Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival

The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.     

Phosphoinositide 3-Kinase Beta Protects Nuclear Envelope Integrity by Controlling RCC1 Localization and Ran Activity

The nuclear envelope (NE) forms a barrier between the nucleus and the cytosol that preserves genomic integrity. The nuclear lamina and nuclear pore complexes (NPCs) are NE components that regulate nuclear events through interaction with other proteins and DNA. Defects in the nuclear lamina are associated with the development of laminopathies. As cells depleted of phosphoinositide 3-kinase beta (PI3Kβ) showed an aberrant nuclear morphology, we studied the contribution of PI3Kβ to maintenance of NE integrity. pik3cb depletion reduced the nuclear membrane tension, triggered formation of areas of lipid bilayer/lamina discontinuity, and impaired NPC assembly. We show that one mechanism for PI3Kβ regulation of NE/NPC integrity is its association with RCC1 (regulator of chromosome condensation 1), the activator of nuclear Ran GTPase. PI3Kβ controls RCC1 binding to chromatin and, in turn, Ran activation. These findings suggest that PI3Kβ regulates the nuclear envelope through upstream regulation of RCC1 and Ran.     

Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

Class Ia phosphoinositide (PI) 3-kinase is a central component in growth factor signaling and is comprised of a p110 catalytic subunit and a regulatory subunit, the most common family of which is derived from the p85alpha gene (Pik3r1). Optimal signaling through the PI 3-kinase pathway depends on a critical molecular balance between the regulatory and catalytic subunits. In wild-type cells, the p85 subunit is more abundant than p110, leading to competition between the p85 monomer and the p85-p110 dimer and ineffective signaling. Heterozygous disruption of Pik3r1 results in increased Akt activity and decreased apoptosis by insulin-like growth factor 1 (IGF-1) through up-regulated phosphatidylinositol (3,4,5)-triphosphate production. Complete depletion of p85alpha, on the other hand, results in significantly increased apoptosis due to reduced PI 3-kinase-dependent signaling. Thus, a reduction in p85alpha represents a novel therapeutic target for enhancing IGF-1/insulin signaling, prolongation of cell survival, and protection against apoptosis.     

A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

Background

The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions.

Methodology/Principal Findings

We show that FGF-2 activates PLCγ1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P₃ using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLCγ1 activation and the PI3K/PLCγ1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)α activation since dowregulation of PKCα expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKCα blocks FGF-2-dependent cell migration.

Conclusion/Significance

These data elucidate the role of PLCγ1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLCγ1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes.     

The PtdIns 3-Kinase/Akt Pathway Regulates Macrophage-Mediated ADCC against B Cell Lymphoma

Macrophages are important effectors in the clearance of antibody-coated tumor cells. However, the signaling pathways that regulate macrophage-induced ADCC are poorly defined. To understand the regulation of macrophage-mediated ADCC, we used human B cell lymphoma coated with Rituximab as the tumor target and murine macrophages primed with IFNγ as the effectors. Our data demonstrate that the PtdIns 3-kinase/Akt pathway is activated during macrophage-induced ADCC and that the inhibition of PtdIns 3-kinase results in the inhibition of macrophage-mediated cytotoxicity. Interestingly, downstream of PtdIns 3-kinase, expression of constitutively active Akt (Myr-Akt) in macrophages significantly enhanced their ability to mediate ADCC. Further analysis revealed that in this model, macrophage-mediated ADCC is dependent upon the release of nitric oxide (NO). However, the PtdIns 3-kinase/Akt pathway does not appear to regulate NO production. An examination of the role of the PtdIns 3-kinase/Akt pathway in regulating conjugate formation indicated that macrophages treated with an inhibitor of PtdIns 3-kinase fail to polarize the cytoskeleton at the synapse and show a significant reduction in the number of conjugates formed with tumor targets. Further, inhibition of PtdIns 3-kinase also reduced macrophage spreading on Rituximab-coated surfaces. On the other hand, Myr-Akt expressing macrophages displayed a significantly greater ability to form conjugates with tumor cells. Taken together, these findings illustrate that the PtdIns 3-kinase/Akt pathway plays a critical role in macrophage ADCC through its influence on conjugate formation between macrophages and antibody-coated tumor cells.     

Phosphoinositide 3-Kinase C2�� Regulates RhoA and the Actin Cytoskeleton through an Interaction with Dbl

The regulation of cell morphology is a dynamic process under the control of multiple protein complexes acting in a coordinated manner. Phosphoinositide 3-kinases (PI3K) and their lipid products are widely involved in cytoskeletal regulation by interacting with proteins regulating RhoGTPases. Class II PI3K isoforms have been implicated in the regulation of the actin cytoskeleton, although their exact role and mechanism of action remain to be established. In this report, we have identified Dbl, a Rho family guanine nucleotide exchange factor (RhoGEF) as an interaction partner of PI3KC2β. Dbl was co-immunoprecipitated with PI3KC2β in NIH3T3 cells and cancer cell lines. Over-expression of Class II phosphoinositide 3-kinase PI3KC2β in NIH3T3 fibroblasts led to increased stress fibres formation and cell spreading. Accordingly, we found high basal RhoA activity and increased serum response factor (SRF) activation downstream of RhoA upon serum stimulation. In contrast, the dominant-negative form of PI3KC2β strongly reduced cell spreading and stress fibres formation, as well as SRF response. Platelet-derived growth factor (PDGF) stimulation of wild-type PI3KC2β over-expressing NIH3T3 cells strongly increased Rac and c-Jun N-terminal kinase (JNK) activation, but failed to show similar effect in the cells with the dominant-negative enzyme. Interestingly, epidermal growth factor (EGF) and PDGF stimulation led to increased extracellular signal-regulated kinase (Erk) and Akt pathway activation in cells with elevated wild-type PI3KC2β expression. Furthermore, increased expression of PI3KC2β protected NIH3T3 from detachment-dependent death (anoikis) in a RhoA-dependent manner. Taken together, these findings suggest that PI3KC2β modulates the cell morphology and survival through a specific interaction with Dbl and the activation of RhoA.     

Gq-coupled Purinergic Receptors Inhibit Insulin-like Growth Factor-I/Phosphoinositide 3-Kinase Pathway-dependent Keratinocyte Migration

Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Gα_(q/11)-coupled-P2Y₂ purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y₂ receptors by extracellular UTP inhibits the IGF-I–induced p110α-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Gα_(q/11)—and not G_(i/o)—independently of phospholipase Cβ. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110α mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I–induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110α mutant, in a Gα(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Gα_(q/11)-coupled receptors, which mediate opposite effects on p110α-PI3K activity and keratinocyte migration.     

Ras and Phosphoinositide 3-Kinase: Partners in Development and Tumorigenesis

Much progress has been made in understanding the myriad of intracellular signalling pathways responsible for control of cell physiology. Signalling downstream of receptor tyrosine kinases (RTKs) is probably the most studied signalling mechanism to date and many of the molecular components and corresponding interactions involved have been delineated. Importantly, deregulation of RTK signalling has been implicated in the formation and maintenance of many human tumours. Two of the pivotal molecular components in RTK signalling, Ras and phosphoinositide 3-kinase (PI 3-kinase), have been shown to bind to each other, leading to the activation of PI 3-kinase. However, in addition to this Ras - PI 3-kinase interaction, first described over a decade ago, several other molecular interactions have more recently been described that appear to mediate the same signal. This has brought into question the physiological relevance of the Ras – PI 3-kinase interaction during RTK signalling. Through disruption of the interaction in a mouse model, we have now confirmed that the interaction is highly functional in vivo both during mammalian development and during Ras-induced tumorigenesis. Many questions still remain: in this Perspective, we explore the remaining uncertainties surrounding the role of this signalling mechanism, as well as the future directions that will likely shed further light on its role within cells.     

Glucose Regulates Rat Beta Cell Number through Age-Dependent Effects on Beta Cell Survival and Proliferation

Background

Glucose effects on beta cell survival and DNA-synthesis suggest a role as regulator of beta cell mass but data on beta cell numbers are lacking. We examined outcome of these influences on the number of beta cells isolated at different growth stages in their population.

Methods

Beta cells from neonatal, young-adult and old rats were cultured serum-free for 15 days. Their number was counted by automated whole-well imaging distinguishing influences on cell survival and on proliferative activity.

Results

Elevated glucose (10–20 versus 5 mmol/l) increased the number of living beta cells from 8-week rats to 30%, following a time- and concentration-dependent recruitment of quiescent cells into DNA-synthesis; a glucokinase-activator lowered the threshold but did not raise total numbers of glucose-recruitable cells. No glucose-induced increase occurred in beta cells from 40-week rats. Neonatal beta cells doubled in number at 5 mmol/l involving a larger activated fraction that did not increase at higher concentrations; however, their higher susceptibility to glucose toxicity at 20 mmol/l resulted in 20% lower living cell numbers than at start. None of the age groups exhibited a repetitively proliferating subpopulation.

Conclusions

Chronically elevated glucose levels increased the number of beta cells from young-adult but not from old rats; they interfered with expansion of neonatal beta cells and reduced their number. These effects are attributed to age-dependent differences in basal and glucose-induced proliferative activity and in cellular susceptibility to glucose toxicity. They also reflect age-dependent variations in the functional heterogeneity of the rat beta cell population.     

Selective Inhibition of Phosphoinositide 3-Kinase p110α Preserves Lymphocyte Function

Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.     

srGAP2 Arginine Methylation Regulates Cell Migration and Cell Spreading through Promoting Dimerization

The Slit-Robo GTPase-activating proteins (srGAPs) are critical for neuronal migration through inactivation of Rho GTPases Cdc42, Rac1, and RhoA. Here we report that srGAP2 physically interacts with protein arginine methyltransferase 5 (PRMT5). srGAP2 localizes to the cytoplasm and plasma membrane protrusion. srGAP2 knockdown reduces cell adhesion spreading and increases cell migration, but has no effect on cell proliferation. PRMT5 binds to the N terminus of srGAP2 (225–538 aa) and methylates its C-terminal arginine residue Arg-927. The methylation mutant srGAP2-R927A fails to rescue the cell spreading rate, is unable to localize to the plasma membrane leading edge, and perturbs srGAP2 homodimer formation mediated by the F-BAR domain. These results suggest that srGAP2 arginine methylation plays important roles in cell spreading and cell migration through influencing membrane protrusion.     

Identification of Novel Piperazinylquinoxaline Derivatives as Potent Phosphoinositide 3-Kinase (PI3K) Inhibitors

Background

Development of small-molecule inhibitors targeting phosphoinositide 3-kinase (PI3K) has been an appealing strategy for the treatment of various types of cancers.

Methodology/Principal Finding

Our approach was to perform structural modification and optimization based on previously identified morpholinoquinoxaline derivative WR1 and piperidinylquinoxaline derivative WR23 with a total of forty-five novel piperazinylquinoxaline derivatives synthesized. Most target compounds showed low micromolar to nanomolar antiproliferative potency against five human cancer cell lines using MTT method. Selected compounds showed potent PI3Kα inhibitory activity in a competitive fluorescent polarization assay, such as compound 22 (IC₅₀ 40 nM) and 41 (IC₅₀: 24 nM), which induced apoptosis in PC3 cells. Molecular docking analysis was performed to explore possible binding modes between target compounds and PI3K.

Conclusions/Significance

The identified novel piperazinylquinoxaline derivatives that showed potent PI3Kα inhibitory activity and cellular antiproliferative potency may be promising agents for potential applications in cancer treatment.     

Control of Cerebellar Long-Term Potentiation by P-Rex-Family Guanine-Nucleotide Exchange Factors and Phosphoinositide 3-Kinase

Background

Long-term potentiation (LTP) at the parallel fibre–Purkinje cell synapse in the cerebellum is a recently described and poorly characterized form of synaptic plasticity. The induction mechanism for LTP at this synapse is considered reciprocal to “classical” LTP at hippocampal CA1 pyramidal neurons: kinases promote increased trafficking of AMPA receptors into the postsynaptic density in the hippocampus, whereas phosphatases decrease internalization of AMPA receptors in the cerebellum. In the hippocampus, LTP occurs in overlapping phases, with the transition from early to late phases requiring the consolidation of initial induction processes by structural re-arrangements at the synapse. Many signalling pathways have been implicated in this process, including PI3 kinases and Rho GTPases.

Principal Findings

We hypothesized that analogous phases are present in cerebellar LTP, and took as the starting point for investigation our recent discovery that P-Rex – a Rac guanine nucleotide exchange factor which is activated by PtdIns(3,4,5)P₃ – is highly expressed in mouse cerebellar Purkinje neurons and plays a role in motor coordination. We found that LTP evoked at parallel fibre synapses by 1 Hz stimulation or by NO donors was not sustained beyond 30 min when P-Rex was eliminated or Rac inhibited, suggesting that cerebellar LTP exhibits a late phase analogous to hippocampal LTP. In contrast, inhibition of PI3 kinase activity eliminated LTP at the induction stage.

Conclusions

Our data suggest that a PI3K/P-Rex/Rac pathway is required for late phase LTP in the mouse cerebellum, and that other PI3K targets, which remain to be discovered, control LTP induction.     

Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication

Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.Hepatitis C virus (HCV) causes liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (52). The HCV genome is a single-stranded RNA molecule where both the 5′ and the 3′ untranslated region (UTR) contain highly conserved RNA structures necessary for polyprotein translation and genome replication (43). The processed polyprotein yields at least three structural proteins and six nonstructural proteins. The structural proteins include the core, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The viral proteins processed by signal peptidases form viral particles that assemble at the endoplasmic reticulum (ER) and/or Golgi bodies and are released from the host cell by viral budding. The structural protein coding regions are separated from nonstructural proteins by the short membrane peptide p7, thought to function as an ion channel (43, 53). The nonstructural proteins NS2, NS3/4A, NS5A, and NS5B are involved in coordinating the intracellular processes of the virus life cycle, including polyprotein processing and viral RNA replication (34).The Luc-1b cell is a human hepatoma cell line (Huh7) that contains a genotype 1b HCV subgenomic replicon, a luciferase reporter, and a neomycin selection marker, allowing HCV replication to be studied both in vitro and in vivo (8, 36). This subgenomic replicon lacks the coding regions for NS2 and the structural proteins but contains the nonstructural proteins in cis, which are required for replication of the viral RNA. Expression of the luciferase gene acts as a surrogate marker for levels of HCV RNA produced in the cell. The goal of the present study was to use this subgenomic HCV replicon to screen siRNA libraries and identify novel host proteins that are involved in HCV replication.A number of cellular pathways and proteins that play critical roles in HCV replication have recently been described (41, 42, 46). In particular, replication of HCV is tied closely to its localization and transport to various internal membranes and to lipid metabolism (2). Most of the HCV proteins appear to be targeted to the surface of the ER and replication complexes appear to be transported to lipid rafts, where RNA replication can occur (2). Infectious virus particle formation occurs in association with lipid droplets, and this process requires the core and NS5A proteins. In addition, cholesterol pathway production of geranylgeranyl-PP is important to geranylate the FBL2 protein, which serves as a membrane anchor for NS5A (62). The hVAP proteins involved in the localization and trafficking between internal membranous structures are known to be associated with the HCV proteins NS5A and NS5B (59). Thus, host factor lipid metabolism and intracellular protein transport are necessary for HCV replication in cells.Targeting host factors that are required for viral replication offers a strategy to overcome viral resistance and may allow treatment for more than one genotype of HCV and/or a related Flaviviridae virus such as Dengue, West Nile, or yellow fever virus. The current standard-of-care treatment for the genotype 1 strain of HCV infection is pegylated interferon alpha plus ribavirin over a 6-month time course with more than half of infected patients being refractory to this treatment (57). In addition to genotype 1, there are at least five naturally occurring genotype variants of HCV that can complicate a patient''s response to therapy when infected with more than one genotype. As well as the development of mutations, the presence of multiple variants coexisting in patients is thought to contribute to the rapid development of resistance (40). A variety of antiviral therapeutic strategies aim to inhibit viral proteins directly with small molecules or siRNAs (13, 31, 33). Although some small molecule approaches have been successful in preclinical studies, small-molecule strategies directed against the viral targets can still be rendered ineffective due to the development of mutant, treatment-resistant viral strains (13, 40). Thus, combination therapies are a necessary approach to treat the many variants of HCV that exist in the patient population.In the present study, a set of 779 SMARTpool small interfering RNAs (siRNAs) targeting the kinome and 4 siRNAs targeting 5,000 druggable genes (20,000 siRNAs) were tested for their ability to block replication of the Luc-1b HCV subgenomic replicon. siRNAs targeting CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a tripartite enzyme that catalyzes the first three steps of pyrimidine biosynthesis, inhibited both the Luc-1b replicon and JFH1-2a virus expression. This activity is consistent with the known inhibitor of this enzyme, leflunomide, which has been shown previously to inhibit both respiratory syncytial virus and HCV (12, 54). siRNAs targeting the mevalonate (diphospho) decarboxylase (MVD) enzyme, which catalyzes the formation of mevalonate, were found to inhibit Luc-1b replication (19). Inhibition of the cholesterol biosynthesis pathway and host cell geranylation has been previously reported to inhibit HCV subgenomic replication (3, 24, 51, 62, 67). siRNA-mediated knockdown of the class III phosphatidylinositol 4-kinases PI4KA and PI4KB inhibited luciferase expression not only for the genotype 1b subgenomic replicons (Luc-1a and Luc-1b) but also for the viral RNA levels of SG-1b, Luc-1b, and Luc-1a. PI4KA knockdown also inhibited Renilla expression in the JFH-1 genotype 2a infectious virus (JFH1-2a), genotype 2a subgenomic replicon (SG-1a), and a genomic and subgenomic genotype 1a replicon (FL-1a and SG-1a). Using the small-molecule inhibitor PIK93 in compound affinity competition experiments and in vitro biochemical assays, we demonstrated PIK93 could bind and inhibit both PI4KA and PI4KB enzymatic activity (58). PIK93 could inhibit luciferase expression in the Luc-1b, Luc-1a, and JFH1-2a infectious virus assays in the submicromolar range. Together, our data suggest that PI4KA and PI4KB regulate HCV replication and that pharmacological inhibition of these enzymes represents a new class of antiviral agents for multiple genotypes of HCV. Finally, since PI4KA and PI4KB are known to regulate protein and lipid transport to and from the ER and Golgi bodies, their function may hold clues as to how movement of HCV replication complexes throughout different organelles is regulated.     

Tetratricopeptide Repeat Domain 9A Negatively Regulates Estrogen Receptor Alpha Activity

Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.