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Abstract Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck squamous carcinoma that is notorious for its high metastatic potential. In this study, we reported that FOXA1 protein was decreased in NPC cells. Loss of FOXA1 is associated with lymph node metastasis and poor prognosis. Silencing FOXA1 in NP69 and C666-1 NPC cells accelerated cell proliferation and migration, while re-expression of FOXA1 has opposite effects. Microarray and RNA-seq analysis revealed that re-expression of FOXA1 in NPC cells reprogrammed the TGF-β-stimulated transcription program, which is characterized by promotion of TGF-β-inducible tumor-suppressive targets but repression of TGF-β-inducible oncogenes expression in NPC cells, leading to restoration of NPC cell sensitivity to TGF-β′s growth-inhibitory effect. BAMBI, a TGF-β responsive tumor suppressor, was induced by FOXA1 in NPC cells. FOXA1 binding on the BAMBI gene facilitated SMAD2/3 binding to the BAMBI promoter via increasing BAMBI associated H3K4me1 and H3K27ac modification. Enforced expression of BAMBI in NPC cells suppressed cell proliferation and invasiveness. Our data suggested that FOXA1 is a master factor in controlling the TGF-β-stimulated transcriptome and a regulator of TGF-β biological functions in NPC oncogenesis. Highlights • We reported the first time that loss of FOXA1 predicts unfavorable prognosis in NPC. • FOXA1 is a bona fide tumor suppressor gene in NPC development. • FOXA1 reprograms TGF-β-stimulated transcription from a metastasis promotive to a growth suppressive program. • FOXA1 cooperates with TGF-β activated SMAD complex to upregulate BAMBI expression in NPC cells. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Liuweiwuling (LWWL) tablets contain a six-herb Chinese formula and are commonly prescribed to facilitate nourishment of the liver and kidneys, clear away toxic materials and activate blood circulation. Administration of LWWL is well known for its protective effects on the liver and its capacity to confer long-term stability in patients exhibiting reduced transaminase levels. Clinical studies have reported that LWWL can also be used for the treatment of liver fibrosis with associated treatment regimens resulting in a concomitant reduction in transforming growth factor β1 (TGF-β1) levels in the serum of patients with hepatic fibrosis. TGF-β1 plays a prominent role in stimulating liver fibrogenesis and this effect is mediated by myofibroblasts (MFB) derived from hepatic stellate cells (HSCs). It is likely that this phenomenon underpins the antifibrotic effects associated with LWWL. Aim The purpose of this study was to investigate the antifibrotic effects and mechanisms pertaining to LWWL. Methods Hepatic fibrosis was induced in rats following bile duct ligation (BDL). Rats that underwent BDL received daily gavage administration of colchicine (0.2 mg/kg per day), LWWL (0.4, 1.6, 6.4 g/kg per day) or PBS (for the control group). Pathological changes in hepatic tissue were examined using hematoxylin and eosin (HE) and sirius red staining. Immunohistochemical analysis was performed to monitor α-SMA and type I collagen (Collagen I) protein expression. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot analyses were used to monitor the expression of genes and proteins in the TGF-β/Smad signaling pathway, including TGF-β1, bone morphogenic protein and activin membrane-bound inhibitor (Bambi), Smad3, phosphorylated Smad3 (p-Smad3) and Smad7. We also monitored the expression of genes and proteins in the nuclear factor-κB (NF-κB) signaling pathway, including NF-κB p65, IκBα and phosphorylation of IκBα (p-IκBα), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1β (IL-1β). Results LWWL dose-dependently inhibited BDL-induced liver injury and hepatic fibrosis in rats. Furthermore, LWWL reduced liver tissue collagen deposition, hydroxyproline content, liver dysfunction and α-SMA expression in BDL-induced hepatic fibrosis rats. Moreover, LWWL markedly prevented activation of the TGF-β/Smad signaling pathway by inhibiting expression of Smad2/3 and phosphorylation of Smad3, and upregulating the expression of Bambi and Smad7. In addition, LWWL regulated the expression of the inflammatory cytokines IL-1β, TNF-α and IL-6 by inhibiting the activation of NF-κB p65 and the phosphorylation of IκBα, and increasing the expression of IκBα. Conclusions LWWL can attenuate BDL-induced hepatic fibrosis in rats, and this effect may be due to modulation of the NF-κB-dependent inflammatory response and activation of HSC and TGF-β/Smad-mediated synthesis and degradation of Collagen I. [ABSTRACT FROM AUTHOR]

Liver fibrosis is characterized by proliferation and activation of hepatic stellate cells (HSCs). Transforming growth factor-β1 (TGFβ1) is crucial for liver fibrogenesis, and gut-derived endotoxin (LPS) also plays an important role in liver fibrogenesis. In the present study, we found that microRNA-146a-5p (miR-146a-5p) could regulate TGFβ1/Smad and LPS/NF-κB/Bambi pathways to attenuate liver fibrosis. Downregulated miR-146a-5p and upregulated level of LPS were found in liver of CCl4-treated rats. On cellular level, expression of miR-146a-5p is reduced during primary rat HSCs naturally activation and changed in response to TGFβ1 and/or LPS stimulation in primary rat HSCs and human HSC line LX-2. Further overexpression of miR-146a-5p suppresses proliferation and activation of HSCs. The underlying mechanism involved that miR-146a-5p directly suppresses profibrogenic effects of TGFβ1 by down-regulating the expression of Smad4 and phosphorylation of Smad2. Moreover, miR-146a-5p indirectly suppresses TGFβ1/Smad pathway by targeting IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor associated factor-6 (TRAF6), two major components of LPS/NF-κB/Bambi pathway, to reduce inhibition of TGFβ pseudoreceptor Bambi. These results indicate that miR-146a-5p abrogate hepatic fibrosis by suppressing both TGFβ/Smad and LPS/NF-κB/Bambi signaling pathway in HSCs and suggest that miR-146a-5p is a potential therapeutic target for liver fibrosis. [ABSTRACT FROM AUTHOR]

Transforming growth factor β (TGF-β), acting as the auto/para endocrine factors, has multi-function in mammalian follicle development. Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is considered as a pseudoreceptor in the TGF-β signal pathway which has the similar extracellular structure of TGF-β receptor but lack of intracellular serine/threonine kinase domains. However, the biological function of BAMBI involved in porcine granulosa cell steroidogenesis remains unknown. This study was thus carried out to explore the effect of BAMBI on the steroidogenesis process in porcine primary granulosa cells. Our results showed overexpression of BAMBI promoted aromatase and StAR, but not P450scc and 3β-HSD mRNA and protein expression levels in porcine primary granulosa cells, and increased the accumulation of estradiol and progesterone in the culture medium. Meanwhile, knockdown endogenous BAMBI decreased the mRNA expression levels of Cyp19a1 and Star and the accumulation levels of estradiol and progesterone. TGF-β1 could decrease Cyp19a1 and Star mRNA expression and estradiol and progesterone production in a dose-dependent manner. Pre-treatment with BAMBI adenovirus reversed TGFβ1-induced downregulation of Cyp19a1 and Star mRNA expression. Moreover, TGF-β1 could induce the phosphorylation of SMAD3 in porcine granulosa cells. Pre-transfected with BAMBI adenovirus also inhibited TGF-β1-induced downregulation of estradiol and progesterone production as well as TGF-β1-induced phosphorylation of SMAD3 in porcine granulosa cells. These findings provided a potential mechanism by which BAMBI could regulate porcine granulosa cell steroidogenesis. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a transforming growth factor β (TGF-β) type I receptor antagonist that negatively regulates TGF-β and bone morphogenetic protein (BMP) signaling. BAMBI has been shown to be regulated by TGF-β signaling; however, whether BAMBI can be regulated by BMP signaling remains to be determined. The aim of this study was to investigate the effect of BMP2 on the regulation of BAMBI expression in human granulosa-lutein cells and the underlying mechanisms. Both primary and immortalized human granulosa-lutein cells were used as research models. Using dual inhibition approaches, our results showed that BMP2 activated SMAD1/5/8 phosphorylation and up-regulated BAMBI mRNA levels, which was reversed by the BMP type I receptor inhibitors, DMH-1 and dorsomorphin, but not by SB431542 (activin/TGF-β type I receptor inhibitor). Moreover, the combined knockdown of SMAD1 and SMAD5 completely abolished the BMP2-induced up-regulation of BAMBI. Similarly, knockdown of SMAD4 reversed the BMP2-induced up-regulation of BAMBI. Pre-treatment with BMP2 inhibited the TGF-β1-induced phosphorylation of SMAD2/3 and up-regulation of MMP2, and these inhibitory effects were reversed by knockdown of endogenous BAMBI. Our findings indicate that BAMBI is a BMP-responsive gene and that BAMBI participates in the negative feedback regulation of TGF-β signaling in the human ovary. [ABSTRACT FROM AUTHOR]

Background & Aims Hepatocytes in which the hepatitis B virus (HBV) is replicating exhibit loss of the chromatin modifying polycomb repressive complex 2 (PRC2), resulting in re-expression of specific, cellular PRC2-repressed genes. Epithelial cell adhesion molecule ( EpCAM ) is a PRC2-repressed gene, normally expressed in hepatic progenitors, but re-expressed in hepatic cancer stem cells (hCSCs). Herein, we investigated the functional significance of EpCAM re-expression in HBV-mediated hepatocarcinogenesis. Methods Employing molecular approaches (transfections, fluorescence-activated cell sorting, immunoblotting, qRT-PCR), we investigated the role of EpCAM-regulated intramembrane proteolysis (RIP) in HBV replicating cells in vitro , and in liver tumors from HBV X/c-myc mice and chronically HBV infected patients. Results EpCAM undergoes RIP in HBV replicating cells, activating canonical Wnt signaling. Transfection of Wnt-responsive plasmid expressing green fluorescent protein (GFP) identified a GFP + population of HBV replicating cells. These GFP + /Wnt + cells exhibited cisplatin- and sorafenib-resistant growth resembling hCSCs, and increased expression of pluripotency genes NANOG , OCT4 , SOX2 , and hCSC markers BAMBI , CD44 and CD133 . These genes are referred as EpCAM RIP and Wnt-induced hCSC-like gene signature. Interestingly, this gene signature is also overexpressed in liver tumors of X/c-myc bitransgenic mice. Clinically, a group of HBV-associated hepatocellular carcinomas was identified, exhibiting elevated expression of the hCSC-like gene signature and associated with reduced overall survival post-surgical resection. Conclusions The hCSC-like gene signature offers promise as prognostic tool for classifying subtypes of HBV-induced HCCs. Since EpCAM RIP and Wnt signaling drive expression of this hCSC-like signature, inhibition of these pathways can be explored as therapeutic strategy for this subtype of HBV-associated HCCs. Lay summary In this study, we provide evidence for a molecular mechanism by which chronic infection by the hepatitis B virus results in the development of poor prognosis liver cancer. Based on this mechanism our results suggest possible therapeutic interventions. [ABSTRACT FROM AUTHOR]

Oxymatrine (OMT) is able to effectively protect against hepatic fibrosis because of its anti-inflammatory property, while the underlying mechanism remains incompletely understood. In this study, forty rats were randomly divided into five groups: control group, model group (carbon tetrachloride, CCl 4 ) and three OMT treatment groups (30, 60, 120 mg/kg). After CCl 4 alone, the fibrosis score was 20.2 ± 0.8, and the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hydroxyproline content, and collagen I expression was elevated, but OMT blunted these parameters. Treatment with OMT prevented CCl 4 -induced increases in expression of pro-inflammatory and pro-fibrotic cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α, meanwhile OMT promoted the expression of anti-inflammatory and anti-fibrotic factors such as interleukin (IL)-10 and bone morphogenetic protein and activin membrane-bound inhibitor (Bambi). Moreover, lipopolysaccharides (LPS) and high mobility group box-1 (HMGB1), which activates Toll-like receptor 4 (TLR4) and modulate hepatic fibrogenesis through hepatic stellate cells (HSCs) or Kupffer cells, were significantly decreased by OMT treatment. These results were further supported by in vitro data. First, OMT suppressed the expression of TLR4 and its downstream pro-inflammatory cytokines, lowered the level of HMGB1, TGF-β1 in macrophages. Then, OMT promoted Bambi expression and thereby inhibited activation of HSCs mediated by transforming growth factor (TGF)-β1. In conclusion, this study showed that OMT could effectively attenuate the CCl 4 -induced hepatic fibrosis, and this effect may be due to modulation of TLR4-dependent inflammatory and TGF-β1 signaling pathways. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β and related growth factors are essential regulators for the development of follicles. Bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI) was reported as a key factor participating in the transforming growth factor-β signal pathway. To investigate the role of BAMBI in porcine granulosa cells, the full length of the BAMBI was cloned from porcine ovarian cDNA. The results of bioinformatics analyses showed that the signaling peptide was located in between positions 20 and 21. The results of online prediction on phosphorylation sites indicate that the sites of Ser, Thr, and Tyr are 9, 1, and 1, respectively. In addition, BAMBI was highly homologous in rodent and livestock. Real-time quantitative polymerase chain reaction (qPCR) indicated that BAMBI was widely expressed in porcine tissues. Immunofluorescence showed that BAMBI was located in both nucleus and cytoplasm. Stimulating the granulosa cells with FSH in vitro could alter BAMBI expression level in a time-dependent manner. Moreover, the expression level declined after treatment with FSH. These results indicated that BAMBI is an FSH-repressed gene in porcine luteinizing granulosa cells and it may be involved in the regulation of ovarian follicle development and oocyte maturation. [ABSTRACT FROM AUTHOR]

Abstract: Left ventricular (LV) pressure overload is a major cause of heart failure. Transforming growth factors-β (TGF-βs) promote LV remodeling under biomechanical stress. BAMBI (BMP and activin membrane-bound inhibitor) is a pseudoreceptor that negatively modulates TGF-β signaling. The present study tests the hypothesis that BAMBI plays a protective role during the adverse LV remodeling under pressure overload. The subjects of the study were BAMBI knockout mice (BAMBI−/−) undergoing transverse aortic constriction (TAC) and patients with severe aortic stenosis (AS). We examined LV gene and protein expression of remodeling-related elements, histological fibrosis, and heart morphology and function. LV expression of BAMBI was increased in AS patients and TAC-mice and correlated directly with TGF-β. BAMBI deletion led to a gain of myocardial TGF-β signaling through canonical (Smads) and non-canonical (TAK1–p38 and TAK1–JNK) pathways. As a consequence, the remodeling response to pressure overload in BAMBI−/− mice was exacerbated in terms of hypertrophy, chamber dilation, deterioration of long-axis LV systolic function and diastolic dysfunction. Functional remodeling associated transcriptional activation of fibrosis-related TGF-β targets, up-regulation of the profibrotic micro-RNA-21, histological fibrosis and increased metalloproteinase-2 activity. Histological remodeling in BAMBI−/− mice involved TGF-βs. BAMBI deletion in primary cardiac fibroblasts exacerbated TGF-β-induced profibrotic responses while BAMBI overexpression in NIH-3T3 fibroblasts attenuated them. Our findings identify BAMBI as a critical negative modulator of myocardial remodeling under pressure overload. We suggest that BAMBI is involved in negative feedback loops that restrain the TGF-β remodeling signals to protect the pressure-overloaded myocardium from uncontrolled extracellular matrix deposition in humans and mice. [Copyright &y& Elsevier]

TLR4-induced mitigation of the BMP down-regulation and activin membrane bound inhibitor (BAMBI) and the consequent enhancement of the transforming growth factor-beta (TGF-β) profibrogenic signaling has not yet been studied in cisplatin (CIS)-induced hepatotoxicity. miRNA-9 and29 have been previously reported to modulate TLR4 signaling via either tempering the expression of nuclear factor kappa-B p50 (NF-κB p50) or downregulation of extracellular matrix genes respectively. Hence we aimed to investigate the involvement of TLR4-induced modulation of TGF-β receptor 1 (TGF-βR1) signaling as well as the implication of miRNA-9 and 29 in CIS-induced hepatotoxicity. Moreover, we examined the ability of the phytochemical; crocin (CROC); to interact with either TLR4 or TGF-βR1 through a molecular docking study and subsequently explore its capability to attenuate CIS-induced hepatotoxicity. CROC pretreatment ameliorated the CIS-induced enhancement of TLR4 and TGF-β signaling and enhanced the expression of BAMBI, miRNA-9 and 29. Accordingly, it may be assumed that the protective effect of CROC against CIS-induce hepatotoxicity is mediated via the crosstalk of TLR4/NF-κBp50 signaling and BAMBI modulation of TGF-β1 activity in addition to the up-regulation of miRNA-9 and 29. These findings came in alignment with our molecular docking results; emphasizing the molecular antagonistic activity of CROC in both TLR4 and TGF-βR1. Image 1 • Phytochemicals potentials in attenuation of cisplatin hepatotoxicity. • The involvement of the cross-talk between TLR4/NF-κBp50 signaling and TGFβ/BAMBI signaling in predisposing CIS-induced hepatotoxicity. • The molecular antagonistic activity of CROC in both TLR4 and TGF-βR1. • The possible role of miRNA-9 and -29 in bestowing CROC hepatoprotective activity. [ABSTRACT FROM AUTHOR]

Both bone morphogenetic protein 2 (BMP2) and WNT/β-catenin signaling promote human trophoblast cell invasion. BMP2 has been shown to up-regulate bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) in granulosa cells. Besides, studies indicate BAMBI is a positive regulator for WNT/β-catenin signaling. However, whether BMP2 can increase BAMBI expression to induce WNT/β-catenin signaling for trophoblast cell invasion is still unknown. To study the roles of BAMBI in BMP2-induced activation of WNT/β-catenin signaling and human trophoblast invasion, we used immortalized human extravillous trophoblast (EVT) cell line (HTR8/SVneo) and primary human EVT cells as study models. Messenger RNA and protein levels of target genes were checked with RT-qPCR and Western blot assay respectively. The function of target proteins was studied via small interfering RNA (siRNA)-mediated knockdown. In addition, trophoblast cell invasiveness was examined by matrigel-coated transwell assays. Our results demonstrate that BMP2 treatment increased BAMBI mRNA levels and the activation of WNT/β-catenin signaling including the raised phosphorylation of GSK3β, the up-regulation of active (non-phosphorylated) β-catenin as well as its downstream target molecule cyclin D1, all of which were totally blocked by the activin receptor-like kinases (ALK) 2/3 inhibitor DMH1 or siRNA-mediated knockdown of BAMBI in HTR8/SVneo cells. Consistently, in primary human EVT cells, BMP2 also induced the up-regulation of BAMBI and the activation of WNT/β-catenin signaling indicated by the increased levels of active β-catenin and cyclin D1, which were completely blocked by BAMBI knockdown. In conclusion, BMP2 promotes the activation of canonical WNT/β-catenin signaling and human trophoblast cell invasion by up-regulating BAMBI. • BMP2 increased BAMBI mRNA levels in HTR8/SVneo and primary human extravillous trophoblast cells. • BMP2 activated canonical WNT/β-catenin signaling which promotes human trophoblast invasion. • DMH1 blocked BMP2-induced up-regulation of BAMBI and activation of WNT/β-catenin signaling. • BAMBI mediated BMP2-induced activation of WNT/β-catenin signaling and trophoblast invasion. [ABSTRACT FROM AUTHOR]

Astrocytic differentiation is developmentally impaired in patients with childhood-onset schizophrenia (SCZ). To determine why, we used genetic gain- and loss-of-function studies to establish the contributions of differentially expressed transcriptional regulators to the defective differentiation of glial progenitor cells (GPCs) produced from SCZ patient-derived induced pluripotent cells (iPSCs). Negative regulators of the bone morphogenetic protein (BMP) pathway were upregulated in SCZ GPCs, including BAMBI, FST, and GREM1, whose overexpression retained SCZ GPCs at the progenitor stage. SMAD4 knockdown (KD) suppressed the production of these BMP inhibitors by SCZ GPCs and rescued normal astrocytic differentiation. In addition, the BMP-regulated transcriptional repressor REST was upregulated in SCZ GPCs, and its KD similarly restored normal glial differentiation. REST KD also rescued potassium-transport-associated gene expression and K+ uptake, which were otherwise deficient in SCZ glia. These data suggest that the glial differentiation defect in childhood-onset SCZ, and its attendant disruption in K+ homeostasis, may be rescued by targeting BMP/SMAD4- and REST-dependent transcription. • Dysregulated BMP signaling restricts the differentiation of SCZ glial progenitor cells • REST is regulated by BMP/SMAD4 signaling and is overexpressed by SCZ GPCs • SMAD4 knockdown rescues normal astrocytic differentiation by SCZ GPCs • REST knockdown rescues K+ transporter gene expression and K+ uptake by SCZ glia Astrocytic differentiation is impaired in childhood-onset schizophrenia (SCZ). Liu et al. report that SMAD4-dependent BMP signaling and REST are upregulated in hiPSC-derived SCZ glia and that SMAD4 and REST knockdown rescue both astroglial differentiation and K+ transport. SCZ astrocytic maturation may thus be rescued by targeting SMAD4- and REST-dependent transcription. [ABSTRACT FROM AUTHOR]

Background & Aims: Development of nonalcoholic steatohepatitis (NASH) involves the innate immune system and is mediated by Kupffer cells and hepatic stellate cells (HSCs). Toll-like receptor 9 (TLR9) is a pattern recognition receptor that recognizes bacteria-derived cytosine phosphate guanine (CpG)–containing DNA and activates innate immunity. We investigated the role of TLR9 signaling and the inflammatory cytokine interleukin-1β (IL-1β) in steatohepatitis, fibrosis, and insulin resistance. Methods: Wild-type (WT), TLR9−/−, IL-1 receptor (IL-1R)−/−, and MyD88−/− mice were fed a choline-deficient amino acid-defined (CDAA) diet for 22 weeks and then assessed for steatohepatitis, fibrosis, and insulin resistance. Lipid accumulation and cell death were assessed in isolated hepatocytes. Kupffer cells and HSCs were isolated to assess inflammatory and fibrogenic responses, respectively. Results: The CDAA diet induced NASH in WT mice, characterized by steatosis, inflammation, fibrosis, and insulin resistance. TLR9−/− mice showed less steatohepatitis and liver fibrosis than WT mice. Among inflammatory cytokines, IL-1β production was suppressed in TLR9−/− mice. Kupffer cells produced IL-1β in response to CpG oligodeoxynucleotide. IL-1β but not CpG-oligodeoxynucleotides, increased lipid accumulation in hepatocytes. Lipid accumulation in hepatocytes led to nuclear factor-κB inactivation, resulting in cell death in response to IL-1β. IL-1β induced fibrogenic responses in HSCs, including secretion of tissue inhibitor of metalloproteinase-1. IL-1R−/− mice had reduced steatohepatitis and fibrosis, compared with WT mice. Mice deficient in MyD88, an adaptor molecule for TLR9 and IL-1R signaling, also had reduced steatohepatitis and fibrosis. TLR9−/−, IL-1R−/−, and MyD88−/− mice had less insulin resistance than WT mice on the CDAA diet. Conclusions: In a mouse model of NASH, TLR9 signaling induces production of IL-1β by Kupffer cells, leading to steatosis, inflammation, and fibrosis. [Copyright &y& Elsevier]