Potassium Channel Silencing by Constitutive Endocytosis and Intracellular Sequestration

Tandem of P domains in a weak inwardly rectifying K⁺ channel 1 (TWIK1) is a K⁺ channel that produces unusually low levels of current. Replacement of lysine 274 by a glutamic acid (K274E) is associated with stronger currents. This mutation would prevent conjugation of a small ubiquitin modifier peptide to Lys-274, a mechanism proposed to be responsible for channel silencing. However, we found no biochemical evidence of TWIK1 sumoylation, and we showed that the conservative change K274R did not increase current, suggesting that K274E modifies TWIK1 gating through a charge effect. Now we rule out an eventual effect of K274E on TWIK1 trafficking, and we provide convincing evidence that TWIK1 silencing results from its rapid retrieval from the cell surface. TWIK1 is internalized via a dynamin-dependent mechanism and addressed to the recycling endosomal compartment. Mutation of a diisoleucine repeat located in its cytoplasmic C terminus (I293A,I294A) stabilizes TWIK1 at the plasma membrane, resulting in robust currents. The effects of I293A,I294A on channel trafficking and of K274E on channel activity are cumulative, promoting even more currents. Activation of serotoninergic receptor 5-HT₁R or adrenoreceptor α2A-AR stimulates TWIK1 but has no effect on TWIK1I293A,I294A, suggesting that G_i protein activation is a physiological signal for increasing the number of active channels at the plasma membrane.     

Nuclear Factor (NF)-��B-dependent Thyroid Hormone Receptor ��1 Expression Controls Dendritic Cell Function via Akt Signaling

Despite considerable progress in our understanding of the interplay between immune and endocrine systems, the role of thyroid hormones and their receptors in the control of adaptive immunity is still uncertain. Here, we investigated the role of thyroid hormone receptor (TR) β¹ signaling in modulating dendritic cell (DC) physiology and the intracellular mechanisms underlying these immunoregulatory effects. Exposure of DCs to triiodothyronine (T³) resulted in a rapid and sustained increase in Akt phosphorylation independently of phosphatidylinositol 3-kinase activation, which was essential for supporting T³-induced DC maturation and interleukin (IL)-12 production. This effect was dependent on intact TRβ¹ signaling as small interfering RNA-mediated silencing of TRβ¹ expression prevented T³-induced DC maturation and IL-12 secretion as well as Akt activation and IκB-ϵ degradation. In turn, T³ up-regulated TRβ¹ expression through mechanisms involving NF-κB, suggesting an autocrine regulatory loop to control hormone-dependent TRβ¹ signaling. These findings were confirmed by chromatin immunoprecipitation analysis, which disclosed a new functional NF-κB consensus site in the promoter region of the TRB1 gene. Thus, a T³-induced NF-κB-dependent mechanism controls TRβ¹ expression, which in turn signals DCs to promote maturation and function via an Akt-dependent but PI3K-independent pathway. These results underscore a novel unrecognized target that regulates DC maturation and function with critical implications in immunopathology at the cross-roads of the immune-endocrine circuits.     

Down-regulation of S1P1 Receptor Surface Expression by Protein Kinase C Inhibition

The sphingosine 1-phosphate receptor type 1 (S1P₁) is important for the maintenance of lymphocyte circulation. S1P₁ receptor surface expression on lymphocytes is critical for their egress from thymus and lymph nodes. Premature activation-induced internalization of the S1P₁ receptor in lymphoid organs, mediated either by pharmacological agonists or by inhibition of the S1P degrading enzyme S1P-lyase, blocks lymphocyte egress and induces lymphopenia in blood and lymph. Regulation of S1P₁ receptor surface expression is therefore a promising way to control adaptive immunity. Hence, we analyzed potential cellular targets for their ability to alter S1P₁ receptor surface expression without stimulation. The initial observation that preincubation of mouse splenocytes with its natural analog sphingosine was sufficient to block Transwell^TM chemotaxis to S1P directed subsequent investigations to the underlying mechanism. Sphingosine is known to inhibit protein kinase C (PKC), and PKC inhibition with nanomolar concentrations of staurosporine, calphostin C, and GF109203X down-regulated surface expression of S1P₁ but not S1P₄ in transfected rat hepatoma HTC₄ cells. The PKC activator phorbol 12-myristate 13-acetate partially rescued FTY720-induced down-regulation of the S1P₁ receptor, linking PKC activation with S1P₁ receptor surface expression. FTY720, but not FTY720 phosphate, efficiently inhibited PKC. Cell-based efficacy was obvious with 10 nm FTY720, and in vivo treatment of mice with 0.3–3 mg/kg/day FTY720 showed increasing concentration-dependent effectiveness. PKC inhibition therefore may contribute to lymphopenia by down-regulating S1P₁ receptor cell surface expression independently from its activation.     

Zinc-induced Neurotoxicity Mediated by Transient Receptor Potential Melastatin 7 Channels

Transient receptor potential melastatin 7 (TRPM7) channels are novel Ca²⁺-permeable non-selective cation channels ubiquitously expressed. Activation of TRPM7 channels has been shown to be involved in cellular Mg²⁺ homeostasis, diseases caused by abnormal magnesium absorption, and in Ca²⁺-mediated neuronal injury under ischemic conditions. Here we show strong evidence suggesting that TRPM7 channels also play an important role in cellular Zn²⁺ homeostasis and in Zn²⁺-mediated neuronal injury. Using a combination of fluorescent Zn²⁺ imaging, small interfering RNA, pharmacological analysis, and cell injury assays, we show that activation of TRPM7 channels augmented Zn²⁺-induced injury of cultured mouse cortical neurons. The Zn²⁺-mediated neurotoxicity was inhibited by nonspecific TRPM7 blockers Gd³⁺ or 2-aminoethoxydiphenyl borate, and by knockdown of TRPM7 channels with small interfering RNA. In addition, Zn²⁺-mediated neuronal injury under oxygen-glucose deprivation conditions was also diminished by silencing TRPM7. Furthermore, we show that overexpression of TRPM7 channels in HEK293 cells increased intracellular Zn²⁺ accumulation and Zn²⁺-induced cell injury, while silencing TRPM7 by small interfering RNA attenuated the Zn²⁺-mediated cell toxicity. Thus, TRPM7 channels may represent a novel target for neurological disorders where Zn²⁺ toxicity plays an important role.     

Differential Functions of ApoER2 and Very Low Density Lipoprotein Receptor in Reelin Signaling Depend on Differential Sorting of the Receptors

ApoER2 and very low density lipoprotein (VLDL) receptor transmit the Reelin signal into target cells of the central nervous system. To a certain extent, both receptors can compensate for each other, and only the loss of both receptors results in the reeler phenotype, which is characterized by a gross defect in the architecture of laminated brain structures. Nevertheless, both receptors also have specific distinct functions, as corroborated by analyses of the subtle phenotypes displayed in mice lacking either ApoER2 or VLDL receptor. The differences in their function(s), however, have not been defined at the cellular level. Here, using a panel of chimeric receptors, we demonstrate that endocytosis of Reelin and the fate of the individual receptors upon stimulation are linked to their specific sorting to raft versus non-raft domains of the plasma membrane. VLDL receptor residing in the non-raft domain endocytoses and destines Reelin for degradation via the clathrin-coated pit/clathrin-coated vesicle/endosome pathway without being degraded to a significant extent. Binding of Reelin to ApoER2, a resident of rafts, leads to the production of specific receptor fragments with specific functions of their own and to degradation of ApoER2 via lysosomes. These features contribute to a receptor-specific fine tuning of the Reelin signal, leading to a novel model that emphasizes negative feedback loops specifically mediated by ApoER2 and VLDL receptor, respectively.     

Soluble Nogo Receptor Down-regulates Expression of Neuronal Nogo-A to Enhance Axonal Regeneration

Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury.     

Nuclear Receptor Liver X Receptor Is O-GlcNAc-modified in Response to Glucose

Post-translational modification of nucleocytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has for the last 25 years emerged as an essential glucose-sensing mechanism. The liver X receptors (LXRs) function as nutritional sensors for cholesterol-regulating lipid metabolism, glucose homeostasis, and inflammation. LXRs are shown to be post-translationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively. In the present study, we show for the first time that LXRα and LXRβ are targets for glucose-hexosamine-derived O-GlcNAc modification in human Huh7 cells. Furthermore, we observed increased hepatic LXRα O-GlcNAcylation in vivo in refed mice and in streptozotocin-induced refed diabetic mice. Importantly, induction of LXRα O-GlcNAcylation in both mouse models was concomitant with increased expression of the lipogenic gene SREBP-1c (sterol regulatory element-binding protein 1c). Furthermore, glucose increased LXR/retinoic acid receptor-dependent activation of luciferase reporter activity driven by the mouse SREBP-1c promoter via the hexosamine biosynthetic pathway in Huh7 cells. Altogether, our results suggest that O-GlcNAcylation of LXR is a novel mechanism by which LXR acts as a glucose sensor affecting LXR-dependent gene expression, substantiating the crucial role of LXR as a nutritional sensor in lipid and glucose metabolism.     

Cyclin D1 Is a Bona Fide Target Gene of NFATc1 and Is Sufficient in the Mediation of Injury-induced Vascular Wall Remodeling

Platelet-derived growth factor BB induced cyclin D1 expression in a time- and nuclear factor of activated T cells (NFAT)-dependent manner in human aortic smooth muscle cells (HASMCs), and blockade of NFATs prevented HASMC DNA synthesis and their cell cycle progression from G₁ to S phase. Selective inhibition of NFATc1 by its small interfering RNA also blocked HASMC proliferation and migration. Characterization of the cyclin D1 promoter revealed the presence of several NFAT binding sites, and the site at nucleotide −1333 was found to be sufficient in mediating platelet-derived growth factor BB-induced cyclin D1 promoter-luciferase reporter gene activity. In addition to its role in cell cycle progression, cyclin D1 mediated HASMC migration in an NFATc1-dependent manner. Balloon injury-induced cyclin D1-CDK4 activity requires NFAT activation, and adenovirus-mediated transduction of cyclin D1 was found to be sufficient to overcome the blockade effect of NFATs by VIVIT on balloon injury-induced vascular wall remodeling events, including smooth muscle cell migration from the medial to luminal region, their proliferation in the intimal region, and neointima formation. Together, these results provide more mechanistic evidence for the role of NFATs, particularly NFATc1, in the regulation of HASMC proliferation and migration as well as vascular wall remodeling. NFATc1 could be a potential therapeutic target against the renarrowing of artery after angioplasty.     

Involvement of the Calcium-sensing Receptor in Human Taste Perception

By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as “kokumi taste” and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist γ-glutamyl peptides, including GSH (γ-Glu-Cys-Gly) and γ-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca²⁺, protamine, polylysine, l-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception.     

Inositol 1,4,5-Trisphosphate 3-Kinase-A Is a New Cell Motility-promoting Protein That Increases the Metastatic Potential of Tumor Cells by Two Functional Activities

Cellular migration is an essential prerequisite for metastatic dissemination of cancer cells. This study demonstrates that the neuron/testis-specific F-actin-targeted inositol 1,4,5-trisphosphate 3-kinase-A (ITPKA) is ectopically expressed in different human tumor cell lines and during tumor progression in the metastatic tumor model Balb-neuT. High expression of ITPKA increases invasive migration in vitro and metastasis in a xenograft SCID mouse model. Mechanistic studies show that ITPKA promotes migration of tumor cells by two different mechanisms as follows: growth factor independently high levels of ITPKA induce the formation of large cellular protrusions by directly modulating the actin cytoskeleton. The F-actin binding activity of ITPKA stabilizes and bundles actin filaments and thus increases the levels of cellular F-actin. In growth factor-stimulated cells, the catalytically active domain enhances basal ITPKA-induced migration by activating store-operated calcium entry through production of inositol 1,3,4,5-tetrakisphosphate and subsequent inhibition of inositol phosphate 5-phosphatase. These two functional activities of ITPKA stimulating tumor cell migration place the enzyme among the potential targets of anti-metastatic therapy.     

Structure of a Ca2+-myristoyl switch protein that controls activation of a phosphatidylinositol 4-kinase in fission yeast

Neuronal calcium sensor (NCS) proteins transduce Ca2+ signals and are highly conserved from yeast to humans. We determined NMR structures of the NCS-1 homolog from fission yeast (Ncs1), which activates a phosphatidylinositol 4-kinase. Ncs1 contains an α-NH2-linked myristoyl group on a long N-terminal arm and four EF-hand motifs, three of which bind Ca2+, assembled into a compact structure. In Ca2+-free Ncs1, the N-terminal arm positions the fatty acyl chain inside a cavity near the C terminus. The C14 end of the myristate is surrounded by residues in the protein core, whereas its amide-linked (C1) end is flanked by residues at the protein surface. In Ca2+-bound Ncs1, the myristoyl group is extruded (Ca2+-myristoyl switch), exposing a prominent patch of hydrophobic residues that specifically contact phosphatidylinositol 4-kinase. The location of the buried myristate and structure of Ca2+-free Ncs1 are quite different from those in other NCS proteins. Thus, a unique remodeling of each NCS protein by its myristoyl group, and Ca2+-dependent unmasking of different residues, may explain how each family member recognizes distinct target proteins.     

Inhibition of Protein Kinase Akt1 by Apoptosis Signal-regulating Kinase-1 (ASK1) Is Involved in Apoptotic Inhibition of Regulatory Volume Increase

Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-α, or a Fas ligand inhibited both RVI and hypertonicity-induced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1.     

UTP Controls Cell Surface Distribution and Vasomotor Activity of the Human P2Y2 Receptor through an Epidermal Growth Factor Receptor-transregulated Mechanism

Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y₂ receptor (P2Y₂R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-β-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1–10 μm UTP, a selective P2Y₂R agonist, displaced the P2Y₂R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y₂R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y₂R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y₂R in intact human blood vessels.     

Coupling of the Cell Cycle and Apoptotic Machineries in Developing T Cells

Proliferation and apoptosis are diametrically opposite processes. Expression of certain genes like c-Myc, however, can induce both, pointing to a possible linkage between them. Developing CD4⁺CD8⁺ thymocytes are intrinsically sensitive to apoptosis, but the molecular basis is not known. We have found that these noncycling cells surprisingly express many cell cycle proteins. We generated transgenic mice expressing a CDK2 kinase-dead (CDK2-DN) protein in the T cell compartment. Analysis of these mice showed that the CDK2-DN protein acts as a dominant negative mutant in mature T cells as expected, but surprisingly, it acts as a dominant active protein in CD4⁺CD8⁺ thymocytes. The levels of CDK2 kinase activity, cyclin E, cyclin A, and other cell cycle proteins in transgenic CD4⁺CD8⁺ thymocytes are increased. Concurrently, caspase levels are elevated, and apoptosis is significantly enhanced in vitro and in vivo. E2F-1, the unique E2F member capable of inducing apoptosis when overexpressed, is specifically up-regulated in transgenic CD4⁺CD8⁺ thymocytes but not in other T cell populations. These results demonstrate that the cell cycle and apoptotic machineries are normally linked, and expression of cell cycle proteins in developing T cells contributes to their inherent 1sensitivity to apoptosis.     

The Anti-apoptotic Protein BCL2L1/Bcl-xL Is Neutralized by Pro-apoptotic PMAIP1/Noxa in Neuroblastoma, Thereby Determining Bortezomib Sensitivity Independent of Prosurvival MCL1 Expression

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.