Receptor Smad3 Research Articles

Role of Native Probiotic Lactobacillus Species via TGF-β Signaling Pathway Modulation in CRC

Colon microbiome composition in colorectal cancer (CRC) patients undergoes remarkable changes. The present study was designed to assess the impact of Lactobacillus mixture on the regulating the CRC by influencing the transforming growth factor beta (TGF-β) signaling pathway in both in vitro (HT-29 cancer cells) and in vivo (BALB/c mice) models. In this study, the antiproliferative effect of a native potential probiotic Lactobacillus mixture on HT-29 cancer cells was evaluated using the MTT assay method. Also, qRT-PCR was performed to assess the RNA expression level of genes associated with the TGF-β signaling pathway at three levels: receptor, regulatory, and inhibitory SMADs. Finally, the in vivo assays were investigated by three groups of mice: a naive group (PBS), a disease group (azoxymethane [AOM]/ dextran sulfate sodium [DSS] + PBS), and a treatment group (AOM/DSS + Lactobacillus mixture in PBS). The MTT results showed a significant decrease in proliferation of HT-29 cancer cells after 120 h of treatment. Furthermore, qRT-PCR demonstrated the downregulation of the smad2/3 gene expression in HT-29-treated cells and also reduction in the level of smad4 gene expression. In addition, in the mouse model, the tgf-βR1 gene was downregulated in the group treated with AOM/DSS/Lactobacillus, but not the AOM/DSS group. A downregulation of smad4 gene expression was also observed in in vivo models. The obtained results suggest that our novel probiotic Lactobacillus mixture could have a positive impact on the inhibition of the CRC progression by downregulating the TGF-β signaling pathway.

SMAD signaling pathway is disrupted by BPA via the AMH receptor in bovine granulosa cells†.

Significant events that determine oocyte competence occur during follicular growth and oocyte maturation. The anti-Mullerian hormone, a positive predictor of fertility, has been shown to be affected by exposure to endocrine disrupting compounds, such as bisphenol A and S. However, the interaction between bisphenols and SMAD proteins, mediators of the anti-Mullerian hormone pathway, has not yet been elucidated. AMH receptor (AMHRII) and downstream SMAD expression was investigated in bovine granulosa cells treated with bisphenol A, bisphenol S, and then competitively with the anti-Mullerian hormone. Here, we show that 24-h bisphenol A exposure in granulosa cells significantly increased SMAD1, SMAD4, and SMAD5 mRNA expression. No significant changes were observed in AMHRII or SMADs protein expression after 24-h treatment. Following 12-h treatments with bisphenol A (alone or with the anti-Mullerian hormone), a significant increase in SMAD1 and SMAD4 mRNA expression was observed, while a significant decrease in SMAD1 and phosphorylated SMAD1 was detected at the protein level. To establish a functional link between bisphenols and the anti-Mullerian hormone signaling pathway, antisense oligonucleotides were utilized to suppress AMHRII expression with or without bisphenol exposure. Initially, transfection conditions were optimized and validated with a 70% knockdown achieved. Our findings show that bisphenol S exerts its effects independently of the anti-Mullerian hormone receptor, while bisphenol A may act directly through the anti-Mullerian hormone signaling pathway providing a potential mechanism by which bisphenols may exert their actions to disrupt follicular development and decrease oocyte competence.

Acetate controls endothelial-to-mesenchymal transition

Endothelial-to-mesenchymal transition (EndMT), a process initiated by activation of endothelial TGF-β signaling, underlies numerous chronic vascular diseases and fibrotic states. Once induced, EndMT leads to a further increase in TGF-β signaling, thus establishing a positive-feedback loop with EndMT leading to more EndMT. Although EndMT is understood at the cellular level, the molecular basis of TGF-β-driven EndMT induction and persistence remains largely unknown. Here, we show that metabolic modulation of the endothelium, triggered by atypical production of acetate from glucose, underlies TGF-β-driven EndMT. Induction of EndMT suppresses the expression of the enzyme PDK4, which leads to an increase in ACSS2-dependent Ac-CoA synthesis from pyruvate-derived acetate. This increased Ac-CoA production results in acetylation of the TGF-β receptor ALK5 and SMADs 2 and 4 leading to activation and long-term stabilizationof TGF-β signaling. Our results establish the metabolic basis of EndMT persistence and unveil novel targets, such as ACSS2, for the potential treatment of chronic vascular diseases.

Sex-specific meiosis responses to Gsdf in medaka (Oryzias latipes).

The meiotic entry of undifferentiated germ cells is sexually specific and strictly regulated by the testicular or ovarian environment. Germline stem cells with a set of abnormal sex chromosomes and associated autosomes undergo defective meiotic processes and are eventually eliminated by yet to be defined post-transcriptional modifications. Herein, we report the role of gsdf, a member of BMP/TGFβ family uniquely found in teleost, in the regulation of meiotic entry in medaka (Oryzias latipes) via analyses of gametogenesis in gsdf-deficient XX and XY gonads in comparison with their wild-type siblings. Several differentially expressed genes, including the FKB506-binding protein 7 (fkbp7), were significantly upregulated in pubertal gsdf-deficient gonads. The increase in alternative pre-mRNA isoforms of meiotic synaptonemal complex gene sycp3 was visualized using Integrative Genomics Viewer and confirmed by real-time qPCR. Nevertheless, immunofluorescence analysis showed that Sycp3 protein products reduced significantly in gsdf-deficient XY oocytes. Transmission electron microscope observations showed that normal synchronous cysts were replaced by asynchronous cysts in gsdf-deficient testis. Breeding experiments showed that the sex ratio deviation of gsdf-/- XY gametes in a non-Mendelian manner might be due to the non-segregation of XY chromosomes. Taken together, our results suggest that gsdf plays a role in the proper execution of cytoplasmic and nuclear events through receptor Smad phosphorylation and Sycp3 dephosphorylation to coordinate medaka gametogenesis, including sex-specific mitotic divisions and meiotic recombination.

Smad8 Is Increased in Duchenne Muscular Dystrophy and Suppresses miR-1, miR-133a, and miR-133b.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by skeletal muscle instability, progressive muscle wasting, and fibrosis. A major driver of DMD pathology stems from aberrant upregulation of transforming growth factor β (TGFβ) signaling. In this report, we investigated the major transducers of TGFβ signaling, i.e., receptor Smads (R-Smads), in DMD patient skeletal muscle and observed a 48-fold increase in Smad8 mRNA. Smad1, Smad2, Smad3, and Smad5 mRNA were only minimally increased. A similar pattern was observed in the muscle from the mdx5cv mouse. Western blot analysis showed upregulation of phosphorylated Smad1, Smad5, and Smad8 compared to total Smad indicating activation of this pathway. In parallel, we observed a profound diminishment of muscle-enriched microRNAs (myomiRs): miR-1, miR-133a, and miR-133b. The pattern of Smad8 induction and myomiR suppression was recapitulated in C2C12 muscle cells after stimulation with bone morphogenetic protein 4 (BMP4), a signaling factor that we found upregulated in DMD muscle. Silencing Smad8 in C2C12 myoblasts derepressed myomiRs and promoted myoblast differentiation; there was also a concomitant upregulation of myogenic regulatory factors (myogenin and myocyte enhancer factor 2D) and suppression of a pro-inflammatory cytokine (interleukin-6). Our data suggest that Smad8 is a negative regulator of miR-1, miR-133a, and miR-133b in muscle cells and that the BMP4-Smad8 axis is a driver of dystrophic pathology in DMD.

Single-Cell Analysis of Aneurysmal Aortic Tissue in Patients with Marfan Syndrome Reveals Dysfunctional TGF-β Signaling.

The molecular and cellular processes leading to aortic aneurysm development in Marfan syndrome (MFS) remain poorly understood. In this study, we examined the changes of aortic cell populations and gene expression in MFS by performing single-cell RNA sequencing (scRNA seq) on ascending aortic aneurysm tissues from patients with MFS (n = 3) and age-matched non-aneurysmal control tissues from cardiac donors and recipients (n = 4). The expression of key molecules was confirmed by immunostaining. We detected diverse populations of smooth muscle cells (SMCs), fibroblasts, and endothelial cells (ECs) in the aortic wall. Aortic tissues from MFS showed alterations of cell populations with increased de-differentiated proliferative SMCs compared to controls. Furthermore, there was a downregulation of MYOCD and MYH11 in SMCs, and an upregulation of COL1A1/2 in fibroblasts in MFS samples compared to controls. We also examined TGF-β signaling, an important pathway in aortic homeostasis. We found that TGFB1 was significantly upregulated in two fibroblast clusters in MFS tissues. However, TGF-β receptor genes (predominantly TGFBR2) and SMAD genes were downregulated in SMCs, fibroblasts, and ECs in MFS, indicating impairment in TGF-β signaling. In conclusion, despite upregulation of TGFB1, the rest of the canonical TGF-β pathway and mature SMCs were consistently downregulated in MFS, indicating a potential compromise of TGF-β signaling and lack of stimulus for SMC differentiation.

Curcumin Inhibition of TGFβ signaling in bone metastatic breast cancer cells and the possible role of oxidative metabolites

TGFβ signaling promotes progression of bone-metastatic (BMET) breast cancer (BCa) cells by driving tumor-associated osteolysis, a hallmark of BCa BMETs, thus allowing for tumor expansion within bone. Turmeric-derived bioactive curcumin, enriched in bone via local enzymatic deconjugation of inactive circulating curcumin-glucuronides, inhibits osteolysis and BMET progression in human xenograft BCa BMET models by blocking tumoral TGFβ signaling pathways mediating osteolysis. This is a unique antiosteolytic mechanism in contrast to current osteoclast-targeting therapeutics. Therefore, experiments were undertaken to elucidate the mechanism for curcumin inhibition of BCa TGFβ signaling and the application of this finding across multiple BCa cell lines forming TGFβ-dependent BMETs, including a possible role for bioactive curcumin metabolites in mediating these effects. Immunoblot analysis of TGFβ signaling proteins in bone tropic human (MDA-SA, MDA-1833, MDA-2287) and murine (4T1) BCa cells revealed uniform curcumin blockade of TGFβ-induced Smad activation due to down-regulation of plasma membrane associated TGFβR2 and cellular receptor Smad proteins that propagate Smad-mediated gene expression, resulting in downregulation of PTHrP expression, the osteolytic factor driving in vivo BMET progression. With the exception of early decreases in TGFβR2, inhibitory effects appeared to be mediated by oxidative metabolites of curcumin and involved inhibition of gene expression. Interestingly, while not contributing to changes in Smad-mediated TGFβ signaling, curcumin caused early activation of MAPK signaling in all cell lines, including JNK, an effect possibly involving interactions with TGFβR2 within lipid rafts. Treatment with curcumin or oxidizable analogs of curcumin may have clinical relevancy in the management of TGFβ-dependent BCa BMETs.

SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis.

High myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease‐related mutations of SLC39A5 dysregulate the TGF‐β pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA‐seq and qRT‐PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGF‐β signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R‐Smads) via phosphorylation was greatly blocked. SLC39A5 re‐expression reversed the phenotype of TGF‐β signalling and ECM synthesis in the KO cells. The fact that TGF‐β signalling was zinc‐regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGF‐β signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGF‐β signalling. Overall, the SLC39A5 depletion–induced zinc deficiency destabilized Smad proteins, which inhibited the TGF‐β signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development.

Regulation of Fibroblast Activation Protein by Transforming Growth Factor Beta-1 in Glioblastoma Microenvironment.

The proline-specific serine protease fibroblast activation protein (FAP) can participate in the progression of malignant tumors and represents a potential diagnostic and therapeutic target. Recently, we demonstrated an increased expression of FAP in glioblastomas, particularly those of the mesenchymal subtype. Factors controlling FAP expression in glioblastomas are unknown, but evidence suggests that transforming growth factor beta (TGFbeta) can trigger mesenchymal changes in these tumors. Here, we investigated whether TGFbeta promotes FAP expression in transformed and stromal cells constituting the glioblastoma microenvironment. We found that both FAP and TGFbeta-1 are upregulated in glioblastomas and display a significant positive correlation. We detected TGFbeta-1 immunopositivity broadly in glioblastoma tissues, including tumor parenchyma regions in the immediate vicinity of FAP-immunopositive perivascular stromal cells. Wedemonstrate for the first time that TGFbeta-1 induces expression of FAP in non-stem glioma cells, pericytes, and glioblastoma-derived endothelial and FAP+ mesenchymal cells, but not in glioma stem-like cells. In glioma cells, this effect is mediated by the TGFbeta type I receptor and canonical Smad signaling and involves activation of FAP gene transcription. We further present evidence of FAP regulation by TGFbeta-1 secreted by glioma cells. Our results provide insight into the previously unrecognized regulation of FAP expression by autocrine and paracrine TGFbeta-1 signaling in a broad spectrum of cell types present in the glioblastoma microenvironment.

E3 Ubiquitin Ligases: Key Regulators of TGFβ Signaling in Cancer Progression.

Transforming growth factor β (TGFβ) is a secreted growth and differentiation factor that influences vital cellular processes like proliferation, adhesion, motility, and apoptosis. Regulation of the TGFβ signaling pathway is of key importance to maintain tissue homeostasis. Perturbation of this signaling pathway has been implicated in a plethora of diseases, including cancer. The effect of TGFβ is dependent on cellular context, and TGFβ can perform both anti- and pro-oncogenic roles. TGFβ acts by binding to specific cell surface TGFβ type I and type II transmembrane receptors that are endowed with serine/threonine kinase activity. Upon ligand-induced receptor phosphorylation, SMAD proteins and other intracellular effectors become activated and mediate biological responses. The levels, localization, and function of TGFβ signaling mediators, regulators, and effectors are highly dynamic and regulated by a myriad of post-translational modifications. One such crucial modification is ubiquitination. The ubiquitin modification is also a mechanism by which crosstalk with other signaling pathways is achieved. Crucial effector components of the ubiquitination cascade include the very diverse family of E3 ubiquitin ligases. This review summarizes the diverse roles of E3 ligases that act on TGFβ receptor and intracellular signaling components. E3 ligases regulate TGFβ signaling both positively and negatively by regulating degradation of receptors and various signaling intermediates. We also highlight the function of E3 ligases in connection with TGFβ’s dual role during tumorigenesis. We conclude with a perspective on the emerging possibility of defining E3 ligases as drug targets and how they may be used to selectively target TGFβ-induced pro-oncogenic responses.

MicroRNAs regulating TGFβ and BMP signaling in the osteoblast lineage.

This review showcases miRNAs contributing to the regulation of bone forming osteoblasts through their effects on the TGFβ and BMP pathways, with a focus on ligands, receptors and SMAD-mediated signaling. The goal of this work is to provide a basis for broadly understanding the contribution of miRNAs to the modulation of TGFβ and BMP signaling in the osteoblast lineage, which may provide a rationale for potential therapeutic strategies. Therefore, the search strategy for this review was restricted to validated miRNA-target interactions within the canonical TGFβ and BMP signaling pathways; miRNA-target interactions based only bioinformatics are not presented. Specifically, this review discusses miRNAs targeting each of the TGFβ isoforms, as well as BMP2 and BMP7. Further, miRNAs targeting the signaling receptors TGFβR1 and TGFβR2, and those targeting the type 1 BMP receptors and BMPR2 are described. Lastly, miRNAs targeting the receptor SMADs, the common SMAD4 and the inhibitory SMAD7 are considered. Of these miRNAs, the miR-140 family plays a prominent role in inhibiting TGFβ signaling, targeting both ligand and receptor. Similarly, the miR-106 isoforms target both BMP2 and SMAD5 to inhibit osteoblastic differentiation. Many of the miRNAs targeting TGFβ and BMP signaling components are induced during fracture, mechanical unloading or estrogen deprivation. Localized delivery of miRNA-based therapeutics that modulate the BMP signaling pathway could promote bone formation.

Higher intestinal and circulatory lactate associated NOX2 activation leads to an ectopic fibrotic pathology following microcystin co-exposure in murine fatty liver disease

Clinical studies implicated an increased risk of intestinal fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). Our previous studies have shown that microcystin-LR (MC-LR) exposure led to altered gut microbiome and increased abundance of lactate producing bacteria and intestinal inflammation in underlying NAFLD. This led us to further investigate the effects of the MC-LR, a PP2A inhibitor in activating the TGF-β fibrotic pathway in the intestines that might be mediated by increased lactate induced redox enzyme NOX2. Exposure to MC-LR led to higher lactate levels in circulation and in the intestinal content. The higher lactate levels were associated with NOX2 activation in vivo that led to increased Smad2/3-Smad4 co-localization and high alpha-smooth muscle actin (α-SMA) immunoreactivity in the intestines. Mechanistically, primary mouse intestinal epithelial cells treated with lactate and MC-LR separately led to higher NOX2 activation, phosphorylation of TGFβR1 receptor and subsequent Smad 2/3-Smad4 co-localization inhibitable by apocynin (NOX2 inhibitor), FBA (a peroxynitrite scavenger) and DMPO (a nitrone spin trap), catalase and superoxide dismutase. Inhibition of NOX2-induced redox signaling also showed a significant decrease in collagen protein thus suggesting a strong redox signaling induced activation of an ectopic fibrotic manifestation in the intestines. In conclusion, the present study provides mechanistic insight into the role of microcystin in dysbiosis-linked lactate production and subsequently advances our knowledge in lactate-induced NOX2 exacerbation of the cell differentiation and fibrosis in the NAFLD intestines.

O-GlcNAcylation Dampens Dpp/BMP Signaling to Ensure Proper Drosophila Embryonic Development.

BMP (bone morphogenetic protein) signaling activity is precisely controlled by both pathway agonists andantagonists. Here, we identify a previously unrecognized BMP signaling antagonist. We demonstrate that the Drosophila BMP type I receptor Sax(Saxophone) functions as a Dpp (Decapentaplegic) receptor in Drosophila embryos, but that its activity is normally inhibited by the O-linked glycosyltransferase Sxc (Super sex combs). In wild-type embryos, Sax activity is inhibited, and the BMP type I receptor Tkv (Thickveins) is the sole conduit for Dpp. In contrast, in sxc mutants, the Dpp signal istransduced by both Tkv and Sax, and elevated Dpp signaling results in embryonic lethality. We also demonstrate that Sxc O-glycosylates Sax andobserve elevated Dpp signaling in response to maternal restriction of dietary sugar. These findings link fertility to nutritive environment and point to Sax signaling as the nutrient-sensitive branch of BMP signaling.

Design, synthesis and discovery of 2(1H)-quinolone derivatives for the treatment of pulmonary fibrosis through inhibition of TGF-β/smad dependent and independent pathway

Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening and interstitial lung disease with the median survival of only 3–5 years. However, due to the unclear etiology and problems in accurate diagnosis, up to now only two drugs were approved by FDA for the treatment of IPF and their outcome responses are limited. Numerous studies have shown that TGF-β is the most important cytokine in the development of pulmonary fibrosis and plays a role through its downstream signaling molecule TGF-binding receptor Smads protein. In this paper, compounds bearing 2(1H)-quinolone scaffold were designed and their anti-fibrosis effects were evaluated. Of these compounds, 20f was identified as the most active one and could inhibit TGF-β-induced collagen deposition of NRK-49F cells and mouse fibroblasts migration with comparable activity and lower cytotoxicity than nintedanib in vitro. Further mechanism studies indicated that 20f reduced the expression of fibrogenic phenotypic protein α-SMA and collagen Ⅰ by inhibiting the TGF-β/Smad dependent pathways and ERK1/2 and p38 pathways. Moreover, compared with the nintedanib, 20f (100 mg/kg/day, p.o) more effectively alleviated collagen deposition in lung tissue and delayed the destruction of lung tissue structure both in bleomycin-induced prevention and treatment mice pulmonary fibrosis models. The immunohistochemical experiments further showed that 20f could block the expression level of phosphorylated Smad3 in the lung tissue cells, which resulted in its anti-fibrosis effects in vivo. In addition, 20f demonstrated good bioavailability (F = 41.55% vs 12%, compare with nintedanib) and an appropriate elimination half-life (T1/2 = 3.5 h), suggesting that 20f may be a potential drug candidate for the treatment of pulmonary fibrosis.

Maintenance of the Undifferentiated State in Myogenic Progenitor Cells by TGFβ Signaling is Smad Independent and Requires MEK Activation.

Transforming growth factor β (TGFβ) is a pluripotent cytokine and regulates a myriad of biological processes. It has been established that TGFβ potently inhibits skeletal muscle differentiation; however, the molecular mechanism is not clearly defined. Previously, we reported that inhibition of the TGFβ canonical pathway by an inhibitory Smad, Smad7, does not reverse this effect on differentiation, suggesting that activation of receptor Smads (R-Smads) by TGFβ is not responsible for repression of myogenesis. In addition, pharmacological blockade of Smad3 activation by TGFβ did not reverse TGFβ’s inhibitory effect on myogenesis. In considering other pathways, we observed that TGFβ potently activates MEK/ERK, and a pharmacological inhibitor of MEK reversed TGFβ’s inhibitory effect on myogenesis, as indicated by a myogenin promoter-reporter gene, sarcomeric myosin heavy chain accumulation, and phenotypic myotube formation. Furthermore, we found that c-Jun, a known potent repressor of myogenesis, which is coincidently also a down-stream target of MEK/ERK signaling, was phosphorylated and accumulates in the nucleus in response to TGFβ activation. Taken together, these observations support a model in which TGFβ activates a MEK/ERK/c-Jun pathway to repress skeletal myogenesis, maintaining the pluripotent undifferentiated state in myogenic progenitors.

Treadmill exercise promotes the expression of TGF-β1 and Smad3 protein, inhibiting cell apoptosis after cerebral ischemia

Objective To observe the effect of treadmill exercise on the expression of transforming growth factor-β1 (TGF-β1) and its receptor Smad3 protein as well as on cell apoptosis in the ischemic boundary zone, so as to explore how exercise promotes the recovery of neurological function after cerebral ischemia. Methods Thirty adult male Sprague-Dawley rats were randomly divided into a sham group (n=6), a model group (n=12) and an exercise group (n=12). A modified Longa′s method was used to establish an animal model of cerebral ischemia by occluding the right middle cerebral artery in the rats of the model and exercise groups. Those of the sham group were subjected to the same surgical procedure except that no thread was inserted. After 24h the exercise group began treadmill training, while the other two groups were left on the treadmill without training. Modified neurological severity scores (mNSSs) were used to quantify the rats′ neurological functioning on the 3rd, 7th and 14th day after the surgery. The ischemic boundary zone tissue was then dissected to detect the expression of TGF-β1 and Smad3 protein using western blotting. Cell apoptosis was detected by TUNEL staining. Results The average mNSS scores of the exercise group on the 7th and the 14th day were significantly lower than those of the model group at the same time points. The average expression level of TGF-β1 and Smad3 protein in the exercise group was significantly higher than in the model group. The number of TUNEL-positive cells in the exercise group was significantly lower than in the model group on the 14th day. Conclusions Treadmill exercise can improve the recovery of neurological function after cerebral ischemia. It may be partly due to upregulating the expression of TGF-β1 and Smad3 protein, which inhibit cell apoptosis in the ischemic boundary zone. Key words: Cerebral ischemia; Exercise; Transforming growth factor; Smad3 protein; Apoptosis

Memory Decline and Its Reversal in Aging and Neurodegeneration Involve miR-183/96/182 Biogenesis.

Aging is characterized by progressive memory decline that can lead to dementia when associated with neurodegeneration. Here, we show in mice that aging-related memory decline involves defective biogenesis of microRNAs (miRNAs), in particular miR-183/96/182 cluster, resulting from increased protein phosphatase 1 (PP1) and altered receptor SMAD (R-SMAD) signaling. Correction of the defect by miR-183/96/182 overexpression in hippocampus or by environmental enrichment that normalizes PP1 activity restores memory in aged animals. Regulation of miR-183/96/182 biogenesis is shown to involve the neurodegeneration-related RNA-binding proteins TDP-43 and FUS. Similar alterations in miR-183/96/182, PP1, and R-SMADs are observed in the brains of patients with amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD), two neurodegenerative diseases with pathological aggregation of TDP-43. Overall, these results identify new mechanistic links between miR-183/96/182, PP1, TDP-43, and FUS in age-related memory deficits and their reversal.

PO-185 Towards dynamic targeting of TGF-β in metastatic melanoma using intravital microscopy

IntroductionMelanoma patients diagnosed with liver metastasis have a poor prognosis. TGF-β inhibitors give mixed results in clinical trials with metastatic melanoma patients. To more specifically target TGF-β in liver metastasis melanoma patients, it is necessary to unravel the spatio-temporal function of the TGF-β pathway in hepatic colonisation of melanoma cells. TGF-β is a multifunctional cytokine that signals via TGF-β receptors and downstream SMAD effector proteins. SMAD proteins regulate transcription by binding, among others, to CAGA elements in the DNA. It can exert both pro-and anti-tumorigenic functions, depending on cellular context. It acts on tumour cells, as well as the tumour micro-environment and the immune system. In metastatic melanoma cells, TGF-β can stimulate invasion and metastasis. To unravel its exact role during the different processes of metastasis, we will investigate the spatio-temporal patterns of TGF-β signalling during metastatic colonisation using intravital microscopy (IVM).Material and methodsSpatio-temporal patterns of TGF-β signalling will be studied in vitro and in vivo. For our in vivo studies, we are using an experimentally induced liver metastasis model, injecting highly aggressive B16F10 melanoma cells in immune competent C57BL/6 mice. By injecting these tumour cells in the mesenteric vein, cells will be transported directly to the liver, the first capillary network the cells will encounter. An abdominal imaging window will be placed after cell injection to visualise the different steps of metastasis in real-time using IVM. We developed a rapid CAGA12-GFP-based transcriptional reporter, which expresses a fluorophore upon TGF-β receptor activation and SMAD binding. Upon the expression of this reporter in B16F10 cells, activation of the TGF-β pathway can be monitored over time. By genetic manipulation of B16F10 cells to express dominant negative or constitutively active TGF-β receptors, the role of TGF-β can be assessed for the different steps of metastasis.Results and discussionsWe confirmed earlier reports showing that B16F10 cells show a transcriptional CAGA response upon TGF-β3 stimulation. Using our liver metastasis model, the injected B16F10 cells are able to perform the steps of metastasis and form liver metastasis within a short time frame. The use of the rapid TGF-β reporter during intravital imaging will show the involvement of the TGF-β pathway in the different phases of metastasis.ConclusionB16F10 cells have a functional TGF-β pathway and are able to colonise the liver.

Abstract 2685: Targeting TGFβ signaling pathways in bone metastatic breast cancer cells to limit metastatic progression using curcuminoids, a turmeric-derived natural product

Abstract Tumor-cell TGFβ signaling has been demonstrated to drive osteolytic breast cancer bone metastases (BrCa BMET) in mouse models. This established an important link between the microenvironment and tumor cells in bone, wherein osteoclast-mediated bone resorption stimulates the release of TGFβ from bone matrix, which induces tumoral secretion of osteolytic factors, such as parathyroid-related protein (PTHrP) that drive further osteoclast-mediated bone destruction in a positive feedback loop. These findings have prompted interest in the use of pharmacologic TGFβ inhibitors, currently under development, for the treatment of BrCa BMET. Interestingly, studies have demonstrated antagonistic effects of a natural product, turmeric-derived polyphenols called curcuminoids (CURC), on BrCa BMET progression and TGFβ-inducible, Smad-dependent signaling in a TGFβ-dependent human xenograft model (human MDA-MB-231 [MDA-SA]).The studies described here were undertaken to determine whether this inhibitory effect is generalizable to other BrCa cells that form TGFβ-dependent BMETs (human MDA-1833 and MDA-2287; murine 4T1) and to begin to elucidate the mechanism by which CURC block BrCa cell TGFβ signaling. Analogous to prior findings using MDA-SA cells forming BMET that are dependent on TGFβ-inducible tumoral secretion of PTHrP via Smad and non-Smad (specifically, p38) dependent pathways, CURC inhibited TGFβ inducible activation (phosphorylation) of receptor Smad2 (Western) in all TGFβ-dependent BrCa cell lines tested.In MDA-SA cells, CURC inhibition of TGFβ-stimulated Smad2 phosphorylation was time- and dose-dependent (Western), and associated with decreased expression of a Smad-dependent luciferase reporter gene in transiently transfected cells. In both MDA-SA and 4T1 cells, these changes were accompanied by decreased TGFβ-stimulated PTHrP secretion (RIA). These effects in MDA-SA cells were not associated with changes in the expression of canonical TGFβ signaling proteins, such as Smad anchor for receptor activation (SARA), which is involved in Smad phosphorylation, or Smad4, a required cofactor for Smad-mediated gene transcription. Also unchanged were noncanonical TGFβ-stimulated, Smad-independent signaling pathways (e.g., p38, ERK1/2 or JNK). Of note, however, in all cell lines tested, CURC inhibition of Smad2 phosphorylation was accompanied by a decrease in constitutive Smad2 levels, with variable decreases in the ratio of phosphorylated vs. total Smad levels. These results suggest that CURC can specifically and uniformly inhibit Smad-regulated TGFβ signaling in bone metastatic BCa cells forming TGFβ-dependent osteolytic lesions, an effect that may be mediated, at least in part, by changes in constitutive levels of receptor-regulated Smad expression. Citation Format: Andrew Kunihiro, Julia A. Brickey, Jen B. Frye, Janet L. Funk. Targeting TGFβ signaling pathways in bone metastatic breast cancer cells to limit metastatic progression using curcuminoids, a turmeric-derived natural product [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2685.

Transcriptome sequencing analysis reveals the effect of combinative treatment with low‑intensity pulsed ultrasound and magnesium ions on hFOB1.19 human osteoblast cells.

Biodegradable magnesium (Mg) materials are considered ideal as osteosynthesis implants. However, clinical application has proven complex. This is primarily associated with the issue of reducing the extent of implant degradation to a range acceptable for the human body, while simultaneously enhancing osteogenesis or osteoinduction. In the present study, a combination of Mg ions and low-intensity pulsed ultrasound (LIPUS) treatment was applied in hFOB 1.19 human osteoblast cells as a potential strategy to resolve this issue. A total of 7,314 differentially expressed genes (DEGs) and 826 shared DEGs in hFOB1.19 osteoblast cells were identified by microarray analysis following treatment with Mg and/or LIPUS. Gene Ontology analysis demonstrated that among cells treated with a combination of Mg and LIPUS, DEGs were significantly enriched in various functional annotations, including ‘wound healing’, ‘transforming growth factor beta receptor signaling pathway’, ‘transcription, DNA-templated’, ‘receptor complex’, ‘nucleus’, ‘SMAD protein complex’, ‘DNA binding’, ‘metal ion binding’ and ‘GTPase activator activity’. Notably, the transforming growth factor (TGF)-β, mitogen-activated protein kinase (MAPK) and tumor necrosis factor (TNF) signaling pathways were preferentially overrepresented in the Mg and LIPUS combination group, which was subsequently confirmed by reverse transcription-quantitative polymerase chain reaction. Furthermore, genes involved in osteoblast mineralization promotion, including bone morphogenetic protein 6, noggin, bone morphogenetic protein receptor (BMPR)1A, BMPR2 and SMAD 5/8, were significantly upregulated following combination treatment compared with the control group. Genes involved in the promotion of migration, including c-Jun N-terminal kinase, doublecortin, paxillin and Jun proto-oncogene AP-1 transcription factor subunit, were also upregulated in the combination treatment group compared with the control group. The DEG data were supported by morphological observations of the osteoblasts using alizarin red S staining and wound healing assays, which indicated that Mg and LIPUS combinative treatment had a synergistic effect on osteoblast mineralization and migration. Additionally, the combined treatment significantly upregulated metal transporter genes associated with Mg entry, including ATPase Na+/K+-transporting subunit α1, cyclin and CBS domain divalent metal cation transport mediator 2, K+ voltage-gated channel subfamily J member 14, transient receptor potential cation channel (TRP) subfamily M member 7 and TRP subfamily V member 2. In summary, the findings of the present study revealed that combined stimulation with Mg and LIPUS may exhibit a synergistic effect on human osteoblast bone formation through the TGF-β, MAPK and TNF signaling pathways, while also facilitating Mg influx. The present study demonstrated the potential of combinative LIPUS and Mg treatment as a novel therapeutic strategy for enhancing the osteoinduction, biocompatibility and biosafety of biodegradable Mg implants.