Activin Membrane-bound Inhibitor Research Articles

TGF-β and Opioid Receptor Signaling Crosstalk Results in Improvement of Endogenous and Exogenous Opioid Analgesia under Pathological Pain Conditions

Transforming growth factor-β1 (TGF-β1) protects against neuroinflammatory events underlying neuropathic pain. TGF-β signaling enhancement is a phenotypic characteristic of mice lacking the TGF-β pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor), which leads to an increased synaptic release of opioid peptides and to a naloxone-reversible hypoalgesic/antiallodynic phenotype. Herein, we investigated the following: (1) the effects of BAMBI deficiency on opioid receptor expression, functional efficacy, and analgesic responses to endogenous and exogenous opioids; and (2) the involvement of the opioid system in the antiallodynic effect of TGF-β1. BAMBI-KO mice were subjected to neuropathic pain by sciatic nerve crash injury (SNI). Gene (PCR) and protein (Western blot) expressions of μ- and δ-opioid receptors were determined in the spinal cord. The inhibitory effects of agonists on the adenylyl cyclase pathway were investigated. Two weeks after SNI, wild-type mice developed mechanical allodynia and the functionality of μ-opioid receptors was reduced. By this time, BAMBI-KO mice were protected against allodynia and exhibited increased expression and function of opioid receptors. Four weeks after SNI, when mice of both genotypes had developed neuropathic pain, the analgesic responses induced by morphine and RB101 (an inhibitor of enkephalin-degrading enzymes, which increases the synaptic levels of enkephalins) were enhanced in BAMBI-KO mice. Similar results were obtained in the formalin-induced chemical-inflammatory pain model. Subcutaneous TGF-β1 infusion prevented pain development after SNI. The antiallodynic effect of TGF-β1 was naloxone-sensitive. In conclusion, modulation of the endogenous opioid system by TGF-β signaling improves the analgesic effectiveness of exogenous and endogenous opioids under pathological pain conditions.

Acyl-CoA:cholesterol acyltransferase 1 mediates liver fibrosis by regulating free cholesterol accumulation in hepatic stellate cells

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes the conversion of free cholesterol (FC) to cholesterol ester, which prevents excess accumulation of FC. We recently found that FC accumulation in hepatic stellate cells (HSCs) plays a role in progression of liver fibrosis, but the effect of ACAT1 on liver fibrosis has not been clarified. In this study, we aimed to define the role of ACAT1 in the pathogenesis of liver fibrosis. ACAT1-deficient and wild-type mice, or Toll-like receptor 4 (TLR4)(-/-)ACAT1(+/+) and TLR4(-/-)ACAT1(-/-) mice were subjected to bile duct ligation (BDL) for 3 weeks or were given carbon tetrachloride (CCl4) for 4 weeks to induce liver fibrosis. ACAT1 was the major isozyme in mice and human primary HSCs, and ACAT2 was the major isozyme in mouse primary hepatocytes and Kupffer cells. ACAT1 deficiency significantly exaggerated liver fibrosis in the mouse models of liver fibrosis, without affecting the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation. ACAT1 deficiency significantly increased FC levels in HSCs, augmenting TLR4 protein and downregulating expression of transforming growth factor-β (TGFβ) pseudoreceptor Bambi (bone morphogenetic protein and activin membrane-bound inhibitor), leading to sensitization of HSCs to TGFβ activation. Exacerbation of liver fibrosis by ACAT1 deficiency was dependent on FC accumulation-induced enhancement of TLR4 signaling. ACAT1 deficiency exaggerates liver fibrosis mainly through enhanced FC accumulation in HSCs. Regulation of ACAT1 activities in HSCs could be a target for treatment of liver fibrosis.

Transcriptional Repression of the Transforming Growth Factor β (TGF-β) Pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) by Nuclear Factor κB (NF-κB) p50 Enhances TGF-β Signaling in Hepatic Stellate Cells

TLR4 signaling induces down-regulation of the bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI), which enhances TGF-β signaling during hepatic stellate cell (HSC) activation. We investigated the mechanism by which TLR4 signaling down-regulates BAMBI expression in HSCs and found that TLR4- and TNF-α-mediated BAMBI down-regulation is dependent on regulation of BAMBI promoter activity through the interaction with NF-κBp50 and HDAC1 in HSCs. Bambi was predominantly expressed in HSCs, at high levels in quiescent HSCs but at low levels in in vivo-activated and LPS-stimulated HSCs. In human HSCs, BAMBI expression was down-regulated in response to LPS and TNF-α. A BAMBI reporter assay demonstrated that the regulatory element to repress BAMBI transcription is located between 3384 and 1560 bp upstream from the transcription start site. LPS stimulation down-regulated BAMBI expression in cells with NF-κBp65 knockdown. However, it failed to down-regulate BAMBI in cells with inactivation of NF-κB or NF-κBp50 silencing, indicating that NF-κBp50 is a factor for BAMBI down-regulation. ChIP analysis revealed that LPS and TNF-α induced binding of the NF-κBp50/p50 homodimer to the BAMBI promoter region. We also found that HDAC1 is bound to this region as part of the NF-κBp50-HDAC1 complex, repressing transcriptional activity of the BAMBI promoter. Finally, we confirmed that LPS does not repress BAMBI reporter activity using a BAMBI reporter construct with a mutation at 3166 bp upstream of the coding region. In summary, our study demonstrates that LPS- and TNF-α-induced NF-κBp50-HDAC1 interaction represses BAMBI transcriptional activity, which contributes to TLR4-mediated enhancement of TGF-β signaling in HSCs during liver fibrosis.

Gene expression profiling of clear cell papillary renal cell carcinoma: comparison with clear cell renal cell carcinoma and papillary renal cell carcinoma

Clear cell papillary renal cell carcinoma is a distinct variant of renal cell carcinoma that shares some overlapping histological and immunohistochemical features of clear cell renal cell carcinoma and papillary renal cell carcinoma. Although the clear cell papillary renal cell carcinoma immunohistochemical profile is well described, clear cell papillary renal cell carcinoma mRNA expression has not been well characterized. We investigated the clear cell papillary renal cell carcinoma gene expression profile using previously identified candidate genes. We selected 17 clear cell papillary renal cell carcinoma, 15 clear cell renal cell carcinoma, and 13 papillary renal cell carcinoma cases for molecular analysis following histological review. cDNA from formalin-fixed paraffin-embedded tissue was prepared. Quantitative real-time PCR targeting alpha-methylacyl coenzyme-A racemase (AMACR), BMP and activin membrane-bound inhibitor homolog (BAMBI), carbonic anhydrase IX (CA9), ceruloplasmin (CP), nicotinamide N-methyltransferase (NNMT), schwannomin-interacting protein 1 (SCHIP1), solute carrier family 34 (sodium phosphate) member 2 (SLC34A2), and vimentin (VIM) was performed. Gene expression data were normalized relative to 28S ribosomal RNA. Clear cell papillary renal cell carcinoma expressed all eight genes at variable levels. Compared with papillary renal cell carcinoma, clear cell papillary renal cell carcinoma expressed more CA9, CP, NNMT, and VIM, less AMACR, BAMBI, and SLC34A2, and similar levels of SCHIP1. Compared with clear cell renal cell carcinoma, clear cell papillary renal cell carcinoma expressed slightly less NNMT, but similar levels of the other seven genes. Although clear cell papillary renal cell carcinoma exhibits a unique molecular signature, it expresses several genes at comparable levels to clear cell renal cell carcinoma relative to papillary renal cell carcinoma. Understanding the molecular pathogenesis of clear cell papillary renal cell carcinoma will have a key role in future sub-classifications of this unique tumor.

Free cholesterol accumulation in hepatic stellate cells: Mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice

Although nonalcoholic steatohepatitis (NASH) is associated with hypercholesterolemia, the underlying mechanisms of this association have not been clarified. We aimed to elucidate the precise role of cholesterol in the pathophysiology of NASH. C57BL/6 mice were fed a control, high-cholesterol (HC), methionine-choline-deficient (MCD), or MCD+HC diet for 12 weeks or a control, HC, high-fat (HF), or HF+HC diet for 24 weeks. Increased cholesterol intake accelerated liver fibrosis in both the mouse models without affecting the degree of hepatocellular injury or Kupffer cell activation. The major causes of the accelerated liver fibrosis involved free cholesterol (FC) accumulation in hepatic stellate cells (HSCs), which increased Toll-like receptor 4 protein (TLR4) levels through suppression of the endosomal-lysosomal degradation pathway of TLR4, and thereby sensitized the cells to transforming growth factor (TGF)β-induced activation by down-regulating the expression of bone morphogenetic protein and activin membrane-bound inhibitor. Mammalian-cell cholesterol levels are regulated by way of a feedback mechanism mediated by sterol regulatory element-binding protein 2 (SREBP2), maintaining cellular cholesterol homeostasis. Nevertheless, HSCs were sensitive to FC accumulation because the high intracellular expression ratio of SREBP cleavage-activating protein (Scap) to insulin-induced gene (Insig) disrupted the SREBP2-mediated feedback regulation of cholesterol homeostasis in these cells. HSC activation subsequently enhanced the disruption of the feedback system by Insig-1 down-regulation. In addition, the suppression of peroxisome proliferator-activated receptor γ signaling accompanying HSC activation enhanced both SREBP2 and microRNA-33a signaling. Consequently, FC accumulation in HSCs increased and further sensitized these cells to TGFβ-induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH. These characteristic mechanisms of FC accumulation in HSCs are potential targets to treat liver fibrosis in liver diseases including NASH.

IL-1 Induces Proinflammatory Leukocyte Infiltration and Regulates Fibroblast Phenotype in the Infarcted Myocardium

In the infarcted myocardium, activation of the inflammatory cascade clears the wound from dead cells, whereas stimulating matrix degradation and chamber dilation, thus contributing to the development of heart failure. IL-1 is critically involved in the postinfarction inflammatory reaction and mediates adverse dilative remodeling. We hypothesized that IL-1 may regulate postinfarction repair and remodeling through cell-specific actions on leukocytes and fibroblasts. Flow cytometry demonstrated that in mouse infarcts, early recruitment of proinflammatory Ly6C(hi) cells expressing IL-1R1, the signaling receptor for IL-1, was followed by infiltration with cells expressing the decoy receptor, IL-1R2. Increased expression of IL-1R2 may serve to terminate IL-1-driven inflammation after infarction. Loss of IL-1 signaling in IL-1R1 null mice globally attenuated leukocyte recruitment, reducing the number of infiltrating Ly6C(hi) and Ly6C(lo) cells. Nonmyeloid CD11b(-) cells harvested during the inflammatory phase of cardiac repair exhibited marked upregulation of chemokines and cytokines; their inflammatory activation was IL-1R1 dependent. Moreover, IL-1β attenuated TGF-β-induced contractile activity of fibroblasts populating collagen pads, attenuated α-smooth muscle actin expression, and stimulated matrix metalloproteinase synthesis in an IL-1R1-dependent manner. The effects of IL-1 on TGF-β responses in cardiac fibroblasts were not due to direct effects on Smad activation, but were associated with endoglin suppression and accentuated expression of bone morphogenetic protein and activin membrane-bound inhibitor, a negative regulator of TGF-β signaling. IL-1 may orchestrate fibroblast responses in the infarct; early stimulation of fibroblast IL-1R1 signaling during the inflammatory phase may prevent premature activation of a matrix-synthetic contractile phenotype until the wound is cleared, and the infarct microenvironment can support mesenchymal cell growth.

The overexpression of BAMBI and its involvement in the growth and invasion of human osteosarcoma cells

The pseudoreceptor BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor), formerly known as NMA, is an inhibitor of the TGF-β signaling pathway. BAMBI exhibits structural homology to TGF-βRI but lacks an intracellular kinase domain. In most of the high-grade carcinomas, the degree of BAMBI expression is abnormally increased, which leads to the proliferation and metastasis of tumor cells. Recent studies have reported that BAMBI is involved in the Wnt-β-catenin pathway that regulates the proliferation and metastasis of tumor cells. However, little is known about the role of BAMBI and β-catenin in human osteosarcoma. Given the above background, we examined the role of BAMBI in the pathophysiology of osteosarcoma. Using immunohistochemical staining and western blot analysis, the degree of the expression of BAMBI and β-catenin was significantly higher in osteosarcoma specimens compared with normal tissues. With the overexpression of BAMBI, mediated by adenovirus, the degree of invasion and migration was significantly increased and the proliferation of U2-OS osteosarcoma cells was stimulated. Transwell analysis showed that BAMBI increased the invasion of osteosarcoma cells and upregulated the secretion of matrix metalloproteinases (MMPs), which was demonstrated by gelatin zymography. Fluorescence-activated cell sorting (FACS) analysis showed a significant arrest in cell cycle progression at G0/G1 in osteosarcoma cells transfected with siRNA targeting BAMBI. With the overexpression of BAMBI, mediated by the adenovirus, however, there was a decrease in the number of cells at G0/G1. Consistent with the findings that cell growth was increased, BAMBI promoted the transition from G0/G1 to G2/M in the osteosarcoma cells. Our results suggest that BAMBI plays a key role in the pathogenesis and progression of osteosarcoma by regulating the expression of β-catenin and other signaling molecules via the pathways involved in the regulation of the cell cycle. This relationship between BAMBI and its involvement in the regulation of the cell cycle would provide a possibility that the BAMBI may be a new target for gene therapy.

Inhibition of TGF‐β/Smad Signaling by BAMBI Blocks Differentiation of Human Mesenchymal Stem Cells to Carcinoma‐Associated Fibroblasts and Abolishes their Protumor Effects

Bone marrow mesenchymal stem cells (BM-MSCs) have multiple therapeutic potentials for regenerative, anti-inflammatory, and immunomodulatory purposes and also show promise as vehicles for gene therapy of various metastatic cancers based on their tumor-tropic capacity. However, BM-MSCs are also a source of carcinoma-associated fibroblasts (CAFs) and may promote growth and metastasis of cancer. Transforming growth factor β (TGF-β) signaling is required to induce CAF differentiation of mouse BM-MSCs in vivo and can induce expression of some CAF markers in human BM-MSCs in vitro. To determine whether inhibiting TGF-β signaling in human BM-MSCs can block their differentiation to CAFs induced by tumor microenvironments and the consequent protumor effects, we transduced human BM-MSCs with a lentiviral vector encoding bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a decoy TGF-β receptor. BAMBI transduction significantly inhibited TGF-β/Smad signaling and expression of CAF markers in human BM-MSCs treated with TGF-β1 or tumor-conditioned medium or cocultured with cancer cells, but did not alter the stem cell properties and the tumor-tropic property of MSCs. In addition, BAMBI transduction disrupted the cytokine network mediating the interaction between MSCs and breast cancer cells. Consequently, BAMBI transduction abolished protumor effects of BM-MSCs in vitro and in an orthotopic breast cancer xenograft model, and instead significantly inhibited growth and metastasis of coinoculated cancer. These results indicated that TGF-β signaling is essential for differentiation of human BM-MSCs to CAFs in tumor microenvironments and the consequent protumor effects, and inhibiting TGF-β/Smad pathway may improve the safety of MSC-based therapies in cancer patients.

BAMBI (BMP and activin membrane-bound inhibitor) protects the murine heart from pressure-overload biomechanical stress by restraining TGF-β signaling

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.

Chlorogenic acid reduces liver inflammation and fibrosis through inhibition of toll-like receptor 4 signaling pathway

Chlorogenic acid (CGA) is a type of polyphenol with anti-inflammatory, antioxidant activities. Our previous studies showed CGA could efficiently inhibit carbon tetrachloride (CCl4)-induced liver fibrosis in rats. However, the specific underlying mechanism remains unclear. The aim of this study is to investigate the effects of CGA on liver inflammation and fibrosis induced by CCl4 and whether they are related to inhibition of toll-like receptor 4 (TLR4) signaling pathway. Male Sprague-Dawley (SD) rats were administrated CCl4 together with or without CGA for 8 weeks. Histopathological and biochemical analyses were carried out. The mRNA and protein expression levels of proinflammatory and profibrotic mediators were detected by RT-PCR and Western blot, respectively. The levels of serum proinflammatory cytokines were detected by ELISA. CGA significantly attenuated CCl4-induced liver damage and symptoms of liver fibrosis, accompanied by reduced serum transaminase levels, collagen I and α-smooth muscle actin (α-SMA) expression. As compared with the CCl4-treated group, the expression levels of TLR4, myeloid differentiation factor 88 (MyD88), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were reduced in the treatment group of CCl4 and CGA, whereas bone morphogenetic protein and activin membrane-bound inhibitor (Bambi) expression was increased. CGA also suppressed CCl4 induced nuclear factor-κB (NF-κB) activation. Moreover, the hepatic mRNA expression and serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were significantly increased in CCl4-treated rats and attenuated by co-treatment with CGA. Our data indicate that CGA can efficiently inhibit CCl4-induced liver fibrosis in rats and the protective effect may be due to the inhibition of TLR4/MyD88/NF-κB signaling pathway.

Resistance to platinum-based chemotherapy is associated with epithelial to mesenchymal transition in epithelial ovarian cancer

BackgroundThe present study is aimed to identify genetic pathways correlated with chemoresistance in epithelial ovarian cancer (EOC). MethodsWe compared the molecular profiles of 23 tumour biopsies of stage III–IV (training set) at primary surgery, before chemotherapy, to the profile from the same patients at second surgery, after several lines of platinum (Pt)-based chemotherapy when the tumours were resistant. In the hypothesis that identified markers were related to Pt-resistance and to prognosis, we validated this signature in 52 EOC taken at primary surgery (validation set) selected to be either very sensitive to the first line therapy, i.e. not relapsing before one year from the end of therapy, or resistant, i.e. relapsing within 6months from the end of therapy. ResultsIn the training set, we identified a resistance signature indicative of the activation of epithelial to mesenchymal transition (EMT) by transforming growth factor (TGF)-beta pathway. We then validated this signature in 52 EOC taken at primary surgery (validation set). Some genes involved in EMT, such as BMP and activin membrane-bound inhibitor (BAMBI), and mir-141 resulted in association with overall or progression free survival. ConclusionSome genes involved in EMT were associated to overall or progression free survival, suggesting EMT as vital to the resistance mechanisms.

Reference genes to quantify gene expression during oogenesis in a teleost fish

Understanding the molecular events involved in the acquisition of competence during oogenesis is a key step to determine the secret of ‘high quality’ eggs for aquaculture. Quantitative real time polymerase chain reaction (qPCR) is the technique of election to determine changes in transcript abundance in such studies, but choosing reference genes for normalization, in particular during oogenesis, remains a challenge.In the present study, transcription of 6 functionally distinct genes, β actin (ACTB), cathepsin D (CTSD), cathepsin Z (CTSZ), elongation factor 1 α (EEF1A), TATA binding protein (TBP) and tubulin A (TUBA1A) was assessed as normalizers of bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) gene expression in mRNA from Mozambique tilapia oocytes during oogenesis. Reverse transcription was equally efficient and varies little in all samples. Most of the genes considered for reference were stable during early stages of oogenesis but variations were observed during vitellogenesis. A single gene and up to 3 genes were shown to be insufficient for reliable normalization throughout the whole oogenesis. The combination of the genes ACTB, CTSD, EEF1A and CTSZ as reference was found to minimize variation and has the most stable expression pattern between maturation stages.

Transforming Growth Factor-β1 Regulates Cdk5 Activity in Primary Sensory Neurons

In addition to many important roles for Cdk5 in brain development and synaptic function, we reported previously that Cdk5 regulates inflammatory pain signaling, partly through phosphorylation of transient receptor potential vanilloid 1 (TRPV1), an important Na(+)/Ca(2+) channel expressed in primary nociceptive afferent nerves. Because TGF-β regulates inflammatory processes and its receptor is expressed in TRPV1-positive afferents, we studied the cross-talk between these two pathways in sensory neurons during experimental peripheral inflammation. We demonstrate that TGF-β1 increases transcription and protein levels of the Cdk5 co-activator p35 through ERK1/2, resulting in an increase in Cdk5 activity in rat B104 neuroblastoma cells. Additionally, TGF-β1 enhances the capsaicin-induced Ca(2+) influx in cultured primary neurons from dorsal root ganglia (DRG). Importantly, Cdk5 activity was reduced in the trigeminal ganglia and DRG of 14-day-old TGF-β1 knock-out mice, resulting in reduced Cdk5-dependent phosphorylation of TRPV1. The decreased Cdk5 activity is associated with attenuated thermal hyperalgesia in TGF-β1 receptor conditional knock-out mice, where TGF-β signaling is significantly reduced in trigeminal ganglia and DRG. Collectively, our results indicate that active cross-talk between the TGF-β and Cdk5 pathways contributes to inflammatory pain signaling.

Identification of BMP and Activin Membrane-Bound Inhibitor (BAMBI) as a Potent Negative Regulator of Adipogenesis and Modulator of Autocrine/Paracrine Adipogenic Factors

Adipose tissue dysfunction underpins the association of obesity with type 2 diabetes. Adipogenesis is required for the maintenance of adipose tissue function. It involves the commitment and subsequent differentiation of preadipocytes and is coordinated by autocrine, paracrine, and endocrine factors. We previously reported that fibroblast growth factor-1 (FGF-1) primes primary human preadipocytes and Simpson Golabi Behmel syndrome (SGBS) preadipocytes and increases adipogenesis through a cascade involving extracellular signal–related kinase 1/2 (ERK1/2). Here, we aimed to use the FGF-1 system to identify novel adipogenic regulators. Expression profiling revealed bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) as a putative FGF-1 effector. BAMBI is a transmembrane protein and modulator of paracrine factors that regulate adipogenesis, including transforming growth factor (TGF) superfamily members (TGF-β and BMP) and Wnt. Functional investigations established BAMBI as a negative regulator of adipogenesis and modulator of the anti- and proadipogenic effects of Wnt3a, TGF-β1, and BMP-4. Further studies showed that BAMBI expression levels are decreased in a mouse model of diet-induced obesity. Collectively, these findings establish BAMBI as a novel, negative regulator of adipogenesis that can act as a nexus to integrate multiple paracrine signals to coordinate adipogenesis. Alterations in BAMBI may play a role in the (patho)physiology of obesity, and manipulation of BAMBI may present a novel therapeutic approach to improve adipose tissue function.

Transforming Growth Factor-β in Normal Nociceptive Processing and Pathological Pain Models

The transforming growth factor-β (TGF-β) superfamily is a multifunctional, contextually acting family of cytokines that participate in the regulation of development, disease and tissue repair in the nervous system. The TGF-β family is composed of several members, including TGF-βs, bone morphogenetic proteins (BMPs) and activins. In this review, we discuss recent findings that suggest TGF-β function as important pleiotropic modulators of nociceptive processing both physiologically and under pathological painful conditions. The strategy of increasing TGF-β signaling by deleting "BMP and activin membrane-bound inhibitor" (BAMBI), a TGF-β pseudoreceptor, has demonstrated the inhibitory role of TGF-β signaling pathways in normal nociception and in inflammatory and neuropathic pain models. In particular, strong evidence suggests that TGF-β1 is a relevant mediator of nociception and has protective effects against the development of chronic neuropathic pain by inhibiting the neuroimmune responses of neurons and glia and promoting the expression of endogenous opioids within the spinal cord. In the peripheral nervous system, activins and BMPs function as target-derived differentiation factors that determine and maintain the phenotypic identity and circuit assembly of peptidergic nociceptors. In this context, activin is involved in the complex events of neuroinflammation that modulate the expression of pain during wound healing. These findings have provided new insights into the physiopathology of nociception. Moreover, specific members of the TGF-β family and their signaling effectors and modulator molecules may be promising molecular targets for novel therapeutic agents for pain management.

A High-Cholesterol Diet Exacerbates Liver Fibrosis in Mice via Accumulation of Free Cholesterol in Hepatic Stellate Cells

Some studies have indicated that dietary cholesterol has a role in the progression of liver fibrosis. We investigated the mechanisms by which dietary cholesterol might contribute to hepatic fibrogenesis. C57BL/6 mice were fed a high-cholesterol diet or a control diet for 4 weeks; liver fibrosis then was induced by bile-duct ligation or carbon tetrachloride administration. Hepatic stellate cells (HSCs) were isolated from mice fed high-cholesterol diets or from Niemann-Pick type C1-deficient mice, which accumulate intracellular free cholesterol. After bile-duct ligation or carbon tetrachloride administration, mice fed high-cholesterol diets had significant increases in liver fibrosis and activation of HSCs compared with mice fed control diets. There were no significant differences in the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation, between mice fed high-cholesterol or control diets. Levels of free cholesterol were much higher in HSCs from mice fed high-cholesterol diets than those fed control diets. In cultured HSCs, accumulation of free cholesterol in HSCs increased levels of Toll-like receptor 4 (TLR4), leading to down-regulation of bone morphogenetic protein and activin membrane-bound inhibitor (a pseudoreceptor for transforming growth factor [TGF]β); the HSCs became sensitized to TGFβ-induced activation. Liver fibrosis was not aggravated by the high-cholesterol diet in C3H/HeJ mice, which express a mutant form of TLR4; HSCs that express mutant TLR4 were not activated by accumulation of free cholesterol. Dietary cholesterol aggravates liver fibrosis because free cholesterol accumulates in HSCs, leading to increased TLR4 signaling, down-regulation of bone morphogenetic protein and activin membrane-bound inhibitor, and sensitization of HSC to TGFβ. This pathway might be targeted by antifibrotic therapies.

Abstract 2438: BAMBI Is overexpressed in metastatic pancreatic cancers with genetically Intact TGF-β pathways: A potential mechanism to escape TGF-β signaling during metastasis formation

Abstract Introduction: Pancreatic cancer remains a largely fatal disease, with an estimated 95% of patients succumbing within five years of initial diagnosis, many by metastatic spread. We have previously shown that genetic inactivation of DPC4, a central mediator of TGF-β signaling, is strongly associated with widespread metastasis in pancreatic cancer patients at autopsy. However, because some patients with metastatic pancreatic cancer do not have genetic inactivation of DPC4 or other TGF-β members, we investigated the role of alternative mechanisms of TGF-β pathway inactivation in promoting pancreatic cancer metastasis. Methods: Eighteen cell lines that underwent whole exome sequencing in association with the pancreatic cancer genome project or directed sequencing of all members of the TGF-β pathway were used. TGF-β signaling levels were analyzed in each cell line using a luciferase reporter system under the control of a Smad Binding Element (SBE). Quantitative real-time PCR was performed using cDNA from representative cell lines to assess expression levels of nine known TGF-β pathway antagonists; data were analyzed using the ΔΔCt method with normalization to β-actin. Immunohistochemistry was performed on pancreatic cancer tissue microarrays that included 27 matched pairs of primary and metastatic cancer. Staining intensity and protein localization were analyzed in relation to DPC4 status as well as clinicopathologic data. Results: Functional TGF-β signaling was completely abolished in cell lines with known DPC4 inactivation compared to normal controls, and was also decreased in cell lines with an intact TGF-β pathway, to a lesser extent. Quantitative RT-PCR of known TGF-β antagonists in cell lines with genetically intact TGF-β pathways revealed 50-fold overexpression of BMP and Activin Membrane-Bound Inhibitor (BAMBI) in two of seven cell lines (29%). Immunolabeling for BAMBI in human pancreatic cancer tissues indicated that it is highly expressed in a subset (26%) of pancreatic cancers, consistent with the cell line data. BAMBI overexpression was not associated with clinicopathologic features at diagnosis, nor was there a difference in BAMBI expression among DPC4 wild type versus DPC4 inactive carcinomas. However, specifically, BAMBI overexpression was significantly more frequent in metastases from patients with wild type DPC4 compared to those with inactivated DPC4 (p=0.01). Conclusion: Overexpression of BAMBI occurs at an appreciable level in pancreatic cancer cell lines and tissues, and may promote metastasis in the presence of a genetically intact TGF-β pathway. Mechanistic studies are ongoing to clarify the role of BAMBI in pancreatic cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2011-2438

Bone Morphogenetic Protein-2 (BMP-2) Increases Gene Expression of FSH Receptor and Aromatase and Decreases Gene Expression of LH Receptor and StAR in Human Granulosa Cells

A growing body of evidence indicates that bone morphogenetic protein (BMP) cytokines play a key role in female fertility in mammals. BMP-2 is known to be expressed in the ovary of many species. In the present study, we examined the expression and function of BMP-2 in the human ovary. BMP-2 mRNA expression in the human ovary was evaluated by in situ hybridization. Human granulosa cells were obtained from in vitro fertilization patients. Human granulosa cells were cultured with recombinant BMP-2 or human chorionic gonadotrophin (HCG), followed by RNA extraction. BMP-2 expression was detected in granulosa cells of antral follicles but not of corpus luteum. The in vitro study showed that BMP-2 induced follicular stimulating hormone (FSH) receptor and aromatase expression, while decreasing luteinizing hormone (LH) receptor and steroidogenic acute regulatory protein expression in human granulosa cells. HCG decreased gene expression of BMP-2 and increased BMP and activin membrane-bound inhibitor (BAMBI), an antagonist of BMP-2. Expression and disappearance of BMP-2 might contribute to folliculogenesis and luteinization by regulating gonadotropin receptor expression in human granulosa cells. HCG can modulate BMP-2 function by controlling BMP-2 and BAMBI expression.

The TGF-beta-Pseudoreceptor BAMBI is strongly expressed in COPD lungs and regulated by nontypeable Haemophilus influenzae

BackgroundNontypeable Haemophilus influenzae (NTHI) may play a role as an infectious trigger in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available regarding the influence of acute and persistent infection on tissue remodelling and repair factors such as transforming growth factor (TGF)-β.MethodsNTHI infection in lung tissues obtained from COPD patients and controls was studied in vivo and using an in vitro model. Infection experiments were performed with two different clinical isolates. Detection of NTHI was done using in situ hybridization (ISH) in unstimulated and in in vitro infected lung tissue. For characterization of TGF-β signaling molecules a transcriptome array was performed. Expression of the TGF-pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) was analyzed using immunohistochemistry (IHC), ISH and PCR. CXC chemokine ligand (CXCL)-8, tumor necrosis factor (TNF)-α and TGF-β expression were evaluated in lung tissue and cell culture using ELISA.ResultsIn 38% of COPD patients infection with NTHI was detected in vivo in contrast to 0% of controls (p < 0.05). Transcriptome arrays showed no significant changes of TGF-β receptors 1 and 2 and Smad-3 expression, whereas a strong expression of BAMBI with upregulation after in vitro infection of COPD lung tissue was demonstrated. BAMBI was expressed ubiquitously on alveolar macrophages (AM) and to a lesser degree on alveolar epithelial cells (AEC). Measurement of cytokine concentrations in lung tissue supernatants revealed a decreased expression of TGF-β (p < 0.05) in combination with a strong proinflammatory response (p < 0.01).ConclusionsWe show for the first time the expression of the TGF pseudoreceptor BAMBI in the human lung, which is upregulated in response to NTHI infection in COPD lung tissue in vivo and in vitro. The combination of NTHI-mediated induction of proinflammatory cytokines and inhibition of TGF-β expression may influence inflammation induced tissue remodeling.

BAMBI is overexpressed in ovarian cancer and co-translocates with Smads into the nucleus upon TGF-ß treatment

Objective Transforming growth factor beta (TGF-ß) signaling via Smads plays a central role in carcinogenesis. Bmp and activin membrane-bound inhibitor ( BAMBI) was initially described as a pseudoreceptor antagonizing TGF-ß receptor activation, thus impairing signaling. Here we wanted to estimate the role of BAMBI in ovarian cancer. Methods The function of BAMBI was studied using a cell line model and intracellular localization experiments. The impact of BAMBI expression on patient outcome was estimated by real-time PCR and immunohistochemistry. Results We demonstrate for the first time a nuclear co-translocation of BAMBI with Smad2/3 upon TGF-ß treatment. Moreover, overexpression of BAMBI in an in vitro model led to significantly increased proliferation (doubling time − 37.0%, P = 0.010), migration (+ 581.2%, P = 0.004) and resistance to TGF-ß-mediated apoptosis (decrease of apoptosis from 30% in the control cells to 7% in the BAMBI-overexpressing cells). Although— prima facie—this fits to the thesis of BAMBI as a pseudoreceptor, it may also be explained by modulation of TGF-ß signaling in the nucleus, leading to the observed pro-oncogenic properties. The tumor promoting impact of BAMBI mRNA overexpression in vitro could not be confirmed in primary tumor samples, and while nearly all tumor samples showed up-regulation of BAMBI (37.3% 1+, 39.2% 2+, and 16.7% 3+, respectively) compared to undetectable BAMBI in healthy pre- and post-menopausal ovarian epithelia, no impact of BAMBI expression on recurrence free and overall survival could be observed. Conclusion These findings provide new insights into the Smad-mediated pathway by inferring that BAMBI is a novel modulator of TGF-ß signaling.