PCR validation was conducted to analyze the downstream regulatory mechanism of SMAD9. RESULTS: High expression of SMAD9 was specifically induced by the transcription factors including MYCN, PHOX2B, GATA3 and HAND2 at the enhancer region. Genetic suppression of SMAD9 inhibited MYCN-amplified NB cell proliferation and tumorigenicity both in vitro and in vivo. Further studies revealed that SMAD9 bound to the MYCN promoter and transcriptionally regulate MYCN expression, with MYCN reciprocally binding to the SMAD9 enhancer and transactivating SMAD9, thus forming a positive feedback loop along with the MYCN-associated cancer cell cycle. CONCLUSION: This study delineates that SMAD9 forms a positive transcriptional feedback loop with MYCN and represents a unique tumor-dependency for MYCN-amplified neuroblastoma.

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/10T1/2 cells was observed in an in vivo 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.

Whole Exome Sequence Analysis Provides Novel Insights into the Genetic Framework of Childhood-Onset Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) describes a rare, progressive vascular disease caused by the obstruction of pulmonary arterioles, typically resulting in right heart failure. Whilst PAH most often manifests in adulthood, paediatric disease is considered to be a distinct entity with increased morbidity and often an unexplained resistance to current therapies. Recent genetic studies have substantially increased our understanding of PAH pathogenesis, providing opportunities for molecular diagnosis and presymptomatic genetic testing in families. However, the genetic architecture of childhood-onset PAH remains relatively poorly characterised. We sought to investigate a previously unsolved paediatric cohort (n = 18) using whole exome sequencing to improve the molecular diagnosis of childhood-onset PAH. Through a targeted investigation of 26 candidate genes, we applied a rigorous variant filtering methodology to enrich for rare, likely pathogenic variants. This analysis led to the detection of novel PAH risk alleles in five genes, including the first identification of a heterozygous ATP13A3 mutation in childhood-onset disease. In addition, we provide the first independent validation of BMP10 and PDGFD as genetic risk factors for PAH. These data provide a molecular diagnosis in 28% of paediatric cases, reflecting the increased genetic burden in childhood-onset disease and highlighting the importance of next-generation sequencing approaches to diagnostic surveillance.

Novel SAMD9 Mutation in a Patient With Immunodeficiency, Neutropenia, Impaired Anti-CMV Response, and Severe Gastrointestinal Involvement.

Mutations in the Sterile alpha motif domain containing 9 (SAMD9) gene have been described in patients with severe multisystem disorder, MIRAGE syndrome, but also in patients with bone marrow (BM) failure in the absence of other systemic symptoms. The role of hematopoietic stem cell transplantation (HSCT) in the management of the disease is still unclear. Here, we present a patient with a novel mutation in SAMD9 (c.2471 G>A, p.R824Q), manifesting with prominent gastrointestinal tract involvement and immunodeficiency, but without any sign of adrenal insufficiency typical for MIRAGE syndrome. He suffered from severe CMV (cytomegalovirus) infection at 3 months of age, with a delayed development of T lymphocyte functional response against CMV, profound T cell activation, significantly reduced B lymphocyte counts and impaired lymphocyte proliferative response. Cultured T cells displayed slightly lower calcium flux and decreased survival. At the age of 6 months, he developed severe neutropenia requiring G-CSF administration, and despite only mild morphological and immunophenotypical disturbances in the BM, 78% of the BM cells showed monosomy 7 at the age of 18 months. Surprisingly, T cell proliferation after CD3 stimulation and apoptosis of the cells normalized during the follow-up, possibly reflecting the gradual development of monosomy 7. Among other prominent symptoms, he had difficulty swallowing, requiring percutaneous endoscopic gastrostomy (PEG), frequent gastrointestinal infections, and perianal erosions. He suffered from repeated infections and periodic recurring fevers with the elevation of inflammatory markers. At 26 months of age, he underwent HSCT that significantly improved hematological and immunological laboratory parameters. Nevertheless, he continued to suffer from other conditions, and subsequently, he died at day 440 post-transplant due to sepsis. Pathogenicity of this novel SAMD9 mutation was confirmed experimentally. Expression of mutant SAMD9 caused a significant decrease in proliferation and increase in cell death of the transfected cells. Conclusion: We describe a novel SAMD9 mutation in a patient with prominent gastrointestinal and immunological symptoms but without adrenal hypoplasia. Thus, SAMD9 mutations should be considered as cause of enteropathy in pediatric patients. The insufficient therapeutic outcome of transplantation further questions the role of HSCT in the management of patients with SAMD9 mutations and multisystem involvement.

A potential role for SMAD9 in goose follicular selection through regulation of mRNA levels of luteinizing hormone receptor.

The egg production of poultry depends on follicular development and selection. Nonetheless, the mechanism underlying the priority of selecting of hierarchical follicles is completely unknown. SMAD9 is one of the important transcription factors in the BMP/SMAD pathway and is involved in goose follicular initiation. To identify its potential role in determination of the goose follicle hierarchy, we used BMP type I receptor inhibitor LDN-193189 both in vivo and in vitro and found that SMAD9 mRNA expression decreased in the presence of LDN-193189. While the level of SMAD9 mRNA decreased after treatment with LDN-193189, we found that the egg production (7.08 eggs per bird per year) of the animals increased, estradiol (E2) levels significantly increased, but the levels of progesterone (P4) remained unchanged. We also detected a significant increase in luteinizing hormone receptor (LHR) mRNA expression, but no change in follicle-stimulating hormone receptor (FSHR) mRNA amounts. The in vitro experimental results indicated that SMAD9 knockdown by RNA interference noticeably reduced E2 and P4 biosynthesis and FSHR and LHR mRNA expression in goose granulosa cells. Chromatin immunoprecipitation assay of goose granulosa cells revealed that phospho-SMAD9 bound to the LHR promoter and possibly regulated its transcriptional activity. These findings revealed that SMAD9 is differentially expressed in goose follicles, and acts as a key player in the control over goose follicular selection.

Bone-forming peptide-3 induces osteogenic differentiation of bone marrow stromal cells via regulation of the ERK1/2 and Smad1/5/8 pathways.

A bone-remodeling imbalance induced by increased bone resorption and osteoclast formation causes skeletal diseases such as osteoporosis. Induction of osteogenic differentiation of bone marrow stromal cells (BMSCs) leads to bone regeneration. Many researchers have tried to develop new adjuvants as specific stimulators of bone regeneration for therapeutic use in patients with bone resorption. We tried to develop a new adjuvant that has stronger osteogenic differentiation-promoting activity than bone morphogenetic proteins (BMPs). In this study, we identified a new peptide, which we called bone-forming peptide (BFP)-3, derived from the immature precursor of BMP-7. Upon osteogenic differentiation, BMSCs treated with BFP-3 exhibited higher alkaline phosphatase (ALP) activity and mineralization ability and significantly up-regulated expression of osteogenic genes such as ALP, osteocalcin (OC), Osterix, and Runx2 compared with control BMSCs. Furthermore, fluorescence-activated cell sorting (FACS) and immunofluorescence analyses demonstrated that BFP-3 treatment up-regulated CD44 expression. Interestingly, extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad1/5/8 phosphorylation was increased by BFP-3 treatment during osteogenic differentiation. Furthermore, BFP-3-induced osteogenic differentiation was significantly decreased by treatment with ERK1/2- and Smad-specific inhibitors. These results suggest that BFP-3 plays an important role in regulating osteogenic differentiation of BMSCs through increasing levels of osteogenic-inducing factors and regulating the ERK1/2 and Smad1/5/8 signaling pathways. Our finding indicates that BFP-3 may be a potential new therapeutic target for promoting bone formation.

The Immunomodulatory Role of BMP-2 on Macrophages to Accelerate Osteogenesis.

Bone morphogenetic protein-2 (BMP-2) is one of the most frequently used osteogenic factors for osteogenesis. Macrophages, acting as both immune cells and osteoclast precursors, have an indispensable functional input during multiple stages of bone healing. This study aims to investigate the immunoregulatory role of BMP-2 on macrophages and the subsequent effects on osteogenesis. In the subcutaneous implantation study, gelatin sponge incorporated with 20 µg/mL BMP-2 rendered significantly enhanced macrophage infiltration compared with the gelatin sponge control. Further in vitro study using murine macrophage cell line has shown that BMP-2 could function as a potent attractant for macrophage recruitment. The supplementation of BMP-2 dramatically diminished the expression of M1 phenotypic markers, including interleukin (IL)-1ß, IL-6, and iNOS in M1 polarized macrophages, indicating a positive immunoregulatory role of BMP-2 under inflammatory status. In addition, BMP-2 alone could robustly activate macrophages through pSmad1/5/8 signaling pathway and generate a positive feedback loop by increasing the expression of angiogenic factors. Conditioned medium collected from BMP2-stimulated macrophages accelerated the osteogenic differentiation of bone marrow stromal cells. Our finding suggests that BMP-2-induced osteogenesis may also involve the regulation of the local osteoimmune environment. The positive effect of BMP-2 on regulating immune response makes it possible to utilize the immunomodulatory properties of BMP-2 to manipulate the osteoimmune environment for favorable bone regeneration.

Identification of Biomarkers for Schistosoma-Associated Pulmonary Arterial Hypertension Based on RNA-Seq Data of Mouse Whole Lung Tissues.

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

Transforming growth factor-ß1 suppresses bone morphogenetic protein-2-induced mesenchymal-epithelial transition in HSC-4 human oral squamous cell carcinoma cells via Smad1/5/9 pathway suppression.

Squamous cell carcinoma is the most common cancer in the oral cavity. We previously demonstrated that transforming growth factor-ß1 (TGF-ß1) promotes the epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (hOSCC) cells; however, it remains to be clarified whether the TGF-ß superfamily member bone morphogenetic protein (BMP) affects this process in hOSCC cells. Here, we examined the independent and collective effects of TGF-ß1 and BMP-2 on EMT and mesenchymal­epithelial transition (MET) in a panel of four hOSCC cell lines. Notably, we found that HSC-4 cells were the most responsive to BMP-2 stimulation, which resulted in the upregulation of Smad1/5/9 target genes such as the MET inducers ID1 and cytokeratin 9 (CK9). Furthermore, BMP-2 downregulated the mesenchymal marker N-cadherin and the EMT inducer Snail, but upregulated epithelial CK9 expression, indicating that BMP-2 prefers to induce MET rather than EMT. Moreover, TGF-ß1 dampened BMP-2-induced epithelial gene expression by inhibiting Smad1/5/9 expression and phosphorylation. Functional analysis revealed that TGF-ß1 and BMP-2 significantly enhanced HSC-4 cell migration and proliferation, respectively. Collectively, these data suggest that TGF-ß positively regulates hOSCC invasion in the primary tumor, whereas BMP-2 facilitates cancer cell colonization at secondary metastatic sites. Thus, the invasive and metastatic characteristics of hOSCC appear to be reciprocally regulated by BMP and TGF-ß.

Differential expression of TGF-ß superfamily members and role of Smad1/5/9-signalling in chondral versus endochondral chondrocyte differentiation.

Proteins of the transforming-growth-factor-ß (TGF-ß)-superfamily have a remarkable ability to induce cartilage and bone and the crosstalk of TGF-ß - and BMP-signalling pathways appears crucial during chondrocyte development. Aim was to assess the regulation of TGF-ß-superfamily members and of Smad2/3- and Smad1/5/9-signalling during endochondral in vitro chondrogenesis of mesenchymal stromal cells (MSC) relative to chondral redifferentiation of articular chondrocytes (AC) to adjust chondrocyte development of MSC towards a less hypertrophic phenotype. While MSC increased BMP4 and BMP7 and reduced TGFBR2 and TGFBR3-expression during chondrogenesis, an opposite regulation was observed during AC-redifferentiation. Antagonists CHRD and CHL2 rose significantly only in AC-cultures. AC showed higher initial BMP4, pSmad1/5/9 and SOX9 protein levels, a faster (re-)differentiation but a similar decline of pSmad2/3- and pSmad1/5/9-signalling versus MSC-cultures. BMP-4/7-stimulation of MSC-pellets enhanced SOX9 and accelerated ALP-induction but did not shift differentiation towards osteogenesis. Inhibition of BMP-signalling by dorsomorphin significantly reduced SOX9, raised RUNX2, maintained collagen-type-II and collagen-type-X lower and kept ALP-activity at levels reached at initiation of treatment. Conclusively, ALK1,2,3,6-signalling was essential for MSC-chondrogenesis and its prochondrogenic rather than prohypertrophic role may explain why inhibition of canonical BMP-signalling could not uncouple cartilage matrix production from hypertrophy as this was achieved with pulsed PTHrP-application.

Smad8/9 Is Regulated Through the BMP Pathway.

Members of the transforming growth factor-ß (TGF-ß) family function through Smad-dependent and Smad-independent pathways. The Smad-dependent pathway is stimulated through the phosphorylation of receptor-regulated Smad (R-Smad) and inhibited through the dephosphorylation of R-Smad or the gene induction of inhibitory Smad (I-Smad). Little information is available on the regulation of R-Smad gene expression. BMP4 potentiated the up-regulation of Smad8/9 expression in C2C12, H9c2, 3T3-L1, HepG2, B16, and primary fibroblasts. BMP4-induced Smad8/9 expression was cycloheximide-insensitive and LDN-193189-sensitive, suggesting a direct event mediated through BMP type I receptors. BMP4 transcriptionally stimulated the Smad8/9 gene, and BMP-responsive elements (BREs) spanning nt -121 to nt -44 are involved in the up-regulation of Smad8/9 expression in response to BMP4. Phosphorylated Smad1/5/8/9 specifically bound to the BREs of Smad8/9 gene. The present study reveals that Smad8/9 is a unique R-Smad regulated through the BMP pathway at the mRNA level. J. Cell. Biochem. 117: 1788-1796, 2016. © 2016 Wiley Periodicals, Inc.

Discovery of a Small Molecule that Enhances Astrocytogenesis by Activation of STAT3, SMAD1/5/8, and ERK1/2 via Induction of Cytokines in Neural Stem Cells.

Identification of small molecules that direct neural stem cells (NSCs) into specific cell types would be helpful to understand the molecular mechanisms involved in regulation of NSC fate, and facilitate the development of therapeutic applications. In the current study, we developed and screened small molecules that can modulate the fate of NSCs that are derived from rat fetal cortex. Among these compounds, compounds 5 and 6 successfully differentiated NSCs into astrocytes and neurons, respectively. Compound 5 induced astrocytogenesis by increasing expression of interleukin-6, bone morphogenetic protein 2 and leukemia inhibitory factor and through consequent phosphorylation of signal transducer and activator of transcription 3 and Sma- and Mad-related protein 1/5/8 in NSCs. In addition, compound 5 increased the expression of fibroblast growth factor (FGF) 2 and FGF8 which may regulate the branching and morphology of astrocytes. Taken together, our results suggest that these small molecules can serve as a useful tool to study cell fate determination in NSCs and be used as an inexpensive alternative to cytokines to study mechanisms of astrocytogenesis.

Exome Sequencing Reveals Germline SMAD9 Mutation That Reduces Phosphatase and Tensin Homolog Expression and Is Associated With Hamartomatous Polyposis and Gastrointestinal Ganglioneuromas.

Hamartomatous polyposis syndromes (HPS) account for a small but appreciable proportion of inherited gastrointestinal cancer predisposition syndromes; patients with HPS have an increased risk for colon and extracolonic malignancies. We present a unique case of familial juvenile polyposis syndrome associated with gastrointestinal ganglioneuromas of unknown etiology. The patient was tested for HPS-associated genes, but no mutation was detected. Exome sequencing identified a germline heterozygous mutation in SMAD9 (SMAD9(V90M)). This mutation was predicted to be an activating mutation. HEK cells transfected to express SMAD9(V90M) had reduced expression of phosphatase and tensin homolog; this reduction was also observed in a polyp from the patient. We have therefore identified a new susceptibility locus for HPS. Patients with hamartomatous polyposis in the colon associated with ganglioneuromatosis should be referred for genetic assessments.

Gremlin1 preferentially binds to bone morphogenetic protein-2 (BMP-2) and BMP-4 over BMP-7.

Gremlin (Grem1) is a member of the DAN family of secreted bone morphogenetic protein (BMP) antagonists. Bone morphogenetic protein-7 (BMP-7) mediates protective effects during renal fibrosis associated with diabetes and other renal diseases. The pathogenic mechanism of Grem1 during diabetic nephropathy (DN) has been suggested to be binding and inhibition of BMP-7. However, the precise interactions between Grem1, BMP-7 and other BMPs have not been accurately defined. In the present study, we show the affinity of Grem1 for BMP-7 is lower than that of BMP-2 and BMP-4, using a combination of surface plasmon resonance and cell culture techniques. Using kidney proximal tubule cells and HEK (human embryonic kidney)-293 cell Smad1/5/8 phosphorylation and BMP-dependent gene expression as readouts, Grem1 consistently demonstrated a higher affinity for BMP-2>BMP-4>BMP-7. Cell-associated Grem1 did not inhibit BMP-2- or BMP-4-mediated signalling, suggesting that Grem1-BMP-2 binding occurred in solution, preventing BMP receptor activation. These data suggest that Grem1 preferentially binds to BMP-2 and this may be the dominant complex in a disease situation where levels of Grem1 and BMPs are elevated.

KMUP-1 Promotes Osteoblast Differentiation Through cAMP and cGMP Pathways and Signaling of BMP-2/Smad1/5/8 and Wnt/ß-Catenin.

Phosphodiesterase (PDE) inhibitors have been suggested as a possible candidate for the treatment of osteopenia, including osteoporosis. KMUP-1 is a novel xanthine derivative with inhibitory activities on the PDE 3, 4, and 5 iso-enzymes to suppress the degradation of cAMP and cGMP. This study aimed to investigate the effect of KMUP-1 on osteoblast differentiation and the underlying cellular and molecular mechanisms. Primary osteoblasts and osteoblastic MC3T3-E1 cells were examined. KMUP-1 enhanced alkaline phosphatase (ALP) activity and mineralization compared to untreated controls in primary osteoblasts and MC3T3-E1 cells. KMUP-1 also increased the mRNA expression of the osteoblastic differentiation markers, including collagen type 1a, ALP, osteocalcin, osteoprotegerin, BMP-2, and Runx2, a key transcription regulator for osteoblastic differentiation. The osteogenic effect of KMUP-1 was abolished by BMP signaling inhibitor, noggin. Furthermore, we found that KMUP-1 upregulated Smad1/5/8 phosphorylations with subsequent BRE-Luc activation confirmed by transient transfection assay. In addition, KMUP-1 inactivated glycogen synthase kinase-3ß (GSK-3ß), with associated nuclear translocation of ß-catenin. Co-treatment with H89 and KT5823, cAMP and cGMP pathway inhibitors, respectively, reversed the KMUP-1-induced activations of Smad1/5/8, ß-catenin, and Runx2. The findings demonstrate for the first time that KMUP-1 can promote osteoblast maturation and differentiation in vitro via BMP-2/Smad1/5/8 and Wnt/ß-catenin pathways. These effects are mediated, in part, by the cAMP and cGMP signaling. Thus, KMUP-1 may be a novel osteoblast activator and a potential new therapy for osteoporosis.

Colorectal cancer cell-derived microvesicles containing microRNA-1246 promote angiogenesis by activating Smad 1/5/8 signaling elicited by PML down-regulation in endothelial cells.

Emerging studies on circulating microRNAs (miRNAs) or microvesicles (MVs) have shown the potential of them to be novel biomarkers and therapeutic targets for cancer. However, the biological roles of these miRNAs and MVs have not been validated yet. To determine the biological significance of MVs, we used human colorectal cancer cells as the MV donor and endothelial cells (HUVECs) as the MV recipient and demonstrated the transfer of colorectal cancer cell-derived MVs (CRC-MVs) to HUVECs and evaluated the roles of these MVs and their cargo in tumor angiogenesis. Consequently, the incubation of HUVECs with CRC-MVs promoted the proliferation, migration, and tube formation activities of these cells. Among the cargoes shuttled by the MVs, miR-1246 and TGF-ß were considered to be responsible for the pro-angiogenic function of MVs by activating Smad 1/5/8 signaling in the HUVECs. These results suggest that colorectal cancer cells secreted MVs to contribute to tumor angiogenesis.

Identification and characterization of functional Smad8 and Smad4 homologues from Echinococcus granulosus.

Smad family proteins are essential cellular mediators of the transforming growth factor-ß superfamily. In the present study, we identified two members of the Smad proteins, Smad8 and Smad4 homologues (termed as EgSmadE and EgSmadD, respectively), from Echinococcus granulosus, the causative agent of cystic echinococcosis (CE). Phylogenetic analysis placed EgSmadE in the Smad1, 5, and 8 subgroup of the R-Smad sub-family and EgSmadD in the Co-Smad family. Furthermore, EgSmadE and EgSmadD attained a high homology to EmSmadE and EmSmadD of E. multilocularis, respectively. Both EgSmadE and EgSmadD were co-expressed in the larval stages and exhibited the highest transcript levels in activated protoscoleces, and their encoded proteins were co-localized in the sub-tegumental and tegumental layer of the parasite. As shown by yeast two-hybrid and pull-down analysis, EgSmadE displayed a positive binding interaction with EgSmadD. In addition, EgSmadE localized in the nuclei of Mv1Lu cells (mink lung epithelial cells) upon treatment with human TGF-ß1 or human BMP2, indicating that EgSmadE is capable of being translocated into nucleus, in vitro. Our study suggests that EgSmadE and EgSmadD may take part in critical biological processes, including echinococcal growth, development, and parasite-host interaction.

The action of JAK, SMAD and ERK signal pathways on hepcidin suppression by polysaccharides from Angelica sinensis in rats with iron deficiency anemia.

A crude polysaccharide was obtained by water extraction and ethanol precipitation from the root of Angelica sinensis (AS) to investigate its suppressive effect on hepcidin expression in rats with iron deficiency anemia (IDA). In this study, we showed that the Angelica sinensis polysaccharide (ASP) was mainly composed of arabinose, glucose and galactose in a molar ratio of 1 : 2.5 : 7.5, with the molecular weight of 72 900 Da and the sugar content of 95.1%. ASP markedly decreased hepcidin expression by suppressing the expression of JAK1/2, phospho-JAK1/2, phospho-SMAD1/5/8, phospho-ERK1/2, and promoting the expression of SMAD7 in the liver. These findings suggest that ASP can prevent the janus-kinase (JAK), son of mother against decapentaplegic (SMAD) and extracellular signal-regulated kinase (ERK) pathways to down-regulate hepcidin expression in IDA rats, and may be useful for the treatments of diseases induced by hepcidin over-expression.

MicroRNA expression signature for Satb2-induced osteogenic differentiation in bone marrow stromal cells.

Satb2 acts as a potent transcription factor to promote osteoblast differentiation and bone regeneration. Recently, microRNAs (miRNA) have been identified as critical regulators of osteogenic differentiation. This study aimed to identify specific miRNAs and their regulatory roles in the process of Satb2-induced osteogenic differentiation. We studied the differentially expressed miRNAs by Satb2 overexpression in murine bone marrow stromal cells using miRNA microarray. Ten down-regulated miRNAs including miR-27a, miR-125a-5p, and miR-466f-3p, and 18 up-regulated miRNAs including miR-17, miR-20a and miR-210 were found to be differentially expressed and their expression were verified by quantitative real time PCR. The differentially expressed miRNAs were further subjected to gene ontology and KEGG analysis. The highly enriched GOs and KEGG pathway showed target genes of these miRNAs were significantly involved in multiple biological processes (mesenchymal cell differentiation, bone formation, and skeletal development), and several osteogenic pathways (TGF-ß/BMP, MAPK, and Wnt signaling pathway). Finally, miR-27a was selected for target verification and function analysis. BMP2, BMPR1A, and Smad9, members of the TGF-ß/BMP superfamily, which were predicted to be target genes of miR-27a, were confirmed to be significantly up-regulated in Satb2-overexpressing cells by quantitative real time PCR. Overexpression of miR-27a significantly inhibited osteogenesis and repressed BMP2, BMPR1A, and Smad9 expression. In this study, we identified that a number of differentially regulated miRNAs, whose target genes involved in the TGF-ß/BMP signaling pathway, play an important role in the early stage of Satb2-induced osteogenic differentiation.