MiR-29 and MiR-140 regulate TRAIL-induced drug tolerance in lung cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has chemotherapeutic potential as a regulator of an extrinsic apoptotic ligand, but its effect as a drug is limited by innate and acquired resistance. Recent findings suggest that an intermediate drug tolerance could mediate acquired resistance, which has made the main obstacle for limited utility of TRAIL as an anti-cancer therapeutics. We propose miRNA-dependent epigenetic modification drives the drug tolerant state in TRAIL-induced drug tolerant (TDT). Transcriptomic analysis revealed miR-29 target gene activation in TDT cells, showing oncogenic signature in lung cancer. Also, the restored TRAIL-sensitivity was associated with miR-29ac and 140-5p expressions, which is known as tumor suppressor by suppressing oncogenic protein RSK2 (p90 ribosomal S6 kinase), further confirmed in patient samples. Moreover, we extended this finding into 119 lung cancer cell lines from public data set, suggesting a significant correlation between TRAIL-sensitivity and RSK2 mRNA expression. Finally, we found that increased RSK2 mRNA is responsible for NF-κB activation, which we previously showed as a key determinant in both innate and acquired TRAIL-resistance. Our findings support further investigation of miR-29ac and -140-5p inhibition to maintain TRAIL-sensitivity and improve the durability of response to TRAIL in lung cancer.

CREB5 promotes the proliferation and self-renewal ability of glioma stem cells.

Glioblastoma multiforme (GBM) is the most fatal form of brain cancer in humans, with a dismal prognosis and a median overall survival rate of less than 15 months upon diagnosis. Glioma stem cells (GSCs), have recently been identified as key contributors in both tumor initiation and therapeutic resistance in GBM. Both public dataset analysis and direct differentiation experiments on GSCs have demonstrated that CREB5 is more highly expressed in undifferentiated GSCs than in differentiated GSCs. Additionally, gene silencing by short hairpin RNA (shRNA) of CREB5 has prevented the proliferation and self-renewal ability of GSCs in vitro and decreased their tumor forming ability in vivo. Meanwhile, RNA-sequencing, luciferase reporter assay, and ChIP assay have all demonstrated the closely association between CREB5 and OLIG2. These findings suggest that targeting CREB5 could be an effective approach to overcoming GSCs.

Lucidumol A, Purified Directly from Ganoderma lucidum, Exhibits Anticancer Effect and Cellular Inflammatory Response in Colorectal Cancer.

Colorectal cancer (CRC) is a deadly disease regardless of sex, and a few therapeutic approaches have been fully developed at advanced stages, even if some strategies have durable clinical benefits, such as immunotherapy and chemotherapy. Ganoderma lucidum has been recognized as an organism that suppresses tumors and inflammation; however, the molecular mechanisms induced by a triterpenoid in Ganoderma lucidum, Lucidumol A, have not yet been fully explored in CRC and inflammatory responses. To this end, we extracted Lucidumol A from Ganoderma lucidum and analyzed its anticancer effect and anti-inflammatory potential in CRC cell lines and RAW264.7 macrophage-derived cell lines, respectively. A series of in vitro experiments including cell survival, wound healing, and migration assays were performed to determine the role of Lucidumol A in the CRC cell line. We also analyzed inflammatory responses using qRT-PCR, Western Blot, and ELISA in RAW 264.7 macrophaged-derived cell lines exposed to various concentrations of Lucidumol A. Lucidumol A efficiently suppressed the metastatic potential of CRC at very low concentrations. Furthermore, significant anti-inflammatory activities were observed in Lucidumol A-treated RAW264.7 cells through modulation of inflammation-associated marker genes and cytokines. In conclusion, Lucidumol A plays an important role in Ganoderma lucidum-dependent tumor suppression and anti-inflammation, suggesting different strategies to treat CRC patients, and other diseases evoked by proinflammatory cytokines, despite the need to explore further its mechanism of action.

Artificial intelligence approaches to the determinants of women's vaginal dryness using general hospital data.

The aim of this study is to analyse the determinants of women's vaginal dryness using machine learning. Data came from Korea University Anam Hospital in Seoul, Republic of Korea, with 3298 women, aged 40-80 years, who attended their general health check from January 2010 to December 2012. Five machine learning methods were applied and compared for the prediction of vaginal dryness, measured by a Menopause Rating Scale. Random forest variable importance, a performance gap between a complete model and a model excluding a certain variable, was adopted for identifying major determinants of vaginal dryness. In terms of the mean squared error, the random forest (1.0597) was much better than linear regression (17.9043) and artificial neural networks with one, two and three hidden layers (1.7452, 1.7148 and 1.7736, respectively). Based on random forest variable importance, the top-10 determinants of vaginal dryness were menopause age, age, menopause, height, thyroid stimulating hormone, neutrophils, years since menopause, lymphocytes, alkaline phosphatase and blood urea nitrogen. In addition, its top-20 determinants were peak expiratory flow rate, low-density lipoprotein cholesterol, white blood cells, monocytes, cancer antigen 19-9, creatinine, eosinophils, total cholesterol, triglyceride and amylase. Machine learning presents a great decision support system for the prediction of vaginal dryness. For preventing vaginal dryness, preventive measures would be needed regarding early menopause, the thyroid function and systematic inflammation.Impact StatementWhat is already known on this subject? Only a few studies have investigated the risk factors of vaginal dryness in middle-aged women. More research is to be done for finding its various risk factors, identifying its major risk groups and drawing its effective clinical implications.What do the results of this study add? This study is the first machine-learning study to predict women's vaginal dryness and analyse their determinants. The random forest could discuss which factors are more important for the prediction of vaginal dryness. Based on random forest variable importance, menopause age was the most important determinant of vaginal dryness and their association was discovered to be negative in this study. Vaginal dryness was closely associated with the height, rather than the body weight or body mass index. The importance rankings of blood conditions related to systematic inflammation were within the top-20 in this study: neutrophils, lymphocytes, white blood cells, monocytes and eosinophils.What are the implications of these findings for clinical practice and/or further research? Machine learning presents a great decision support system for the prediction of vaginal dryness. For preventing vaginal dryness, preventive measures would be needed regarding early menopause and systematic inflammation.

Machine Learning Approaches to Identify Factors Associated with Women's Vasomotor Symptoms Using General Hospital Data.

BACKGROUND: To analyze the factors associated with women's vasomotor symptoms (VMS) using machine learning. METHODS: Data on 3,298 women, aged 40-80 years, who attended their general health check-up from January 2010 to December 2012 were obtained from Korea University Anam Hospital in Seoul, Korea. Five machine learning methods were applied and compared for the prediction of VMS, measured by the Menopause Rating Scale. Variable importance, the effect of a variable on model performance, was used for identifying the major factors associated with VMS. RESULTS: In terms of the mean squared error, the random forest (0.9326) was much better than linear regression (12.4856) and artificial neural networks with one, two, and three hidden layers (1.5576, 1.5184, and 1.5833, respectively). Based on the variable importance from the random forest, the most important factors associated with VMS were age, menopause age, thyroid-stimulating hormone, and monocyte, triglyceride, gamma glutamyl transferase, blood urea nitrogen, cancer antigen 19-9, C-reactive protein, and low-density lipoprotein cholesterol levels. Indeed, the following variables were ranked within the top 20 in terms of variable importance: cancer antigen 125, total cholesterol, insulin, free thyroxine, forced vital capacity, alanine aminotransferase, forced expired volume in 1 second, height, homeostatic model assessment for insulin resistance, and carcinoembryonic antigen. CONCLUSION: Machine learning provides an invaluable decision support system for the prediction of VMS. For managing VMS, comprehensive consideration is needed regarding thyroid function, lipid profile, liver function, inflammation markers, insulin resistance, monocyte count, cancer antigens, and lung function.

Methanol Extract of Mongolian Iris bungei Maxim. Stimulates 3T3-L1 Adipocyte Differentiation.

Iris bungei Maxim. (IB), which is native to China and Mongolia, is used as a traditional medicine for conditions such as inflammation, cancer, and bacterial infections. However, the effects of Iris bungei Maxim. on adipocyte differentiation have not been studied. In the present study, we first demonstrated the molecular mechanisms underlying the adipogenic activity of the methanol extract of Mongolian I. bungei Maxim. (IB). IB significantly enhanced intracellular lipid accumulation and adipocyte differentiation in 3T3-L1 preadipocytes in a concentration-dependent manner. Moreover, IB markedly stimulated the expression of genes related to adipogenesis such as peroxisome proliferator-activated receptor γ, adiponectin, and aP2. In addition, we also observed that IB induces lipogenic genes such as fatty acid synthase, sterol regulatory element binding protein 1c, stearoyl-CoA desaturase, and acetyl-CoA carboxylase. Interestingly IB regulated adipocyte differentiation in both the early and middle stages. Taken together, these adipogenic and lipogenic effects of IB suggest its efficacy for the prevention and/or treatment of type 2 diabetes.

Antarctic Freshwater Microalga, Micractinium simplicissimum, Suppresses Inflammation.

Inflammation mediated by the innate immune system is the organism's protective mechanism against infectious environmental risk factors. Uncontrolled acute inflammation can become chronic, contributing to various chronic inflammatory diseases such as arthritis, asthma, autoimmune diseases, and atherosclerosis. Although microalgae are increasingly receiving attention as a source of bioactive molecules with therapeutic potential for various human diseases, the underlying mechanisms are not yet well understood. In the present study, we investigated the molecular mechanisms underlying the anti-inflammatory and anti-aging activities of ethanol extracts of Antarctic freshwater microalga Micractinium simplicissimum. Using RAW 264.7 macrophages, microalgal extracts exerted anti-inflammatory activity by regulating the major inflammatory indicators including cyclooxy-genase (COX)-2, interleukin (IL)-6, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α and nitric oxide (NO). Besides, we observed the anti-aging activity of the microalgal extract by suppressing MMP-1 production in human dermal fibroblast. Taken together, these data suggest that anti-inflammatory and anti-aging activities of Antarctic freshwater microalga, Micractinium simplicissimum, can provide new clues to understanding the molecular link between inflammation and diseases, and be a potential anti-inflammatory agent.

Fhit induces the reciprocal suppressions between Lin28/Let-7 and miR-17/92miR.

Objective: Fhit gene is known as a genome "caretaker" and frequently inactivated by deletion or hypermethylation on the promoter in several cancers. In spite of several lines of evidence, the exact mechanism underlying Fhit-induced biology is relatively less studied. This study will focus the role of Fhit in regulating Lin28 and microRNAs (miRNAs) loop. Material and Methods: To this end, we employed Fhit overexpressing isogenic cell lines to conduct miRNA nanostring array, and differentially expressed miRNAs were identified. Using real-time PCR and Western blot analysis, expression levels of Lin28b or miRNAs were investigated in response to the overexpression of Fhit gene in H1299 lung cancer cells. Results: A series of in vitro including gene nanostring analyses revealed that Lin28B protein was induced by Fhit gene overexpression, which consequently suppressed Let-7 miRNAs. Also, we found that miRNAs in miR-17/92 clusters are redundantly increased and there is an inverse correlation between Let-7 and miR-17/92 clusters in Fhit-expressing cells. Also, a series of in vitro experiments suggests that ELF-1- and/or STAT1-dependent Lin28b regulation is responsible for Let-7 induction in Fhit-expressing cancer cells. Conclusions: Based on the same experimental system proving that Fhit gene has a robust role in suppressing tumor progression and epithelial-mesenchymal transition, our data show that Fhit mediates the negative feedback between Lin28/Let-7 axis and miR-17/-92 miRNA although the physiological relevance of current interesting observation should be further investigated.

TAZ Is a Negative Regulator of PPARγ Activity in Adipocytes and TAZ Deletion Improves Insulin Sensitivity and Glucose Tolerance.

Insulin resistance is a major factor in obesity-linked type 2 diabetes. PPARγ is a master regulator of adipogenesis, and small molecule agonists, termed thiazolidinediones, are potent therapeutic insulin sensitizers. Here, we studied the role of transcriptional co-activator with PDZ-binding motif (TAZ) as a transcriptional co-repressor of PPARγ. We found that adipocyte-specific TAZ knockout (TAZ AKO) mice demonstrate a constitutively active PPARγ state. Obese TAZ AKO mice show improved glucose tolerance and insulin sensitivity compared to littermate controls. PPARγ response genes are upregulated in adipose tissue from TAZ AKO mice and adipose tissue inflammation was also decreased. In vitro and in vivo mechanistic studies revealed that the TAZ-PPARγ interaction is partially dependent on ERK-mediated Ser112 PPARγ phosphorylation. As adipocyte PPARγ Ser112 phosphorylation is increased in obesity, repression of PPARγ activity by TAZ could contribute to insulin resistance. These results identify TAZ as a new factor in the development of obesity-induced insulin resistance.

Knockdown of Ant2 Reduces Adipocyte Hypoxia And Improves Insulin Resistance in Obesity.

Decreased adipose tissue oxygen tension and increased HIF-1α expression can trigger adipose tissue inflammation and dysfunction in obesity. Our current understanding of obesity-associated decreased adipose tissue oxygen tension is mainly focused on changes in oxygen supply and angiogenesis. Here, we demonstrate that increased adipocyte O2 demand, mediated by ANT2 activity, is the dominant cause of adipocyte hypoxia. Deletion of adipocyte Ant2 improves obesity-induced intracellular adipocyte hypoxia by decreasing obesity-induced adipocyte oxygen demand, without effects on mitochondrial number or mass, or oligomycin-sensitive respiration. This led to decreased adipose tissue HIF-1α expression and inflammation with improved glucose tolerance and insulin resistance in both a preventative or therapeutic setting. Our results suggest that ANT2 may be a target for the development of insulin sensitizing drugs and that ANT2 inhibition might have clinical utility.

Expansion of Islet-Resident Macrophages Leads to Inflammation Affecting ß Cell Proliferation and Function in Obesity.

The nature of obesity-associated islet inflammation and its impact on ß cell abnormalities remains poorly defined. Here, we explore immune cell components of islet inflammation and define their roles in regulating ß cell function and proliferation. Islet inflammation in obese mice is dominated by macrophages. We identify two islet-resident macrophage populations, characterized by their anatomical distributions, distinct phenotypes, and functional properties. Obesity induces the local expansion of resident intra-islet macrophages, independent of recruitment from circulating monocytes. Functionally, intra-islet macrophages impair ß cell function in a cell-cell contact-dependent manner. Increased engulfment of ß cell insulin secretory granules by intra-islet macrophages in obese mice may contribute to restricting insulin secretion. In contrast, both intra- and peri-islet macrophage populations from obese mice promote ß cell proliferation in a PDGFR signaling-dependent manner. Together, these data define distinct roles and mechanisms for islet macrophages in the regulation of islet ß cells.

Adipose Tissue Macrophage-Derived Exosomal miRNAs Can Modulate In Vivo and In Vitro Insulin Sensitivity.

MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis.

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding.

Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with ß-arrestin binding to M1 muscarinic acetylcholine receptors (M1Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M1R, ß-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, ß-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.

Extracellular ATP protects pancreatic duct epithelial cells from alcohol-induced damage through P2Y1 receptor-cAMP signal pathway.

Extracellular adenosine-5'-triphosphate (ATP) regulates cell death and survival of neighboring cells. The detailed effects are diverse depending on cell types and extracellular ATP concentration. We addressed the effect of ATP on ethanol-induced cytotoxicity in epithelial cells, the cell type that experiences the highest concentrations of alcohol. Using pancreatic duct epithelial cells (PDEC), we found that a micromolar range of ATP reverses all intracellular toxicity mechanisms triggered by exceptionally high doses of ethanol and, thus, improves cell viability dramatically. Out of the many purinergic receptors expressed in PDEC, the P2Y1 receptor was identified to mediate the protective effect, based on pharmacological and siRNA assays. Activation of P2Y1 receptors increased intracellular cyclic adenosine monophosphate (cAMP). The protective effect of ATP was mimicked by forskolin and 8-Br-cAMP but inhibited by a protein kinase A (PKA) inhibitor, H-89. Finally, ATP reverted leakiness of PDEC monolayers induced by ethanol and helped to maintain epithelial integrity. We suggest that purinergic receptors reduce extreme alcohol-induced cell damage via the cAMP signal pathway in PDEC and some other types of cells.

Noradrenaline upregulates T-type calcium channels in rat pinealocytes.

KEY POINTS: The mammalian pineal gland is a neuroendocrine organ that responds to circadian and seasonal rhythms. Its major function is to secrete melatonin as a hormonal night signal in response to nocturnal delivery of noradrenaline from sympathetic neurons. Culturing rat pinealocytes in noradrenaline for 24 h induced a low-voltage activated transient Ca(2+) current whose pharmacology and kinetics corresponded to a CaV3.1 T-type channel. The upregulation of the T-type Ca(2+) current is initiated by ß-adrenergic receptors, cyclic AMP and cyclic AMP-dependent protein kinase. Messenger RNA for CaV3.1 T-type channels is significantly elevated by noradrenaline at 8 h and 24 h. The noradrenaline-induced T-type channel mediated an increased Ca(2+) entry and supported modest transient electrical responses to depolarizing stimuli, revealing the potential for circadian regulation of pinealocyte electrical excitability and Ca(2+) signalling. ABSTRACT: Our basic hypothesis is that mammalian pinealocytes have cycling electrical excitability and Ca(2+) signalling that may contribute to the circadian rhythm of pineal melatonin secretion. This study asked whether the functional expression of voltage-gated Ca(2+) channels (CaV channels) in rat pinealocytes is changed by culturing them in noradrenaline (NA) as a surrogate for the night signal. Channel activity was assayed as ionic currents under patch clamp and as optical signals from a Ca(2+)-sensitive dye. Channel mRNAs were assayed by quantitative polymerase chain reaction. Cultured without NA, pinealocytes showed only non-inactivating L-type dihydropyridine-sensitive Ca(2+) current. After 24 h in NA, additional low-voltage activated transient Ca(2+) current developed whose pharmacology and kinetics corresponded to a T-type CaV3.1 channel. This change was initiated by ß-adrenergic receptors, cyclic AMP and protein kinase A as revealed by pharmacological experiments. mRNA for CaV3.1 T-type channels became significantly elevated, but mRNA for another T-type channel and for the major L-type channel did not change. After only 8 h of NA treatment, the CaV3.1 mRNA was already elevated, but the transient Ca(2+) current was not. Even a 16 h wait without NA following the 8 h NA treatment induced little additional transient current. However, these cells were somehow primed to make transient current as a second NA exposure for only 60 min sufficed to induce large T-type currents. The NA-induced T-type channel mediated an increased Ca(2+) entry during short depolarizations and supported modest transient electrical responses to depolarizing stimuli. Such experiments reveal the potential for circadian regulation of excitability.

Apoptotic damage of pancreatic ductal epithelia by alcohol and its rescue by an antioxidant.

Alcohol abuse is a major cause of pancreatitis. However alcohol toxicity has not been fully elucidated in the pancreas and little is known about the effect of alcohol on pancreatic ducts. We report the molecular mechanisms of ethanol-induced damage of pancreatic duct epithelial cells (PDEC). Ethanol treatment for 1, 4, and 24 h resulted in cell death in a dose-dependent manner. The ethanol-induced cell damage was mainly apoptosis due to generation of reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (MMP), and activation of caspase-3 enzyme. The antioxidant N-acetylcysteine (NAC) attenuated these cellular responses and reduced cell death significantly, suggesting a critical role for ROS. Acetaldehyde, a metabolic product of alcohol dehydrogenase, induced significant cell death, depolarization of MMP, and caspase-3 activation as ethanol and this damage was also averted by NAC. Reverse transcription-polymerase chain reaction revealed the expression of several subtypes of alcohol dehydrogenase and acetaldehyde dehydrogenase. Nuclear magnetic resonance spectroscopy data confirmed the accumulation of acetaldehyde in ethanol-treated cells, suggesting that acetaldehyde formation can contribute to alcohol toxicity in PDEC. Finally, ethanol increased the leakage of PDEC monolayer which was again attenuated by NAC. In conclusion, ethanol induces apoptosis of PDEC and thereby may contribute to the development of alcohol-induced pancreatitis.

Characterization of store-operated Ca2+ channels in pancreatic duct epithelia.

Store-operated Ca2+ channels (SOCs) are activated by depletion of intracellular Ca2+ stores following agonist-mediated Ca2+ release. Previously we demonstrated that Ca2+ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca2+ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca2+ influx as Ca2+ was released from intracellular stores. Direct measurement of [Ca2+] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP3 or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La3+ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca2+ entry through SOCs fills internal Ca2+ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca2+ such as salt and mucin secretion from the exocrine pancreatic ducts.

Actin cytoskeleton controls movement of intracellular organelles in pancreatic duct epithelial cells.

In most eukaryotic cells, microtubules and filamentous actin (F-actin) provide tracks on which intracellular organelles move using molecular motors. Here we report that cytoplasmic movement of both mitochondria and lysosomes is slowed by F-actin meshwork formation in pancreatic duct epithelial cells (PDEC). Mitochondria and lysosomes were labeled with fluorescent Mitotracker Red CMXRos and Lysotracker Red DND-99, respectively, and their movements were monitored using epi-fluorescence and confocal microscopy. Mitochondria and lysosomes moving actively at rest stopped rapidly within several seconds after an intracellular Ca(2+) rise induced by activation of P2Y(2) purinergic receptors. The 'freezing' of the organelles was inhibited by blocking the Ca(2+) rise or by pretreatment with latrunculin B, an inhibitor of F-actin formation. Indeed, this freezing effect on the organelles was accompanied by the formation of F-actin in the whole cytoplasm as stained with Alexa 488-phalloidin in fixed PDEC. For real-time monitoring of F-actin formation in live cells, we expressed sGFP-fimbrin actin binding domain2 (fABD2) in PDEC. Rapid recruitment of the fluorescent probe near the nucleus and lysosomes suggested dense F-actin formation around intracellular structures. The development of F-actin paralleled that of organelle freezing. We conclude that rapid Ca(2+)-dependent F-actin formation physically restrains intracellular organelles and reduces their mobility non-selectively in PDEC.

Adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element-binding protein 1c regulates mouse adiponectin expression.

Adiponectin is exclusively expressed in differentiated adipocytes and plays an important role in regulating energy homeostasis, including the glucose and lipid metabolism associated with increased insulin sensitivity. However, the control of adiponectin gene expression in adipocytes is poorly understood. We show here that levels of adiponectin mRNA and protein are reduced in the white adipose tissue of ob/ob and db/db mice and that there is a concomitant reduction of the adipocyte determination- and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein 1c (SREBP1c) transcription factor. To determine whether ADD1/SREBP1c regulates adiponectin gene expression, we isolated and characterized the mouse adiponectin promoter. Analysis of the adiponectin promoter revealed putative binding sites for the adipogenic transcription factors ADD1/SREBP1c, peroxisomal proliferator-activated receptor gamma and CCAAT enhancer-binding proteins. DNase I footprinting and chromatin immunoprecipitation analyses revealed that ADD1/SREBP1c binds in vitro and in vivo to the proximal promoter containing sterol regulatory element (SRE) motifs. A luciferase reporter containing the promoter was transactivated by ADD1/SREBP1c, and introduction of SRE mutations into the construct abolished transactivation. Adenoviral overexpression of ADD1/SREBP1c also elevated adiponectin mRNA and protein levels in 3T3-L1 adipocytes. Furthermore, insulin stimulated adiponectin mRNA expression in adipocytes and augmented transactivation of the adiponectin promoter by ADD1/SREBP1c. Taken together, these data indicate that ADD1/SREBP1c controls adiponectin gene expression in differentiated adipocytes.

Functional characterization of the human resistin promoter with adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element binding protein 1c and CCAAT enhancer binding protein-alpha.

Recent studies with murine models propose that resistin would be a possible mediator to link between obesity and insulin resistance. Although it has been reported that resistin is highly expressed and secreted by adipocytes, transcription factors that are involved in resistin gene expression have not been well characterized. To investigate the molecular mechanisms of resistin gene expression, we cloned and characterized the human resistin promoter. Sequence analysis of the resistin promoter revealed several putative binding sites for adipogenic transcription factors including adipocyte determination- and differentiation-dependent factor 1 (ADD1)/sterol regulatory element binding protein 1c (SREBP1c) and CCAAT enhancer binding protein-alpha (C/EBP alpha). EMSA and chromatin immunoprecipitation assays demonstrated that ADD1/SREBP1c binds to the human resistin promoter in vitro and in vivo. Expression of ADD1/SREBP1c transactivated the luciferase reporter gene activity, the promoter region of which contains a human resistin promoter in a sterol regulatory element (SRE)-dependent manner. Furthermore, ectopic expression of ADD1/SREBP1c by adenovirus significantly increased the expression of resistin mRNA in adipocytes. Human resistin promoter was also activated by C/EBP alpha expression, although ectopic expression of both transcription factors did not show any synergistic effects on the activation of resistin promoter. Together, these data suggest that ADD1/SREBP1c and C/EBP alpha may play discrete roles in the regulation of the resistin gene expression.