Department of Pharmacology and Toxicology Works

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Nerve growth factor/p75 neurotrophin receptor–mediated sensitization of rat sensory neurons depends on membrane cholesterol
(Elsevier, 2013-09-17) Zhang, YH; Khanna, R; Nicol, Grant D.; Department of Pharmacology and Toxicology, IU School of Medicine
Nerve growth factor (NGF) is an important mediator in the initiation of the inflammatory response and NGF via activation of the p75 neurotrophin receptor (p75(NTR)) and downstream sphingomyelin signaling leads to significant enhancement of the excitability of small-diameter sensory neurons. Because of the interaction between sphingomyelin and cholesterol in creating membrane liquid-ordered domains known as membrane or lipid rafts, we examined whether neuronal NGF-induced sensitization via p75(NTR) was dependent on the integrity of membrane rafts. Here, we demonstrate that the capacity of NGF to enhance the excitability of sensory neurons may result from the interaction of p75(NTR) with its downstream signaling partner(s) in membrane rafts. Two agents known to disrupt membrane rafts, edelfosine and methyl-β-cyclodextrin (MβCD), block the increase in excitability produced by NGF. In contrast, treatment with MβCD containing saturated amounts of cholesterol does not alter the capacity of NGF to augment excitability. In addition, adding back MβCD with cholesterol restored the NGF-induced sensitization in previously cholesterol-depleted neurons, suggesting that cholesterol and the structural integrity of rafts are key to promoting NGF-mediated sensitization. Using established protocols to isolate detergent-resistant membranes, both p75(NTR) and the neuronal membrane raft marker, flotillin, localize to raft fractions. These results suggest that downstream signaling partners interacting with p75(NTR) in sensory neurons are associated with membrane raft signaling platforms.
Methylmercury exposure increases lipocalin related (lpr) and decreases activated in blocked unfolded protein response (abu) genes and specific miRNAs in Caenorhabditis elegans
(Elsevier, 2013-10-24) Rudgalvyte, Martina; VanDuyn, Natalia; Aarnio, Vuokko; Heikkinen, Liisa; Peltonen, Juhani; Lakso, Merja; Nass, Richard; Wong, Garry; Department of Pharmacology and Toxicology, School of Medicine
Methylmercury (MeHg) is a persistent environmental and dietary contaminant that causes serious adverse developmental and physiologic effects at multiple cellular levels. In order to understand more fully the consequences of MeHg exposure at the molecular level, we profiled gene and miRNA transcripts from the model organism Caenorhabditis elegans. Animals were exposed to MeHg (10µM) from embryo to larval 4 (L4) stage and RNAs were isolated. RNA-seq analysis on the Illumina platform revealed 541 genes up- and 261 genes down-regulated at a cutoff of 2-fold change and false discovery rate-corrected significance q < 0.05. Among the up-regulated genes were those previously shown to increase under oxidative stress conditions including hsp-16.11 (2.5-fold), gst-35 (10.1-fold), and fmo-2(58.5-fold). In addition, we observed up-regulation of 6 out of 7 lipocalin related (lpr) family genes and down regulation of 7 out of 15 activated in blocked unfolded protein response (abu) genes. Gene Ontology enrichment analysis highlighted the effect of genes related to development and organism growth. miRNA-seq analysis revealed 6–8 fold down regulation of mir-37-3p, mir-41-5p, mir-70-3p, and mir-75-3p. Our results demonstrate the effects of MeHg on specific transcripts encoding proteins in oxidative stress responses and in ER stress pathways. Pending confirmation of these transcript changes at protein levels, their association and dissocation characteristics with interaction partners, and integration of these signals, these findings indicate broad and dynamic mechanisms by which MeHg exerts its harmful effects.
Expression of the essential Kinase PfCDPK1 from Plasmodium falciparum in Toxoplasma gondii facilitates the discovery of novel antimalarial drugs
(American Society for Microbiology, 2014-05) Gaji, Rajshekhar Y.; Checkley, Lisa; Reese, Michael L.; Ferdig, Michael T.; Arrizabalaga, Gustavo; Pharmacology and Toxicology, School of Medicine
We have previously shown that genetic disruption of Toxoplasma gondii calcium-dependent protein kinase 3 (TgCDPK3) affects calcium ionophore-induced egress. We examined whether Plasmodium falciparum CDPK1 (PfCDPK1), the closest homolog of TgCDPK3 in the malaria parasite P. falciparum, could complement a TgCDPK3 mutant strain. PfCDPK1 is essential and plays critical roles in merozoite development, motility, and secretion. We show that expression of PfCDPK1 in the TgCDPK3 mutant strain rescues the egress defect. This phenotypic complementation requires the localization of PfCDPK1 to the plasma membrane and kinase activity. Interestingly, PfCDPK1-expressing Toxoplasma becomes more sensitive to egress inhibition by purfalcamine, a potent inhibitor of PfCDPK1 with low activity against TgCDPK3. Based on this result, we tested eight small molecules previously determined to inhibit the kinase activity of recombinant PfCDPK1 for their abilities to inhibit ionophore-induced egress in the PfCDPK1-expressing strain. While two of these chemicals did not inhibit egress, we found that six drugs affected this process selectively in PfCDPK1-expressing Toxoplasma. Using mutant versions of PfCDPK1 and TgCDPK3, we show that the selectivities of dasatinib and PLX-4720 are regulated by the gatekeeper residue in the ATP binding site. Importantly, we have confirmed that the three most potent inhibitors of egress in the PfCDPK1-expressing strain effectively kill P. falciparum. Thus, we have established and validated a recombinant strain of Toxoplasma that can be used as a surrogate for the discovery and analysis of PfCDPK1-specific inhibitors that can be developed as antimalarials.
Tetrodotoxin-resistant sodium channels in sensory neurons generate slow resurgent currents that are enhanced by inflammatory mediators
(Society for Neuroscience, 2014-05-21) Tan, Zhi-Yong; Piekarz, Andrew D.; Priest, Birgit T.; Knopp, Kelly L.; Krajewski, Jeffrey L.; McDermott, Jeff S.; Nisenbaum, Eric S.; Cummins, Theodore R.; Pharmacology and Toxicology, School of Medicine
Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the β4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel β4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the β4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.
Roles of c-FLIP in Apoptosis, Necroptosis, and Autophagy
(OMICS, 2013) Safa, Ahmad R.; Department of Pharmacology and Toxicology, IU School of Medicine
Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) is a major antiapoptotic protein and an important cytokine and chemotherapy resistance factor that suppresses cytokine- and chemotherapyinduced apoptosis. c-FLIP is expressed as long (c-FLIPL), short (c-FLIPS), and c-FLIPR splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 and TRAIL receptor 5 (DR5). This interaction in turn prevents Death-Inducing Signaling Complex (DISC) formation and subsequent activation of the caspase cascade. c-FLIPL and c-FLIPS are also known to have multifunctional roles in various signaling pathways, as well as activating and/ or upregulating several cytoprotective and pro-survival signaling proteins including Akt, ERK, and NF-κB. In addition to its role in apoptosis, c-FLIP is involved in programmed necroptosis (necrosis) and autophagy. Necroptosis is regulated by the Ripoptosome, which is a signaling intracellular cell death platform complex. The Ripoptosome contains receptor-interacting protein-1/Receptor-Interacting Protein-3 (RIP1), caspase-8, caspase-10, FADD, and c-FLIP isoforms involved in switching apoptotic and necroptotic cell death. c-FLIP regulates the Ripoptosome; in addition to its role in apoptosis, it is therefore also involved in necrosis. c-FLIPL attenuates autophagy by direct acting on the autophagy machinery by competing with Atg3 binding to LC3, thereby decreasing LC3 processing and inhibiting autophagosome formation. Upregulation of c-FLIP has been found in various tumor types, and its silencing has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. This review focuses on (1) the anti-apoptotic role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and chemotherapy drug resistance, as well as its roles in necrosis and autophagy, and (2) modulation of c-FLIP expression as a means to enhance apoptosis and modulate necrosis and autophagy in cancer cells.
Determinants of 14-3-3σ dimerization and function in drug and radiation resistance
(2013-11) Li, Zhaomin; Peng, Hui; Qin, Li; Qi, Jing; Zuo, Xiaobing; Liu, Jing-Yuan; Zhang, Jian-Ting; Department of Pharmacology and Toxicology, IU School of Medicine
Many proteins exist and function as homodimers. Understanding the detailed mechanism driving the homodimerization is important and will impact future studies targeting the “undruggable” oncogenic protein dimers. In this study, we used 14-3-3σ as a model homodimeric protein and performed a systematic investigation of the potential roles of amino acid residues in the interface for homodimerization. Unlike other members of the conserved 14-3-3 protein family, 14-3-3σ prefers to form a homodimer with two subareas in the dimeric interface that has 180° symmetry. We found that both subareas of the dimeric interface are required to maintain full dimerization activity. Although the interfacial hydrophobic core residues Leu12 and Tyr84 play important roles in 14-3-3σ dimerization, the non-core residue Phe25 appears to be more important in controlling 14-3-3σ dimerization activity. Interestingly, a similar non-core residue (Val81) is less important than Phe25 in contributing to 14-3-3σ dimerization. Furthermore, dissociating dimeric 14-3-3σ into monomers by mutating the Leu12, Phe25, or Tyr84 dimerization residue individually diminished the function of 14-3-3σ in resisting drug-induced apoptosis and in arresting cells at G2/M phase in response to DNA-damaging treatment. Thus, dimerization appears to be required for the function of 14-3-3σ.
Voltage-Gated Sodium Channel in Grasshopper Mice Defends Against Bark Scorpion Toxin
(AAAS, American Association for the Advancement of Science, 2013-10-25) Rowe, Ashlee H.; Xiao, Yucheng; Rowe, Matthew P.; Cummins, Theodore R.; Zakon, Harold H.; Department of Pharmacology & Toxicology, School of Medicine
Painful venoms are used to deter predators. Pain itself, however, can signal damage and thus serves an important adaptive function. Evolution to reduce general pain responses, although valuable for preying on venomous species, is rare, likely because it comes with the risk of reduced response to tissue damage. Bark scorpions capitalize on the protective pain pathway of predators by inflicting intensely painful stings. However, grasshopper mice regularly attack and consume bark scorpions, grooming only briefly when stung. Bark scorpion venom induces pain in many mammals (house mice, rats, humans) by activating the voltage-gated Na+ channel Nav1.7, but has no effect on Nav1.8. Grasshopper mice Nav1.8 has amino acid variants that bind bark scorpion toxins and inhibit Na+ currents, blocking action potential propagation and inducing analgesia. Thus, grasshopper mice have solved the predator-pain problem by using a toxin bound to a nontarget channel to block transmission of the pain signals the venom itself is initiating.
Human β-galactoside α-2,3-sialyltransferase (ST3Gal III) attenuated Taxol-induced apoptosis in ovarian cancer cells by downregulating caspase-8 activity
(Springer US, 2009-11) Huang, Su; Day, Travis W.; Choi, Mi-Ran; Safa, Ahmad R.; Department of Pharmacology & Toxicology, School of Medicine
Taxol triggers apoptosis in a variety of cancer cells, but it also upregulates cytoprotective proteins and/or pathways that compromise its therapeutic efficacy. In this report, we found that Taxol treatment resulted in caspase-8-dependent apoptosis in SKOV3 human ovarian cancer cells. Moreover, Taxol-induced apoptosis was associated with caspase-3 activation. Interestingly, Taxol treatment upregulated α-2,3-sialyltransferase (ST3Gal III) expression and forced expression of ST3Gal III attenuated Taxol-induced apoptosis. Furthermore, ST3Gal III overexpression inhibited Taxol-ttiggered caspase-8 activation, indicating that ST3Gal III upregulation produces cellular resistance to Taxol and hence reduces the efficacy of Taxol therapy.
4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) targets mRNA of the c-FLIP variants and induces apoptosis in MCF-7 human breast cancer cells
(Springer US, 2010-09) Bijangi-Vishehsaraei, Khadijeh; Huang, Su; Safa, Ahmad R.; Saadatzadeh, Mohammad Reza; Murphy, Michael P.; Department of Pharmacology & Toxicology, School of Medicine
Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor for the tumor necrosis factor-related apoptosis-inducing ligand TRAIL and in drug resistance in human malignancies. c-FLIP is an antagonist of caspases-8 and -10, which inhibits apoptosis and is expressed as long (c-FLIPL) and short (c-FLIPS) splice forms. c-FLIP is often overexpressed in various human cancers, including breast cancer. Several studies have shown that silencing c-FLIP by specific siRNAs sensitizes cancer cells to TRAIL and anticancer agents. However, systemic use of siRNA as a therapeutic agent is not practical at present. In order to reduce or inhibit c-FLIP expression, small molecules are needed to allow targeting c-FLIP without inhibiting caspases-8 and -10. We used a small molecule inhibitor of c-FLIP, 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), and show that CMH, but not its inactive analog, downregulated c-FLIPL and c-FLIPS mRNA and protein levels, caused poly(ADP-ribose) polymerase (PARP) degradation, reduced cell survival, and induced apoptosis in MCF-7 breast cancer cells. These results revealed that c-FLIP is a critical apoptosis regulator that can serve as a target for small molecule inhibitors that downregulate its expression and serve as effective targeted therapeutics against breast cancer cells.
GCN2-like eIF2α kinase manages the amino acid starvation response in Toxoplasma gondii
(Elsevier, 2014-02) Konrad, Christian; Wek, Ronald C.; Sullivan, William J., Jr.; Department of Pharmacology and Toxicology, IU School of Medicine
The apicomplexan protozoan Toxoplasma gondii is a significant human and veterinary pathogen. As an obligate intracellular parasite, Toxoplasma depends on nutrients provided by the host cell and needs to adapt to limitations in available resources. In mammalian cells, translational regulation via GCN2 phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) is a key mechanism for adapting to nutrient stress. Toxoplasma encodes two GCN2-like protein kinases, TgIF2K-C and TgIF2K-D. We previously showed that TgIF2K-D phosphorylates T. gondii eIF2α (TgIF2α) upon egress from the host cell, which enables the parasite to overcome exposure to the extracellular environment. However, the function of TgIF2K-C remained unresolved. To determine the functions of TgIF2K-C in the parasite, we cloned the cDNA encoding TgIF2K-C and generated knockout parasites of this TgIF2α kinase to study its function during the lytic cycle. The TgIF2K-C knockout did not exhibit a fitness defect compared with parental parasites. However, upon infection of human fibroblasts that were subsequently cultured in glutamine-free medium, the intracellular TgIF2K-C knockout parasites were impeded for induced phosphorylation of TgIF2α and showed a 50% reduction in the number of plaques formed compared with parental parasites. Furthermore, we found that this growth defect in glutamine-free media was phenocopied in parasites expressing only a non-phosphorylatable TgIF2α (TgIF2α-S71A), but not in a TgIF2K-D knockout. These studies suggest that Toxoplasma GCN2-like kinases TgIF2K-C and TgIF2K-D evolved to have distinct roles in adapting to changes in the parasite’s environment.

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