Dioleoylphosphoethanolamine Retains Cell Surface GLUT4 by Inhibiting PKCα-Driven Internalization.

BACKGROUND/AIMS: Phosphatidylethanolamine, a component of the plasma membrane, regulates diverse cellular processes. The present study investigated the role of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in the trafficking of the glucose transporter GLUT4 and the glucose homeostasis. METHODS: Monitoring of GLUT4 trafficking, GLUT4 internalization assay, and glucose uptake assay were carried out using differentiated 3T3-L1-GLUT4myc adipocytes. Akt1/2 and PKC isozymes were knocked-down by transfecting each siRNA. Cell-free PKC assay and in situ PKCα assay with a FRET probe were carried out. Oral glucose tolerance test (OGTT) was performed using BKS.Cg-+Lepdb/+Lebdb/Jcl mice, an animal model of type 2 diabetes mellitus (DM). RESULTS: DOPE increased cell surface localization of the glucose transporter GLUT4 in differentiated 3T3-L1-GLUT4myc adipocytes, regardless of Akt activation. Likewise, PKCα deficiency increased cell surface localization of GLUT4, that occludes the effect of DOPE. DOPE clearly suppressed phorbol 12-myristate 13-acetate-induced PKCα activation in the cell-free and in situ PKC assay. DOPE and PKCα deficiency cancelled endocytic internalization of GLUT4 localized on the plasma membrane after insulin stimulation. DOPE significantly enhanced glucose uptake into cells. A similar effect was obtained by knocking-down PKCα, that occludes the effect of DOPE. In OGTT, oral administration with DOPE effectively restricted an increase in the blood glucose levels after glucose loading in type 2 DM model mice. CONCLUSION: The results of the present study show that DOPE retains cell surface GLUT4 by suppressing PKCα-driven endocytic internalization of GLUT4, to enhance glucose uptake into cells and restrict an increase in the blood glucose levels after glucose loading in type 2 DM.

PKCε Increases Extracellular Elastin and Fibulin-5/DANCE in Dermal Fibroblasts.

BACKGROUND/AIMS: In the earlier study, the selective PKCε activator DCP-LA increased elastic fibres in the dermis of HR-1 hairless mice. As a process of elastic fibre formation, tropoelastin, an elastin monomer, is secreted into the extracellular space. Secreted tropoelastin is delivered to the microfibrils by fibulin-5/developmental arteries and neural crest epidermal growth factor-like (DANCE) and undergoes self-association. Then, tropoelastin assembles around the microfibrils, growing into elastin and elastic fibres by lysyl oxidase (LOX)- or LOX-like (LOXL)-mediated cross-linking. The present study was conducted to understand the mechanism underlying DCP-LA-induced increase in elastin/elastic fibre. METHODS: Western blotting, immunocytochemistory, and real-time reverse transcription-polymerase chain reaction (RT-PCR) were carried out in cultured human dermal fibroblasts. PKCε, mammalian target of rapamycin complex (mTOR), and p70 S6 kinase (S6K) were knocked-down by transfecting each siRNA. RESULTS: DCP-LA increased elastin and fibulin-5/DANCE in a treatment time (6-24 h)- and a bell-shaped concentration (1 nM-1 µM)-dependent manner in the culture medium of human dermal fibroblasts. DCP-LA markedly increased elastic fibres in the extracellular space of cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was abolished by a PKC inhibitor or knocking-down PKCε. DCP-LA did not affect expression of mRNAs for tropoelastin and fiblin-5/DANCE in cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was not inhibited by the protein synthesis inhibitor cycloheximide or by knocking-down mTOR and S6K. DCP-LA never increased extracellular elastin in the presence of elastase, that breaks down elastin. An inhibitor of matrix metalloproteinase 9, that degrades multiple extracellular matrix components including elastin, had no effect on the basal levels and the DCP-LA-induced increase levels of extracellular elastin. CONCLUSION: The results of the present study indicate that PKCε, activated by DCP-LA, increases elastin and fibulin-5/DANCE in the extracellular space of cultured fibroblasts by the mechanism independent of transcriptional and translational modulation or inhibition of elastolysis.

Profilin facilitates PKCε activation by accelerating ATP supply.

Profilin catalyzes the exchange of actin-bound ADP to ATP. The present study investigated the role of profilin in PKCε activation. Profilin associated with PKCε in differentiated PC-12 cells under the basal conditions, which was inhibited by the PKC inhibitor GF109203X. The selective PKCε activator DCP-LA markedly increased the association, which was clearly prevented by GF109203X. The basal PKC activity in PC-12 cells was attenuated by knocking-down profilin, while the basal activities of PKA and CaMKII were not affected. DCP-LA enhanced the PKC activity to approximately 3.5 folds of the basal levels, and the effect was suppressed by knocking-down profilin. In the cell-free system, PKCε was not activated by profilin alone. DCP-LA activated PKCε in an ATP concentration (2-500 µM)-dependent manner, and addition of profilin shifted the ATP concentration/DCP-LA-induced PKCε activity relation curve to the left (the direction of lower ATP concentrations). Taken together, the results of the present study indicate that profilin binds to activated PKCε and facilitates PKCε activation by accelerating ATP supply to PKCε.

IL-33 Acts to Express Schaffer Collateral/CA1 LTP and Regulate Learning and Memory by Targeting MyD88.

Interleukin-33 (IL-33) is recognized to transmit a signal through a heterodimeric receptor complex ST2/interleukin-1 receptor accessory protein (IL-1RAcP) bearing activation of myeloid differentiation factor 88 (MyD88). High-frequency stimulation to the Schaffer collateral induced long-term potentiation (LTP) in the CA1 region of hippocampal slices from wild-type control mice. Schaffer collateral/CA1 LTP in IL-33-deficient mice was significantly suppressed, which was neutralized by application with IL-33. Similar suppression of the LTP was found with MyD88-deficient mice but not with ST2-deficient mice. In the water maze test, the acquisition latency in IL-33-deficient and MyD88-deficient mice was significantly prolonged as compared with that in wild-type control mice. Moreover, the retention latency in MyD88-deficient mice was markedly prolonged. In contrast, the acquisition and retention latencies in ST2-deficient mice were not affected. Taken together, these results show that IL-33 acts to express Schaffer collateral/CA1 LTP relevant to spatial learning and memory in a MyD88-dependent manner and that the LTP might be expressed through an IL-1R1/IL-1RAcP-MyD88 pathway in the absence of ST2.

N-Ethylmaleimide Dissociates α7 ACh Receptor from a Complex with NSF and Promotes Its Delivery to the Presynaptic Membrane.

N-Ethylmaleimide (NEM)-sensitive factor (NSF) associates with soluble NSF attachment protein (SNAP), that binds to SNAP receptors (SNAREs) including syntaxin, SNAP25, and synaptobrevin. The complex of NSF/SNAP/SNAREs plays a critical role in the regulation of vesicular traffic. The present study investigated NEM-regulated α7 ACh receptor translocation. NSF associated with ß-SNAP and the SNAREs syntaxin 1 and synaptobrevin 2 in the rat hippocampus. NSF also associated with the α7 ACh receptor subunit, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, and the γ-aminobutyric acid A (GABAA) receptor γ2 subunit. NEM, an inhibitor of NSF, significantly dissociated the α7 ACh receptor subunit from a complex with NSF and increased cell surface localization of the receptor subunit, but such effect was not obtained with the GluA1, GluA2 or γ2 subunits. NEM, alternatively, dissociated synaptobrevin 2 from an assembly of NSF/ß-SNAP/syntaxin 1/synaptobrevin 2. NEM significantly increased the rate of nicotine-triggered AMPA receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, in rat hippocampal slices. The results of the present study indicate that NEM releases the α7 ACh receptor subunit and synaptobrevin 2 from an assembly of α7 ACh receptor subunit/NSF/ß-SNAP/syntaxin 1/synaptobrevin 2, thereby promoting delivery of the α7 ACh receptor subunit to presynaptic membrane.

DCP-LA Exerts an Antiaging Action on the Skin.

The present study assessed the possibility for the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) as an antiaging compound for the skin by assaying senescence-associated ß-galactosidase (SA-ß-Gal), a biomarker of senescence and cell viability. The nitric oxide (NO) donor sodium nitroprusside (SNP) increased in SA-ß-Gal-positive cells in cultured human fibroblasts and mouse keratinocytes, and DCP-LA significantly inhibited the effect of SNP. Moreover, SNP induced cell death in cultured mouse keratinocytes, and DCP-LA significantly prevented NO stress-induced death of keratinocytes. Taken together, these results indicate that DCP-LA exerts an antiaging action on the skin.

Phosphatidylinositol Induces Caspase-Independent Apoptosis of Malignant Pleural Mesothelioma Cells by Accumulating AIF in the Nucleus.

BACKGROUND/AIMS: Phosphatidylinositol (PI) regulates a variety of cell processes. The present study investigated the antitumor action of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol)(DOPI) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo-inositol)(DPPI) on human malignant pleural mesothelioma (MPM) cell lines such as NCI-H28, NCI-H2052, NCI-H2452, and MSTO-211H cells. METHODS: MTT assay, TUNEL staining, flow cytometry using propidium iodide (PI) and annexin V (AV), enzymatic caspase assay, and nuclear staining using DAPI were carried out, and mitochondrial membrane potentials and intracellular distribution of apoptosis-inducing factor (AIF) were monitored in cells with and without the siRNA silencing the Bid-targeted gene. RESULTS: Both DOPI and DPPI reduced cell viability for all the investigated MPM cell lines in a concentration (0.01-100 µM)-dependent manner. DOPI and DPPI significantly increased TUNEL-positive cells and the population of PI-negative/AV-positive and PI-positive/AV-positive cells, corresponding to early apoptosis and late apoptosis/secondary necrosis, respectively. DOPI and DPPI perturbed mitochondrial membrane potentials in MSTO-211H cells, but no significant activation of caspase-3, -4, -8, and -9 was obtained. DOPI and DPPI upregulated expression of Bid in MSTO-211H cells. DOPI and DPPI significantly increased nuclear localization of AIF without affecting expression of the mRNAs and proteins in MSTO-211H cells, which was inhibited by knocking-down Bid. In the DAPI staining, nuclear fragmentation and condensation were found. CONCLUSION: The results of the present study indicate that DOPI and DPPI facilitate Bid-mediated AIF release from the mitochondria, to accumulate AIF in the nucleus and induce caspase-independent apoptosis of MPM cells.

DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain.

BACKGROUND/AIMS: The linoleic acid derivative DCP-LA selectively and directly activates PKCε. The present study aimed at understanding the mechanism of DCP-LA-induced PKCε activation. METHODS: Point mutation in the C2-like domain on PKCε was carried out, and each kinase activity was monitored in PC-12 cells using a föerster resonance energy transfer (FRET) probe with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) at the N- and C-terminal ends of PKCε, respectively, or in the cell-free systems using a reversed phase high-performance liquid chromatography (HPLC). Intracellular PKCε mobilization was monitored in PC-12 cells using mRuby-conjugated PKCε. DCP-LA binding to PKCε was assayed using a fluorescein conjugated to DCP-LA at the carboxyl-terminal end (Fluo-DCP). Uptake of DCP-LA into cells was measured in PC-12 ells. RESULTS: In the FRET analysis, DCP-LA decreased the ratio of YFP signal intensity/CFP signal intensity in PC-12 cells and in the cell-free kinase assay, DCP-LA increased area of phosphorylated PKC substrate peptide, indicating DCP-LA-induced PKCε activation. These effects were significantly suppressed by replacing Arg50 and Ile89 by Ala or Asn in the C2-like domain of PKCε. In the fluorescent cytochemistry, DCP-LA did not affect intracellular PKCε distribution. In the cell-free binding assay, Fluo-DCP, that had no effect on the potential for PKCε activation, bound to PKCε, and the binding was inhibited only by mutating Ile89. Extracellularly applied DCP-LA was taken up into cells in a concentration-dependent manner. Although no activation was obtained in the cell-free kinase assay, the broad PKC activator PMA activated PKCε in PC-12 cells in association with translocation towards the cell surface, which was inhibited by mutating I89A. CONCLUSION: Unlike PMA DCP-LA activates cytosolic PKCε by binding to the phosphatidylserine binding/associating sites Arg50 and Ile89, possibly at the carboxyl-terminal end and the cyclopropane rings, respectively.

Adenosine deaminase inhibitor EHNA exhibits a potent anticancer effect against malignant pleural mesothelioma.

BACKGROUND/AIMS: Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor and an effective therapy has been little provided as yet. The present study investigated the possibility for the adenosine deaminase (ADA) inhibitor EHNA as a target of MPM treatment. METHODS: MTT assay, TUNEL staining, monitoring of intracellular adenosine concentrations, and Western blotting were carried out in cultured human MPM cell lines without and with knocking-down ADA. The in vivo effect of EHNA was assessed in mice inoculated with NCI-H2052 MPM cells. RESULTS: EHNA induced apoptosis of human MPM cell lines in a concentration (0.01-1 mM)- and treatment time (24-48 h)-dependent manner, but such effect was not obtained with another ADA inhibitor pentostatin. EHNA increased intracellular adenosine concentrations in a treatment time (3-9 h)-dependent manner. EHNA-induced apoptosis of MPM cells was mimicked by knocking-down ADA, and the effect was neutralized by the adenosine kinase inhibitor ABT-702. EHNA clearly suppressed tumor growth in mice inoculated with NCI-H2052 MPM cells. CONCLUSION: The results of the present study show that EHNA induces apoptosis of MPM cells by increasing intracellular adenosine concentrations, to convert to AMP, and effectively prevents MPM cell proliferation. This suggests that EHNA may be useful for treatment of the tragic neoplasm MPM.

Amyloid-ß peptide increases cell surface localization of α7 ACh receptor to protect neurons from amyloid ß-induced damage.

Amyloid-ß peptide 1-42 (Aß1-42) reduced PC-12 cell viability in a concentration (1-10 µM)- and treatment time (48-72 h)-dependent manner. Nicotine prevented Aß1-42-induced PC-12 cell death, but conversely, the α7 ACh receptor antagonist α-bungarotoxin enhanced Aß1-42-induced cell toxicity. Extracellularly applied Aß1-42 significantly increased cell surface localization of α7 ACh receptor in PC-12 cells as compared with that for non-treated control cells. Cell surface localization of α7 ACh receptor in the brain of 5xFAD mouse, an animal model of Alzheimer's disease (AD), apparently increased in an age (1-12 months)-dependent manner in association with increased accumulation of Aß1-42 in the plasma membrane component. Taken together, these results indicate that Aß1-42 promotes translocation of α7 ACh receptor towards the cell surface and that α7 ACh receptor rescues neuronal cells from Aß1-42-induced damage.

Acute restraint stress impairs induction of long-term potentiation by activating GSK-3ß.

The present study investigated the effect of acute restraint stress on long-term potentiation (LTP) and the underlying mechanism. Induction of Schaffer collateral-CA1 LTP was suppressed in hippocampal slices from mice with 1-h restraint stress. Cell surface localization of the N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2 was not affected in the hippocampus from mice with 1-h restraint stress. Phosphorylation of Akt at Ser473, but not at Thr308, and phosphorylation of GSK-3ß at Ser9, but not at Tyr216, were significantly inhibited in the hippocampus with 1-h restraint stress. Taken together, the results of the present study show that acute restraint stress impairs induction of LTP by enhancing GSK-3ß activity following suppressed Akt activity, without affecting cell surface localization of the NMDA and AMPA receptor subunits.

Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway.

1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE) induces both necrosis/necroptosis and apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells. The present study was conducted to understand the mechanism for DAPE-induced apoptosis of NCI-H28 cells. DAPE induced caspase-independent apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells, and the effect of DAPE was prevented by antioxidants or an inhibitor of NADPH oxidase (NOX). DAPE generated reactive oxygen species (ROS) and inhibited activity of thioredoxin (Trx) reductase (TrxR). DAPE decreased an association of apoptosis signal-regulating kinase 1 (ASK1) with thioredoxin (Trx), thereby releasing ASK1. DAPE activated p38 mitogen-activated protein kinase (MAPK), which was inhibited by an antioxidant or knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was also prevented by knocking-down ASK1. Taken together, the results of the present study indicate that DAPE stimulates NOX-mediated ROS production and suppresses TrxR activity, resulting in the decrease of reduced Trx and the dissociation of ASK1 from a complex with Trx, allowing sequential activation of ASK1 and p38 MAPK, to induce apoptosis of NCI-H28 MPM cells.

Rac1 and ROCK are implicated in the cell surface delivery of GLUT4 under the control of the insulin signal mimetic diDCP-LA-PE.

The phosphatidylethanolamine derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidylethanolamine (diDCP-LA-PE) promoted GLUT4 translocation to the cell surface in differentiated 3T3-L1-GLUT4myc adipocytes through a pathway along a phosphatidylinositol 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, that mimics insulin signaling. Moreover, diDCP-LA-PE-induced GLUT4 translocation was suppressed by inhibitors of the Rho GTPase Rac1 and Rho-associated coiled-coil-containing protein kinase (ROCK) or knocking-down Rac1 and ROCK1. The results of the present study show that Rac1 and ROCK are critical for regulation of GLUT4 trafficking by diDCP-LA-PE as well as insulin.

Irinotecan induces cell cycle arrest, but not apoptosis or necrosis, in Caco-2 and CW2 colorectal cancer cell lines.

Irinotecan, a topoisomerase I inhibitor, is clinically used as an anticancer drug. The present study investigated the anticancer effect of irinotecan on p53-negative Caco-2 and p53-positive CW2 human colorectal cancer cell lines. Cell viability for both Caco-2 and CW2 cells was little affected by treatment with irinotecan at concentrations ranging from 0.3 to 30 µmol/l for 24-48 h. Irinotecan did not increase the number of TUNEL-positive cells and did not affect the population of propidium iodide (PI)-positive and annexin V-negative cells, corresponding to primary necrosis, or that of PI-positive and annexin-positive cells, corresponding to late apoptosis/secondary necrosis, in either of the two cell lines. In the cell cycle analysis, irinotecan significantly increased the proportions at the S and G2/M phases of cell cycling in parallel with a decreased population at the G1 phase in both cell lines. Irinotecan significantly inhibited tumor growth in mice inoculated with CW2 cells. Taken together, these results indicate that irinotecan induces cell cycle arrest, but not apoptosis or necrosis, both in Caco-2 and CW2 cells, leading to suppression of cell proliferation.

Newly synthesized anticancer drug HUHS1015 is effective on malignant pleural mesothelioma.

The newly synthesized naftopidil analogue HUHS1015 reduced cell viability in malignant pleural mesothelioma cell lines MSTO-211H, NCI-H28, NCI-H2052, and NCI-H2452, with the potential greater than that for the anticancer drugs paclitaxel or cisplatin at concentrations higher than 30 µM. HUHS1015 induced both necrosis and apoptosis of MSTO-211H and NCI-H2052 cells. HUHS1015 upregulated expression of mRNAs for Puma, Hrk, and Noxa in MSTO-211H and NCI-H2052 cells, suggesting HUHS1015-induced mitochondrial apoptosis. HUHS1015 clearly suppressed tumor growth in mice inoculated with NCI-H2052 cells. Taken together, the results of the present study indicate that HUHS1015 could be developed as an effective anticancer drug for treatment of malignant pleural mesothelioma.

Dipalmitoleoylphosphoethanolamine as a PP2A enhancer obstructs insulin signaling by promoting Ser/Thr dephosphorylation of Akt.

BACKGROUND/AIMS: The phospholipid phosphatidylethanolamine is implicated in the regulation of a variety of cellular processes. The present study investigated the effect of phosphatidylethanolamines such as 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE) on protein phosphatases, Akt1/2 activity, GLUT4 mobilizations, and glucose uptake into cells. METHODS: Activity of protein phosphatase 2A (PP2A) was assayed under the cell-free conditions, and Western blotting, intracellular GLUT4 trafficking, and glucose uptake into cells were monitored using differentiated 3T3-L1-GLUT4myc adipocytes. RESULTS: Of the investigated phosphatidylethanolamines, DLPE and DPPE significantly enhanced PP2A activity. DPPE inhibited insulin-induced phosphorylation of Akt1/2 at Thr308/309 and Ser473/474 in differentiated 3T3-L1-GLUT4myc adipocytes. DPPE also inhibited insulin-stimulated GLUT4 translocation to the cell surface and reduced insulin-stimulated glucose uptake into adipocytes. CONCLUSION: The results of the present study indicate that the PP2A enhancer DPPE obstructs insulin signaling by promoting serine/threonine dephosphorylation of Akt1/2, resulting in the suppression of GLUT4 translocation to the cell surface and glucose uptake into adipocytes.

DCP-LA-phosphatidylinositol and its enantiomer exhibit different bioactivities.

BACKGROUND/AIMS: The present study was conducted to understand biochemical and biological characteristics of the phosphatidylinositol (PI) derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-Sn-glycero-3-phosphatidyl-D-1-inositol (diDCP-LA-PI) and its enantiomer 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-Sn-glycero-3-phosphatidyl-L-1-inositol (diDCP-LA-PIe), with 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) on the α and ß position. METHODS: Activities of protein kinase C (PKC) and protein phosphatases such as protein phosphatase 1 (PP1), PP2A, and protein tyrosine phosphatase 1B (PTP1B) were assayed under the cell-free conditions and in PC-12 cells. Akt1/2 activity was monitored by quantifying phosphorylation at Thr308/309 and Ser473/474 in PC-12 cells. RESULTS: diDCP-LA-PI significantly activated PKCα, -ßΙ, -δ, and -ε, to an extent greater than that for diDCP-LA-PIe. diDCP-LA-PI still activated PKC in PC-12 cells, with the potential higher than that for diDCP-LA-PIe. Both diDCP-LA-PI and diDCP-LA-PIe reduced PP1 activity to a similar extent (30% of basal levels). diDCP-LA-PI enhanced PP2A activity to 180% of basal levels, while diDCP-LA-PIe had no effect. Drastic inhibition of PTP1B was obtained with diDCP-LA-PI and diDCP-LA-PIe, the extent reaching nearly 0% of basal levels. diDCP-LA-PI and diDCP-LA-PIe increased phosphorylation of Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells in the presence and absence of the PP2A inhibitor okadaic acid, respectively. CONCLUSION: The results of the present study show that diDCP-LA-PI and diDCP-LA-PIe exhibit different bioactivities with the different potentials each other.

PTP1B inhibition causes Rac1 activation by enhancing receptor tyrosine kinase signaling.

BACKGROUND/AIMS: The present study investigated the signaling pathway underlying Rac1 activation induced by the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). METHODS: Activity of protein tyrosine phosphatase 1B (PTP1B) was assayed under cell-free conditions. Western blot was carried out to quantify phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt in PC-12 cells. Rac1 activity was monitored in the föerster resonance energy transfer (FRET) analysis using living and fixed PC-12 cells. RESULTS: DCP-LA markedly suppressed PTP1B activity in a concentration (100 pM-100 µM)-dependent manner. In the DCP-LA binding assay, fluorescein-conjugated DCP-LA produced a single fluorescent signal band at 60 kDa, corresponding to the molecule of PTP1B, and the signal was attenuated or abolished by co-treatment or pretreatment with non-conjugated DCP-LA. DCP-LA significantly enhanced nerve growth factor (NGF)-stimulated phosphorylation of IRS-1 at Tyr1222 and Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells. In the FRET analysis, DCP-LA significantly enhanced NGF-stimulated Rac1 activation, which is abrogated by the phosphatidylinositol 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1) inhibitor BX912, or the Akt inhibitor MK2206. CONCLUSION: The results of the present study show that DCP-LA-induced PTP1B inhibition, possibly through its direct binding, causes Rac1 activation by enhancing a pathway along a receptor tyrosine kinase (RTK)/IRS-1/PI3K/Akt/Rac1 axis.

Lithium potentiates GSK-3ß activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation.

Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3ß. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li2CO3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li2CO3 did not affect PI3K-mediated PI(3,4,5)P3 production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3ß activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3ß in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li2CO3 on Akt1-induced phosphorylation of GSK-3ß at Ser9, but otherwise Li2CO3 significantly reduced GSK-3ß-mediated phosphorylation of ß-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3ß in an Akt-independent manner. In rat hippocampal slices Li2CO3 significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3ß at Ser9, and ß-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3ß activity.