Comparison of the reverse bevel versus Franseen type endoscopic ultrasound needle.

BACKGROUND: Reverse bevel (RB) needle is widely used for endoscopic ultrasound fine needle biopsy (EUS-FNB). A 3-plane symmetrical needle with Franseen geometry (FG) has recently become available. AIM: To compare the clinical efficacy of FG to that of RB needle. METHODS: A retrospective cohort study of all adult patients who underwent EUS-FNB for solid and mixed lesions either with 22G RB needle or 22G FG needle between January 2016 and February 2019 was undertaken. All cytology slides were reviewed by an independent gastrointestinal cytopathologist blinded to the needle used and the initial cytology report. The primary and secondary outcomes were to assess the sample adequacy using Euro-cytology criteria and the number of cell clusters, respectively. RESULTS: Two hundred and twenty six procedures were included in the study. RB needle was used in 128 procedures and FG needle in 98 procedures. The baseline characteristics of both groups were comparable. On multivariable analysis, FG needle (P = 0.02) and location of the lesion (P < 0.01) were independently associated with adequate tissue. Further, the use of FG needle (P = 0.04) and the size of the lesion (P = 0.02) were independently associated with acquisition of increased number of cell clusters. CONCLUSION: FG needle is superior to RB needle in acquiring adequate tissue and attaining higher number of cell clusters for solid and mixed lesions.

Bone biology: insights from osteogenesis imperfecta and related rare fragility syndromes.

The limited accessibility of bone and its mineralized nature have restricted deep investigation of its biology. Recent breakthroughs in identification of mutant proteins affecting bone tissue homeostasis in rare skeletal diseases have revealed novel pathways involved in skeletal development and maintenance. The characterization of new dominant, recessive and X-linked forms of the rare brittle bone disease osteogenesis imperfecta (OI) and other OI-related bone fragility disorders was a key player in this advance. The development of in vitro models for these diseases along with the generation and characterization of murine and zebrafish models contributed to dissecting previously unknown pathways. Here, we describe the most recent advances in the understanding of processes involved in abnormal bone mineralization, collagen processing and osteoblast function, as illustrated by the characterization of new causative genes for OI and OI-related fragility syndromes. The coordinated role of the integral membrane protein BRIL and of the secreted protein PEDF in modulating bone mineralization as well as the function and cross-talk of the collagen-specific chaperones HSP47 and FKBP65 in collagen processing and secretion are discussed. We address the significance of WNT ligand, the importance of maintaining endoplasmic reticulum membrane potential and of regulating intramembrane proteolysis in osteoblast homeostasis. Moreover, we also examine the relevance of the cytoskeletal protein plastin-3 and of the nucleotidyltransferase FAM46A. Thanks to these advances, new targets for the development of novel therapies for currently incurable rare bone diseases have been and, likely, will be identified, supporting the important role of basic science for translational approaches.

BDNF activates an NFI-dependent neurodevelopmental timing program by sequestering NFATc4.

We show that BDNF regulates the timing of neurodevelopment via a novel mechanism of extranuclear sequestration of NFATc4 in Golgi. This leads to accelerated derepression of an NFI temporal occupancy gene program in cerebellar granule cells that includes Bdnf itself, revealing an autoregulatory loop within the program driven by BDNF and NFATc4.

Neuritin Up-regulates Kv4.2 α-Subunit of Potassium Channel Expression and Affects Neuronal Excitability by Regulating the Calcium-Calcineurin-NFATc4 Signaling Pathway.

Neuritin is an important neurotrophin that regulates neural development, synaptic plasticity, and neuronal survival. Elucidating the downstream molecular signaling is important for potential therapeutic applications of neuritin in neuronal dysfunctions. We previously showed that neuritin up-regulates transient potassium outward current (IA) subunit Kv4.2 expression and increases IA densities, in part by activating the insulin receptor signaling pathway. Molecular mechanisms of neuritin-induced Kv4.2 expression remain elusive. Here, we report that the Ca(2+)/calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) c4 axis is required for neuritin-induced Kv4.2 transcriptional expression and potentiation of IA densities in cerebellum granule neurons. We found that neuritin elevates intracellular Ca(2+) and increases Kv4.2 expression and IA densities; this effect was sensitive to CaN inhibition and was eliminated in Nfatc4(-/-) mice but not in Nfatc2(-/-) mice. Stimulation with neuritin significantly increased nuclear accumulation of NFATc4 in cerebellum granule cells and HeLa cells, which expressed IR. Furthermore, NFATc4 was recruited to the Kv4.2 gene promoter loci detected by luciferase reporter and chromatin immunoprecipitation assays. More importantly, data obtained from cortical neurons following adeno-associated virus-mediated overexpression of neuritin indicated that reduced neuronal excitability and increased formation of dendritic spines were abrogated in the Nfatc4(-/-) mice. Together, these data demonstrate an indispensable role for the CaN/NFATc4 signaling pathway in neuritin-regulated neuronal functions.

Biotinylation: a novel posttranslational modification linking cell autonomous circadian clocks with metabolism.

Circadian clocks are critical modulators of metabolism. However, mechanistic links between cell autonomous clocks and metabolic processes remain largely unknown. Here, we report that expression of the biotin transporter slc5a6 gene is decreased in hearts of two distinct genetic mouse models of cardiomyocyte-specific circadian clock disruption [i.e., cardiomyocyte-specific CLOCK mutant (CCM) and cardiomyocyte-specific BMAL1 knockout (CBK) mice]. Biotinylation is an obligate posttranslational modification for five mammalian carboxylases: acetyl-CoA carboxylase α (ACCα), ACCß, pyruvate carboxylase (PC), methylcrotonyl-CoA carboxylase (MCC), and propionyl-CoA carboxylase (PCC). We therefore hypothesized that the cardiomyocyte circadian clock impacts metabolism through biotinylation. Consistent with decreased slc5a6 expression, biotinylation of all carboxylases is significantly decreased (10-46%) in CCM and CBK hearts. In association with decreased biotinylated ACC, oleate oxidation rates are increased in both CCM and CBK hearts. Consistent with decreased biotinylated MCC, leucine oxidation rates are significantly decreased in both CCM and CBK hearts, whereas rates of protein synthesis are increased. Importantly, feeding CBK mice with a biotin-enriched diet for 6 wk normalized myocardial 1) ACC biotinylation and oleate oxidation rates; 2) PCC/MCC biotinylation (and partially restored leucine oxidation rates); and 3) net protein synthesis rates. Furthermore, data suggest that the RRAGD/mTOR/4E-BP1 signaling axis is chronically activated in CBK and CCM hearts. Finally we report that the hepatocyte circadian clock also regulates both slc5a6 expression and protein biotinylation in the liver. Collectively, these findings suggest that biotinylation is a novel mechanism by which cell autonomous circadian clocks influence metabolic pathways.

Reciprocal autoregulation by NFI occupancy and ETV1 promotes the developmental expression of dendrite-synapse genes in cerebellar granule neurons.

Nuclear Factor One (NFI) transcription factors regulate temporal gene expression required for dendritogenesis and synaptogenesis via delayed occupancy of target promoters in developing cerebellar granule neurons (CGNs). Mechanisms that promote NFI temporal occupancy have not been previously defined. We show here that the transcription factor ETV1 directly binds to and is required for expression and NFI occupancy of a cohort of NFI-dependent genes in CGNs maturing in vivo. Expression of ETV1 is low in early postnatal cerebellum and increases with maturation, mirroring NFI temporal occupancy of coregulated target genes. Precocious expression of ETV1 in mouse CGNs accelerated onset of expression and NFI temporal occupancy of late target genes and enhanced Map2(+) neurite outgrowth. ETV1 also activated expression and NFI occupancy of the Etv1 gene itself, and this autoregulatory loop preceded ETV1 binding and activation of other coregulated target genes in vivo. These findings suggest a potential model in which ETV1 activates NFI temporal binding to a subset of late-expressed genes in a stepwise manner by initial positive feedback regulation of the Etv1 gene itself followed by activation of downstream coregulated targets as ETV1 expression increases. Sequential transcription factor autoregulation and subsequent binding to downstream promoters may provide an intrinsic developmental timer for dendrite/synapse gene expression.

Dysregulated Inflammatory Signaling upon Charcot-Marie-Tooth Type 1C Mutation of SIMPLE Protein.

Endosomal trafficking is a key mechanism to modulate signal propagation and cross talk. Ubiquitin adaptors, along with endosomal sorting complex required for transport (ESCRT) complexes, are also integrated to terminate ligand-receptor activation in late endosomes and multivesicular bodies (MVBs). Within these pathways, we recently demonstrated that the protein SIMPLE is a novel player in MVB regulation. SIMPLE is also clinically important and its mutation accounts for the Charcot-Marie-Tooth type 1C (CMT1C) disease. MVB defects of mutation and deletion of SIMPLE, however, are distinct. Here, we show that MVB defects found in mutation but not deletion of SIMPLE lead to impaired turnover and accumulation of ESCRT-0 protein Hrs punctain late endosomes. We further uncover increased colocalization of ubiquitin ligase TRAF6 and Hrs in late endosomes. Upon stimulation with interkeukin-1 or transforming growth factor , prolonged activation of p38 kinase/JNK is detected, while nuclear accumulation of NF-κB and phosphorylation of SMAD2 is reduced with CMT1C mutation. The aberrant kinetics we observed in inflammatory signaling may contribute to increased tumor susceptibility and changes in the levels of chemokines/cytokines that result from CMT1C mutation. We propose that altered endosomal trafficking due to malformations of MVBs and subsequent atypical signaling kinetic may account for a toxic gain of function in CMT1C pathogenesis.

Cardiomyocyte-specific BMAL1 plays critical roles in metabolism, signaling, and maintenance of contractile function of the heart.

Circadian clocks are cell autonomous, transcriptionally based, molecular mechanisms that confer the selective advantage of anticipation, enabling cells/organs to respond to environmental factors in a temporally appropriate manner. Critical to circadian clock function are 2 transcription factors, CLOCK and BMAL1. The purpose of the present study was to reveal novel physiologic functions of BMAL1 in the heart, as well as to determine the pathologic consequences of chronic disruption of this circadian clock component. To address this goal, we generated cardiomyocyte-specific Bmal1 knockout (CBK) mice. Following validation of the CBK model, combined microarray and in silico analyses were performed, identifying 19 putative direct BMAL1 target genes, which included a number of metabolic (e.g., ß-hydroxybutyrate dehydrogenase 1 [Bdh1]) and signaling (e.g., the p85α regulatory subunit of phosphatidylinositol 3-kinase [Pik3r1]) genes. Results from subsequent validation studies were consistent with regulation of Bdh1 and Pik3r1 by BMAL1, with predicted impairments in ketone body metabolism and signaling observed in CBK hearts. Furthermore, CBK hearts exhibited depressed glucose utilization, as well as a differential response to a physiologic metabolic stress (i.e., fasting). Consistent with BMAL1 influencing critical functions in the heart, echocardiographic, gravimetric, histologic, and molecular analyses revealed age-onset development of dilated cardiomyopathy in CBK mice, which was associated with a severe reduction in life span. Collectively, our studies reveal that BMAL1 influences metabolism, signaling, and contractile function of the heart.

Circadian regulation of myocardial sarcomeric Titin-cap (Tcap, telethonin): identification of cardiac clock-controlled genes using open access bioinformatics data.

Circadian rhythms are important for healthy cardiovascular physiology and are regulated at the molecular level by a circadian clock mechanism. We and others previously demonstrated that 9-13% of the cardiac transcriptome is rhythmic over 24 h daily cycles; the heart is genetically a different organ day versus night. However, which rhythmic mRNAs are regulated by the circadian mechanism is not known. Here, we used open access bioinformatics databases to identify 94 transcripts with expression profiles characteristic of CLOCK and BMAL1 targeted genes, using the CircaDB website and JTK_Cycle. Moreover, 22 were highly expressed in the heart as determined by the BioGPS website. Furthermore, 5 heart-enriched genes had human/mouse conserved CLOCK:BMAL1 promoter binding sites (E-boxes), as determined by UCSC table browser, circadian mammalian promoter/enhancer database PEDB, and the European Bioinformatics Institute alignment tool (EMBOSS). Lastly, we validated findings by demonstrating that Titin cap (Tcap, telethonin) was targeted by transcriptional activators CLOCK and BMAL1 by showing 1) Tcap mRNA and TCAP protein had a diurnal rhythm in murine heart; 2) cardiac Tcap mRNA was rhythmic in animals kept in constant darkness; 3) Tcap and control Per2 mRNA expression and cyclic amplitude were blunted in Clock(Δ19/Δ19) hearts; 4) BMAL1 bound to the Tcap promoter by ChIP assay; 5) BMAL1 bound to Tcap promoter E-boxes by biotinylated oligonucleotide assay; and 6) CLOCK and BMAL1 induced tcap expression by luciferase reporter assay. Thus this study identifies circadian regulated genes in silico, with validation of Tcap, a critical regulator of cardiac Z-disc sarcomeric structure and function.

Comparison of acute hepatitis E infection outcome in patients with and without chronic hepatitis B infection: a 10 year retrospective study in three regional hospitals in Hong Kong.

BACKGROUND: Acute hepatitis E virus (HEV) infection has recently become the commonest cause of acute viral hepatitis in Hong Kong (HK) with majority of HEV belonging to genotype 4. Studies from China have shown that acute hepatitis E patients with underlying chronic hepatitis B virus (HBV) infection may have a worse outcome than those without. In India where genotype 1 is more prevalent, superinfection with hepatitis E on patients with chronic liver disease including chronic hepatitis B infection can cause liver decompensation. OBJECTIVES: The study aims to determine the clinical outcome of acute hepatitis E infection in patients with and without chronic hepatitis B infection in HK. STUDY DESIGN: The rates of liver failure, liver-related mortality and all-cause mortality will be compared between acute hepatitis E patients with and without chronic hepatitis B. Analysis was performed using the Statistical Package for the Social Science (SPSS version 12.0). All statistical tests were two-sided, and statistical significance was taken as p<0.05. RESULTS: Chronic HBV carriers with acute HEV infection were found to have higher liver failure rate, liver-related mortality and all-cause mortality, but the results did not reach statistical significance. Chronic HBV carriers were found to have statistically significantly lower admission ALT level, lower day 28 serum albumin level and higher day 28 serum ALT level. CONCLUSIONS: A prospective study with sufficient sample size is needed to confirm whether the clinical outcome of patients with chronic HBV infection is worse compared with patients who were not chronic HBV carriers.

Role of extracellular signal-regulated kinase 5 in adipocyte signaling.

Increased adiposity due to energy imbalance is a critical factor of the epidemic crisis of obesity and type II diabetes. In addition to the obvious role in energy storage, regulatory factors are secreted from adipose depots to control appetite and cellular homeostasis. Complex signaling cross-talks within adipocyte are also evident due to the metabolic and immune nature of adipose depots. Here, we uncover a role of extracellular signal-regulated kinase 5 (ERK5) in adipocyte signaling. We find that deletion of ERK5 in adipose depots (adipo-ERK5(-/-)) increases adiposity, in part, due to increased food intake. Dysregulated secretion of adipokines, leptin resistance, and impaired glucose handling are also found in adipo-ERK5(-/-) mice. Mechanistically, we show that ERK5 impinges on transcription factor NFATc4. Decreased phosphorylation at the conserved gate-keeping Ser residues and increased nuclear localization of NFATc4 are found in adipo-ERK5(-/-) mice. We also find attenuated PKA activation in adipo-ERK5(-/-) mice. In response to stimulation of ß-adrenergic G-protein-coupled receptor, we find decreased NFATc4 phosphorylation and impaired PKA activation in adipo-ERK5(-/-) mice. Reduced cAMP accumulation and increased phosphodiesterase activity are also found. Together, these results demonstrate integration of ERK5 with NFATc4 nucleo-cytoplasmic shuttling and PKA activation in adipocyte signaling.

Chronic exercise downregulates myocardial myoglobin and attenuates nitrite reductase capacity during ischemia-reperfusion.

The infarct sparing effects of exercise are evident following both long-term and short-term training regimens. Here we compared the infarct-lowering effects of nitrite therapy, voluntary exercise, and the combination of both following myocardial ischemia-reperfusion (MI/R) injury. We also compared the degree to which each strategy increased cardiac nitrite levels, as well as the effects of each strategy on the nitrite reductase activity of the heart. Mice subjected to voluntary wheel running (VE) for 4weeks displayed an 18% reduction in infarct size when compared to sedentary mice, whereas mice administered nitrite therapy (25mg/L in drinking water) showed a 53% decrease. However, the combination of VE and nitrite exhibited no further protection than VE alone. Although the VE and nitrite therapy mice showed similar nitrite levels in the heart, cardiac nitrite reductase activity was significantly reduced in the VE mice. Additionally, the cardiac protein expression of myoglobin, a known nitrite reductase, was also reduced after VE. Further studies revealed that cardiac NFAT activity was lower after VE due to a decrease in calcineurin activity and an increase in GSK3ß activity. These data suggest that VE downregulates cardiac myoglobin levels by inhibiting calcineurin/NFAT signaling. Additionally, these results suggest that the modest infarct sparing effects of VE are the result of a decrease in the hearts ability to reduce nitrite to nitric oxide during MI/R.

Mutation of SIMPLE in Charcot-Marie-Tooth 1C alters production of exosomes.

Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) account for the rare autosomal-dominant demyelination in CMT1C patients. Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by perplexity about the role of SIMPLE, which is expressed in multiple cell types. Here we show that SIMPLE resides within the intraluminal vesicles of multivesicular bodies (MVBs) and inside exosomes, which are nanovesicles secreted extracellularly. Targeting of SIMPLE to exosomes is modulated by positive and negative regulatory motifs. We also find that expression of SIMPLE increases the number of exosomes and secretion of exosome proteins. We engineer a point mutation on the SIMPLE allele and generate a physiological mouse model that expresses CMT1C-mutated SIMPLE at the endogenous level. We find that CMT1C mouse primary embryonic fibroblasts show decreased number of exosomes and reduced secretion of exosome proteins, in part due to improper formation of MVBs. CMT1C patient B cells and CMT1C mouse primary Schwann cells show similar defects. Together the data indicate that SIMPLE regulates the production of exosomes by modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome-mediated intercellular communications may place an overwhelming burden on the nervous system and account for CMT1C molecular pathogenesis.

Temporal regulation of nuclear factor one occupancy by calcineurin/NFAT governs a voltage-sensitive developmental switch in late maturing neurons.

Dendrite and synapse development are critical for establishing appropriate neuronal circuits, and disrupted timing of these events can alter neural connectivity. Using microarrays, we have identified a nuclear factor I (NFI)-regulated temporal switch program linked to dendrite formation in developing mouse cerebellar granule neurons (CGNs). NFI function was required for upregulation of many synapse-related genes as well as downregulation of genes expressed in immature CGNs. Chromatin immunoprecipitation analysis revealed that a central feature of this program was temporally regulated NFI occupancy of late-expressed gene promoters. Developing CGNs undergo a hyperpolarizing shift in membrane potential, and depolarization inhibits their dendritic and synaptic maturation via activation of calcineurin (CaN) (Okazawa et al., 2009). Maintaining immature CGNs in a depolarized state blocked NFI temporal occupancy of late-expressed genes and the NFI switch program via activation of the CaN/nuclear factor of activated T-cells, cytoplasmic (NFATc) pathway and promotion of late-gene occupancy by NFATc4, and these mechanisms inhibited dendritogenesis. Conversely, inhibition of the CaN/NFATc pathway in CGNs maturing under physiological nondepolarizing conditions upregulated the NFI switch program, NFI temporal occupancy, and dendrite formation. NFATc4 occupied the promoters of late-expressed NFI program genes in immature mouse cerebellum, and its binding was temporally downregulated with development. Further, NFI temporal binding and switch gene expression were upregulated in the developing cerebellum of Nfatc4 (-/-) mice. These findings define a novel NFI switch and temporal occupancy program that forms a critical link between membrane potential/CaN and dendritic maturation in CGNs. CaN inhibits the program and NFI occupancy in immature CGNs by promoting NFATc4 binding to late-expressed genes. As maturing CGNs become more hyperpolarized, NFATc4 binding declines leading to onset of NFI temporal binding and the NFI switch program.

Ablation of calcineurin Aß reveals hyperlipidemia and signaling cross-talks with phosphodiesterases.

Insulin resistance, hyperlipidemia, and cardiovascular complications are common dysregulations of metabolic syndrome. Transplant patients treated with immunosuppressant drugs such as cyclosporine A (CsA), an inhibitor of calcineurin phosphatase, frequently develop similar metabolic complications. Although calcineurin is known to mediate insulin sensitivity by regulating ß-cell growth and adipokine gene transcription, its role in lipid homeostasis is poorly understood. Here, we examined lipid homeostasis in mice lacking calcineurin Aß (CnAß(-/-)). We show that mice lacking calcineurin Aß are hyperlipidemic and develop age-dependent insulin resistance. Hyperlipidemia found in CnAß(-/-) mice is, in part, due to increased lipolysis in adipose tissues, a process mediated by ß-adrenergic G-protein-coupled receptor signaling pathways. CnAß(-/-) mice also exhibit additional pathophysiological phenotypes caused by the potentiated GPCR signaling pathways. A cell autonomous mechanism with sustained cAMP/PKA activation is found in CnAß(-/-) mice or upon CsA treatment to inhibit calcineurin. Increased PKA activation and cAMP accumulation in CnAß(-/-) mice, however, are sensitive to phosphodiesterase inhibitor. Indeed, we show that calcineurin regulates degradation of phosphodiesterase 3B, in addition to phosphodiesterase 4D. These results establish a role for calcineurin in lipid homeostasis. These data also indicate that potentiated cAMP signaling pathway may provide an alternative molecular pathogenesis for the metabolic complications elicited by CsA in transplant patients.

Neuritin activates insulin receptor pathway to up-regulate Kv4.2-mediated transient outward K+ current in rat cerebellar granule neurons.

Neuritin is a new neurotrophic factor discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin also plays multiple roles in the process of neural development and synaptic plasticity. The receptors for binding neuritin and its downstream signaling effectors, however, remain unclear. Here, we report that neuritin specifically increases the densities of transient outward K(+) currents (I(A)) in rat cerebellar granule neurons (CGNs) in a time- and concentration-dependent manner. Neuritin-induced amplification of I(A) is mediated by increased mRNA and protein expression of Kv4.2, the main α-subunit of I(A). Exposure of CGNs to neuritin markedly induces phosphorylation of ERK (pERK), Akt (pAkt), and mammalian target of rapamycin (pmTOR). Neuritin-induced I(A) and increased expression of Kv4.2 are attenuated by ERK, Akt, or mTOR inhibitors. Unexpectedly, pharmacological blockade of insulin receptor, but not the insulin-like growth factor 1 receptor, abrogates the effect of neuritin on I(A) amplification and Kv4.2 induction. Indeed, neuritin activates downstream signaling effectors of the insulin receptor in CGNs and HeLa. Our data reveal, for the first time, an unanticipated role of the insulin receptor in previously unrecognized neuritin-mediated signaling.

Elucidating protein: DNA complex by oligonucleotide DNA affinity purification.

Transcription factors recruit a wide variety of associated co-factors to regulate gene expression. These co-factors include protein kinases, phosphatases, deacetylases, methylases, and ubiquitin ligases, etc. To identify novel protein kinases associated with transcription factor NFAT, we took advantage of the increased ability of DNA binding and used an oligonucleotide affinity-binding approach. Coupling with in-gel kinase assays to detect phosphotransferase activity, we were able to identify p90 ribosomal S6 kinase (RSK) and p70 S6 kinase (S6K) that are present in the NFAT:DNA complex. We further demonstrated that RSK and S6K binds to and physically interacts with NFATc4. Similar oligonucleotide affinity-binding approach can be coupled with other enzymatic reactions, such as dephosphorylation, deacetylation, methylation, ubiquitination, etc. Mass spectrometry can also be carried out to systemically identify these transcription co-factors in the protein:DNA complex. Lastly, gene-specific enhancer elements can also be devised based on their respective sequence to identify distinctive protein:DNA complexes.

Assay for protein modification by poly-ADP-ribose in vitro.

The enzymatic function of poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) is central to many of its function as a component of DNA repair machinery, modulator of gene transcription, and cell differentiation. While the auto-modification domain of PARP has been shown to be a primary acceptor site of poly-ADP ribose (pADPr), other DNA binding nuclear proteins are also modified by pADPr. It is -generally accepted that pADPr polymer is built upon the carboxyl side chain of specific Glu, Asp, and/or Lys residues within the target protein. Identification of the unique amino acid acceptors of pADPr in these nuclear proteins is an active area of study. Because of the heterogeneity of pADPr chain on modified -protein targets, the resulting modified proteins have unpredictable final masses, making it difficult to -identify acceptor amino acids. Using recombinant proteins, in vitro pADP ribosylation assay and mass spectrometry, we have been able to identify conserved Glu residue in transcription factor NFAT that is enzymatically modified in vitro with pADPr by PARP-1. We discuss this protocol here as a model approach for identifying pADPr acceptors in other nuclear proteins.

NFATc1 regulation of TRAIL expression in human intestinal cells.

TNF-related apoptosis-inducing ligand (TRAIL; Apo2) has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT) participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187 (Io) increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA), an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter.