Transfected CHO Cells Research Articles

Importance of NPA motifs in the expression and function of water channel aquaporin-1

The asparagine-proline-alanine sequences (NPA motifs) are highly conserved in aquaporin water channel family. Crystallographic studies of AQP1 structure demonstrated that the two NPA motifs are in the narrow central constriction of the channel, serving to bind water molecules for selective and efficient water passage. To investigate the importance of the two NPA motifs in the structure, function and biogenesis of aquaporin water channels, we generated AQP1 mutations with NPA1 deletion, NPA2 deletion and NPA1,2 double deletion. The coding sequences of the three mutated cDNAs were subcloned into the mammalian expression vector pcDNA3.1 to form expression plasmids. We established stably transfected CHO cell lines expressing these AQP1 mutants. Immunofluorescence indicated that all the three mutated AQP1 proteins are expressed normally on the plasma membrane of stably transfected CHO cells, suggesting that deletion of NPA motifs does not influence the expression and intracellular processing of AQP1. Functional analysis demonstrated that NPA1 or NPA2 deletion reduced AQP1 water permeability by 49.6% and 46.7%, respectively, while NPA1,2 double deletion had little effect on AQP1 water permeability. These results provide evidence that NPA motifs are important for water permeation but not essential for the expression, intracellular processing and the basic structure of AQP1 water channel.

Caveolin-1 Triggers T-cell Activation via CD26 in Association with CARMA1

CD26 is a widely distributed 110-kDa cell surface glycoprotein with an important role in T-cell costimulation. We demonstrated previously that CD26 binds to caveolin-1 in antigen-presenting cells, and following exogenous CD26 stimulation, Tollip and IRAK-1 disengage from caveolin-1 in antigen-presenting cells. IRAK-1 is then subsequently phosphorylated to up-regulate CD86 expression, resulting in subsequent T-cell proliferation. However, it is unclear whether caveolin-1 is a costimulatory ligand for CD26 in T-cells. Using soluble caveolin-1-Fc fusion protein, we now show that caveolin-1 is the costimulatory ligand for CD26, and that ligation of CD26 by caveolin-1 induces T-cell proliferation and NF-kappaB activation in a T-cell receptor/CD3-dependent manner. We also demonstrated that the cytoplasmic tail of CD26 interacts with CARMA1 in T-cells, resulting in signaling events that lead to NF-kappaB activation. Ligation of CD26 by caveolin-1 recruits a complex consisting of CD26, CARMA1, Bcl10, and IkappaB kinase to lipid rafts. Taken together, our findings provide novel insights into the regulation of T-cell costimulation via the CD26 molecule.

Crim1 is involved in adhesion and migration events during development

We investigated the role Crim1, a type-I transmembrane protein containing Chordin-like cysteine-rich repeat domains, plays in development. To this end, we utilised a gene-trap line, KST264, hypomorphic for Crim1. KST264/KST264 mice die perinatally and display phenotypes indicative of adhesion and migration defects. These include skin “blebbing” from 12.5dpc, where the Crim1-expressing epidermis separates from the underlying dermis. From 15.5dpc, KST264/KST264 mice display renal defects, including enlarged, simplified glomerular capillaries and effacement of the Crim1-expressing podocytes, reminiscent of alpha 3 integrin “knock-out” mice and suggestive of defects in adhesion to the glomerular basement membrane. Furthermore, migration assays on primary cultures of epicardia from the hearts of KST264/KST264 embryos indicate that cells hypomorphic for Crim1 demonstrate increased migration. Conversely, stable transfected CHO cells over-expressing Crim1 showed a decreased propensity to migrate in different assays. Moreover, Crim1 over-expressing cell lines displayed increased adhesion to of collagen and laminin. We conclude that Crim1 mediates cell adhesion and/or migration in development and that this may reflect a direct interaction with ECM components. This research was supported by the National Health and Medical Research Council of Australia (grant # 455972).

The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide

There is a great demand for the improvement of mammalian cell production systems such that they can compete economically with their prokaryotic counterparts. Of a number of parameters that need to be explored to accomplish this we have tested the effects of different signal peptides on the synthesis and secretion of Gaussia princeps luciferase in mammalian cells. A series of plasmids were transfected into CHO cells where the coding region for the marine luciferase was fused to the signal peptide coding regions derived from different sources. Both cell extracts and medium samples were analysed for luciferase activity. When the native Gaussia luciferase signal sequence in the vector was substituted by that from human interleukin-2 or albumin then the amount of active recombinant protein produced was substantially reduced, both in transiently and stably transfected cells. Western blotting showed that enzyme activity and protein levels mirrored one another. The major decrease in luciferase activity was shown not to be a result of decreased mRNA levels, indicating the involvement of a post-transcriptional event. When the coding region of human endostatin was fused to that of the Gaussia luciferase signal peptide then an elevated level of secreted endostatin was observed compared to when that of the albumin signal peptide was used. Stable transfection of HepG2 cells with the different signal peptide constructs gave essentially the same results as seen in CHO cells. The overall results indicate that the choice of signal peptide can be imperative to ensure an optimal synthesis and secretion of a recombinant protein in a mammalian cell culture system.

Erythropoietin Fusion Proteins Comprised of Identical Head-to-Tail Repeats with Enhanced Biological Activity.

Recombinant human erythropoietin (EPO, epoetin) is used widely for treatment of chronic anemia due to renal failure, cancer, and other causes. However, considerably high and frequent doses of EPO are required to maintain therapeutic effectiveness, since it has a relatively short in vivo half-life. Thus, alternatives with higher efficacy and/or longer half-life are being developed. We have shown previously that EPO-dimers, either produced by chemical cross-linking of monomeric EPO or expressed as a recombinant fusion protein from COS cells, exhibit enhanced biological properties in vitro and in vivo (Sytkowski, et.al. Proc. Natl. Acad. Sci. USA 95, 1184; Sytkowski, et.al. J. Biol. Chem. 274, 24773). We now report increased activities of EPO-dimer fusion protein and EPO-trimer fusion protein comprised of identical head-to-tail repeats and a 15 or 20-amino acid linker (for dimer), or 17-amino acid linkers (for trimer) produced from stably transfected CHO cells. EPO-fusion proteins were expressed under a CMV promoter with a signal peptide present on the first monomer coding sequence. The EPO-dimer fusion protein was connected with either three or four repeats of Gly-Gly-Gly-Gly-Ser as a 15 or 20-amino acid linker sequence, respectively. The expression levels of EPO-dimer fusion protein from cloned CHO cells to supernatant of protein-free medium ranged from 4 to 40 mg/L determined by EPO-ELISA, and from 2.0×105 to 4.5×106 IU/L determined by in vitro bioassay. We selected clones producing EPO-dimer fusion protein with the greatest extent of glycosylation, as indicated by SDS-PAGE and isoelectric focusing. Subcutaneous injection of mice with three doses of EPO-dimer fusion protein resulted in percent increases in mean hematocrit of 32.6% (300 IU/kg) or 18.2% (100 IU/kg), while equivalent unit doses of EPO-monomer increased mean hematocrit by 12.5% (300 IU/kg) or 6.4% (100 IU/kg). Moreover, a single dose of EPO-dimer fusion protein (100 IU/kg) increased their mean hematocrit by 4.3% within 7 days, while an equivalent unit dose of EPO-monomer had no effect. Importantly, three doses of EPO-trimer fusion protein increased their mean hematocrit by 8.83% per IU injected, which was much greater than that observed with EPO-monomer (0.69%) or EPO-dimer fusion protein (1.81%). The results show that EPO-fusion proteins exhibit biological activities superior to those of EPO-monomer, suggesting important therapeutic advantages.

Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH)

To study structure–activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine α and β subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked α and β subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHβ subunit was genetically fused to the amino end of the α subunit. This gene was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Serial dilutions of pituitary derived native eLH and the single chain reLH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from five colts was processed for Leydig cell cultures. Increasing doses of reLH were incubated with equine Leydig cells for 24 h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either reLH ( n = 5) or saline ( n = 3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared to the saline control. Therefore, the single chain recombinant will be effective for a variety of structure–function analyses and for breeding management in the horse.

K-Space Image Correlation Spectroscopy: A Method for Accurate Transport Measurements Independent of Fluorophore Photophysics

We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or “blinking”. Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities that agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of α5-integrin/enhanced green fluorescent protein constructs in a transfected CHO cell imaged on a total internal reflection fluorescence microscope using charge-coupled device area detection.

Electrophysiological effects of brompheniramine on cardiac ion channels and action potential

Some antihistamines (mainly terfenadine and astemizole) have been demonstrated to cause QT interval prolongation and, in some cases, torsade-de-pointes. We investigated the cardiac electrophysiological effects of brompheniramine, a conventional antihistamine. Brompheniramine was reported to prolong QT interval in isolated hearts. To evaluate the electrophysiological effects of brompheniramine, we used whole-cell patch clamp techniques in human ether-a-go-go related gene (hERG)-stably transfected CHO cells, the SCN5A sodium channel transiently transfected CHO cells, and rat myocytes and conventional microelectrode recording techniques in isolated guinea pig papillary muscles. As for the I hERG, the IC 50 value of brompheniramine was found to be 0.90 ± 0.14 μM with a Hill coefficient ( n H) of 1.75 ± 0.42. Action potential duration at 90% repolarization (APD 90) was slightly prolonged by brompheniramine at 10 and 100 μM, but APD 50 was shortened by 100 μM. Moreover, despite the potent hERG current block, reductions of the V max and total amplitude of action potential were observed at high concentrations of brompheniramine. The change in action potential parameters and poor correlations between hERG and APD assay indicated additional effects of brompheniramine on non-hERG channels. In agreement with this hypothesis, the inhibition of I Na (IC 50 values: 21.26 ± 2.52 μM) and I Ca (IC 50 values: 16.12 ± 9.43 μM) by brompheniramine was observed. The results of this study suggest that brompheniramine may possess classes III, Ib and IV properties, especially at high concentrations and that additional studies on non-hERG channels will be necessary to elucidate the complex electrophysiological effects of brompheniramine on the heart.

Microencapsulation of Engineered Cells to Deliver Sustained High Circulating Levels of Interleukin-6 to Study Hepatocellular Carcinoma Progression

Interlukin-6 (IL-6) is a pleitropic cytokine that plays a central role in normal and abnormal hepatic function and response. The aims of the current study were to determine the viability of using cell encapsulation technology to introduce a genetically modified xenogeneic (CHO) cell population to elevate circulating levels of rhIL-6 in a rat model and determine the effects of sustained high rhIL-6 levels on hepatocellular carcinoma (HCC) progression in vivo. An alginate matrix was combined with transfected CHO cells, selected for their ability to synthesize rhIL-6, and used to generate uniform alginate-cell beads. Once encapsulated transfected cells continued to undergo replication, formed colonies within the bead, and synthesized/released large quantities of rhIL-6 into culture medium in vitro. Intraperitoneal implantation of beads into rats resulted in significantly increased circulating and intrahepatic levels of rhIL-6 up to 4 days postimplantation. Prolonged implantation led to the escape of CHO cells from the bead, resulting in a host response and CHO cell death within the bead. Subsequently CHO-IL-6 encapsulated cells were implanted into rats previously inoculated intrahepatically with the H4IIE HCC cell line. These studies demonstrated the maintenance of high circulating/intrahepatic rhIL-6 levels in this model. Despite significantly increased rhIL-6, this technique did not significantly alter the rate of net tumor progression. However, Stat3 activity was significantly increased in both normal liver and HCC tissue resected from animals implanted with CHO-IL-6 cells. Collectively these data demonstrate the short-term viability of using cell encapsulation technology to generate high levels of active circulating and intrahepatic cytokines and raise the possibility of modifying specific signal transduction cascades identified to be important during tumor progression.

Identification and characterization of five-transmembrane isoforms of human vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide receptors

The seven-transmembrane (7TM) G-protein-coupled neuroendocrine receptors VPAC1 (HGNC approved gene symbol VIPR1) and VPAC2 (HGNC approved gene symbol VIPR2) are expressed in different tissues and involved in the regulation of important biological functions. We now report the identification and characterization of novel five-transmembrane(5TM) forms of both human VPAC1 and human VPAC2. These alternatively spliced variant mRNAs result from the skipping of exons 10/11, spanning the third intracellular loop, the fourth extracellular loop, and the transmembrane regions 6 and 7, producing in-frame 5TM receptors predicted to lack a G-protein-binding motif. RT-PCR showed that these 5TM receptors are differentially expressed in transformed and normal cells. Translation of the 5TM protein was demonstrated by transfection and expression in CHO cells. Following agonist stimulation, differential signaling of the 7TM versus 5TM forms was shown both for the activation of adenylate cyclase and for tyrosine phosphorylation. The identification of these splice variants in various cells and their expression and differential signal transduction compared to the 7TM form suggest that these novel receptors have biological relevance.

Control of Culture Environment for Improved Polyethylenimine-Mediated Transient Production of Recombinant Monoclonal Antibodies by CHO Cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39 mg L(-1). Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended "activated hypothermic synthesis".

The key residues in the immunodominant region 353–363 of human thyroid peroxidase were identified

Auto-antibodies (aAbs) to thyroid peroxidase (TPO) interact with a restricted immunodominant region (IDR) divided into two overlapping regions A and B. Among the five major regions structuring the IDR/B, regions 210-225, 353-363, 549-563, 713-720 and 766-775, region 353-363 constitutes an important anchor point for the binding of TPO-specific aAbs in sera from Hashimoto's and Graves' patients. We combined site-directed mutagenesis and expression of TPO mutants in stably transfected CHO cells to precisely define the critical residues in that region. By using flow cytometry and ELISA, we identified four amino acid residues, H353, D358, S359 and R361, that contribute to the interaction between human TPO and anti-TPO aAbs. This identification of these contributing amino acid residues in the IDR allowed us to more precisely depict contours of the IDR.

Comparison of HCMV IE and EF-1 Promoters for the Stable Expression of β-Subunit of Hexosaminidase in CHO Cell Lines

The ability to use in vitro mutagenesis and transfection techniques to generate stable cell lines that highly express a mutant protein associated with a metabolic disease has been an important technique in linking genotype to clinical phenotype (Ozkara and Sandhoff, 2003). One group of metabolic diseases is GM2 gangliosidosis (AB-variant, Tay-Sachs and Sandhoff diseases). These diseases are characterized by the accumulation of GM2 ganglioside, primarily in the lysosome of neuronal tissue, leading to progressive neurodegeneration. Tay-Sachs and Sandhoff diseases are caused by defects in the HEXA gene, which encodes the α-subunit, and the HEXB gene, which encodes the β-subunit of heterodimeric N-acetyl-β-hexosaminidase (Hex) (EC 3.2.1.52), respectively (Gravel et al., 2001; Mahuran, 1999). The AB-variant is associated with defects in the GM2 activator protein, a substrate-specific cofactor for Hex A. The second major Hex isozyme in normal human tissue is Hex B, a homodimer of β-subunits. In this study, we introduced a cDNA encoding the β-subunit of human Hex (βcDNA) into three different eukaryotic expression vectors containing two different promoters, in order to establish which produces the highest expression levels of Hex B activity and β-protein in permanently transfected CHO cells. Our results indicate that the type of promoter employed can be critical in the establishment of highly expressing cell lines.

The Cholera Toxin A1 3 Subdomain Is Essential for Interaction with ADP-Ribosylation Factor 6 and Full Toxic Activity but Is Not Required for Translocation from the Endoplasmic Reticulum to the Cytosol

Cholera toxin (CT) moves from the plasma membrane to the endoplasmic reticulum (ER) by retrograde vesicular traffic. In the ER, the catalytic CTA1 polypeptide dissociates from the rest of the toxin and enters the cytosol by a process that involves the quality control mechanism of ER-associated degradation (ERAD). The cytosolic CTA1 then ADP ribosylates Gsalpha, resulting in adenylate cyclase activation and intoxication of the target cell. It is hypothesized that the C-terminal A1(3) subdomain of CTA1 plays two crucial roles in the intoxication process: (i) it contains a hydrophobic domain that triggers the ERAD mechanism and (ii) it facilitates interaction with the cytosolic ADP-ribosylation factors (ARFs) that serve as allosteric activators of CTA1. In this study, we examined the role(s) of the CTA1(3) subdomain in CT intoxication. Full-length CTA1 constructs and truncated CTA1 constructs lacking the A1(3) subdomain were generated and used to conduct two-hybrid studies of interactions with ARF6, in vitro enzyme assays, in vivo toxicity assays, and in vivo processing/degradation assays. Direct, plasmid-mediated expression of CTA1 constructs in the ER or cytosol of transfected CHO cells was used to perform the in vivo assays. With these methods, we found that the A1(3) subdomain of CTA1 is important both for interaction with ARF6 and for full expression of enzyme activity in vivo. Surprisingly, however, the A1(3) subdomain was not required for ERAD-mediated passage of CTA1 from the ER to the cytosol. A possible alternative trigger for CTA1 to activate the ERAD mechanism is discussed.

The ASIC1a antagonist PcTX‐1 reduces fear‐related behavior

Anxiety disorders are the most common psychiatric disorder. Current medications often fail to achieve remission. Identifying novel pharmacological targets in animal models of fear may lead to new therapies. We found that the acid sensing ion channel (ASIC1a) plays an important role in fear-related behavior. ASIC1a is located at synapses, where it may be activated by extracellular protons, and modulates synaptic plasticity. In mice, deletion of the ASIC1a gene significantly reduces the fear response to predator odor, open spaces, and Pavlovian fear conditioning. We hypothesized that ASIC1a antagonists might act similarly. To test this hypothesis, we used the recently identified ASIC1a antagonist PcTX-1. When we found that PcTX-1 blocks ASIC1a-mediated currents in transfected CHO cells, we tested its effect on fear-related behavior. We administered PcTX-1 or artificial cerebrospinal fluid into mice by intracerebroventricular cannula and assessed the fear-response to the predator odor trimethylthiazoline (TMT) and in the open field test. Consistent with an anxiolytic effect, in wild type mice PcTX-1 greatly diminished TMT-evoked freezing and increased center time in the open field. PcTX-1 had little or no effect on fear-related behavior in the ASIC1a null mice. Thus, inhibition of ASIC1a with PcTX-1 attenuates fear response in mice. These data suggest that pharmacological inhibition of ASIC1a may provide a novel way to reduce anxiety in patients.

Aquaporin‐1 as a NO transporter.

NO is thought to diffuse freely across cell membranes without need for a specific transporter. The water channel aquaporin-1 (AQP-1) transports CO2 and NH3 in addition to water and is highly expressed in cells that produce and are targets for NO. Consequently, we hypothesized that AQP-1 transports NO across lipid bilayers. We transfected CHO cells with an AQP-1-expressing vector or a control vector and measured NO influx with DAF2-DA. NO influx was initiated by creating a 5 μM NO gradient. When using a NO donor to create the gradient, AQP-1 increased NO influx from 10.2±3.4 to 32.5±6.9 fluorescence units (F.U)/sec (p<0.03) and NO correlated with water permeability (r = 0.70, n = 71). DMSO and HgCl2, inhibitors of AQP-1, blunted AQP-1-stimulated NO influx by 64±11% and 71±3% respectively (p<0.001). When using 5 μM NO gas, AQP-1 increased NO influx from 13.7±0.9 to 37.8±3.3 F.U/sec (p<0.03) and DMSO blunted this effect by 49±11% (p<0.03). AQP-1-dependent NO transport was concentration dependent with a K1/2 of 0.54μM. Influx saturated at 3 μM NO. Reconstituted purified AQP-1 in lipid vesicles increased NO influx from 5.3±1.1 to 22.1±6.2 F.U/sec (p<0.024). We conclude that AQP-1 transports NO across lipid bilayers. NO transport by AQP-1 may allow cells to control intracellular NO levels and their effects.

Heteromultimerization of ASIC and ENaC channels determined by FRET microscopy

High grade glioma cells express constitutively active inward Na+ currents that are inhibited by amiloride and psalmotoxin 1, implicating Epithelial Na+ channels (ENaCs) and Acid Sensitive Ion Channels (ASICs) as core components of the active channel. ASIC and ENaC subunits have been detected in normal glia and glioma cells using rtPCR, but the functional subunit composition of the glioma Na+ channel remains unknown. In order to determine if ASIC and ENaC subunits intermix to form heteromeric channels, intersubunit associations were measured using confocal fluorescence resonant energy transfer (FRET) microscopy. Human ASIC (1b, 2a) and ENaC (α, β, γ, δ ) ion channel subunits have been fused with the fluorescent fusion proteins eCFP and eYFP at both N- and C-termini; these constructs form functional ASIC and ENaC channels in Xenopus oocytes or transiently transfected CHO cells. FRET efficiencies measured in transiently transfected CHO cells follow: eCFP fused directly to eYFP, 27.5% ± 3.8% (positive control); eCFP cotransfected with eYFP, 6.6% ± 3.6% (negative control); ASIC1-eCFP or ASIC2-eCFP cotransfected with ASIC1, ASIC2, αENaC, or γ ENaC fused to eYFP, > 20%; ASIC1-eCFP or ASIC2-eCFP cotransfected with either βENaC or δ ENaC, < 12% (error is standard deviation from >15 cells from >3 transfections). These results suggest heteromultimerization of ASIC and ENAC subunits, particularly between ASIC1, ASIC2, αENaC, and γ ENaC. Heteromeric channel assembly has also been demonstrated by immunoprecipitation and western blot analysis. Supported by NIH grants CA101952 and DK37206.

Calcium-permeable Acid-sensing Ion Channel Is a Molecular Target of the Neurotoxic Metal Ion Lead

Acid-sensing ion channels (ASICs) are emerging as fundamental players in the regulation of neural plasticity and in pathological conditions. Here we showed that lead (Pb2+), a well known neurotoxic metal ion, reversibly and concentration-dependently inhibited ASIC currents in the acutely dissociated spinal dorsal horn and hippocampal CA1 neurons of rats. In vitro expression of ASIC subunits in combination demonstrated that both ASIC1 and -3 subunits were sensitive to Pb2+. Mechanistically, Pb2+ reduced the pH sensitivity of ASICs independent of membrane voltage change. Moreover, Pb2+ inhibited the ASIC-mediated membrane depolarization and the elevation of intracellular Ca2+ concentration. In addition, we compared the effect of Pb2+ with that of Ca2+ or amiloride to explore the possible interactions of Pb2+ and Ca2+ in regulating ASICs, and we found that Pb2+ inhibited ASIC currents independent of the amiloride/Ca2+ blockade. Because ASIC1b and -3 subunits are mainly expressed in peripheral neurons, our data identified ASIC1a-containing Ca2+-permeable ASIC as a novel central target of Pb2+ action, which may contribute to Pb2+ neurotoxicity.

The BTB-kelch Protein LZTR-1 Is a Novel Golgi Protein That Is Degraded upon Induction of Apoptosis

Members of the BTB-kelch superfamily play important roles during fundamental cellular processes, such as the regulation of cell morphology, migration, and gene expression. The BTB-kelch protein LZTR-1 is deleted in the majority of DiGeorge syndrome patients and is believed to act as a transcriptional regulator. However, functional and expression profiling studies of LZTR-1 have not been performed thus far. Therefore, we examined the subcellular localization and function of LZTR-1 to gain insights into its biological role. Analysis of the primary structure of the protein revealed six N-terminal kelch motifs and two BTB/POZ domains at the C terminus within LZTR-1. Confocal analysis of the subcellular distribution of LZTR-1 using the Golgi markers GM130, Golgin-97, and TGN46 identified a localization of LZTR-1 exclusively on the cytoplasmic surface of the Golgi network that is mediated by its second BTB/POZ domain. In contrast to most other BTB-kelch proteins, LZTR-1 did not co-localize with actin. Treatment with brefeldin A did not lead to redistribution of LZTR-1 to the endoplasmic reticulum but caused its relocalization in dispersed, punctuated structures that were also positive for GM130. These data demonstrate that LZTR-1 is a Golgi matrix-associated protein. Upon induction of apoptosis, LZTR-1 was phosphorylated on tyrosine residues and subsequently degraded; that could be rescued partially by the addition of the caspase inhibitor Z-VAD-fmk and the proteasome inhibitors lactacystin and MG132. Taken together, our experiments identify LZTR-1 as the first BTB-kelch protein that exclusively localizes to the Golgi network, and the binding of LZTR-1 to the Golgi complex is mediated by its second BTB/POZ domain.

Niacin induces PPARγ expression and transcriptional activation in macrophages via HM74 and HM74a-mediated induction of prostaglandin synthesis pathways

HM74 and HM74a have been identified as receptors for niacin. HM74a mediates the pharmacological anti-lipolytic effects of niacin in adipocytes by reducing intracellular cyclic AMP (cAMP) and inhibiting release of free fatty acids into the circulation. In macrophages, niacin induces peroxisome proliferator-activated receptor γ (PPARγ)-dependent and cAMP-dependent expression of genes mediating reverse cholesterol transport, although via an unidentified receptor. We describe constitutive expression of HM74a mRNA and hypoxia- and IFNγ-inducible expression of HM74 and HM74a in human monocytic cell lines and primary cells in culture. In U937 cells niacin-induced expression of 15-deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2), the most potent endogenous ligand of PPARγ. Both niacin and the structurally distinct HM74/HM74a ligand acifran-induced nuclear expression of PPARγ protein and enhanced PPARγ transcriptional activity. Niacin-induced PPARγ transcriptional activity was pertussis toxin sensitive and required activity of phospholipase A 2 (EC 3.1.1.4), cyclo-oxygenase (EC 1.14.99.1) and prostaglandin D 2 synthase (EC 5.3.99.2). Niacin also induced PPARγ transcriptional activity in HM74 and HM74a CHO cell transfectants, although not in vector-only control cells. This was sensitive to pertussis toxin and to inhibition of phoshoplipase A 2 and cyclo-oxygenase activity. Additionally, niacin increased intracellular cAMP in U937 via a pertussis toxin and cyclo-oxygenase-sensitive mechanism. These results indicate that HM74 and HM74a can mediate macrophage responses to niacin via activation of the prostaglandin synthesis pathway and induction and activation of PPARγ. This suggests a novel mechanism(s) mediating the clinical effects of pharmacological doses of niacin.