Activation Of Smad Signaling Research Articles

TGF-β downstream Smad3 and MAPK signaling are mutually antagonistic in liver progenitor cells

Introduction: Liver progenitor cells (LPCs) are a subpopulation of cells which are always found in chronic injuried liver, and are contribute to liver fibrosis and liver cancer initiation under the circumstance of chronic inflammation. However, the precise role of LPCs in responding to chronic inflammatory signals are remain elusive. Method: We observed the effects of TGF-β, one of the most commonly detected cytokine in chronic hepatitis, in LPCs, by using CCK-8, F-actin staining, western blot, and transwell analyses in liver progenitoc cell lines (WB-F344 and LE/6). Western blot and luciferase reporter analyses were utilized to measure the activation of Smad and MAPK signaling in LPCs. We then overexpressed the expression of Smad3 in LPCs by adenovirus carrying wild type Smad3, mutated Smad3 at specific serine phosphorylation site respectively, and observed their downstream effects. Result: In LPCs, TGF-β showed limited cytostatic and epithelial-mesenchymal transition (EMT) effects, and TGF-β activated both Smad and MAPK (Erk, JNK and p38 MAPK) signaling. TGFβ-Smad signaling was the inducer of cytostasis and EMT, whereas TGFβ-MAPK signaling showed opposite effects. Moreover, the activity of TGF-β downstream Smad and MAPK signaling were mutually restricted. Mechansitically, TGF-β activated Smad signaling through serine phosphorylation of both C-terminal and linker region of Smad2 and Smad3 in LPCs, and TGFβ-MAPK signaling inhibited the phosphorylation of Smad3 at C-terminal, but it reinforced the phosphorylation of Smad3 at the linker region. Phosphorylation of Smad3 at C-terminal contributed to TGF-β induced cytostasis and EMT effects, whereas the phosphorylation of Smad3 at the linker region inhibited these effects in LPCs. Conclusion: TGF-β downstream Smad and MAPK signaling were mutually antagonistic in LPCs through phosphorylation of Smad3 at different serine sites, and this antagonism may help LPCs to survive in the circumstance of chronic inflammation. Additionally, imbalanced antagonisitc Smad3 and MAPK signaling may contribute to the malignant transformation of LPCs.

Magnesium isoglycyrrhizinate ameliorates high fructose-induced liver fibrosis in rat by increasing miR-375-3p to suppress JAK2/STAT3 pathway and TGF-β1/Smad signaling.

Increasing evidence has demonstrated that excessive fructose intake induces liver fibrosis. Epithelial-mesenchymal transition (EMT) driven by transforming growth factor-β1 (TGF-β1)/mothers against decapentaplegic homolog (Smad) signaling activation promotes the occurrence and development of liver fibrosis. Magnesium isoglycyrrhizinate is clinically used as a hepatoprotective agent to treat liver fibrosis, but its underlying molecular mechanism has not been identified. Using a rat model, we found that high fructose intake reduced microRNA (miR)-375-3p expression and activated the janus-activating kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) cascade and TGF-β1/Smad signaling, which is consistent with the EMT and liver fibrosis. To further verify these observations, BRL-3A cells and/or primary rat hepatocytes were exposed to high fructose and/or transfected with a miR-375-3p mimic or inhibitor or treated with a JAK2 inhibitor, and we found that the low expression of miR-375-3p could induce the JAK2/STAT3 pathway to activate TGF-β1/Smad signaling and promote the EMT. Magnesium isoglycyrrhizinate was found to ameliorate high fructose-induced EMT and liver fibrosis in rats. More importantly, magnesium isoglycyrrhizinate increased miR-375-3p expression to suppress the JAK2/STAT3 pathway and TGF-β1/Smad signaling in these animal and cell models. This study provides evidence showing that magnesium isoglycyrrhizinate attenuates liver fibrosis associated with a high fructose diet.

Cerium-doped whitlockite nanohybrid scaffolds promote new bone regeneration via SMAD signaling pathway

The development of nanoscience, nanohybrid technology and molecular biology provide an important strategy in the construction of novel bone scaffolds. Nanosized whitlockite (WH) and trace cerium (Ce) element exist widely in human bones; however their positive functions on bone cells/tissues still remain unknown. Inspired by the chemical components and nanostructures of bones, Ce-doped WH/chitosan (Ce-WH/CS) nanohybrid scaffolds were developed for new bone regeneration. The Ce-WH/CS scaffolds exhibited interconnected macropores with sizes of ∼300 μm, which facilitated cell adhesion and bone tissue in-growth. Ce-WH nanoparticles with sizes of 10–50 nm were dispersed on macroporous walls. The WH/CS scaffolds with or without doping Ce3+ ions had non-toxicity to human adipose derived stromal cell (hADSCs). Interestingly, the Ce3+ ions in the Ce-WH/CS scaffolds significantly improved osteogenic activity via activated SMAD signaling pathway, and up-regulated the expression of osteogenic-related genes including osteocalcin (OCN), osterix (OSX) and collagen 1A1 (COL1A1). Rat cranial defect models further demonstrated that Ce-WH/CS scaffolds remarkably accelerated material biodegradation and bone tissue growth compared to WH/CS scaffolds. The exciting finding revealed the positive functions of Ce-WH nanoparticles on bone tissues, and provided a promising platform for new bone regeneration.

P6 - Redox Modification of the Cytoskeleton During Cell Adhesion and Migration

Actin cytoskeleton assembly and organization are modulated by reactive oxygen species and the cellular redox environment. Focal adhesions link the cytoskeleton to the extracellular matrix and play an important role in growth and migration of vascular smooth muscle cells. We have previously shown that NADPH oxidase Nox4, a H2O2-producing enzyme, and its binding partner, polymerase delta interacting protein 2 (Poldip2), regulate turnover of focal adhesions. Downstream targets of Nox4 include Hic-5 and Hsp27, as well as the small GTPase, Rho A. Activation of Smad signaling by TGFp, which is known to strengthen focal adhesions, is Nox4-dependent, as is upregulation of Hsp27 and Hic-5 by TGFp. Similarly, overexpression of Poldip2 activates RhoA to prevent focal adhesion dissolution, while dominant-negative RhoA reverses the effects of Nox4/Poldip2 on focal adhesions. More recently, we discovered that H2O2 levels increase during integrin-mediated cell attachment due to activation of NOX4. Attachment induces F-actin oxidation by sulfenylation, and depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibits adhesion-induced F-actin oxidation. Of importance, the binding of F-actin to vinculin, a protein present in focal adhesion complexes that regulates focal adhesion maturation, is inhibited after transfection of an oxidation-resistant actin mutant. Silencing of Poldip2 or NOX4 also impairs actin-vinculin interaction. Taken together, our work has identified a major role for Poldip2/Nox4 in cytoskeletal organization, focal adhesion turnover and smooth muscle cell migration.

Rhynchophylline attenuates allergic bronchial asthma by inhibiting transforming growth factor-β1-mediated Smad and mitogen-activated protein kinase signaling transductions in vivo and in vitro.

Rhynchophylline (Rhy) is a major active component of Uncaria rhynchophylla and exhibits the potential to inhibit the proliferation of airway smooth muscle cells (ASMCs). In the current study, it was hypothesized that Rhy serves a key role in the anti-asthma effect of Uncaria rhynchophylla by inhibiting transforming growth factor-β1 (TGF-β1)-mediated activation of Smad and mitogen-activated protein kinase (MAPK) signaling. Allergic asthma was induced in mice using ovalbumin (OVA), and the effect of Rhy treatment on inflammatory and allergic responses in the bronchoalveolar lavage fluid (BALF) and serum of mice was determined. Subsequently, the changes in TGF-β1-induced Smad and MAPK signaling following Rhy administration were detected to determine the mechanism associated with this treatment. In addition, TGF-β1 was employed to induce hyperplasia of ASMCs, and the effect of Rhy on proliferation of ASMCs, and Smad and MAPK signaling in vitro was also assessed. The administration of Rhy attenuated the recruitment of eosinophils in BALF induced by OVA, which was associated with the suppressed production of immunoglobulin E, interleukin (IL)-13, IL-4 and IL-5. At the molecular level, the administration of Rhy suppressed the expression levels of TGF-β1, Smad4, p-Smad2 and p-Smad3, while it induced the expression of Smad7, indicating the inhibitory effect of Rhy on TGF-β1-mediated Smad and MAPK signaling. Furthermore, Rhy inhibited the proliferation of ASMCs and, similar to the results of the in vivo assay, it blocked the pro-hyperplasia signaling transduction in vitro. In conclusion, the current study demonstrated the anti-asthma effect of Rhy, which depended on the inhibition of TGF-β1-mediated Smad and MAPK signaling.

Chitotriosidase enhances TGFβ-Smad signaling and uptake of β-amyloid in N9 microglia

TGFβ-Smad signaling is involved in the modulation of β-amyloid (Aβ) clearance in microglia. This signaling is impaired in the brain of Alzheimer's disease (AD). Chitotriosidase (CHIT1) is elevated in the cerebrospinal fluid and peripheral blood of AD patients, and has been reported to augment TGFβ signaling in fibroblast and T cells. In this study, we investigated the role of CHIT1 in TGFβ-Smad signaling and Aβ phagocytosis in N9 microglia. We found that CHIT1 significantly enhanced TGFβ1-induced expression of TβRI (TGFβ receptor I) and activation of Smad signaling. CHIT1 did not affect Aβ uptake in microglia by itself, but did enhance TGFβ1-induced phagocytosis of Aβ, which was blocked by pretreatment with SB431542 (TβRI inhibitor). These results indicate that CHIT1 may play a protective role in Aβ clearance by enhancing TGFβ signaling in microglia.

Sphingosine kinase 2 cooperating with Fyn promotes kidney fibroblast activation and fibrosis via STAT3 and AKT

Sphingosine kinases (Sphks) are the rate-limiting enzymes in the conversion of sphingosine to biologically active sphingosine-1-phosphate. The present study aimed to determine the role of Sphk2 and its downstream targets in renal fibroblast activation and interstitial fibrosis. In the kidney interstitium of patients with renal fibrosis, Sphk2high-expressing cells (mainly interstitial fibroblasts) were significantly elevated and highly correlated with disease progression in patients. In a murine model of renal interstitial fibrosis, Sphk2 was upregulated in the kidney of wild-type mice in response to disease progression. Importantly, Sphk2-knockout (KO) mice exhibited significantly lower levels of extracellular matrix (ECM) production and a suppressed inflammatory response in the kidney tissues, compared to those in their wild-type counterparts, whereas the expression of TGF-β1 was unaffected. TGF-β1 effectively upregulated Sphk2 expression in the renal interstitial fibroblast line, NRK-49F, independent of canonical Smad signaling activation. Furthermore, siRNA-mediated Sphk2 knockdown or suppression of Sphk2 activity by ABC294640 exposure effectively attenuated AKT and STAT3 activation and ECM production, but had no effects on Smad2 and Smad3 activation. Sphk2 phosphorylated Fyn to activate downstream STAT3 and AKT, thereby promoting ECM synthesis. Therefore, our findings indicate that targeting Sphk2-Fyn-STAT3/AKT signaling pathway may be a novel therapeutic approach for renal fibrosis.

Lack of microRNA-155 ameliorates renal fibrosis by targeting PDE3A/TGF-β1/Smad signaling in mice with obstructive nephropathy.

Although microRNA-155 (miR-155) is implicated in the pathogenesis of several fibrotic diseases, information regarding its functional role in renal fibrosis is limited. The current study aims to investigate the effects of miR-155 on renal fibrosis in unilateral ureteral occlusion (UUO) mice. MiR-155 level was significantly increased in renal tissues of UUO mice and TGF-β1-treated HK2 cells. Masson's trichrome staining showed that delivery of adeno-associated virus encoding miR-155 inhibitor led to a decrease in renal fibrosis induced by UUO. The increased expression of plasminogen activator inhibitor type 1, collagen III and collagen IV was also inhibited after miR-155 inhibition. In addition, miR-155 knockdown also prevented TGF-β1-induced epithelial-mesenchymal transition, concomitantly with a restoration of E-cadherin expression and a decrease of vimentin expression. Computational analysis revealed that miR-155 directly targets at 3'UTR of PDE3A. Overexpression of miR-155 suppressed the luciferase activity and protein expression of PDE3A, whereas inhibition of miR-155 increased PDE3A luciferase activity and expression. Furthermore, miR-155 inhibited TGF-β1-induced the increase of TGF-β1 expression and Smad-2/3 phosphorylation in HK2 cells. In contrast, knockdown of PDE3A reversed the effect of miR-155 inhibition on TGF-β1 expression. This study demonstrates that knockdown of miR-155 attenuates renal fibrosis via inhibiting TGF-β1/Smad signaling activation by targeting the upstream molecule PDE3A. This study suggests that miR-155 inhibition may be a novel therapeutic approach for preventing fibrotic kidney diseases.

Abstract 100: PRDM16 is a Novel Regulator of Cardiac Hypertrophy, Remodeling and Mitochondrial Dynamics

Elucidation of the mechanisms that cause cardiac hypertrophy is essential for developing novel therapeutic strategies to prevent or reverse these prevalent conditions. Prdm16/Mel1 (PRDM16) belongs to the Prdm family of transcription factors involved in cell fate determination and development. PRDM16 deletion, found in humans with 1p36 deletion syndrome, has been associated with two main cardiomyopathies: left ventricle non-compaction (LVNC) and dilated cardiomyopathy. Nonetheless, its role in the heart and the underlying mechanisms by which these mutations contribute to the development of cardiomyopathy are unknown. We sought to directly assess the role of Prdm16 in the developing and adult heart by generating both a germline (cPRDM16) and a tamoxifen-inducible (ciPRDM16) cardiomyocyte-specific Prdm16 knockout mice. cPRDM16KO mice died shortly after birth, showing grossly abnormal ventricles that were reduced in size and often exhibiting a cleft at their apex, consistent with LVCN. Both heterozygous mice (cPRDM16KOhet) and ciPRDM16KO are viable and displayed markedly increased heart size, cardiomyocyte cross sectional area, collagen deposition and expression of genes involved in cardiac hypertrophy and ECM production. We observed an increased in TGF-β receptors mRNA expression as well as activation of Smad signaling in the hearts of cPRDM16KO neonates, cPRDM16 heterozygous, and ciPRDM16 KO mice. Interestingly, we observed a systematic reduction in Drp1 phosphorylation on Ser616 (activation) in the hearts of cPRDM16KO and cPRDM16het mice. The reduction in Drp1 phosphorylation was associated with more tubular mitochondria in cPRDM16KO mice. Interestingly, by 25 weeks, cPRDM16het mice accumulated more mitochondrial proteins, despite exhibiting no change in PGC1α. Together, these data reveal previously unknown roles for Prdm16 in protection against cardiac hypertrophy and fibrosis through the suppression of TGF-β/Smad signaling in the heart. They also reveal roles for PRDM16 in the modulation of mitochondrial dynamics and clearance in the heart, a process that likely involves the fission protein Drp1.

Abstract 2096: Multicellular modeling and identification of protein ligand-mediated and exosome-mediated crosstalk signaling cascades in the heterogeneous ovarian tumor microenvironment

Abstract Ovarian cancer is the most lethal gynecologic malignancy in the US. While research efforts have been dedicated to identify disease related genes and mutations in cancer cells, the stroma-tumor interactions in the tumor microenvironment and their roles in disease progression is relatively unexplored. The identification of stroma-tumor crosstalk networks with prognostic value presents a unique opportunity for developing new treatment strategies. To capture such an opportunity, a multicellular computational modeling platform, the Cell-Cell Communication Explorer (CCCExplorer), has been developed to identify novel ligand- and exosome-mediated crosstalk networks among different cell types within tumor microenvironment. With transcriptome profiling data generated from laser microdissected ovarian cancer cells and cancer associated fibroblasts (CAFs) in high-grade serous ovarian caner tissue as inputs, CCCExplorer identified TGF-β–dependent and TGF-β–independent Smad signaling networks as protein ligand-mediated crosstalk signaling cascades activated in CAFs associated with poor patient survival rates. Validation studies by co-culturing ovarian cancer cells with CAFs indicated that activation of Smad signaling in CAFs promoted aggressive phenotypes of ovarian cancer cells while inhibition of Smad signaling in CAFs suppressed ovarian cancer progression both in vitro and in vivo. On the other hand, CCCExplorer used transcriptome data generated from exosomes isolated from CAFs and normal fibroblasts (NFs) as well as from CAF- and NF-derived exosomes treated ovarian cancer cells to compute and predict crosstalk cascades mediated by fibroblast-derived exosomes. Our results showed that CAF-derived exosomal microRNAs and LncRNAs promoted signaling cascade activation and the subsequent increase of chemoresistance, motility and proliferation in ovarian cancer cells. After functional validation of predicted crosstalk cascades, CCCExplorer can further be used to query underlying drug information databases to identify and rank candidate drug agents that could target cancer progression-associated crosstalk between ovarian cancer cells and CAFs. Our findings demonstrate a powerful tool at the multi-cellular level of heterogeneous tumor microenvironment in which a computational platform can be used to understand, model, visualize, predict, and target crosstalk signaling cascades and coarse-grained behavior of heterogeneous tumor microenvironment. We are applying CCCExplorer to uncover activated crosstalk networks among ovarian cancer cells and stromal cells and to discover drug compounds targeting these pathways, potentially leading to faster cures for ovarian cancer after the validation and confirmation using in vitro and in vivo models. Citation Format: Tsz-Lun Yeung, Jianting Sheng, Samuel C. Mok, Stephen T. C. Wong. Multicellular modeling and identification of protein ligand-mediated and exosome-mediated crosstalk signaling cascades in the heterogeneous ovarian tumor microenvironment [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 2096.

Systematic Identification of Druggable Epithelial-Stromal Crosstalk Signaling Networks in Ovarian Cancer.

BackgroundBulk tumor tissue samples are used for generating gene expression profiles in most research studies, making it difficult to decipher the stroma–cancer crosstalk networks. In the present study, we describe the use of microdissected transcriptome profiles for the identification of cancer–stroma crosstalk networks with prognostic value, which presents a unique opportunity for developing new treatment strategies for ovarian cancer.MethodsTranscriptome profiles from microdissected ovarian cancer–associated fibroblasts (CAFs) and ovarian cancer cells from patients with high-grade serous ovarian cancer (n = 70) were used as input data for the computational systems biology program CCCExplorer to uncover crosstalk networks between various cell types within the tumor microenvironment. The crosstalk analysis results were subsequently used for discovery of new indications for old drugs in ovarian cancer by computational ranking of candidate agents. Survival analysis was performed on ovarian tumor–bearing Dicer/Pten double-knockout mice treated with calcitriol, a US Food and Drug Administration–approved agent that suppresses the Smad signaling cascade, or vehicle control (9–11 mice per group). All statistical tests were two-sided.ResultsActivation of TGF-β-dependent and TGF-β-independent Smad signaling was identified in a particular subtype of CAFs and was associated with poor patient survival (patients with higher levels of Smad-regulated gene expression by CAFs: median overall survival = 15 months, 95% confidence interval [CI] = 12.7 to 17.3 months; vs patients with lower levels of Smad-regulated gene expression: median overall survival = 26 months, 95% CI = 15.9 to 36.1 months, P = .02). In addition, the activated Smad signaling identified in CAFs was found to be targeted by repositioning calcitriol. Calcitriol suppressed Smad signaling in CAFs, inhibited tumor progression in mice, and prolonged the median survival duration of ovarian cancer–bearing mice from 36 to 48 weeks (P = .04).ConclusionsOur findings suggest the feasibility of using novel multicellular systems biology modeling to identify and repurpose known drugs targeting cancer–stroma crosstalk networks, potentially leading to faster and more effective cures for cancers.

Hypoxia Promotes Ectopic Adhesion Ability of Endometrial Stromal Cells via TGF-β1/Smad Signaling in Endometriosis.

Hypoxia plays a vital role in the progression of endometriosis. Additionally, integrin-mediated aberrant adhesion is also essential for establishment of endometriotic lesions. In this study, we sought to determine the function of hypoxia in integrin-mediated adhesion of endometrial stromal cells (ESCs) in endometriosis. The expressions of adhesion molecule integrins (integrin α5, integrin αV, integrin β3, and integrin β5) were determined in 15 normal endometria and 15 paired eutopic and ectopic endometria by immunohistochemistry. Thirteen primary ESCs from patients with peritoneal endometriosis in the proliferative phase were cultured under a hypoxic (1% O2) or normoxic (21% O2) environment, and the expression levels of hypoxia-inducible factor (HIF)-1α, transforming growth factor (TGF)-β1, and integrins were detected by quantitative reverse transcription polymerase chain reaction and western blot. The alteration of integrins in endometriotic mouse models were also explored. Our results demonstrated that HIF-1α and integrins were highly expressed in ESCs of endometriotic lesions compared with ESCs of eutopic and normal endometrium. Hypoxia treatment significantly increased ESC adhesion abilities and integrin expression, which were positively correlated with TGF-β1 expression. Both TGF-β1 and hypoxia enhanced ESC adhesion properties, whereas hypoxia combined with TGF-β1 receptor inhibitor inhibited ESC adhesion. Knockdown of HIF-1α attenuated TGF-β1/Smad signaling activation and integrin expression and reduced ESC adhesion. Higher expression levels of HIF-1α, TGF-β1, and integrins were detected in endometriotic cysts from mice models. Our findings provide a novel insight of endometriosis that the hypoxic microenvironment stimulates ESCs to produce excessive TGF-β1 and activates the TGF-β1/Smad signaling pathway, thus enhancing integrin expression and the adhesion ability of ESCs.

Poricoic acid ZA, a novel RAS inhibitor, attenuates tubulo-interstitial fibrosis and podocyte injury by inhibiting TGF-β/Smad signaling pathway

BackgroundThe pathogenesis of tubulo-interstitial fibrosis and glomerulosclerosisis was characterized by cellular hypertrophy, extracellular matrix accumulation and podocyte detachment. Poricoic acid ZA (PZA) is a tetracyclic triterpenoid compound extracted from the surface layer of Poria cocos (LPC), which have been used extensively for diuretic and renoprotective effects. MethodsThe anti-fibrotic effect of PZA is investigated in HK-2 cells and podocytes induced by TGF-β1 and angiotensin II (ANGII). qRT-PCR, siRNA, immunofluorescence staining, co-immunoprecipitation and Western blot analyses are used to evaluate the expression of RAS signaling, TGF-β/Smad pathway, epithelial-to-mesenchymal transition (EMT) and podocyte markers. ResultsPZA restores the mRNA and protein expression of EMT in HK-2 cells. Specific TGF-β1-siRNA efficiently blocks ANGII-induced protein expression of TGF-β1 and further inhibits activated Smad signaling. PZA significantly attenuates up-regulation of angiotensinogen, renin, ACE and AT1. Further, PZA reverses up-regulation of TGFβRII and suppresses Smad proteins. Simultaneously, PZA inhibits the protein interaction of TGF-β receptor and Smads and PZA also inhibits activated RAS and TGF-β/Smad signaling cascade and up-regulates protein expression of podocyte markers and mitigates podocyte injury. ConclusionsThis study demonstrated the beneficial role of PZA in renal fibrosis and podocyte injury. Our study highlighted that PZA inhibits RAS and further suppresses TGF-β/Smad pathway through inhibiting Smad2/3 phosphorylation via blocking Smad2/3-TGFβRI protein interaction. PZA is implicated in activation of RAS/TGF-β/Smad axis in HK-2 cells and podocytes. PZA could be considered as a novel RAS inhibitor for treating CKD.

TGF-β1 targets Smad, p38 MAPK, and PI3K/Akt signaling pathways to induce PFKFB3 gene expression and glycolysis in glioblastoma cells.

In human cancers, transforming growth factor-β1 (TGF-β1) plays a dual role by acting as both a tumor suppressor and a promoter of tumor metastasis. Although TGF-β1 contributes to the metabolic reprogramming of cancer cells and tumor-associated stromal cells, little is known of the molecular mechanisms connecting this cytokine with enhanced glycolysis. PFKFB3 is a homodymeric bifunctional enzyme, belonging to the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, that controls the conversion of fructose-6-phosphate (Fru-6-P) to fructose-2,6-bisphosphate (Fru-2,6-P2 ). This metabolite is important for the dynamic regulation of glycolytic flux by allosterically activating phosphofructokinase-1, a rate-limiting enzyme in glycolysis. The PFKFB3 gene is involved in cell proliferation via its role in carbohydrate metabolism. Here, we studied the mechanisms connecting TGF-β1, glucose metabolism, and PFKFB3 in glioblastoma cell lines. We demonstrate that TGF-β1 upregulates PFKFB3 mRNA and protein expression resulting in an increase in fructose 2,6-bisphosphate concentration, glucose uptake, glycolytic flux and lactate production. Moreover, these increases in PFKFB3 mRNA and protein expression and Fru-2,6-P2 concentration were reduced when the Smad3, p38 mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways were inhibited. We demonstrate that inhibition of PFKFB3 activity with 3PO or siRNA-mediated knockdown of PFKFB3 significantly eliminated the capacity of the T98G cells to form colonies by TGF-β1, one of the hallmarks of transformation. Taken together, these results show that TGF-β1 induces PFKFB3 expression through activation of the p38 MAPK and PI3K/Akt signaling pathways that complement and converge with early activation of Smad signaling. This suggests that PFKFB3 induction by TGF-β1 can be one of the main mechanisms mediating the reprogramming of glioma cells.

Advanced oxidation protein products induce hepatocyte epithelial-mesenchymal transition via a ROS-dependent, TGF-β/Smad signaling pathway.

Epithelial-mesenchymal transition (EMT) occurs during the progression of liver fibrosis in response to chronic liver injury. However, the molecular mechanism underlying the regulation of hepatocyte EMT remains unclear. The aim of this study was to determine whether advanced oxidation protein products (AOPP) had an effect on hepatocyte EMT. The human L02 hepatocyte cell line and hepatocytes from normal Sprague-Dawley rats were challenged with AOPP treatment in both in vitro and in vivo studies. The expression of cell and molecular markers of EMT in L02 hepatocytes were studied using Western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Hepatocyte migratory potential was analyzed using a wound healing assay. Intracellular reactive oxygen species (ROS) were detected using the dichlorofluorescein (DCF) assay. In liver tissue sections, expression of EMT markers was evaluated using immunohistochemistry, and collagen was assessed using histochemical staining with Masson's trichrome. The findings were that AOPP treatment resulted in EMT in hepatocytes, which was associated with reduced expression of E-cadherin, increased expression of vimentin, increased deposition of collagen protein, and enhanced cell migration in vivo and in vitro. AOPP was also found to promote migration in L02 cells, and to promote the production of ROS and the activation of TGF-βR and Smad signaling. Inhibition of the generation of intracellular ROS and TGF-β receptor blocking could reverse AOPP-induced EMT in hepatocytes. This study has identified a novel mechanism in the regulation of hepatocyte EMT, and the findings may have implications for the control of liver fibrosis.

Abstract 977: Phenotypic analysis of single-cell breast cancer inhibition data reveals insights into EMT

Abstract Background: A leading model of cancer metastasis is epithelial-to-mesenchymal transition (EMT). We sought to determine whether single-cell inhibition data targeting potential mediators of EMT could uncover mechanistic insights into the EMT process. Methods: EMT was artificially induced on Py2T murine breast cancer cells by TGFb treatment. Additionally, a unique drug inhibitor was added to each well of a multiplexed CyTOF experiment. 37 transcription factors and cell surface markers were measured in each cell to assess epithelial and mesenchymal states, SMAD, AKT, and MAPK signaling activity, cell cycle regulation, and apoptosis pathway activation. The final single-cell dataset consisted of 300 inhibition and control conditions (cell populations), which we aimed to characterize in relation to one another with respect to effect on EMT. Analyzing the similarity between drug inhibitions amounts to a novel type of clustering problem that involves computing the similarity between diverse cell populations generated by each inhibitor. Traditional methods for comparing cell populations are not robust to the intra-population heterogeneity we observed amongst cells undergoing EMT. Thus, we developed Phenotypic Earth Mover’s Distance (PhEMD). This method for comparing cell populations leverages the insight that only a limited number of “cell subtypes” (e.g. mesenchymal, epithelial, transitional) are observed in unperturbed and perturbed EMT. By classifying each cell as one of these distinct subtypes using community-detection based clustering, PhEMD represents an inhibition or control condition as its relative abundance of each cell subtype. It then uses Earth Mover’s Distance (EMD) to compare two relative abundance distributions (i.e. heterogeneous cell populations). PhEMD thus derives a single value representing the dissimilarity between two inhibition conditions. Using PhEMD as measure of dissimilarity between each pair of inhibition and control conditions, we constructed an inhibitor-inhibitor graph and used graph clustering to identify groups of inhibitors that had similar effects to one another. Results: PhEMD analysis revealed that MEK, EGFR and Src inhibitors significantly halted EMT by generating far fewer mesenchymal cells and maintaining a large epithelial cell subpopulation. PhEMD also revealed that different mTOR, PI3K, and Akt inhibitors tended to have similar effects to one another and collectively generated a distinct, transitional cell subpopulation with altered pS6 expression. The pS6 dysregulation may be explained by the fact that ribosomal protein S6 is downstream of PI3K/AKT/mTOR, and the intermediate levels of both E-cadherin and vimentin suggest that this subset of cells may have been halted mid-transition. Finally, several Aurora Kinase and CDK inhibitors resulted in a relatively high percentage of apoptotic cells, suggesting these kinases may be important cell cycle regulators in the context of EMT. Citation Format: William S. Chen, Nevena Zivanovic, Dana Pe'er, Bernd Bodenmiller, Smita Krishnaswamy. Phenotypic analysis of single-cell breast cancer inhibition data reveals insights into EMT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 977. doi:10.1158/1538-7445.AM2017-977

BmpR1A is a major type 1 BMP receptor for BMP-Smad signaling during skull development

Craniosynostosis is caused by premature fusion of one or more sutures in an infant skull, resulting in abnormal facial features. The molecular and cellular mechanisms by which genetic mutations cause craniosynostosis are incompletely characterized, and many of the causative genes for diverse types of syndromic craniosynostosis have not yet been identified. We previously demonstrated that augmentation of BMP signaling mediated by a constitutively active BMP type IA receptor (ca-BmpR1A) in neural crest cells (ca1A hereafter) causes craniosynostosis and superimposition of heterozygous null mutation of Bmpr1a rescues premature suture fusion (ca1A;1aH hereafter). In this study, we superimposed heterozygous null mutations of the other two BMP type I receptors, Bmpr1b and Acvr1 (ca1A;1bH and ca1A;AcH respectively hereafter) to further dissect involvement of BMP-Smad signaling. Unlike caA1;1aH, ca1A;1bH and ca1A;AcH did not restore the craniosynostosis phenotypes. In our in vivo study, Smad-dependent BMP signaling was decreased to normal levels in mut;1aH mice. However, BMP receptor-regulated Smads (R-Smads; pSmad1/5/9 hereafter) levels were comparable between ca1A, ca1A;1bH and ca1A;AcH mice, and elevated compared to control mice. Bmpr1a, Bmpr1b and Acvr1 null cells were used to examine potential mechanisms underlying the differences in ability of heterozygosity for Bmpr1a vs. Bmpr1b or Acvr1 to rescue the mut phenotype. pSmad1/5/9 level was undetectable in Bmpr1a homozygous null cells while pSmad1/5/9 levels did not decrease in Bmpr1b or Acvr1 homozygous null cells. Taken together, our study indicates that different levels of expression and subsequent activation of Smad signaling differentially contribute each BMP type I receptor to BMP-Smad signaling and craniofacial development. These results also suggest differential involvement of each type 1 receptor in pathogenesis of syndromic craniosynostoses.

Transforming Growth Factor-β Receptors and Smads: Regulatory Complexity and Functional Versatility.

Transforming growth factor (TGF)-β family proteins control cell physiology, proliferation, and growth, and direct cell differentiation, thus playing key roles in normal development and disease. The mechanisms of how TGF-β family ligands interact with heteromeric complexes of cell surface receptors to then activate Smad signaling that directs changes in gene expression are often seen as established. Even though TGF-β-induced Smad signaling may be seen as a linear signaling pathway with predictable outcomes, this pathway provides cells with a versatile means to induce different cellular responses. Fundamental questions remain as to how, at the molecular level, TGF-β and TGF-β family proteins activate the receptor complexes and induce a context-dependent diversity of cell responses. Among the areas of progress, we summarize new insights into how cells control TGF-β responsiveness by controlling the TGF-β receptors, and into the key roles and versatility of Smads in directing cell differentiation and cell fate selection.

MG53-Mediated Protection in Heart Valve Biology

Utilizing both overexpression and extracellular application, we aim to target MG53, an essential cell membrane repair protein, to valvular cells for treatment of tissue injuries associated with valvular heart disease (VHD). Membrane repair is important to maintain normal cellular physiology, and disruption of this process can result in disease in a number of different tissues. With the four heart valves experiencing a tremendous amount of mechanical stress, we have utilized microelectrode needle penetration and glass bead damage assays to assess the ability of MG53 to protect valvular interstitial cells (VICs) from physical injury. Furthermore, we have begun in vitro and in vivo studies to investigate the ability of MG53 to protect against the biological and physiological changes associated with VHD. To date, we have observed MG53 expression in valvular tissue and MG53-mediated protection against membrane damage through both live cell imaging and lactate dehydrogenase (LDH) release. Ongoing experiments are further characterizing mechanisms of protection against valvular fibrosis and calcification and assessing the effects of MG53 in in vivo models of VHD. Specifically, we have found that, in addition to protecting against cell membrane injury, recombinant human MG53 (rhMG53) can enter VICs and suppress TGF-beta-dependent activation of Smad and MAPK signaling associated with fibrocalcific valvular remodeling. We anticipate that, once all of our aims are completed, our experiments will greatly contribute to the study of cell biology, regenerative medicine, and cardiovascular disease, the number one cause of death in the United States and worldwide.

Emodin suppresses silica-induced lung fibrosis by promoting Sirt1 signaling via direct contact.

Pulmonary silicosis is characterized by lung fibrosis, which leads to impairment of pulmonary function; the specific mechanism remains to be fully elucidated Emodin shows antifibrotic effects in several organs with fibrosis, however, it has not been investigated in pulmonary silicosis. In the present study, the possible mechanism of lung fibrosis and the antifibrotic effect of emodin in silica inhalation-induced lung fibrosis were investigated. Pulmonary silica particle inhalation was used to induce lung fibrosis in mice. Emodin and or the sirtuin 1 (Sirt1) inhibitor, nicotinamide, were used to treat the modeled animals. Pulmonary function was assessed using an occlusion method. The deposition of collagen I and α-smooth muscle actin (SMA) in the lung tissue were detected using fluorescence staining; transforming growth factor-β1 (TGF-β1) in the bronchoalveolar lavage fluid (BALF) was examined using an enzyme-linked immunosorbent assay; TGF-β1/Sirt1/small mothers against decapentaplegic (Smad) signaling activation in lung tissue was also examined. The molecular contacts between emodin were evaluated using liquid chromatography-mass spectrometry analysis. The deposition of collagen I and α-SMA in lung tissues were found to be elevated following silica exposure, however, this was relieved by emodin treatment. The pulmonary function of the animals was impaired by silica inhalation, and this was improved by emodin administration. However, the therapeutic effects of emodin on lung fibrosis were impaired by nicotinamide administration. The levels of TGF-β1 in the BALF and lung tissue were elevated by silica inhalation, however, they were not affected by either emodin or nicotinamide treatment. Additionally, emodin was found to increase the expression level of Sirt1, which decreased the level of deacetylated Smad3 to attenuate collagen deposition. Furthermore, the data suggested that there was direct binding between emodin and Sirt1. Sirt1-regulated TGF-β1/Smad signaling was involved in silica inhalation-induced lung fibrosis. Emodin attenuated this lung fibrosis to improve pulmonary function by targeting Sirt1, which regulated TGF-β1/Smad fibrotic signaling.