9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes

It is well established that intracellular calcium ([Ca²⁺]_i) controls the inotropic state of the myocardium, and evidence mounts that a “Ca²⁺ clock” controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSC_Ca) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSC_Ca that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca²⁺ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca²⁺ oscillations followed by an overall increase in [Ca²⁺]_i. The latter occurs also in HL-1 cells in Ca²⁺-free solution and after depletion of sarcoplasmic reticulum Ca²⁺ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130–150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca²⁺ oscillations followed by a compensatory increase in [Ca²⁺]_i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.     

An N-terminal Nuclear Export Signal Regulates Trafficking and Aggregation of Huntingtin (Htt) Protein Exon 1

Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity.     

Intratumoral,not circulating,endothelial progenitor cells share genetic aberrations with glial tumor cells

Literature data indicate that glioma stem cells may give rise to both tumor cells and endothelial progenitor cells (EPCs). Malignant glioma patients usually have increased levels of circulating (EPCs) and these cells are known to contribute to the glioma neovasculature. In this study we compared the intratumoral and circulating EPCs of glioma patients for a set of common glioma genotypical aberrations (amplification of EGFR; deletion of PTEN and aneusomy of chromosomes 7 and 10). We found that the EPCs present in the tumor tissues, not the circulating EPCs, share genetic aberrations with the tumor cells. EPCs with EGFR amplification were found in 46% and with PTEN deletion in 36% of the cases. EPCs with polysomy 7 and monosomy 10 were detected in 56% and 38% of the cases while centrosomal abnormalities in EPCs were found in 68% of the cases. The presence of genetic aberrations of glioma cells in intratumoral EPCs may point to transdifferentiation of glioma stem cells into EPCs. However, the tissue specific CD133 splice variant of blood EPCs was detected in the glioma tissues but not in control brains, suggestive of a blood origin of at least part of the intratumoral EPCs. The findings highlight the complexity of the cellular constituents of glioma neovascularization which should be taken into account when developing anti‐angiogenic strategies for gliomas. J. Cell. Physiol. 228: 1383–1390, 2013. © 2012 Wiley Periodicals, Inc.     

The tumor suppressor CDKN3 controls mitosis

Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2^pThr-161 at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKβ phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.     

Involvement of CaMK‐IIδ and gelsolin in Cd2+‐dependent cytoskeletal effects in mesangial cells

Cadmium is a toxic metal with pleiotropic effects on cell death and survival. The mesangial cell is particularly responsive to Cd's effects on kinase signaling pathways and cytoskeletal dynamics. Here we show that CaMK‐II is a participant in the cytoskeletal effects of Cd²⁺. A major mesangial cell isoform, CaMK‐IIδ, was identified in pellets of DNase I pull‐downs and cytosolic immunoprecipitates of G‐actin. CaMK‐IIδ was also present in Triton X‐100‐insoluble cytoskeletal preparations and translocated to the cytoskeleton in a concentration‐dependent manner in Cd‐treated cells. Translocation was suppressed by KN93, an inhibitor of CaMK‐II phosphorylation. In vitro actin polymerization studies indicated that recombinant CaMK‐IIδ sequestered actin monomer. Cytoskeletal preparations from Cd‐treated cells decrease the rate of polymerization, but KN93 co‐treatment prevents this effect. Over‐expressed CaMK‐IIδ also translocated to the cytoskeleton upon Cd exposure, and this was prevented by KN93. Conversely, siRNA silencing of CaMK‐IIδ increases the effect of cytoskeletal extracts on actin polymerization, and abrogates the effect of Cd. The actin capping and severing protein, gelsolin, translocates to the cytoskeleton in the presence of Cd²⁺, dependent upon the phosphorylation of CaMK‐II, and is recovered together with actin and CaMK‐IIδ in G‐actin pull‐downs and F‐actin sedimentation. Translocation is accompanied by generation of a 50 kDa gelsolin fragment whose appearance is prevented by KN93 and CaMK‐IIδ silencing. We conclude that cytoskeletal effects of Cd in mesangial cells are partially mediated by Cd‐dependent activation of CaMK‐IIδ, binding of CaMK‐IIδ and gelsolin to actin filaments, and cleavage of gelsolin. J. Cell. Physiol. 228: 78–86, 2013. © 2012 Wiley Periodicals, Inc.     

Biosynthesis,isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.     

In vivo effects of anacetrapib on preβ HDL: improvement in HDL remodeling without effects on cholesterol absorption

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol and lowers LDL cholesterol in dyslipidemic patients. We previously demonstrated that ANA increases macrophage-to-feces reverse cholesterol transport and fecal cholesterol excretion in hamsters, and increased preβ HDL-dependent cholesterol efflux via ABCA1 in vitro. However, the effects of ANA on in vivo preβ HDL have not been characterized. In vitro, ANA inhibited the formation of preβ, however in ANA-treated dyslipidemic hamsters, preβ HDL levels (measured by two-dimensional gel electrophoresis) were increased, in contrast to in vitro findings. Because changes in plasma preβ HDL have been proposed to potentially affect markers of cholesterol absorption with other CETP inhibitors, a dual stable isotope method was used to directly measure cholesterol absorption in hamsters. ANA treatment of hamsters (on either dyslipidemic or normal diet) had no effect on cholesterol absorption, while dalcetrapib-treated hamsters displayed an increase in cholesterol absorption. Taken together, these data support the notion that ANA promotes preβ HDL functionality in vivo, with no effects on cholesterol absorption.     

IL‐1β and TNF‐α induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase

Glutaminase 1 is the main enzyme responsible for glutamate production in mammalian cells. The roles of macrophage and microglia glutaminases in brain injury, infection, and inflammation are well documented. However, little is known about the regulation of neuronal glutaminase, despite neurons being a predominant cell type of glutaminase expression. Using primary rat and human neuronal cultures, we confirmed that interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), two pro‐inflammatory cytokines that are typically elevated in neurodegenerative disease states, induced neuronal death and apoptosis in vitro. Furthermore, both intracellular and extracellular glutamate levels were significantly elevated following IL‐1β and/or TNF‐α treatment. Pre‐treatment with N‐Methyl‐d ‐aspartate (NMDA) receptor antagonist MK‐801 blocked cytokine‐induced glutamate production and alleviated the neurotoxicity, indicating that IL‐1β and/or TNF‐α induce neurotoxicity through glutamate. To determine the potential source of excess glutamate production in the culture during inflammation, we investigated the neuronal glutaminase and found that treatment with IL‐1β or TNF‐α significantly upregulated the kidney‐type glutaminase (KGA), a glutaminase 1 isoform, in primary human neurons. The up‐regulation of neuronal glutaminase was also demonstrated in situ in a murine model of HIV‐1 encephalitis. In addition, IL‐1β or TNF‐α treatment increased the levels of KGA in cytosol and TNF‐α specifically increased KGA levels in the extracellular fluid, away from its main residence in mitochondria. Together, these findings support neuronal glutaminase as a potential component of neurotoxicity during inflammation and that modulation of glutaminase may provide therapeutic avenues for neurodegenerative diseases.