Extracellular vesicles in systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis is a chronic pediatric inflammatory disease of unknown etiology, characterized by fever, rash, hepatosplenomegaly, serositis, and arthritis. We hypothesized that intercellular communication, mediated by extracellular vesicles, contributes to systemic juvenile idiopathic arthritis pathogenesis and that the number and cellular sources of extracellular vesicles would differ between inactive and active states of systemic juvenile idiopathic arthritis and healthy controls. We evaluated plasma from healthy pediatric controls and patients with systemic juvenile idiopathic arthritis with active systemic flare or inactive disease. We isolated extracellular vesicles by size exclusion chromatography and determined total extracellular vesicle abundance and size distribution using microfluidic resistive pulse sensing. Cell-specific extracellular vesicle subpopulations were measured by nanoscale flow cytometry. Isolated extracellular vesicles were validated using a variety of ways, including nanotracking and cryo-electron microscopy. Extracellular vesicle protein content was analyzed in pooled samples using mass spectrometry. Total extracellular vesicle concentration did not significantly differ between controls and patients with systemic juvenile idiopathic arthritis. Extracellular vesicles with diameters <200 nm were the most abundant, including the majority of cell-specific extracellular vesicle subpopulations. Patients with systemic juvenile idiopathic arthritis had significantly higher levels of extracellular vesicles from activated platelets, intermediate monocytes, and chronically activated endothelial cells, with the latter significantly more elevated in active systemic juvenile idiopathic arthritis relative to inactive disease and controls. Protein analysis of isolated extracellular vesicles from active patients showed a proinflammatory profile, uniquely expressing heat shock protein 47, a stress-inducible protein. Our findings indicate that multiple cell types contribute to altered extracellular vesicle profiles in systemic juvenile idiopathic arthritis. The extracellular vesicle differences between systemic juvenile idiopathic arthritis disease states and healthy controls implicate extracellular vesicle-mediated cellular crosstalk as a potential driver of systemic juvenile idiopathic arthritis disease activity.

Mapping transcriptional heterogeneity and metabolic networks in fatty livers at single-cell resolution.

Non-alcoholic fatty liver disease is a heterogeneous disease with unclear underlying molecular mechanisms. Here, we perform single-cell RNA sequencing of hepatocytes and hepatic non-parenchymal cells to map the lipid signatures in mice with non-alcoholic fatty liver disease (NAFLD). We uncover previously unidentified clusters of hepatocytes characterized by either high or low srebp1 expression. Surprisingly, the canonical lipid synthesis driver Srebp1 is not predictive of hepatic lipid accumulation, suggestive of other drivers of lipid metabolism. By combining transcriptional data at single-cell resolution with computational network analyses, we find that NAFLD is associated with high constitutive androstane receptor (CAR) expression. Mechanistically, CAR interacts with four functional modules: cholesterol homeostasis, bile acid metabolism, fatty acid metabolism, and estrogen response. Nuclear expression of CAR positively correlates with steatohepatitis in human livers. These findings demonstrate significant cellular differences in lipid signatures and identify functional networks linked to hepatic steatosis in mice and humans.

Candesartan, an angiotensin-II receptor blocker, ameliorates insulin resistance and hepatosteatosis by reducing intracellular calcium overload and lipid accumulation.

Insulin resistance is a major contributor to the pathogenesis of several human diseases, including type 2 diabetes, hypertension, and hyperlipidemia. Notably, insulin resistance and hypertension share common abnormalities, including increased oxidative stress, inflammation, and organelle dysfunction. Recently, we showed that excess intracellular Ca2+, a known pathogenic factor in hypertension, acts as a critical negative regulator of insulin signaling by forming Ca2+-phosphoinositides that prevent the membrane localization of AKT, a key serine/threonine kinase signaling molecule. Whether preventing intracellular Ca2+ overload improves insulin sensitivity, however, has not yet been investigated. Here, we show that the antihypertensive agent candesartan, compared with other angiotensin-II receptor blockers, has previously unrecognized beneficial effects on attenuating insulin resistance. We found that candesartan markedly reduced palmitic acid (PA)-induced intracellular Ca2+ overload and lipid accumulation by normalizing dysregulated store-operated channel (SOC)-mediated Ca2+ entry into cells, which alleviated PA-induced insulin resistance by promoting insulin-stimulated AKT membrane localization and increased the phosphorylation of AKT and its downstream substrates. As pharmacological approaches to attenuate intracellular Ca2+ overload in vivo, administering candesartan to obese mice successfully decreased insulin resistance, hepatic steatosis, dyslipidemia, and tissue inflammation by inhibiting dysregulated SOC-mediated Ca2+ entry and ectopic lipid accumulation. The resulting alterations in the phosphorylation of key signaling molecules consequently alleviate impaired insulin signaling by increasing the postprandial membrane localization and phosphorylation of AKT. Thus, our findings provide robust evidence for the pleiotropic contribution of intracellular Ca2+ overload in the pathogenesis of insulin resistance and suggest that there are viable approved drugs that can be repurposed for the treatment of insulin resistance and hypertension.

High-Phytate Diets Increase Amyloid ß Deposition and Apoptotic Neuronal Cell Death in a Rat Model.

Amyloid-ß (Aß) accumulation in the hippocampus is an essential event in the pathogenesis of Alzheimer's disease. Insoluble Aß is formed through the sequential proteolytic hydrolysis of the Aß precursor protein, which is cleaved by proteolytic secretases. However, the pathophysiological mechanisms of Aß accumulation remain elusive. Here, we report that rats fed high-phytate diets showed Aß accumulation and increased apoptotic neuronal cell death in the hippocampus through the activation of the amyloidogenic pathway in the hippocampus. Immunoblotting and immunohistochemical analyses confirmed that the overexpression of BACE1 ß-secretase, a critical enzyme for Aß generation, exacerbated the hippocampal Aß accumulation in rats fed high-phytate diets. Moreover, we identified that parathyroid hormone, a physiological hormone responding to the phytate-mediated dysregulation of calcium and phosphate homeostasis, plays an essential role in the transcriptional activation of the Aß precursor protein and BACE1 through the vitamin D receptor and retinoid X receptor axis. Thus, our findings suggest that phytate-mediated dysregulation of calcium and phosphate is a substantial risk factor for elevated Aß accumulation and apoptotic neuronal cell death in rats.

Mitochondria-Rich Extracellular Vesicles Rescue Patient-Specific Cardiomyocytes From Doxorubicin Injury: Insights Into the SENECA Trial.

BACKGROUND: Anthracycline-induced cardiomyopathy (AIC) is a significant source of morbidity and mortality in cancer survivors. The role of mesenchymal stem cells (MSCs) in treating AIC was evaluated in the SENECA trial, a Phase 1 National Heart, Lung, and Blood Institute-sponsored study, but the mechanisms underpinning efficacy in human tissue need clarification. OBJECTIVES: The purpose of this study was to perform an in vitro clinical trial evaluating the efficacy and putative mechanisms of SENECA trial-specific MSCs in treating doxorubicin (DOX) injury, using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iCMs) generated from SENECA patients. METHODS: Patient-specific iCMs were injured with 1 µmol/L DOX for 24 hours, treated with extracellular vesicles (EVs) from MSCs by either coculture or direct incubation and then assessed for viability and markers of improved cellular physiology. MSC-derived EVs were separated into large extracellular vesicles (L-EVs) (>200 nm) and small EVs (<220nm) using a novel filtration system. RESULTS: iCMs cocultured with MSCs in a transwell system demonstrated improved iCM viability and attenuated apoptosis. L-EVs but not small EVs recapitulated this therapeutic effect. L-EVs were found to be enriched in mitochondria, which were shown to be taken up by iCMs. iCMs treated with L-EVs demonstrated improved contractility, reactive oxygen species production, ATP production, and mitochondrial biogenesis. Inhibiting L-EV mitochondrial function with 1-methyl-4-phenylpyridinium attenuated efficacy. CONCLUSIONS: L-EV-mediated mitochondrial transfer mitigates DOX injury in patient-specific iCMs. Although SENECA was not designed to test MSC efficacy, consistent tendencies toward a positive effect were observed across endpoints. Our results suggest a mechanism by which MSCs may improve cardiovascular performance in AIC independent of regeneration, which could inform future trial design evaluating the therapeutic potential of MSCs.

Isthmin-1 is an adipokine that promotes glucose uptake and improves glucose tolerance and hepatic steatosis.

With the increasing prevalence of type 2 diabetes and fatty liver disease, there is still an unmet need to better treat hyperglycemia and hyperlipidemia. Here, we identify isthmin-1 (Ism1) as an adipokine and one that has a dual role in increasing adipose glucose uptake while suppressing hepatic lipid synthesis. Ism1 ablation results in impaired glucose tolerance, reduced adipose glucose uptake, and reduced insulin sensitivity, demonstrating an endogenous function for Ism1 in glucose regulation. Mechanistically, Ism1 activates a PI3K-AKT signaling pathway independently of the insulin and insulin-like growth factor receptors. Notably, while the glucoregulatory function is shared with insulin, Ism1 counteracts lipid accumulation in the liver by switching hepatocytes from a lipogenic to a protein synthesis state. Furthermore, therapeutic dosing of recombinant Ism1 improves diabetes in diet-induced obese mice and ameliorates hepatic steatosis in a diet-induced fatty liver mouse model. These findings uncover an unexpected, bioactive protein hormone that might have simultaneous therapeutic potential for diabetes and fatty liver disease.

ß-propeller phytase hydrolyzes insoluble Ca(2+)-phytate salts and completely abrogates the ability of phytate to chelate metal ions.

Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate's binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the ß-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.

Isolation, culture, and functional analysis of hepatocytes from mice with fatty liver disease.

We present a protocol for isolating hepatocytes from mice with established non-alcoholic fatty liver disease. This protocol consists of liver perfusion using a peristaltic pump, followed by a modified 25% and 90% Percoll gradient centrifugation protocol to capture lipid-laden hepatocytes that are usually lost using traditional isolation protocols. This protocol enables simultaneous isolation of normal and lipid-filled hepatocytes. Lipid-filled hepatocytes can be used in cell culture systems to study drug metabolism, hepatotoxicity, or glucose and lipid metabolism. For complete details on the use and execution of this protocol, please refer to Sharabi et al. (2017) and Kegel et al. (2016).

Regulation of Energy Metabolism by Receptor Tyrosine Kinase Ligands.

Metabolic diseases, such as diabetes, obesity, and fatty liver disease, have now reached epidemic proportions. Receptor tyrosine kinases (RTKs) are a family of cell surface receptors responding to growth factors, hormones, and cytokines to mediate a diverse set of fundamental cellular and metabolic signaling pathways. These ligands signal by endocrine, paracrine, or autocrine means in peripheral organs and in the central nervous system to control cellular and tissue-specific metabolic processes. Interestingly, the expression of many RTKs and their ligands are controlled by changes in metabolic demand, for example, during starvation, feeding, or obesity. In addition, studies of RTKs and their ligands in regulating energy homeostasis have revealed unexpected diversity in the mechanisms of action and their specific metabolic functions. Our current understanding of the molecular, biochemical and genetic control of energy homeostasis by the endocrine RTK ligands insulin, FGF21 and FGF19 are now relatively well understood. In addition to these classical endocrine signals, non-endocrine ligands can govern local energy regulation, and the intriguing crosstalk between the RTK family and the TGFß receptor family demonstrates a signaling network that diversifies metabolic process between tissues. Thus, there is a need to increase our molecular and mechanistic understanding of signal diversification of RTK actions in metabolic disease. Here we review the known and emerging molecular mechanisms of RTK signaling that regulate systemic glucose and lipid metabolism, as well as highlighting unexpected roles of non-classical RTK ligands that crosstalk with other receptor pathways.

High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss.

Phosphate overload contributes to mineral bone disorders that are associated with crystal nephropathies. Phytate, the major form of phosphorus in plant seeds, is known as an indigestible and of negligible nutritional value in humans. However, the mechanism and adverse effects of high-phytate intake on Ca2+ and phosphate absorption and homeostasis are unknown. Here, we show that excessive intake of phytate along with a low-Ca2+ diet fed to rats contributed to the development of crystal nephropathies, renal phosphate wasting, and bone loss through tubular dysfunction secondary to dysregulation of intestinal calcium and phosphate absorption. Moreover, Ca2+ supplementation alleviated the detrimental effects of excess dietary phytate on bone and kidney through excretion of undigested Ca2+-phytate, which prevented a vicious cycle of intestinal phosphate overload and renal phosphate wasting while improving intestinal Ca2+ bioavailability. Thus, we demonstrate that phytate is digestible without a high-Ca2+ diet and is a risk factor for phosphate overloading and for the development of crystal nephropathies and bone disease.

The conserved metalloprotease invadolysin is present in invertebrate haemolymph and vertebrate blood.

We identified invadolysin, a novel essential metalloprotease, for functions in chromosome structure, cell proliferation and migration. Invadolysin also plays an important metabolic role in insulin signalling and is the only protease known to localise to lipid droplets, the main lipid storage organelle in the cell. In silico examination of the protein sequence of invadolysin predicts not only protease and lipase catalytic motifs, but also post-translational modifications and the secretion of invadolysin. Here we show that the protease motif of invadolysin is important for its role in lipid accumulation, but not in glycogen accumulation. The lipase motif does not appear to be functionally important for the accumulation of lipids or glycogen. Post-translational modifications likely contribute to modulating the level, localisation or activity of invadolysin. We identified a secreted form of invadolysin in the soluble fraction of invertebrate hemolymph (where we observe sexually dimorphic forms) and also vertebrate plasma, including in the extracellular vesicle fraction. Biochemical analysis for various post-translational modifications demonstrated that secreted invadolysin is both N- and O-glycosylated, but not apparently GPI-linked. The discovery of invadolysin in the extracellular milieu suggests a role for invadolysin in normal organismal physiology.

A context-specific circadian clock in adipocyte precursor cells modulates adipogenesis.

The circadian clock is an intricate molecular network that paces a variety of physiological process to ~ 24 hour day/night cycles. Whereas the central circadian clock in the brain is primarily entrained by light signals, peripheral circadian clocks, which are in most cells in the body, receive cues not only from the central pacemaker but also endocrine and other systemic and tissue-specific signals. Prior studies have connected peripheral circadian clocks to metabolism, primarily with studies focused on the robust clock in the liver that responds to feeding/fasting cycles. Adipose tissue is also critical for metabolism and adipocytes have circadian clocks. Yet, the role of the circadian clock in adipocytes is poorly understood. Here we describe our studies that revealed components of the circadian clock in primary adipocyte precursor cells (APCs) in mice. We made the surprising discovery of a particularly prominent role for the circadian gene Period 3 (Per3) in the APC clock. Furthermore, we elucidated that Per3 directly regulates an output pathway of the APC clock to modulate the expression of the Kruppel-like factor 15 (Klf15) gene. Finally, we discovered that this clock-Klf15 pathway regulates adipogenesis in APCs. These finding have important implications for our understanding of adipose tissue biology and metabolism and, we speculate, will generate opportunities to develop novel therapeutic strategies based on the context-specific features of the circadian clock in APCs.

Isl1ß Overexpression With Key ß Cell Transcription Factors Enhances Glucose-Responsive Hepatic Insulin Production and Secretion.

Adenoviral gene transfer of key ß cell developmental regulators including Pdx1, Neurod1, and Mafa (PDA) has been reported to generate insulin-producing cells in the liver. However, PDA insulin secretion is transient and glucose unresponsive. Here, we report that an additional ß cell developmental regulator, insulin gene enhancer binding protein splicing variant (Isl1ß), improved insulin production and glucose-responsive secretion in PDA mice. Microarray gene expression analysis suggested that adenoviral PDA transfer required an additional element for mature ß cell generation, such as Isl1 and Elf3 in the liver. In vitro promoter analysis indicated that splicing variant Isl1, or Isl1ß, is an important factor for transcriptional activity of the insulin gene. In vivo bioluminescence monitoring using insulin promoter-luciferase transgenic mice verified that adenoviral PDA + Isl1ß transfer produced highly intense luminescence from the liver, which peaked at day 7 and persisted for more than 10 days. Using insulin promoter-GFP transgenic mice, we further confirmed that Isl1ß supplementation to PDA augmented insulin-producing cells in the liver, insulin production and secretion, and ß cell‒related genes. Finally, the PDA + Isl1ß combination ameliorated hyperglycemia in diabetic mice for 28 days and enhanced glucose tolerance and responsiveness. Thus, our results suggest that Isl1ß is a key additional transcriptional factor for advancing the generation of insulin-producing cells in the liver in combination with PDA.

MafB Is Critical for Glucagon Production and Secretion in Mouse Pancreatic α Cells In Vivo.

The MafB transcription factor is expressed in pancreatic α and ß cells during development but becomes exclusive to α cells in adult rodents. Mafb-null (Mafb-/- ) mice were reported to have reduced α- and ß-cell numbers throughout embryonic development. To further analyze the postnatal function of MafB in the pancreas, we generated endocrine cell-specific (MafbΔEndo ) and tamoxifen-dependent (MafbΔTAM ) Mafb knockout mice. MafbΔEndo mice exhibited reduced populations of insulin-positive (insulin+) and glucagon+ cells at postnatal day 0, but the insulin+ cell population recovered by 8 weeks of age. In contrast, the Arx+ glucagon+ cell fraction and glucagon expression remained decreased even in adulthood. MafbΔTAM mice, with Mafb deleted after pancreas maturation, also demonstrated diminished glucagon+ cells and glucagon content without affecting ß cells. A decreased Arx+ glucagon+ cell population in MafbΔEndo mice was compensated for by an increased Arx+ pancreatic polypeptide+ cell population. Furthermore, gene expression analyses from both MafbΔEndo and MafbΔTAM islets revealed that MafB is a key regulator of glucagon expression in α cells. Finally, both mutants failed to respond to arginine, likely due to impaired arginine transporter gene expression and glucagon production ability. Taken together, our findings reveal that MafB is critical for the functional maintenance of mouse α cells in vivo, including glucagon production and secretion, as well as in development.

Co-chaperone BAG2 Determines the Pro-oncogenic Role of Cathepsin B in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is considered incurable with currently available treatments, highlighting the need for therapeutic targets and predictive biomarkers. Here, we report a unique role for Bcl-2-associated athanogene 2 (BAG2), which is significantly overexpressed in TNBC, in regulating the dual functions of cathepsin B as either a pro- or anti-oncogenic enzyme. Silencing BAG2 suppresses tumorigenesis and lung metastasis and induces apoptosis by increasing the intracellular mature form of cathepsin B, whereas BAG2 expression induces metastasis by blocking the auto-cleavage processing of pro-cathepsin B via interaction with the propeptide region. BAG2 regulates pro-cathepsin B/annexin II complex formation and facilitates the trafficking of pro-cathespin-B-containing TGN38-positive vesicles toward the cell periphery, leading to the secretion of pro-cathepsin B, which induces metastasis. Collectively, our results uncover BAG2 as a regulator of the oncogenic function of pro-cathepsin B and a potential diagnostic and therapeutic target that may reduce the burden of metastatic breast cancer.

ß-Cell-Specific Mafk Overexpression Impairs Pancreatic Endocrine Cell Development.

The MAF family transcription factors are homologs of v-Maf, the oncogenic component of the avian retrovirus AS42. They are subdivided into 2 groups, small and large MAF proteins, according to their structure, function, and molecular size. MAFK is a member of the small MAF family and acts as a dominant negative form of large MAFs. In previous research we generated transgenic mice that overexpress MAFK in order to suppress the function of large MAF proteins in pancreatic ß-cells. These mice developed hyperglycemia in adulthood due to impairment of glucose-stimulated insulin secretion. The aim of the current study is to examine the effects of ß-cell-specific Mafk overexpression in endocrine cell development. The developing islets of Mafk-transgenic embryos appeared to be disorganized with an inversion of total numbers of insulin+ and glucagon+ cells due to reduced ß-cell proliferation. Gene expression analysis by quantitative RT-PCR revealed decreased levels of ß-cell-related genes whose expressions are known to be controlled by large MAF proteins. Additionally, these changes were accompanied with a significant increase in key ß-cell transcription factors likely due to compensatory mechanisms that might have been activated in response to the ß-cell loss. Finally, microarray comparison of gene expression profiles between wild-type and transgenic pancreata revealed alteration of some uncharacterized genes including Pcbd1, Fam132a, Cryba2, and Npy, which might play important roles during pancreatic endocrine development. Taken together, these results suggest that Mafk overexpression impairs endocrine development through a regulation of numerous ß-cell-related genes. The microarray analysis provided a unique data set of differentially expressed genes that might contribute to a better understanding of the molecular basis that governs the development and function of endocrine pancreas.

Generation of insulin-producing cells from the mouse liver using ß cell-related gene transfer including Mafa and Mafb.

Recent studies on the large Maf transcription factors have shown that Mafb and Mafa have respective and distinctive roles in ß-cell development and maturation. However, whether this difference in roles is due to the timing of the gene expression (roughly, expression of Mafb before birth and of Mafa after birth) or to the specific function of each gene is unclear. Our aim was to examine the functional differences between these genes that are closely related to ß cells by using an in vivo model of ß-like cell generation. We monitored insulin gene transcription by measuring bioluminescence emitted from the liver of insulin promoter-luciferase transgenic (MIP-Luc-VU) mice. Adenoviral gene transfers of Pdx1/Neurod/Mafa (PDA) and Pdx1/Neurod/Mafb (PDB) combinations generated intense luminescence from the liver that lasted for more than 1 week and peaked at 3 days after transduction. The peak signal intensities of PDA and PDB were comparable. However, PDA but not PDB transfer resulted in significant bioluminescence on day 10, suggesting that Mafa has a more sustainable role in insulin gene activation than does Mafb. Both PDA and PDB transfers ameliorated the glucose levels in a streptozotocin (STZ)-induced diabetic model for up to 21 days and 7 days, respectively. Furthermore, PDA transfer induced several gene expressions necessary for glucose sensing and insulin secretion in the liver on day 9. However, a glucose tolerance test and liver perfusion experiment did not show glucose-stimulated insulin secretion from intrahepatic ß-like cells. These results demonstrate that bioluminescence imaging in MIP-Luc-VU mice provides a noninvasive means of detecting ß-like cells in the liver. They also show that Mafa has a markedly intense and sustained role in ß-like cell production in comparison with Mafb.

Loss of TBK1 induces epithelial-mesenchymal transition in the breast cancer cells by ERα downregulation.

Estrogen receptor α (ERα) is the pivotal regulator of proliferation and differentiation in mammary epithelia, where it serves as a crucial prognostic marker and therapeutic target in breast cancer. In this study, we show that the loss of the kinase TANK-binding kinase 1 (TBK1) induces epithelial-mesenchymal transition in ERα-positive breast cancer cells by downregulating ERα expression. TBK1 was overexpressed in ERα-positive breast cancers, where it was associated with distant metastasis-free survival in patients, whereas it was underexpressed in ERα-negative breast cancers. TBK1 silencing decreased expression of epithelial markers and increased expression of mesenchymal markers in ERα-positive breast cancer cells, enhancing tumor growth and lung metastasis in vivo in a manner associated with downregulation of ERα expression. Mechanistically, TBK1 silencing reduced FOXO3A binding to the ERα promoter by inducing the translocation of phosphorylated FOXO3A from the nucleus to the cytoplasm. Thus, our results indicate that the loss of TBK1 expression parallels the loss of ERα expression, in turn helping drive an aggressive breast cancer phenotype.

DRAK2 participates in a negative feedback loop to control TGF-ß/Smads signaling by binding to type I TGF-ß receptor.

TGF-ß1 is a multifunctional cytokine that mediates diverse biological processes. However, the mechanisms by which the intracellular signals of TGF-ß1 are terminated are not well understood. Here, we demonstrate that DRAK2 serves as a TGF-ß1-inducible antagonist of TGF-ß signaling. TGF-ß1 stimulation rapidly induces DRAK2 expression and enhances endogenous interaction of the type I TGF-ß receptor with DRAK2, thereby blocking R-Smads recruitment. Depletion of DRAK2 expression markedly augmented the intensity and the extent of TGF-ß1 responses. Furthermore, a high level of DRAK2 expression was observed in basal-like and HER2-enriched breast tumors and cell lines, and depletion of DRAK2 expression suppressed the tumorigenic ability of breast cancer cells. Thus, these studies define a function for DRAK2 as an intrinsic intracellular antagonist participating in the negative feedback loop to control TGF-ß1 responses, and aberrant expression of DRAK2 increases tumorigenic potential, in part, through the inhibition of TGF-ß1 tumor suppressor activity.