Human Embryonic Kidney 293A Cells Research Articles

Positive Allosteric Modulators of Metabotropic Glutamate Receptor 5 as Tool Compounds to Study Signaling Bias.

Positive allosteric modulation of metabotropic glutamate subtype 5 (mGlu5) receptor has emerged as a potential new therapeutic strategy for the treatment of schizophrenia and cognitive impairments. However, positive allosteric modulator (PAM) agonist activity has been associated with adverse side effects, and neurotoxicity has also been observed for pure PAMs. The structural and pharmacological basis of therapeutic versus adverse mGlu5 PAM in vivo effects remains unknown. Thus, gaining insights into the signaling fingerprints, as well as the binding kinetics of structurally diverse mGlu5 PAMs, may help in the rational design of compounds with desired properties. We assessed the binding and signaling profiles of N-methyl-5-(phenylethynyl)pyrimidin-2-amine (MPPA), 3-cyano-N-(2,5-diphenylpyrazol-3-yl)benzamide (CDPPB), and 1-[4-(4-chloro-2-fluoro-phenyl)piperazin-1-yl]-2-(4-pyridylmethoxy)ethenone [compound 2c, a close analog of 1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone] in human embryonic kidney 293A cells stably expressing mGlu5 using Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and receptor internalization assays. Of the three allosteric ligands, only CDPPB had intrinsic agonist efficacy, and it also had the longest receptor residence time and highest affinity. MPPA was a biased PAM, showing higher positive cooperativity with orthosteric agonists in ERK1/2 phosphorylation and Ca2+ mobilization over IP1 accumulation and receptor internalization. In primary cortical neurons, all three PAMs showed stronger positive cooperativity with (S)-3,5-dihydroxyphenylglycine (DHPG) in Ca2+ mobilization over IP1 accumulation. Our characterization of three structurally diverse mGlu5 PAMs provides further molecular pharmacological insights and presents the first assessment of PAM-mediated mGlu5 internalization. SIGNIFICANCE STATEMENT: Enhancing metabotropic glutamate receptor subtype 5 (mGlu5) activity is a promising strategy to treat cognitive and positive symptoms in schizophrenia. It is increasingly evident that positive allosteric modulators (PAMs) of mGlu5 are not all equal in preclinical models; there remains a need to better understand the molecular pharmacological properties of mGlu5 PAMs. This study reports detailed characterization of the binding and functional pharmacological properties of mGlu5 PAMs and is the first study of the effects of mGlu5 PAMs on receptor internalization.

Differential effects of cotreatment of the antibiotic rifampin with host-directed therapeutics in reducing intracellular Staphylococcus aureus infection.

BackgroundChronic infection by Staphylococcus aureus drives pathogenesis in important clinical settings, such as recurrent pulmonary infection in cystic fibrosis and relapsing infection in osteomyelitis. Treatment options for intracellular S. aureus infection are limited. Rifampin, a lipophilic antibiotic, readily penetrates host cell membranes, yet monotherapy is associated with rapid antibiotic resistance and development of severe adverse events. Antibiotic cotreatment can reduce this progression, yet efficacy diminishes as antibiotic resistance develops. ML141 and simvastatin inhibit S. aureus invasion through host-directed rather than bactericidal mechanisms.ObjectiveTo determine whether cotreatment of ML141 or of simvastatin with rifampin would enhance rifampin efficacy.MethodsAssays to assess host cell invasion, host cell viability, host cell membrane permeability, and bactericidal activity were performed using the human embryonic kidney (HEK) 293-A cell line infected with S. aureus (29213) and treated with vehicle control, simvastatin, ML141, rifampin, or cotreatment of simvastatin or ML141 with rifampin.ResultsWe found cotreatment of ML141 with rifampin reduced intracellular infection nearly 85% when compared to the no treatment control. This decrease more than doubled the average 40% reduction in response to rifampin monotherapy. In contrast, cotreatment of simvastatin with rifampin failed to improve rifampin efficacy. Also, in contrast to ML141, simvastatin increased propidium iodide (PI) positive cells, from an average of 10% in control HEK 293-A cells to nearly 20% in simvastatin-treated cells, indicating an increase in host cell membrane permeability. The simvastatin-induced increase was reversed to control levels by cotreatment of simvastatin with rifampin.ConclusionTaken together, rifampin efficacy is increased through host-directed inhibition of S. aureus invasion by ML141, while efficacy is not increased by simvastatin. Considerations regarding novel therapeutic approaches may be dependent on underlying differences in pharmacology.

Heterozygous Mutation (Q459R) in the Calcium-Sensing Receptor Gene Causes Familial Hypocalciuric Hypercalcemia 1 (FHH1).

Several heterozygous loss-of-function mutations in the calcium-sensing receptor gene (CASR) leading to elevated ionized serum calcium and familial hypocalciuric hypercalcemia 1 (FHH1) have been characterized. Few mutations are not pathogenic, and previous studies suggested that the Q459R mutation does not result in an FHH1 phenotype. We identified a family with a heterozygous CASR Q459R mutation and characterized their calcium homeostasis and the pathophysiological mechanisms of a homozygous and heterozygous Q459R mutation in vitro. The index patient and her family had clinical, biochemical, and genetic analyses performed. In vitro functional characterization of homozygous and heterozygous (Q459R) mutations was conducted by determining CaSR cell-surface expression and inositol monophosphate (IP1) signaling in transiently transfected human embryonic kidney 293A (HEK293A) cells. All 3 heterozygous carriers had mild asymptomatic hypercalcemia, hypocalciuria, and 2 had elevated serum parathyroid hormone (PTH). In vitro characterization in HEK293A cells revealed that CASR Q459R is a loss-of-function mutation with no impact on cell-surface expression. Cells with the homozygous Q459R genotype had significantly reduced calcium potency of IP1 signaling compared to wild type, whereas the heterozygous Q459R also had lower calcium potency albeit not significantly different from wild type. A loss-of-function Q459R mutation in CASR in a family caused FHH1 characterized by elevated ionized calcium and PTH and low calcium excretion. The marked presence of CaSR at the membrane and inhibition of IP1 signaling in vitro suggest that calcimimetics may be functional in patients with this mutation, which seems to be a mild loss-of-function mutation associated with autosomal dominant transmission of FHH1.

The anti-tumor activity and mechanism of alkaloids from Aconitum szechenyianum Gay.

In the current study, the anti-tumor activity of Aconitum szechenyianum Gay alkaloids (ASA) and its mechanism of action were investigated. The result showed that ASA could induce apoptosis in HepG-2, Hela and A549 cells but not in normal human embryonic kidney 293A cells, and its apoptotic effect on A549 cells was stronger than those of HepG-2 and Hela cells. Moreover, the following study showed that ASA could up-regulate the expression levels of p38 and phosphorylated p38 MAPK, suggesting ASA-induced apoptosis was associated with the p38 MAPK mediated pathway. Furthermore, ASA could up-regulate TNF-R1 and DR5 via activation of p38 MAPK, thereby activating caspase 8, revealing the death receptor pathway was also involved in this process. In addition, ASA could led to a loss in the mitochondrial out membrane potential, up-regulate p53, phosphorylated p53 and Bax, down-regulate Bcl-2, release cytochrome c from the mitochondria to the cytoplasm, and activate caspase-9 and caspase-3 in A549 cells, which revealed that ASA could also induce apoptosis through the mitochondria mediated pathway. These results suggested that ASA played the anti-tumor role through the activation of p38 MAPK-, death receptor-, mitochondria- and caspase-dependent apoptotic pathways.

Introduction of silent mutations in a codon-optimized xylanase (xynB) results in enhanced protein expression in HEK293A cells

Xylanase is used extensively to improve feed conversion rates to enhance the performance of poultry and pigs. By expressing xylanase in simple-stomached animals, new breeds of genetically modified animals with enhanced feed conversion rates may be obtained. However, expression of heterologous proteins derived from lower organisms in mammalian cells is usually inefficient. When common codons of a ''one amino acid-one codon"-optimized xylanase from Streptomyces olivaceoviridis were replaced with rare codons, xylanase expression in human embryonic kidney 293A cells increased by 1.4- to 2.3-fold as determined by flow cytometry, western blot and enzymatic activity assay. Quantitative RT-PCR assay indicated that the enhanced expression could not be attributed to altered mRNA levels. This study provides an alternative strategy for improving expression levels of heterologous proteins in mammalian cells, which is potentially helpful for generating genetically modified animals with enhanced feed conversion ability.

α1A-Adrenergic Receptor Induces Activation of Extracellular Signal-Regulated Kinase 1/2 through Endocytic Pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α1A-adrenergic receptor (α1A-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α1A-AR. α1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31–8220 (a PKC inhibitor) inhibited α1B-AR- but not α1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α1A-AR-induced ERK1/2 activation, which is independent of Gq/PLC/PKC signaling.

Evaluation of cholesterol depletion as a marker of nephrotoxicity in vitro for novel β-cyclodextrin derivatives

Cyclodextrins (CDs) have been shown to improve physicochemical and biopharmaceutical properties of drugs when low solubility and low safety limit their use in the pharmaceutical field. Recently, we have developed new multi-substituted-β-CDs, hydroxypropyl-sulfobutyl ether-β-cyclodextrin (HPn-SBEm-β-CD). HPn-SBEm-β-CD exhibit low hemolysis, good solubility, strong inclusion ability, and an appropriate average molecular weight. In this study, we chose two products of HPn-SBEm-β-CD (HP3-SBE2-β-CD and HP2-SBE3-β-CD) and compared their effects to sulfobutyl ether-β-cyclodextrin (SBE7-β-CD), methyl-β-cyclodextrin (M-β-CD) and 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD). We evaluated viability, membrane damage, induction of apoptosis and necrosis, cholesterol depletion, and morphological changes in human embryonic kidney 293A cells (HEK293A) in vitro. CDs caused a reduction of cell viability and increased LDH levels in a concentration-dependent manner. The effect of HP3-SBE2-β-CD or HP2-SBE3-β-CD on cell viability, membrane damage, and the induction of apoptosis and necrosis resembled that of SBE7-β-CD, whereas the effects were significantly lower for M-β-CD or DM-β-CD. HP3-SBE2-β-CD and HP2-SBE3-β-CD exhibited morphological changes at high concentrations. In conclusion, the results showed that cholesterol depletion may be as a marker for evaluating the cytotoxicity of novel β-CD derivatives. These results will provide useful information for HPn-SBEm-β-CD as a promising safe adjuvant for intravenous administration in the future.

A Microarray-based Approach Identifies ADP Ribosylation Factor-like Protein 2 as a Target of microRNA-16

microRNAs (miRNAs) are generally thought to negatively regulate the expression of their target genes by mRNA degradation or by translation repression. Here we show an efficient way to identify miRNA target genes by screening alterations in global mRNA levels following changes in miRNA levels. In this study, we used mRNA microarrays to measure global mRNA expression in three cell lines with increased or decreased levels of miR-16 and performed bioinformatics analysis based on multiple target prediction algorithms. For further investigation among the predicted miR-16 target genes, we selected genes that show an expression pattern opposite to that of miR-16. One of the candidate target genes that may interact with miR-16, ADP-ribosylation factor-like protein 2 (ARL2), was further investigated. First, ARL2 was deduced to be an ideal miR-16 target by computational predictions. Second, ARL2 mRNA and protein levels were significantly abolished by treatment with miR-16 precursors, whereas a miR-16 inhibitor increased ARL2 mRNA and protein levels. Third, a luciferase reporter assay confirmed that miR-16 directly recognizes the 3'-untranslated region (3'-UTR) of ARL2. Finally, we showed that miR-16 could regulate proliferation and induce a significant G0/G1 cell cycle arrest, which was due at least in part, to the down-regulation of ARL2. In summary, the present study suggests that integrating global mRNA profiling and bioinformatics tools may provide the basis for further investigation of the potential targets of a given miRNA. These results also illustrate a novel function of miR-16 targeting ARL2 in modulating proliferation and cell cycle progression.

Design of Fluorescent Bradykinin Analogs: Application to Imaging of B2Receptor-Mediated Agonist Endocytosis and Trafficking and Angiotensin-Converting Enzyme

The known structure-activity relationship and docking models for peptide ligands of the bradykinin B(2) receptor indicate a certain tolerance to N-terminal extension. We took advantage of this by generating two fluorescent bradykinin analogs containing 5(6)-carboxyfluorescein (CF) optionally used with the ε-aminocaproyl spacer condensed at the N terminus of the agonist. Pharmacological studies indicated that CF-bradykinin was virtually inactive as a B(2) receptor ligand and agonist, whereas CF-ε-aminocaproyl-bradykinin (CF-εACA-BK) was 400- to 1000-fold less potent than bradykinin (competition of [(3)H]bradykinin binding to B(2) receptors, contractility of the human isolated umbilical vein). Nevertheless, CF-εACA-BK (5 μM) was taken up by human embryonic kidney 293a cells expressing recombinant B(2) receptors, but not by those cotreated with an antagonist or expressing a truncated receptor that is pharmacologically intact but not phosphorylable. A higher-affinity CF-conjugated peptide, the antagonist CF-εACA-d-Arg-[Hyp(3),Igl(5),d-Igl(7),Oic(8)]-bradykinin (B-10380), labeled both intact and truncated receptor forms at the cell surface. The fluorescent agonist CF-εACA-BK was found in vesicles positive for β-arrestin(1), Rab5, and Rab7, then apparently degraded as a function of time because the fluorescence was transferred from the vesicles to the cytosol in a vesicular-ATPase-dependent process (3 h). The ectopeptidase angiotensin-converting enzyme (ACE) is a major kininase. The binding affinity of CF-εACA-BK for this carboxydipeptidase is identical to that of bradykinin ([(3)H]enalaprilat displacement assay). Recombinant ACE is essentially a plasma membrane protein in CF-εACA-BK imaging of intact cells. Micromolar CF-εACA-BK is a probe for the two major physiological targets of bradykinin, the B(2) receptor and ACE. As an agonist, it is subjected to β-arrestin-mediated endocytosis, trafficking, and subsequent ligand degradation.

A Novel Protein Kinase A-independent, β-Arrestin-1-dependent Signaling Pathway for p38 Mitogen-activated Protein Kinase Activation by β2-Adrenergic Receptors

A growing body of evidence has demonstrated that p38 mitogen-activated protein kinase (MAPK) has a crucial role in various physiological and pathological processes mediated by beta(2)-adrenergic receptors (beta(2)-ARs). However, the detailed mechanism of beta(2)-ARs-induced p38 MAPK activation has not yet been fully defined. The present study demonstrates a novel kinetic model of p38 MAPK activation induced by beta(2)-ARs in human embryonic kidney 293A cells. The beta(2)-AR agonist isoproterenol induced a time-dependent biphasic phosphorylation of p38 MAPK: the early phase peaked at 10 min, and was followed by a delayed phase that appeared at 90 min and was sustained for 6 h. Interestingly, inhibition of the cAMP/protein kinase A (PKA) pathway failed to affect the early phosphorylation but abolished the delayed activation. By contrast, silencing of beta-arrestin-1 expression by small interfering RNA inhibited the early phase activation of p38 MAPK. Furthermore, the NADPH oxidase complex is a downstream target of beta-arrestin-1, as evidenced by the fact that isoproterenol-induced Rac1 activation was also suppressed by beta-arrestin-1 knockdown. In addition, early phase activation of p38 MAPK was prevented by inactivation of Rac1 and NADPH oxidase by pharmacological inhibitors, overexpression of a dominant negative mutant of Rac1, and p47(phox) knockdown by RNA interference. Of note, we demonstrated that only early activation of p38 MAPK is involved in isoproterenol-induced F-actin rearrangement. Collectively, these data suggest that the classic cAMP/PKA pathway is responsible for the delayed activation, whereas a beta-arrestin-1/Rac1/NADPH oxidase-dependent signaling is a heretofore unrecognized mechanism for beta(2)-AR-mediated early activation of p38 MAPK.

A Protein Kinase Cε-Anti-apoptotic Kinase Signaling Complex Protects Human Vascular Endothelial Cells against Apoptosis through Induction of Bcl-2

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.

Fibroblast Growth Factor-2 Is a Downstream Mediator of Phosphatidylinositol 3-Kinase-Akt Signaling in 14,15-Epoxyeicosatrienoic Acid-induced Angiogenesis

To determine the efficacy of cytochrome P450 2C9 metabolites of arachidonic acid, viz. 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), in inducing angiogenesis, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All four EETs stimulated HDMVEC tube formation and migration in a dose-dependent manner. Because 14,15-EET was found to be slightly more efficacious than 5,6-, 8,9-, and 11,12-EETs in stimulating HDMVEC tube formation and migration, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 14,15-EET stimulated Akt and S6K1 phosphorylation in Src- and phosphatidylinositol 3-kinase (PI3K)-dependent manner in HDMVECs. Inhibition of Src and PI3K-Akt-mTOR signaling by both pharmacological and dominant-negative mutant approaches suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. In addition, 14,15-EET induced the expression of fibroblast growth factor-2 (FGF-2) in Src- and PI3K-Akt-dependent and mTOR-independent manner in HDMVECs. Neutralizing anti-FGF-2 antibodies completely suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. Together, these results show for the first time that Src and PI3K-Akt signaling via targeting in parallel with FGF-2 expression and mTOR-S6K1 activation plays an indispensable role in 14,15-EET-induced angiogenesis.

Polyethylene glycosylation prolongs the circulatory stability of recombinant human butyrylcholinesterase

Previous studies in rodents and non-human primates have demonstrated that pretreatment of animals with cholinesterases could provide significant protection against organophosphate (OP) nerve agent toxicity. Gene delivery/therapy is emerging as an approach to achieve high-level expression of proteins in vivo that are very similar to their native counterparts. Recently, adenoviral (Ad) vectors have proven to be excellent vehicles for delivering genes to cells in vitro and in vivo. In this study, we explored the use of the newly designed AdenoVATOR system for the expression of recombinant human butyrylcholinesterase (rHu BChE) in human embryonic kidney 293A (HEK-293A) cells. In these cells, rHu BChE was expressed as mostly tetrameric form by the simultaneous expression of proline-rich attachment domain. By optimizing the culture conditions, 1.5–2.0 U/ml of rHu BChE could be expressed in HEK-293A cells. Recombinant Hu BChE was purified to homogeneity by ammonium sulfate fractionation followed by affinity column chromatography using procainamide Sepharose and cobalt Sepharose gels. The enzymatic and physico-chemical properties of purified rHu BChE were similar to those of native serum-derived Hu BChE. To determine the suitability of this preparation for use as an antidote against highly toxic nerve agents, its pharmacokinetics were evaluated in mice. Recombinant Hu BChE exhibited a mean residence time of 18.3 h which was 2.5-fold shorter than that observed for native Hu BChE in mice. However, rHu BChE chemically modified with polyethyleneglycol (PEG) displayed a mean residence time of 36.2 h suggesting that PEG-modification can prolong the circulatory stability of rHu BChE. The efficacy of Ad-Hu BChE to induce the production of therapeutic levels of bioscavenger in vivo is under evaluation.

Overexpression of Kinin B1 Receptors Induces Hypertensive Response to Des-Arg9-bradykinin and Susceptibility to Inflammation

We demonstrated that rat kinin B(1) receptors displayed a ligand-independent constitutive activity, assessed through inositol phosphate production in transiently or stably transfected human embryonic kidney 293A cells. Substitution of Ala for Asn(130) in the third transmembrane domain resulted in additional constitutive activation of the B(1) receptor. The constitutively active mutant N130A receptor could be further activated by the B(1) receptor agonist des-Arg(9)-bradykinin. To gain insights into the physiological function of the B(1) receptor, we have generated transgenic mice overexpressing wild-type and constitutively active mutant receptors under the control of human cytomegalovirus immediately early gene enhancer/promoter. The rat B(1) receptor transgene expression was detected in the aorta, brain, heart, lung, liver, kidney, uterus, and prostate of transgenic mice by reverse transcription-polymerase chain reaction/Southern blot analysis. Transgenic mice were fertile and normotensive. Overexpression of B(1) receptors exacerbated paw edema induced by carrageenan and rendered transgenic mice more susceptible to lipopolysaccharide-induced endotoxic shock. Interestingly, the hemodynamic response to kinins was altered in transgenic mice, with des-Arg(9)-bradykinin inducing blood pressure increase when intravenously administered. Our study supports an important role for B(1) receptors in modulating inflammatory responses and for the first time demonstrates that B(1) receptors mediate a hypertensive response to des-Arg(9)-bradykinin.