Molecular Pharmacology current issue

Adenylyl Cyclase-A-kinase Anchoring Protein Complexes: The Next Dimension in cAMP Signaling [MINIREVIEW]

Dessauer, C. W. — Fri, 16 Oct 2009 10:34:50 PDT

The formation of multiprotein complexes is a repeated theme in biology ranging from the regulation of the extracellular signal-regulated kinase and cAMP signaling pathways to the formation of postsynaptic density complexes or tight junctions. A-kinase anchoring proteins (AKAPs) are well known for their ability to scaffold protein kinase A and components upstream and downstream of cAMP production, including G protein-coupled receptors, cAMP-dependent Rap-exchange factors, and phosphodiesterases. Specific adenylyl cyclase (AC) isoforms have also been identified as components of AKAP complexes, namely AKAP79, Yotiao, and mAKAP. In this review, we summarize recent evidence for AC-AKAP complexes and requirements for compartmentalization of cAMP signaling. The ability of AKAPs to assemble intricate feedback loops to control spatiotemporal aspects of cAMP signaling adds yet another dimension to the classic cAMP pathway.

An Ion Channel Hypothesis to Explain Divergent Cardiovascular Safety of Cyclooxygenase-2 Inhibitors: The Answer to a Hotly Debated Puzzle? [PERSPECTIVE]

Shapiro, M. S. — Fri, 16 Oct 2009 10:34:50 PDT

Cyclooxygenase inhibitors represented extremely promising novel anti-inflammatory drugs until one of them, rofecoxib (Vioxx), was found to be associated with increased cardiovascular morbidity; however, another such drug, celecoxib (Celebrex), suffers far less from this side effect for unknown reasons and is still widely used. In this issue, Brueggemann et al. (page p. 1053) suggest a hypothesis. Celecoxib, but not rofecoxib, is shown to act as an "opener" of voltage-gated KCNQ5 K⁺ channels and a blocker of "L-type" Ca²⁺ channels, causing a reduction in the excitability and contractility of vascular smooth-muscle cells (VSMCs). Furthermore, VSMC tone is shown to be selectively reduced by celecoxib, resulting in dilation of blood vessels and reduction in systemic blood pressure, suggesting that the reduced work load on the heart may counteract any other deleterious effects of this class of drugs. Here, these findings are discussed in light of the role of KCNQ K⁺ channels in control of excitability in general, the "lipid imbalance theory" of cyclooxygenase-2 risks, and the potential for novel therapeutic modalities for cardiovascular disease focused on ion channels in vascular smooth muscle.

Identification of Compounds that Correlate with ABCG2 Transporter Function in the National Cancer Institute Anticancer Drug Screen [ARTICLES]

Fri, 16 Oct 2009 10:34:50 PDT

ABCG2 is an ATP-binding cassette transporter that counts multiple anticancer compounds among its substrates and is believed to regulate oral bioavailability as well as serve a protective role in the blood-brain barrier, the maternal-fetal barrier, and hematopoietic stem cells. We sought to determine whether novel compounds that interact with the transporter could be identified through analysis of cytotoxicity profiles recorded in the NCI Anticancer Drug Screen database. A flow cytometric assay was used to measure ABCG2 function in the 60 cell lines and generate a molecular profile for COMPARE analysis. This strategy identified >70 compounds with Pearson correlation coefficients (PCCs) >0.4, where reduced drug sensitivity correlated with ABCG2 expression, as well as >120 compounds with PCCs < –0.4, indicating compounds to which ABCG2 expression conferred greater sensitivity. Despite identification of known single nucleotide polymorphisms in the ABCG2 gene in a number of the cell lines, omission of these lines from the COMPARE analysis did not affect PCCs. Available compounds were subjected to validation studies to confirm interaction with the transporter, including flow cytometry, [¹²⁵I]IAAP binding, and cytotoxicity assays, and interaction was documented in 20 of the 27 compounds studied. Although known substrates of ABCG2 such as mitoxantrone or topotecan were not identified, we characterized three novel substrates—5-hydroxypicolinaldehyde thiosemicarbazone (NSC107392), (E)-N-(1-decylsulfanyl-3-hydroxypropan-2-yl)-3-(6-methyl-2,4-dioxo-1H-pyrimidin-5-yl)prop-2-enamide (NSC265473), and 1,2,3,4,7-pentahydroxy-1,3,4,4a,5,11b-hexahydro[1,3]dioxolo[4,5-j]phenanthridin-6(2H)-one [NSC349156 (pancratistatin)]—and four compounds that inhibited transporter function—2-[methyl(2-pyridin-2-ylethyl)-amino]fluoren-9-one hydroiodide (NSC24048), 5-amino-6-(7-amino-5,8-dihydro-6-methoxy-5,8-dioxo-2-quinolinyl)-4-(2-hydroxy-3,4-dimethoxyphenyl)-3-methyl-2-pyridinecarboxylic acid, methyl ester (NSC45384), (17β)-2,4-dibromo-estra-1,3,5(10)-triene-3,17-diol (NSC103054), and methyl N-(pyridine-4-carbonylamino)carbamodithioate (NSC636795). In summary, COMPARE analysis of the NCI drug screen database using the ABCG2 functional profile was able to identify novel substrates and transporter-interacting compounds.

Targeting of the Akt-Nuclear Factor-{kappa}B Signaling Network by [1-(4-Chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), a Novel Indole-3-Carbinol Derivative, in a Mouse Model of Hepatocellular Carcinoma [ARTICLES]

Fri, 16 Oct 2009 10:34:50 PDT

Constitutive activation of Akt and nuclear factor-B (NF-B) represents major cellular abnormalities associated with the development and progression of hepatocellular carcinoma (HCC). Based on the structure of indole-3-carbinol, a chemopreventive phytochemical, we developed a novel derivative, [1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), that exhibits higher potency in inducing apoptosis by targeting the Akt-NF-B signaling network. This study was aimed at assessing the antitumor activity of OSU-A9 using both in vitro and in vivo models of HCC, a malignancy in which the Akt-NF-B signaling network plays major roles in pathogenesis and therapeutic resistance. Our data show that OSU-A9 was 100 times more potent than indole-3-carbinol in suppressing the viability of Hep3B, Huh7, and PLC5 HCC cells with IC₅₀ values ranging from 2.8 to 3.2 µM. OSU-A9 interfered with the interplay between Akt- and NF-B-mediated oncogenic signaling, leading to changes in the functional status of diverse signaling effectors involved in cell cycle progression, apoptosis, angiogenesis, and metastasis. The in vivo efficacy of OSU-A9 was assessed in nude mice bearing luciferase-expressing Hep3B xenograft tumors. Daily oral treatments with OSU-A9 at 25 or 50 mg/kg for 56 days suppressed tumor growth by 67 and 80%, respectively, which was correlated with changes in intratumoral biomarkers pertinent to Akt-NF-B signaling, and without apparent toxicity or evidence of hepatic biotransformation enzyme induction. Together, these findings indicate that OSU-A9 is a potent, orally bioavailable inhibitor of the Akt-NF-B signaling network with a broad spectrum of antitumor activity that includes targets regulating multiple aspects of HCC pathogenesis and progression.

Potential Use of Cetrimonium Bromide as an Apoptosis-Promoting Anticancer Agent for Head and Neck Cancer [ARTICLES]

Fri, 16 Oct 2009 10:34:50 PDT

A potential therapeutic agent for human head and neck cancer (HNC), cetrimonium bromide (CTAB), was identified through a cell-based phenotype-driven high-throughput screen (HTS) of 2000 biologically active or clinically used compounds, followed by in vitro and in vivo characterization of its antitumor efficacy. The preliminary and secondary screens were performed on FaDu (hypopharyngeal squamous cancer) and GM05757 (primary normal fibroblasts), respectively. Potential hit compounds were further evaluated for their anticancer specificity and efficacy in combination with standard therapeutics on a panel of normal and cancer cell lines. Mechanism of action, in vivo antitumor efficacy, and potential lead compound optimizations were also investigated. In vitro, CTAB interacted additively with radiation and cisplatin, two standard HNC therapeutic agents. CTAB exhibited anticancer cytotoxicity against several HNC cell lines, with minimal effects on normal fibroblasts; a selectivity that exploits cancer-specific metabolic aberrations. The central mode of cytotoxicity was mitochondria-mediated apoptosis via inhibition of H⁺-ATP synthase activity and mitochondrial membrane potential depolarization, which in turn was associated with reduced intracellular ATP levels, caspase activation, elevated sub-G₁ cell population, and chromatin condensation. In vivo, CTAB ablated tumor-forming capacity of FaDu cells and delayed growth of established tumors. Thus, using an HTS approach, CTAB was identified as a potential apoptogenic quaternary ammonium compound possessing in vitro and in vivo efficacy against HNC models.

Plasma Membrane Nucleolin Is a Receptor for the Anticancer Aptamer AS1411 in MV4-11 Leukemia Cells [ARTICLES]

Fri, 16 Oct 2009 10:34:50 PDT

AS1411 is a DNA aptamer that is in phase II clinical trials for relapsed or refractory acute myeloid leukemia and for renal cell carcinoma. AS1411 binds to nucleolin, a protein that is overexpressed in the cytoplasm and on the plasma membrane of some tumor cells compared with normal cells. Studies were performed to determine whether cell surface nucleolin is a receptor for AS1411 in the acute myeloid leukemia cell line MV4-11. Biotinylation of MV4-11 cell surface proteins followed by immunoblotting of the biotinylated proteins showed that full-length (106 kDa) and truncated forms of nucleolin were present on the cell surface. In contrast, K-562 cells, which are 4-fold less sensitive than MV4-11 cells to AS1411, showed no full-length nucleolin and lesser amounts of the truncated forms of nucleolin on the cell surface. Incubation of MV4-11 cells with [³²P]AS1411 and immunoprecipitation of the plasma membrane fraction with anti-nucleolin antibody demonstrated the presence of [³²P]AS1411-nucleolin complexes. Anti-nucleolin antibody inhibited binding of fluorescein isothiocyanate (FITC)-AS1411 to plasma membrane nucleolin 56 ± 10% SE (P < 0.01) compared with cells incubated with FITC-AS1411 only. Cellular uptake of [³²P]AS1411 into MV4-11 cells was blocked by a 20-fold excess of unlabeled AS1411 but not by a 20-fold excess of the biologically inactive oligonucleotide CRO-26. Uptake was approximately 3-fold faster into MV4-11 cells than into K-562 cells. Partial knockdown of plasma membrane and cytosolic nucleolin in MCF-7 cells resulted in a 3-fold decrease in AS1411 uptake. These results provide evidence that plasma membrane nucleolin is a functional receptor for AS1411 in MV4-11 cells.

Altered G-Protein Coupling in an mGluR6 Point Mutant Associated with Congenital Stationary Night Blindness [ARTICLES]

Beqollari, D., Betzenhauser, M. J., Kammermeier, P. J. — Fri, 16 Oct 2009 10:34:50 PDT

The highly specialized metabotropic glutamate receptor type 6 (mGluR6) is postsynaptically localized and expressed only in the dendrites of ON bipolar cells. Upon activation of mGluR6 by glutamate released from photoreceptors, a nonselective cation channel is inhibited, causing these cells to hyperpolarize. Mutations in this gene have been implicated in the development of congenital stationary night blindness type 1 (CSNB1). We investigated five known mGluR6 point mutants that lead to CSNB1 to determine the molecular mechanism of each phenotype. In agreement with other studies, four mutants demonstrated trafficking impairment. However, mGluR6 E775K (E781K in humans) suggested no trafficking or signaling deficiencies measured by our initial assays. Most importantly, our results indicate a switch in G-protein coupling, in which E775K loses G_o coupling but retains coupling to G_i, which may explain the phenotype.

Bid and Calpains Cooperate to Trigger Oxaliplatin-Induced Apoptosis of Cervical Carcinoma HeLa Cells [ARTICLES]

Fri, 16 Oct 2009 10:34:50 PDT

The Bcl-2 homology 3-only protein Bid is an important mediator of death receptor-induced apoptosis. Recent reports and this study suggest that Bid may also mediate genotoxic drug-induced apoptosis of various human cancer cells. Here, we characterized the role of Bid and the mechanism of Bid activation during oxaliplatin-induced apoptosis of HeLa cervical cancer cells. Small hairpin RNA-mediated silencing of Bid protected HeLa cells against both death receptor- and oxaliplatin-induced apoptosis. Expression of a Bid mutant in which caspase-8 cleavage site was mutated (D59A) reactivated oxaliplatin-induced apoptosis in Bid-deficient cells but failed to reactivate death receptor-induced apoptosis, suggesting that caspase-8-mediated Bid cleavage did not contribute to oxaliplatin-induced apoptosis. Overexpression of bcl-2 or treatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone abolished caspase-2, -8, -9, and -3 activation as well as Bid cleavage in response to oxaliplatin, suggesting that Bid cleavage occurred downstream of mitochondrial permeabilization and was predominantly mediated by caspases. We also detected an early activation of calpains in response to oxaliplatin. Calpain inhibition reduced Bid cleavage, mitochondrial depolarization, and activation of caspase-9, -3, -2, and -8 in response to oxaliplatin. Further experiments, however, suggested that Bid cleavage by calpains was not a prerequisite for oxaliplatin-induced apoptosis: single-cell imaging experiments using a yellow fluorescent protein-Bid-cyan fluorescent protein probe demonstrated translocation of full-length Bid to mitochondria that was insensitive to calpain or caspase inhibition. Moreover, calpain inhibition showed a potent protective effect in Bid-silenced cells. In conclusion, our data suggest that calpains and Bid act in a cooperative, but mutually independent, manner to mediate oxaliplatin-induced apoptosis of HeLa cells.

tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance [ARTICLES]

Zhang, H., Lin, H.-l., Walker, V. J., Hamdane, D., Hollenberg, P. F. — Fri, 16 Oct 2009 10:34:51 PDT

We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a K_I of 0.44 µM and k_inact of 0.12 min^–1. The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 Å away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by ~20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4.

Interactions of Histamine H1-Receptor Agonists and Antagonists with the Human Histamine H4-Receptor [ARTICLES]

Deml, K.-F., Beermann, S., Neumann, D., Strasser, A., Seifert, R. — Fri, 16 Oct 2009 10:34:51 PDT

The human histamine H₄-receptor (hH₄R) possesses high constitutive activity and, like the human H₁-receptor (hH₁R), is involved in the pathogenesis of type-I allergic reactions. The study aims were to explore the value of dual H₁/H₄R antagonists as antiallergy drugs and to address the question of whether H₁R ligands bind to hH₄R. In an acute murine asthma model, the H₁R antagonist mepyramine and the H₄R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ 7777120) exhibited synergistic inhibitory effects on eosinophil accumulation in the bronchoalveolar lavage fluid. At the hH₄R expressed in Sf9 insect cells, 18 H₁R antagonists and 22 H₁R agonists showed lower affinity to hH₄R than to hH₁R as assessed in competition binding experiments. For a small number of H₁R antagonists, hH₄R partial agonism was observed in the steady-state GTPase assay. Most compounds were neutral antagonists or inverse agonists. Twelve phenylhistamine-type hH₁R partial agonists were also hH₄R partial agonists. Four histaprodifen-type hH₁R partial agonists were hH₄R inverse agonists. Dimeric histaprodifen was a more efficacious hH₄R inverse agonist than the reference compound thioperamide. Suprahistaprodifen was the only histaprodifen acting as hH₄R partial agonist. Suprahistaprodifen was docked into the binding pocket of inactive and active hH₄R models in two different orientations, predominantly stabilizing the active state of hH₄R. Collectively, the synergistic effects of H₁R and H₄R antagonists in an acute asthma model and the overlapping interaction of structurally diverse H₁R ligands with hH₁R and hH₄R indicate that the development of dual H₁R/H₄R antagonists is a worthwhile and technically feasible goal for the treatment of type-I allergic reactions.

Functional Characterization of Human Cytochrome P450 2S1 Using a Synthetic Gene-Expressed Protein in Escherichia coli [ARTICLES]

Bui, P. H., Hankinson, O. — Fri, 16 Oct 2009 10:34:51 PDT

Human cytochrome P450 2S1 was recently identified and shown to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and hypoxia. It is highly expressed in epithelial cells of tissues that are exposed to the environment and in many tumors of epithelial origin. The biological function of CYP2S1 has not yet been determined, although its possible role in carcinogen metabolism has been suggested. In this report, we investigated its ability to metabolize carcinogens. To obtain a large quantity of active enzyme for substrate screening, we overexpressed CYP2S1 in Escherichia coli (200 nM culture), using a synthetic gene approach. High-level expression allowed us to achieve purification of CYP2S1 with high specific content and purity (16 nmol/mg). Despite high-level expression, we found that CYP2S1 was not readily reduced by cytochrome P450 reductase, and thus no activity was found using NADPH. However, the oxidative activity of CYP2S1 was supported by cumene hydroperoxide or H₂O₂, such that CYP2S1 oxidized many important environmental carcinogens, including benzo[a]pyrene, 9,10-dihydro-benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene-7,8-dihydrodiol, aflatoxin B1, naphthalene, and styrene, with high turnover. Most substrates tested were converted to detoxification products, except in the case of benzo[a]pyrene-7,8-dihydrodiol, which was converted into the very potent carcinogenic metabolite 7,8-dihydrodiol-trans-9,10-epoxide at a relatively efficient rate (K_m = 12.4 ± 2 µM, turnover = 2.3 min^–1). This metabolite formation was also supported both in vitro and in vivo by fatty acid hydroperoxides described in the accompanying report (p. 1044). Together, these data indicate that CYP2S1 contributes to the metabolism of environmental carcinogens via an NADPH independent activity.

Fatty Acid Hydroperoxides Support Cytochrome P450 2S1-Mediated Bioactivation of Benzo[a]pyrene-7,8-dihydrodiol [ARTICLES]

Bui, P. H., Hsu, E. L., Hankinson, O. — Fri, 16 Oct 2009 10:34:51 PDT

In the accompanying report (p. 1031), we showed that a novel dioxin-inducible cytochrome P450, CYP2S1, efficiently metabolizes benzo[a]pyrene-trans-7,8-dihydrodiol (BaP-7,8-diol) into the highly mutagenic and carcinogenic benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BaP-diol-t-epoxide), using cumene hydroperoxide in lieu of NADPH/O₂. Lipid hydroperoxide-supported P450 oxidation has been reported in several cases. However, it has not yet been described for the bioactivation of BaP-7,8-diol. In this report, we demonstrate that CYP2S1 can use various fatty acid hydroperoxides to support epoxidation of BaP-7,8-diol at a much higher rate than with cumene hydroperoxide. Kinetic analyses with several fatty acid hydroperoxides revealed that 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) was the most potent oxidant tested (K_m, 3.4 ± 0.8 µM; turnover, 4.51 ± 0.13 min^–1), followed by 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K_m, 2.8 ± 0.7 µM; turnover, 3.7 ± 0.1 min^–1), 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (K_m, 2.7 ± 0.8 µM; turnover, 3.69 ± 0.09 min^–1), and 15S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K_m, 11.6 ± 0.3 µM; turnover, 0.578 ± 0.030 min^–1). The antioxidant butylated hydroxyanisole inhibited CYP2S1-catalyzed epoxidation by 100%, suggesting that epoxidation proceeds by a free radical mechanism. Other cytochromes P450, including CYP1A1, CYP1B1, CYP1A2, and CYP3A4, were also able to epoxidize BaP-7,8-diol using various fatty acid hydroperoxides, although at slower rates than CYP2S1. The cytotoxicity of BaP-7,8-diol significantly increased in mammalian cells overexpressing CYP2S1, and BaP-diol-t-epoxide formation in these cells also increased in the presence of 13-HpODE. Together, these results suggest that fatty acid hydroperoxides can serve as physiological cofactors in supporting in vivo CYP2S1-catalyzed oxidation of BaP-7,8-diol, and that fatty acid hydroperoxides and CYP2S1 may play important roles in benzo[a]pyrene-induced carcinogenesis.

Differential Effects of Selective Cyclooxygenase-2 Inhibitors on Vascular Smooth Muscle Ion Channels May Account for Differences in Cardiovascular Risk Profiles [ARTICLES]

Brueggemann, L. I., Mackie, A. R., Mani, B. K., Cribbs, L. L., Byron, K. L. — Fri, 16 Oct 2009 10:34:51 PDT

Celecoxib, rofecoxib, and diclofenac are clinically used cyclooxygenase-2 (COX-2) inhibitors, which have been under intense scrutiny because long-term rofecoxib (Vioxx; Merck, Whitehouse Station, NJ) treatment was found to increase the risk of adverse cardiovascular events. A differential risk profile for these drugs has emerged, but the underlying mechanisms have not been fully elucidated. We investigated the effects of celecoxib, rofecoxib, and diclofenac on ionic currents and calcium signaling in vascular smooth muscle cells (VSMCs) using patch-clamp techniques and fura-2 fluorescence and on arterial constriction using pressure myography. Celecoxib, but not rofecoxib or diclofenac, dramatically enhanced KCNQ (K_v7) potassium currents and suppressed L-type voltage-sensitive calcium currents in A7r5 rat aortic smooth muscle cells (native KCNQ currents or overexpressed human KCNQ5 currents) and freshly isolated rat mesenteric artery myocytes. The effects of celecoxib were concentration-dependent within the therapeutic concentration range, and were reversed on washout. Celecoxib, but not rofecoxib, also inhibited calcium responses to vasopressin in A7r5 cells and dilated intact or endothelium-denuded rat mesenteric arteries. A celecoxib analog, 2,5-dimethyl-celecoxib, which does not inhibit COX-2, mimicked celecoxib in its enhancement of vascular KCNQ5 currents, suppression of L-type calcium currents, and vasodilation. We conclude that celecoxib inhibits calcium responses in VSMCs by enhancing KCNQ5 currents and suppressing L-type calcium currents, which ultimately reduces vascular tone. These effects are independent of its COX-2 inhibitory actions and may explain the differential risk of cardiovascular events in patients taking different drugs of this class.

Vitamin D3 and Its Nuclear Receptor Increase the Expression and Activity of the Human Proton-Coupled Folate Transporter [ARTICLES]

Eloranta, J. J., Hiller, C., Hausler, S., Stieger, B., Kullak-Ublick, G. A. — Fri, 16 Oct 2009 10:34:51 PDT

Folates are essential for nucleic acid synthesis and are particularly required in rapidly proliferating tissues, such as intestinal epithelium and hemopoietic cells. Availability of dietary folates is determined by their absorption across the intestinal epithelium, mediated by the proton-coupled folate transporter (PCFT) at the apical enterocyte membranes. Whereas transport properties of PCFT are well characterized, regulation of PCFT gene expression remains less elucidated. We have studied the mechanisms that regulate PCFT promoter activity and expression in intestine-derived cells. PCFT mRNA levels are increased in Caco-2 cells treated with 1,25-dihydroxyvitamin D₃ (vitamin D₃) in a dose-dependent fashion, and the duodenal rat Pcft mRNA expression is induced by vitamin D₃ ex vivo. The PCFT promoter region is transactivated by the vitamin D receptor (VDR) and its heterodimeric partner retinoid X receptor- (RXR) in the presence of vitamin D₃. In silico analyses predicted a VDR response element (VDRE) in the PCFT promoter region –1694/–1680. DNA binding assays showed direct and specific binding of the VDR:RXR heterodimer to the PCFT(–1694/–1680), and chromatin immunoprecipitations verified that this interaction occurs within living cells. Mutational promoter analyses confirmed that the PCFT(–1694/–1680) motif mediates a transcriptional response to vitamin D₃. In functional support of this regulatory mechanism, treatment with vitamin D₃ significantly increased the uptake of [³H]folic acid into Caco-2 cells at pH 5.5. In conclusion, vitamin D₃ and VDR increase intestinal PCFT expression, resulting in enhanced cellular folate uptake. Pharmacological treatment of patients with vitamin D₃ may have the added therapeutic benefit of enhancing the intestinal absorption of folates.

Three Epigenetic Drugs Up-Regulate Homeobox Gene Rhox5 in Cancer Cells through Overlapping and Distinct Molecular Mechanisms [ARTICLES]

Li, Q., Bartlett, D. L., Gorry, M. C., O'Malley, M. E., Guo, Z. S. — Fri, 16 Oct 2009 10:34:51 PDT

Epigenetic therapy of cancer using inhibitors of DNA methyltransferases (DNMT) or/and histone deacetylases (HDACs) has shown promising results in preclinical models and is being investigated in clinical trials. Homeodomain proteins play important roles in normal development and carcinogenesis. In this study, we demonstrated for the first time that an epigenetic drug could up-regulate homeobox genes in the reproductive homeobox genes on chromosome X (Rhox) family, including murine Rhox5, Rhox6, and Rhox9 and human RhoxF1 and RhoxF2 in breast, colon, and other types of cancer cells. We examined the molecular mechanisms underlining selective induction of Rhox5 in cancer cells by three epigenetic drugs: 5-aza-2'-deoxycytidine (DAC; decitabine), arsenic trioxide (ATO), and MS-275 [entinostat; N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide]. DAC induced Rhox5 mRNA expression from both distal promoter (Pd) and proximal promoter, whereas MS-275 and ATO induced gene expression from the Pd only. DAC and ATO inhibited both DNMT1 and DNMT3B protein expression, whereas MS-275 significantly reduced DNMT3B protein. In contrast to DAC, neither MS-275 nor ATO induced DNA demethylation on the Pd region. All three drugs led to enhanced acetylation of histones H3 and H4 at the promoter region. The occupancy of the activating histone mark dimethylated lysine 4 of H3 at Pd was enhanced by DAC and MS-275 but not ATO. Because they modulate gene expression with different potencies through shared and distinct epigenetic mechanisms, these epigenetic drugs may possess great potential in different applications for epigenetic therapy of cancer and other diseases.

Caveolin-1 and Lipid Microdomains Regulate Gs Trafficking and Attenuate Gs/Adenylyl Cyclase Signaling [ARTICLES]

Fri, 16 Oct 2009 10:34:51 PDT

Lipid rafts and caveolae are specialized membrane microdomains implicated in regulating G protein-coupled receptor signaling cascades. Previous studies have suggested that rafts/caveolae may regulate β-adrenergic receptor/G_s signaling, but underlying molecular mechanisms are largely undefined. Using a simplified model system in C6 glioma cells, this study disrupts rafts/caveolae using both pharmacological and genetic approaches to test whether caveolin-1 and lipid microdomains regulate G_s trafficking and signaling. Lipid rafts/caveolae were disrupted in C6 cells by either short-term cholesterol chelation using methyl-β-cyclodextrin or by stable knockdown of caveolin-1 and -2 by RNA interference. In imaging studies examining G_s-GFP during signaling, stimulation with the βAR agonist isoproterenol resulted in internalization of G_s-GFP; however, this trafficking was blocked by methyl-β-cyclodextrin or by caveolin knockdown. Caveolin knockdown significantly decreased G_s localization in detergent insoluble lipid raft/caveolae membrane fractions, suggesting that caveolin localizes a portion of G_s to these membrane microdomains. Methyl-β-cyclodextrin or caveolin knockdown significantly increased isoproterenol or thyrotropin-stimulated cAMP accumulation. Furthermore, forskolin- and aluminum tetrafluoride-stimulated adenylyl cyclase activity was significantly increased by caveolin knockdown in cells or in brain membranes obtained from caveolin-1 knockout mice, indicating that caveolin attenuates signaling at the level of G_s/adenylyl cyclase and distal to GPCRs. Taken together, these results demonstrate that caveolin-1 and lipid microdomains exert a major effect on G_s trafficking and signaling. It is suggested that lipid rafts/caveolae are sites that remove G_s from membrane signaling cascades and caveolins might dampen globally G_s/adenylyl cyclase/cAMP signaling.

High-Throughput Screening Reveals a Small-Molecule Inhibitor of the Renal Outer Medullary Potassium Channel and Kir7.1 [ARTICLES]

Fri, 16 Oct 2009 10:34:51 PDT

The renal outer medullary potassium channel (ROMK) is expressed in the kidney tubule and critically regulates sodium and potassium balance. The physiological functions of other inward rectifying K⁺ (Kir) channels expressed in the nephron, such as Kir7.1, are less well understood in part due to the lack of selective pharmacological probes targeting inward rectifiers. In an effort to identify Kir channel probes, we performed a fluorescence-based, high-throughput screen (HTS) of 126,009 small molecules for modulators of ROMK function. Several antagonists were identified in the screen. One compound, termed VU590, inhibits ROMK with submicromolar affinity, but has no effect on Kir2.1 or Kir4.1. Low micromolar concentrations inhibit Kir7.1, making VU590 the first small-molecule inhibitor of Kir7.1. Structure-activity relationships of VU590 were defined using small-scale parallel synthesis. Electrophysiological analysis indicates that VU590 is an intracellular pore blocker. VU590 and other compounds identified by HTS will be instrumental in defining Kir channel structure, physiology, and therapeutic potential.

Regulation of the CYP3A4 and CYP3A7 Promoters by Members of the Nuclear Factor I Transcription Factor Family [ARTICLES]

Riffel, A. K., Schuenemann, E., Vyhlidal, C. A. — Fri, 16 Oct 2009 10:34:51 PDT

Despite the established interindividual variability and ontogeny of the CYP3A enzymes, the most abundant phase I drug-metabolizing enzymes in human liver and intestine, the mechanisms that regulate basal expression remain poorly understood. Electrophoretic mobility shift assays using nuclear proteins extracted from human prenatal and postnatal liver samples identified multiple, developmentally distinct nuclear factor I (NFI)-containing protein complexes from human liver bound to sequences from the CYP3A4 (–243/–220) and CYP3A7 (–242/–219) proximal promoters. In addition, a hepatocyte nuclear factor (HNF) 3-containing complex from prenatal liver interacted with CYP3A7–242/–219 but not CYP3A4–243/–220. Cotransfection of HepG2 cells with a CYP3A4 proximal promoter construct and expression vectors for the NFI isoforms NFIA1.1, NFIB2, NFIC1, NFIC2, and NFIX1 enhanced the expression of luciferase activity. In contrast, cotransfection of NFIB2, NFIC1, NFIC2, NFIX1, and NFIX2 reduced the expression of luciferase under the control of the CYP3A7 gene promoter. Mutagenesis of the NFI/HNF3 binding sites in the CYP3A7 and CYP3A4 proximal promoters suggests that regulation of basal promoter activity by members of the NFI transcription factor family occur via multiple mechanisms. These results demonstrate that members of the NFI transcription factor family regulate CYP3A4 and CYP3A7 basal expression in an isoform- and promoter-specific manner.

Mechanisms Underlying Activation of Soluble Guanylate Cyclase by the Nitroxyl Donor Angeli's Salt [ARTICLES]

Fri, 16 Oct 2009 10:34:51 PDT

Nitroxyl (HNO) may be formed endogenously by uncoupled nitric-oxide (NO) synthases, enzymatic reduction of NO or as product of vascular nitroglycerin bioactivation. The established HNO donor Angeli's salt (trioxodinitrate, AS) causes cGMP-dependent vasodilation through activation of soluble guanylate cyclase (sGC). We investigated the mechanisms underlying this effect using purified sGC and cultured endothelial cells. AS (up to 0.1 mM) had no significant effect on sGC activity in the absence of superoxide dismutase (SOD) or dithiothreitol (DTT). In the presence of SOD, AS caused biphasic sGC activation (apparent EC₅₀ ~10 nM, maximum at 1 µM) that was accompanied by the formation of NO. DTT (2 mM) inhibited the effects of <10 µM AS but led to sGC activation and NO release at 0.1 mM AS even without SOD. AS had no effect on ferric sGC, excluding activation of the oxidized enzyme by HNO. The NO scavenger carboxy-PTIO inhibited endothelial cGMP accumulation induced by AS in the presence but not in the absence of SOD (EC₅₀ ~50 nM and ~16 µM, respectively). Carboxy-PTIO (0.1 mM) inhibited the effect of ≤10 µM AS in the presence of SOD but caused NO release from 0.1 mM AS in the absence of SOD. These data indicate that AS activates sGC exclusively via NO, formed either via SOD-catalyzed oxidation of HNO or through a minor AS decomposition pathway that is unmasked in the presence of HNO scavenging thiols.

Protein Kinase A and B-Raf Mediate Extracellular Signal-Regulated Kinase Activation by Thyrotropin [ARTICLES]

Vuchak, L. A., Tsygankova, O. M., Prendergast, G. V., Meinkoth, J. L. — Fri, 16 Oct 2009 10:34:51 PDT

Thyrotropin (TSH) regulates thyroid cell proliferation and function through cAMP-mediated signaling pathways that activate protein kinase A (PKA) and Epac/Rap1. The respective roles of PKA versus Epac/Rap1 in TSH signaling remain unclear. We set out to determine whether PKA and/or Rap1 mediate extracellular signal-regulated kinase (ERK) activation by TSH. Neither blocking Rap1 activity nor silencing the expression of Rap1 impaired TSH or forskolin-induced ERK activation in Wistar rat thyroid cells. Direct activation of Epac1 failed to stimulate ERK activity in starved cells, suggesting that Epac-induced Rap1 activity is not coupled to ERK activation in rat thyroid cells. By contrast, PKA activity was required for cAMP-stimulated ERK phosphorylation and was sufficient to increase ERK phosphorylation in starved cells. Expression of dominant-negative Ras inhibited ERK activation by TSH, forskolin, and N⁶-monobutyryl (6MB)-cAMP, a selective activator of PKA. Silencing the expression of B-Raf also inhibited ERK activation by TSH, forskolin, and 6MB-cAMP, but not that stimulated by insulin or serum. Depletion of B-Raf impaired TSH-induced DNA synthesis, indicating a functional role for B-Raf in TSH-regulated proliferation. Collectively, these results position PKA, Ras, and B-Raf as upstream regulators of ERK activation and identify B-Raf as a selective target of cAMP-elevating agents in thyroid cells. These data provide the first evidence for a functional role for B-Raf in TSH signaling.