Anti-apoptotic effects of Z alpha-1 antitrypsin in human bronchial epithelial cells.

α1-antitrypsin (α1-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of α1-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z α1-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved.

Control, M variant α1-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-κB activation and induced expression of a selection of pro- and anti-apoptotic genes.

Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-κB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-κB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies.

The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

Mechanisms underlying the metabolic actions of galegine that contribute to weight loss in mice

Background and purpose: Galegine and guanidine, originally isolated from Galega officinalis, led to the development of the biguanides. The weight-reducing effects of galegine have not previously been studied and the present investigation was undertaken to determine its mechanism(s) of action.

Experimental approach: Body weight and food intake were examined in mice. Glucose uptake and acetyl-CoA carboxylase activity were studied in 3T3-L1 adipocytes and L6 myotubes and AMP activated protein kinase (AMPK) activity was examined in cell lines. The gene expression of some enzymes involved in fat metabolism was examined in 3T3-L1 adipocytes.

Key results: Galegine administered in the diet reduced body weight in mice. Pair-feeding indicated that at least part of this effect was independent of reduced food intake. In 3T3-L1 adipocytes and L6 myotubes, galegine (50 µm-3 mm) stimulated glucose uptake. Galegine (1–300 µm) also reduced isoprenaline-mediated lipolysis in 3T3-L1 adipocytes and inhibited acetyl-CoA carboxylase activity in 3T3-L1 adipocytes and L6 myotubes. Galegine (500 µm) down-regulated genes concerned with fatty acid synthesis, including fatty acid synthase and its upstream regulator SREBP. Galegine (10 µm and above) produced a concentration-dependent activation of AMP activated protein kinase (AMPK) in H4IIE rat hepatoma, HEK293 human kidney cells, 3T3-L1 adipocytes and L6 myotubes.

Conclusions and implications: Activation of AMPK can explain many of the effects of galegine, including enhanced glucose uptake and inhibition of acetyl-CoA carboxylase. Inhibition of acetyl-CoA carboxylase both inhibits fatty acid synthesis and stimulates fatty acid oxidation, and this may to contribute to the in vivo effect of galegine on body weight.

Short Isoforms of the Cold Receptor TRPM8 Inhibit Channel Gating by Mimicking Heat Action Rather than Chemical Inhibitors

Transient receptor potential (TRP) channels couple various environmental factors to changes in membrane potential, calcium influx, and cell signaling. They also integrate multiple stimuli through their typically polymodal activation. Thus, although the TRPM8 channel has been extensively investigated as the major neuronal cold sensor, it is also regulated by various chemicals, as well as by several short channel isoforms. Mechanistic understanding of such complex regulation is facilitated by quantitative single-channel analysis. We have recently proposed a single-channel mechanism of TRPM8 regulation by voltage and temperature. Using this gating mechanism, we now investigate TRPM8 inhibition in cell-attached patches using HEK293 cells expressing TRPM8 alone or coexpressed with its short sM8-6 isoform. This is compared with inhibition by the chemicals N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC) and clotrimazole or by elevated temperature. We found that within the seven-state single-channel gating mechanism, inhibition of TRPM8 by short sM8-6 isoforms closely resembles inhibition by increased temperature. In contrast, inhibition by BCTC and that by clotrimazole share a different set of common features. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

A novel dual-fluorescence strategy for functionally validating microRNA targets in 3-prime untranslated regions: regulation of the inward rectifier potassium channel Kir2.1 by miR-212.

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 (KCNJ2) which is dysregulated in cardiac and vascular disorders. The 3'UTR was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known downregulator of Kir2.1 expression, and was used to investigate targeting of the Kir2.1 3'UTR by miR-212. Red/green ratio was lower in miR-212-expressing cells compared to non-targeting controls, an effect that was attenuated by mutating the predicted target site. MiR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

Clathrin is spindle-associated but not essential for mitosis

BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

METHODOLOGY/PRINCIPAL FINDINGS: Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

CONCLUSIONS/SIGNIFICANCE: We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors.

Subcellular localization of legionella Dot/Icm effectors

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.