Addiction to Golgi-resident PI4P synthesis in chromosome 1q21.3-amplified lung adenocarcinoma cells.

A chromosome 1q21.3 region that is frequently amplified in diverse cancer types encodes phosphatidylinositol (PI)-4 kinase IIIß (PI4KIIIß), a key regulator of secretory vesicle biogenesis and trafficking. Chromosome 1q21.3-amplified lung adenocarcinoma (1q-LUAD) cells rely on PI4KIIIß for Golgi-resident PI-4-phosphate (PI4P) synthesis, prosurvival effector protein secretion, and cell viability. Here, we show that 1q-LUAD cells subjected to prolonged PI4KIIIß antagonist treatment acquire tolerance by activating an miR-218-5p-dependent competing endogenous RNA network that up-regulates PI4KIIα, which provides an alternative source of Golgi-resident PI4P that maintains prosurvival effector protein secretion and cell viability. These findings demonstrate an addiction to Golgi-resident PI4P synthesis in a genetically defined subset of cancers.

Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death.

Phosphatidylinositol 3-phosphate (PI(3)P) levels in Plasmodium falciparum correlate with tolerance to cellular stresses caused by artemisinin and environmental factors. However, PI(3)P function during the Plasmodium stress response was unknown. Here, we used PI3K inhibitors and antimalarial agents to examine the importance of PI(3)P under thermal conditions recapitulating malarial fever. Live cell microscopy using chemical and genetic reporters revealed that PI(3)P stabilizes the digestive vacuole (DV) under heat stress. We demonstrate that heat-induced DV destabilization in PI(3)P-deficient P. falciparum precedes cell death and is reversible after withdrawal of the stress condition and the PI3K inhibitor. A chemoproteomic approach identified PfHsp70-1 as a PI(3)P-binding protein. An Hsp70 inhibitor and knockdown of PfHsp70-1 phenocopy PI(3)P-deficient parasites under heat shock. Furthermore, PfHsp70-1 downregulation hypersensitizes parasites to heat shock and PI3K inhibitors. Our findings underscore a mechanistic link between PI(3)P and PfHsp70-1 and present a novel PI(3)P function in DV stabilization during heat stress.

The C-terminal acidic motif of Phafin2 inhibits PH domain binding to phosphatidylinositol 3-phosphate.

Changes in membrane curvature are required to control the function of subcellular compartments; malfunctions of such processes are associated with a wide range of human diseases. Membrane remodeling often depends upon the presence of phosphoinositides, which recruit protein effectors for a variety of cellular functions. Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns3P)-binding effector involved in endosomal and lysosomal membrane-associated signaling. Both the Phafin2 PH and the FYVE domains bind PtdIns3P, although their redundant function in the protein is unclear. Through a combination of lipid-binding assays, we found that, unlike the FYVE domain, recognition of the PH domain to PtdIns3P requires a lipid bilayer. Using site-directed mutagenesis and truncation constructs, we discovered that the Phafin2 FYVE domain is constitutive for PtdIns3P binding, whereas PH domain binding to PtdIns3P is autoinhibited by a conserved C-terminal acidic motif. These findings suggest that binding of the Phafin2 PH domain to PtdIns3P in membrane compartments occurs through a highly regulated mechanism. Potential mechanisms are discussed throughout this report.

The Legionella effector RavD binds phosphatidylinositol-3-phosphate and helps suppress endolysosomal maturation of the Legionella-containing vacuole.

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein-lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

Development of Three Orthogonal Assays Suitable for the Identification and Qualification of PIKfyve Inhibitors.

FYVE-type zinc finger-containing phosphoinositide kinase (PIKfyve) catalyzes the formation of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol 3-phosphate (PI(3)P). PIKfyve has been implicated in multiple cellular processes, and its role in the regulation of toll-like receptor (TLR) pathways and the production of proinflammatory cytokines has sparked interest in developing small-molecule PIKfyve inhibitors as potential therapeutics to treat autoimmune and inflammatory diseases. We developed three orthogonal assays to identify and qualify small-molecule inhibitors of PIKfyve: (1) a purified component microfluidic enzyme assay that measures the conversion of fluorescently labeled PI(3)P to PI(3,5)P2 by purified recombinant full-length human 6His-PIKfyve (rPIKfyve); (2) an intracellular protein stabilization assay using the kinase domain of PIKfyve expressed in HEK293 cells; and (3) a cell-based functional assay that measures the production of interleukin (IL)-12p70 in human peripheral blood mononuclear cells stimulated with TLR agonists lipopolysaccharide and R848. We determined apparent Km values for both ATP and labeled PI(3)P in the rPIKfyve enzyme assay and evaluated the enzyme's ability to use phosphatidylinositol as a substrate. We also tested four reference compounds in the three assays and showed that together these assays provide a platform that is suitable to select promising inhibitors having appropriate functional activity and confirmed cellular target engagement to advance into preclinical models of inflammation.

Inhibition of phosphatidylinositol 3,5-bisphosphate production has pleiotropic effects on various membrane trafficking routes in Arabidopsis.

Phosphoinositides play an important role in various membrane trafficking events in eukaryotes. One of them, however, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], has not been studied widely in plants. Using a combination of fluorescent reporter proteins and the PI(3,5)P2-specific inhibitor YM202636, here we demonstrated that in Arabidopsis thaliana, PI(3,5)P2 affects various membrane trafficking events, mostly in the post-Golgi routes. We found that YM201636 treatment effectively reduced PI(3,5)P2 concentration not only in the wild type but also in FAB1A-overexpressing Arabidopsis plants. In particular, reduced PI(3,5)P2 levels caused abnormal membrane dynamics of plasma membrane proteins, AUX1 and BOR1, with different trafficking patterns. Secretion and morphological characteristics of late endosomes and vacuoles were also affected by the decreased PI(3,5)P2 production. These pleiotropic defects in the post-Golgi trafficking events were caused by the inhibition of PI(3,5)P2 production. This effect is probably mediated by the inhibition of maturation of FAB1-positive late endosomes, thereby impairing late endosome function. In conclusion, our results imply that in Arabidopsis, late endosomes are involved in multiple post-Golgi membrane trafficking routes including not only vacuolar trafficking and endocytosis but also secretion.

Metastasis suppressor 1 regulates neurite outgrowth in primary neuron cultures.

Metastasis suppressor 1 (MTSS1) or missing in metastasis (MIM) is an actin- and membrane-binding protein with tumor suppressor functions. MTSS1 is important for cell morphology, motility, metastasis. The role of MTSS1 in cell morphology has been widely investigated in non-neuronal tissues; however the role of MTSS1 in neurite outgrowth remains unclear. Here we investigated the effect of MTSS1 on neurite outgrowth in primary cerebellar granule and hippocampal neurons of mouse. We found that overexpression of MTSS1 in cerebellar granule neurons significantly enhanced dendrite elaboration but inhibited axon elongation. This phenotype was significantly reduced by deletion of the Wiskott-Aldrich homology 2 (WH2) motif and point mutation in the insulin receptor substrate p53 (IRSp53) and MIM/MTSS1 homology (IMD) domain. Furthermore, inhibition of Rac1 activity or blocking of phosphatidyl inositol phosphates (PIPs) signaling decreased the effect of MTSS1 markedly. In accordance with the over-expression data, knockdown of MTSS1 in cerebellar granule neurons could increase the axon length but decrease the dendrite length and the number of dendrites. In addition, MTSS1 knock down in embryonic hippocampal neurons suppressed neurite branching and reduced dendrite length. Our findings have demonstrated that MTSS1 modulates neuronal morphology, possibly through a Rac1-PIPs signaling pathway.

The function of phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) explored using a specific inhibitor that targets the PI5P-binding site.

NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P4K assays in vitro showed that NIH-12848 inhibited PI5P4Kγ with an IC50 of approximately 1 µM but did not inhibit the α and ß PI5P4K isoforms at concentrations up to 100 µM. A lack of inhibition of PI5P4Kγ ATPase activity suggested that NIH-12848 does not interact with the enzyme's ATP-binding site and direct exploration of binding using hydrogen-deuterium exchange (HDX)-MS (HDX-MS) revealed the putative PI5P-binding site of PI5P4Kγ to be the likely region of interaction. This was confirmed by a series of mutation experiments which led to the identification of a single PI5P4Kγ amino acid residue that can be mutated to its PI5P4Ks α and ß homologue to render PI5P4Kγ resistant NIH-12848 inhibition. NIH-12848 (10 µM) was applied to cultured mouse principal kidney cortical collecting duct (mpkCCD) cells which, we show, express PI5P4Kγ that increases when the cells grow to confluence and polarize. NIH-12848 inhibited the translocation of Na⁺/K⁺-ATPase to the plasma membrane that occurs when mpkCCD cells grow to confluence and also prevented reversibly their forming of 'domes' on the culture dish. Both these NIH-12848-induced effects were mimicked by specific RNAi knockdown of PI5P4Kγ, but not that of PI5P4Ks α or ß. Overall, the data reveal a probable contribution of PI5P4Kγ to the development and maintenance of epithelial cell functional polarity and show that NIH-12848 is a potentially powerful tool for exploring the cell physiology of PI5P4Ks.

NS5A inhibitors impair NS5A-phosphatidylinositol 4-kinase IIIα complex formation and cause a decrease of phosphatidylinositol 4-phosphate and cholesterol levels in hepatitis C virus-associated membranes.

The hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIIIα complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIIIα-targeting inhibitor. In addition, both the NS5A and PI4KIIIα classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIIIα. Because of the similarities between the effects induced by treatment with PI4KIIIα or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIIIα complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIIIα complex.

Engineering ePTEN, an enhanced PTEN with increased tumor suppressor activities.

The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a key regulator of cell proliferation, survival, and migration and the enzyme that dephosphorylates it, phosphatase and tensin homolog (PTEN), is an important tumor suppressor. As excess PIP3 signaling is a hallmark of many cancers, its suppression through activation of PTEN is a potential cancer intervention. Using a heterologous expression system in which human PTEN-GFP is expressed in Dictyostelium cells, we identified mutations in the membrane-binding regulatory interface that increase the recruitment of PTEN to the plasma membrane due to enhanced association with PI(4,5)P2. We engineered these into an enhanced PTEN (ePTEN) with approximately eightfold increased ability to suppress PIP3 signaling. Upon expression in human cells, ePTEN decreases PIP3 levels in the plasma membrane; phosphorylation of AKT, a major downstream event in PIP3 signaling; and cell proliferation and migration. Thus, the activation of PTEN can readjust PIP3 signaling and may serve as a feasible target for anticancer therapies.

Small chemicals with inhibitory effects on PtdIns(3,4,5)P3 binding of Btk PH domain.

Phosphatidylinositol-3,4-5-triphosphates (PtdIns(3,4,5)P3) formed by phosphoinositide-3-kinase (PI3K) had been known as a signaling molecule that plays important roles in diverse cellular processes such as cell signaling, metabolism, cell differentiation, and apoptosis. PtdIns(3,4,5)P3 regulates diverse cellular processes by recruiting effector proteins to the specific cellular locations for correct functions. In this study, we reported the inhibitory effect of small chemicals on the interaction between PtdIns(3,4,5)P3-Btk PH domain. Small chemicals were synthesized based on structural similarity of PtdInsP head-groups, and tested the inhibitory effects in vitro via surface plasmon resonance (SPR). As a result, the chemical 8 showed highest inhibitory effect with 17µM of IC50 value. To elucidate diverse inhibitory effects of different small chemicals we employed in silico docking experiment using molecular modeling and simulation. The result of docking experiments showed chemical 8 has more hydrogen bonding with the residues in PtdIns(3,4,5)P3 binding site of Btk PH domain than others. Overall, our studies demonstrate the efficient approach to develop lipid binding inhibitors, and further we can use these chemicals to regulate effector proteins. In addition, our study would provide new insight that lipid binding domain may be the attractive therapeutic targets to treat severe human diseases.

Role of phosphatidylinositol 3,4,5-trisphosphate in cell signaling.

Many lipids present in cellular membranes are phosphorylated as part of signaling cascades and participate in the recruitment, localization, and activation of downstream protein effectors. Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) is one of the most important second messengers and is capable of interacting with a variety of proteins through specific PtdIns(3,4,5)P3 binding domains. Localization and activation of these effector proteins controls a myriad of cellular functions including cell survival, proliferation, cytoskeletal rearrangement, and gene expression. Aberrations in the production and metabolism of PtdIns(3,4,5)P3 have been implicated in many human diseases including cancer, diabetes, inflammation, and heart disease. This chapter provides an overview of the role of PtdIns(3,4,5)P3 in cellular regulation and the implications of PtdIns(3,4,5)P3 dysregulation in human diseases. Additionally, recent attempts at targeting PtdIns(3,4,5)P3 signaling via small molecule inhibitors are summarized.

Inhibition of cell migration by PITENINs: the role of ARF6.

We have reported previously the development of small-molecule phosphatidylinositol-3,4,5-trisphosphate (PIP3) antagonists (PITs) that block pleckstrin homology (PH) domain interaction, including activation of Akt, and show anti-tumor potential. Here we show that the same molecules inhibit growth factor-induced actin remodeling, lamellipodia formation and, ultimately, cell migration and invasion, consistent with an important role of PIP3 in these processes. In vivo, a PIT-1 analog displays significant inhibition on tumor angiogenesis and metastasis. ADP ribosylation factor 6 (ARF6) was recently identified as an important mediator of cytoskeleton and cell motility, which is regulated by PIP3-dependent membrane translocation of the guanine nucleotide exchange factors (GEFs), such as ADP-ribosylation factor nucleotide binding site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1). We demonstrate that PITs inhibit PIP3/ARNO or GRP1 PH domain binding and membrane localization, resulting in the inhibition of ARF6 activation. Importantly, we show that expression of the constitutively active mutant of ARF6 attenuates inhibition of lamellipodia formation and cell migration by PITs, confirming that inhibition of ARF6 contributes to inhibition of these processes by PITs. Overall, our studies demonstrate the feasibility of developing specific small-molecule targeting PIP3 binding by PH domains as potential anticancer agents that can simultaneously interfere with cancer development at multiple points.

Targeting phospshatidylinositol 3-kinase signaling with novel phosphatidylinositol 3,4,5-triphosphate antagonists.

The critical role of phopshatidylinositol-3-kinase (PtdIns3K) signaling in the regulation of a wide range of cellular functions, including cell survival and proliferation, autophagy, metabolism and cell migration, is well recognized. Activation of PtdIns3K leads to the generation of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P 3). PtdIns(3,4,5)P 3 activates a complex signaling network controlling these diverse cellular functions through binding to Pleckstrin Homology (PH) domains of the effector proteins. We have recently described a new structural class of nonphosphoinositide small molecule inhibitors targeting binding of PtdIns(3,4,5) P 3 to PH domain targets. Using an in vitro PtdIns(3,4,5)P 3-PH domain binding assay, we identified two distinct PtdIns(3,4,5)P 3 antagonists, PIT-1 and PIT-2. Further cellular analysis revealed that both PITs inhibit PtdIns(3,4,5) P 3-dependent signaling mediated by Akt kinase, leading to the induction of apoptosis, metabolic stress and autophagy. An improved PIT-1 analog, DM-PIT-1, displays significant anticancer activity in the mouse syngeneic 4T1 breast cancer model in vivo. Discovery of PITs as well as other PtdIns(3,4,5)P 3 antagonists recently described by other laboratories suggest the possibility of targeting a key universal PtdIns(3,4,5)P 3/PH domain binding step in the PtdIns3K pathway using heterologous small molecule modulators.

Phosphoinositides direct equine infectious anemia virus gag trafficking and release.

Phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2) ], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P(2) is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P(2) is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with compartments bearing these PIs was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound phosphatidylinositol 3-phosphate [PI(3)P] with higher affinity than PI(4,5)P(2) as revealed by nuclear magnetic resonance (NMR) spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in phosphatidylinositol 3,5-biphosphate [PI(3,5)P(2) ]. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P(2) from PI(3)P, caused Gag to colocalize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by coexpression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P(2) from the plasma membrane. However, coexpression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that PI-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release.

Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments.

The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) vesicles. PI(4)P inhibition did not prevent vesicle tethering but significantly reduced formation of soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes between vesicle and Golgi SNARE proteins. Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors. Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments. Together, the data presented here indicate that PI(4)P is required for the SNARE-dependent fusion stage of COPII vesicles with the Golgi complex.

Small molecule inhibition of phosphatidylinositol-3,4,5-triphosphate (PIP3) binding to pleckstrin homology domains.

The PI3-kinase (PI3K) pathway regulates many cellular processes, especially cell metabolism, cell survival, and apoptosis. Phosphatidylinositol-3,4,5-trisphosphate (PIP3), the product of PI3K activity and a key signaling molecule, acts by recruiting pleckstrin-homology (PH) domain-containing proteins to cell membranes. Here, we describe a new structural class of nonphosphoinositide small molecule antagonists (PITenins, PITs) of PIP3-PH domain interactions (IC(50) ranges from 13.4 to 31 µM in PIP3/Akt PH domain binding assay). PITs inhibit interactions of a number of PIP3-binding PH domains, including those of Akt and PDK1, without affecting several PIP2-selective PH domains. As a result, PITs suppress the PI3K-PDK1-Akt pathway and trigger metabolic stress and apoptosis. A PIT-1 analog displayed significant antitumor activity in vivo, including inhibition of tumor growth and induction of apoptosis. Overall, our studies demonstrate the feasibility of developing specific small molecule antagonists of PIP3 signaling.

U73122, an aminosteroid phospholipase C inhibitor, is a potent inhibitor of cardiac phospholipase D by a PIP2-dependent mechanism.

The aminosteroid 1-[6-({17beta-3-methoxyestra-1,3,5(10)-trien-17-yl}amino)hexyl]-1H-pyrrole-2,5-dione (U73122) has been extensively used as a pharmacologic inhibitor of phospholipase C (PLC). The inhibitory effect of U73122 on PLC activity is most likely the result of decreased availability of phosphatidylinositol 4,5-bisphosphate (PIP2), the substrate of the PLC signal transduction pathway, rather than direct inhibition of the enzyme. PIP2 is a phospholipid with pleiotropic cellular functions, including a pivotal role in regulating cardiac phospholipase D (PLD) signal transduction. Here, we hypothesized that U73122 acts as an inhibitor of cardiac PLD activity by interference with PIP2. U73122 concentration-dependently inhibited PLD activity in rat myocardial membranes. The inhibitory effect of U73122 was significantly attenuated when assayed on solubilized PLD activity and was completely restored if solubilized PLD activity was assayed in the presence of PIP2. U73122 had no inhibitory effect on purified PLD indicating that the substance does not interact with PLD directly. These data highlight a mechanism of action of U73122 as an inhibitor of myocardial PLD by interaction with PIP2 as a cofactor for optimal PLD activity. Hence, studies using U73122 as a specific inhibitor of PLC have to take into account that PLD may be involved in some of the effects ascribed to PLC.

Activation of native TRPC1/C5/C6 channels by endothelin-1 is mediated by both PIP3 and PIP2 in rabbit coronary artery myocytes.

We investigate activation mechanisms of native TRPC1/C5/C6 channels (termed TRPC1 channels) by stimulation of endothelin-1 (ET-1) receptor subtypes in freshly dispersed rabbit coronary artery myocytes using single channel recording and immunoprecipitation techniques. ET-1 evoked non-selective cation channel currents with a unitary conductance of 2.6 pS which were not inhibited by either ET(A) or ET(B) receptor antagonists, respectively BQ-123 and BQ788, when administered separately. However, in the presence of both antagonists, ET-1-evoked channel activity was abolished indicating that both ET(A) and ET(B) receptor stimulation activate this conductance. Stimulation of both ET(A) and ET(B) receptors evoked channel activity which was inhibited by the protein kinase C (PKC) inhibitor chelerythrine and by anti-TRPC1 antibodies indicating that activation of both receptor subtypes causes TRPC1 channel activation by a PKC-dependent mechanism. ET(A) receptor-mediated TRPC1 channel activity was selectively inhibited by phosphoinositol-3-kinase (PI-3-kinase) inhibitors wortmannin (50 nM) and PI-828 and by antibodies raised against phosphoinositol-3,4,5-trisphosphate (PIP(3)), the product of PI-3-kinase-mediated phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Moreover, exogenous application of diC8-PIP(3) stimulated PKC-dependent TRPC1 channel activity. These results indicate that stimulation of ET(A) receptors evokes PKC-dependent TRPC1 channel activity through activation of PI-3-kinase and generation of PIP(3). In contrast, ET(B) receptor-mediated TRPC1 channel activity was inhibited by the PI-phospholipase C (PI-PLC) inhibitor U73122. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), an analogue of diacylglycerol (DAG), which is a product of PI-PLC, also activated PKC-dependent TRPC1 channel activity. OAG-induced TRPC1 channel activity was inhibited by anti-phosphoinositol-4,5-bisphosphate (PIP(2)) antibodies and high concentrations of wortmannin (20 microM) which depleted tissue PIP(2) levels. In addition exogenous application of diC8-PIP(2) activated PKC-dependent TRPC1 channel activity. These data indicate that stimulation of ET(B) receptors evokes PKC-dependent TRPC1 activity through PI-PLC-mediated generation of DAG and requires a permissive role of PIP(2). In conclusion, we provide the first evidence that stimulation of ET(A) and ET(B) receptors activate native PKC-dependent TRPC1 channels through two distinct phospholipids pathways involving a novel action of PIP(3), in addition to PIP(2), in rabbit coronary artery myocytes.

In vitro cytotoxicity of novel pro-apoptotic agent DM-PIT-1 in PEG-PE-based micelles alone and in combination with TRAIL.

The purpose of this study was to develope and characterize a micellar formulations of N-{[(2-hydroxy-5- nitrophenyl)amino]carbonothioyl}-3,5-dimethylbenzamide (DM-PIT-1)-a new small molecule non-lipid antagonist of phopshotidylinositol-3.4.5-triphopshate and inhibitor of the PI3-kinase pathway. Micelle-forming PEG(2000)-PE was used to solubilize DM-PIT-1. To improve the specificity of the micellar DM-PIT-1, cancer-targeting anti-nucleosomal mAb2C5 antibodies as well as Tumor necrosis factor- Related Apoptosis-Inducing Ligand (TRAIL) were attached to the surface of polymeric micelles. DM-PIT-1 was effectively incorporated (> 70%) into 14-16 nm micelles, which had a negative surface zeta potential of 4-5 mV. Micellar DM-PIT-1 demonstrated high in vitro cytotoxicity against various cancer cells. An improved potency of the dual-activity DM-PIT-1/TRAIL combination nanoparticles in inducing death of TRAIL-resistant cancer cells was shown. Efficacy of the TRAIL therapy was enhanced by combining it with the 2C5 antibody cancer-targeted micellar form of DM-PIT-1. In conclusion, DM-PIT-1 micellar preparations can be used for targeted combination therapy against TRAIL-resistant cancers.