The pleckstrin homology domain of phospholipase D1 accelerates EGFR endocytosis by increasing the expression of the Rab5 effector, rabaptin-5

Endocytosis is differentially regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) and phospholipase D (PLD). However, the relationship between HIF-1alpha and PLD in endocytosis is unknown. HIF-1alpha is degraded through the prolyl hydroxylase (PHD)/von Hippel-Lindau (VHL) ubiquitination pathway in an oxygen-dependent manner. Here, we show that PLD1 recovers the decrease in epidermal growth factor receptor (EGFR) endocytosis induced by HIF-1alpha independent of lipase activity via the Rab5-mediated endosome fusion pathway. EGF-induced interaction of PLD1 with HIF-1alpha, PHD and VHL may contribute to EGFR endocytosis. The pleckstrin homology domain (PH) of PLD1 itself promotes degradation of HIF-1alpha, then accelerates EGFR endocytosis via upregulation of rabaptin-5 and suppresses tumor progression. These findings reveal a novel role of the PLD1-PH domain as a positive regulator of endocytosis and provide a link between PLD1 and HIF-1alpha in the EGFR endocytosis pathway.

Phospholipase D2 promotes degradation of hypoxia-inducible factor-1alpha independent of lipase activity

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key transcriptional mediator that coordinates the expression of various genes involved in tumorigenesis in response to changes in oxygen tension. The stability of HIF-1alpha protein is determined by oxygen-dependent prolyl hydroxylation, which is required for binding of the von Hippel-Lindau protein (VHL), the recognition component of an E3 ubiquitin ligase that targets HIF-1alpha for ubiquitination and degradation. Here, we demonstrate that PLD2 protein itself interacts with HIF-1alpha, prolyl hydroxylase (PHD) and VHL to promote degradation of HIF-1alpha via the proteasomal pathway independent of lipase activity. PLD2 increases PHD2-mediated hydroxylation of HIF-1alpha by increasing the interaction of HIF-1alpha with PHD2. Moreover, PLD2 promotes VHL-dependent HIF-1alpha degradation by accelerating the association between VHL and HIF-1alpha. The interaction of the pleckstrin homology domain of PLD2 with HIF-1alpha also promoted degradation of HIF-1alpha and decreased expression of its target genes. These results indicate that PLD2 negatively regulates the stability of HIF-1alpha through the dynamic assembly of HIF-1alpha, PHD2 and VHL.

Euodia sutchuenensis Dode extract stimulates osteoblast differentiation via Wnt/beta-catenin pathway activation

The Wnt/beta-catenin pathway has a role in osteoblast differentiation and bone formation. We screened 100 plant extracts and identified an extract from Euodia sutchuenensis Dode (ESD) leaf and young branch as an effective activator of the Wnt/beta-catenin pathway. ESD extract increased beta-catenin levels and beta-catenin nuclear accumulation in murine primary osteoblasts. The ESD extract also increased mRNA levels of osteoblast markers, including RUNX2, BMP2 and COL1A1, and enhanced alkaline phosphatase (ALP) activity in murine primary osteoblasts. Both ESD extract-induced beta-catenin increment and ALP activation were abolished by beta-catenin knockdown, confirming that the Wnt/beta-catenin pathway functions in osteoblast differentiation. ESD extract enhanced terminal osteoblast differentiation as shown by staining with Alizarin Red S and significantly increased murine calvarial bone thickness. This study shows that ESD extract stimulates osteoblast differentiation via the Wnt/beta-catenin pathway and enhances murine calvarial bone formation ex vivo.

Phospholipase D activates HIF-1-VEGF pathway via phosphatidic acid

Growth factor-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), generating phosphatidic acid (PA) which may act as a second messenger during cell proliferation and survival. Therefore, PLD is believed to play an important role in tumorigenesis. In this study, a potential mechanism for PLD-mediated tumorigenesis was explored. Ectopic expression of PLD1 or PLD2 in human glioma U87 cells increased the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) protein. PLD-induced HIF-1 activation led to the secretion of vascular endothelial growth factor (VEGF), a HIF-1 target gene involved in tumorigenesis. PLD induction of HIF-1alpha was significantly attenuated by 1-butanol which blocks PA production by PLD, and PA per se was able to elevate HIF-1alpha protein level. Inhibition of mTOR, a PA-responsive kinase, reduced the levels of HIF-1alpha and VEGF in PLD-overexpressed cells. Epidermal growth factor activated PLD and increased the levels of HIF-1alpha and VEGF in U87 cells. A specific PLD inhibitor abolished expression of HIF-1alpha and secretion of VEGF. PLD may utilize HIF-1-VEGF pathway for PLD-mediated tumor cell proliferation and survival.

Inhibition of phospholipase D2 induces autophagy in colorectal cancer cells

Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.

Phospholipase D inhibitor enhances radiosensitivity of breast cancer cells

Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.

The hydrophobic amino acids involved in the interdomain association of phospholipase D1 regulate the shuttling of phospholipase D1 from vesicular organelles into the nucleus

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidic acid. PLD is localized in most cellular organelles, where it is likely to play different roles in signal transduction. PLD1 is primarily localized in vesicular structures such as endosomes, lysosomes and autophagosomes. However, the factors defining its localization are less clear. In this study, we found that four hydrophobic residues present in the N-terminal HKD catalytic motif of PLD1, which is involved in intramolecular association, are responsible for vesicular localization. Site-directed mutagenesis of the residues dramatically disrupted vesicular localization of PLD1. Interestingly, the hydrophobic residues of PLD1 are also involved in the interruption of its nuclear localization. Mutation of the residues increased the association of PLD1 with importin-beta, which is known to mediate nuclear importation, and induced the localization of PLD1 from vesicles into the nucleus. Taken together, these data suggest that the hydrophobic amino acids involved in the interdomain association of PLD1 are required for vesicular localization and disturbance of its nuclear localization.

Rebamipide-induced downregulation of phospholipase D inhibits inflammation and proliferation in gastric cancer cells

Rebamipide a gastroprotective drug, is clinically used for the treatment of gastric ulcers and gastritis, but its actions on gastric cancer are not clearly understood. Phospholipase D (PLD) is overexpressed in various types of cancer tissues and has been implicated as a critical factor in inflammation and carcinogenesis. However, whether rebamipide is involved in the regulation of PLD in gastric cancer cells is not known. In this study, we showed that rebamipide significantly suppressed the expression of both PLD1 and PLD2 at a transcriptional level in AGS and MKN-1 gastric cancer cells. Downregulation of PLD expression by rebamipide inhibited its enzymatic activity. In addition, rebamipide inhibited the transactivation of nuclear factor kappa B (NFkappaB), which increased PLD1 expression. Rebamipide or PLD knockdown significantly suppressed the expression of genes involved in inflammation and proliferation and inhibited the proliferation of gastric cancer cells. In conclusion, rebamipide-induced downregulation of PLD may contribute to the inhibition of inflammation and proliferation in gastric cancer.

Triptolide-induced suppression of phospholipase D expression inhibits proliferation of MDA-MB-231 breast cancer cells

In spite of the importance of phospholipase D (PLD) in cell proliferation and tumorigenesis, little is known about the molecules regulating PLD expression. Thus, identification of small molecules inhibiting PLD expression would be an important advance for PLD-mediated physiology. We examined one such here, denoted "Triptolide", which was identified in a chemical screen for inhibitors of PLD expression using cell assay system based on measurement of PLD promoter activity. Triptolide significantly suppressed the expression of both PLD1 and PLD2 with sub-microM potency in MDA-MB-231 breast cancer cells as analyzed by promoter assay and RT-PCR. Moreover, triptolide abolished the protein level of PLD in a time and dose-dependent manner. Triptolide-induced PLD1 downregulation was also observed in all the cancer cells examined, suggesting a general phenomenon detected in various cancer cells. Decrease of PLD expression by triptolide suppressed both basal and PMA-induced PLD activity. In addition, triptolide inhibited activation of NFkappaB which increased PLD1 expression. Ultimately, downregulation of PLD by triptolide inhibited proliferation of breast cancer cells. Taken together, we demonstrate that triptolide suppresses the expression of PLD via inhibition of NFkappaB activation and then decreases cell proliferation.

The expression and cellular localization of phospholipase D isozymes in the developing mouse testis

To examine the involvement of phospholipase D (PLD)isozymes in postnatal testis development, the expression ofPLD1 and PLD2 was examined in the mouse testis atpostnatal weeks 1, 2, 4, and 8 using Western blot analysisand immunohistochemistry. The expression of both PLD1and PLD2 increased gradually with development frompostnatal week 1 to 8. Immunohistochemically, PLDimmunoreactivity was detected in some germ cells in thetestis and interstitial Leydig cells at postnatal week 1.PLD was mainly detected in the spermatocytes andresidual bodies of spermatids in the testis after 8 weeksafter birth. The intense immunostaining of PLD in Leydigcells remained unchanged by postnatal week 8. Thesefindings suggest that PLD isozymes are involved in thespermatogenesis of the mouse testis.

Phospholipase D is activated and phosphorylated by casein kinase-II in human U87 astroglioma cells

Elevated expression of protein casein kinase II (CKII) stimulated basal phospholipase D (PLD) activity as well as PMA-induced PLD activation in human U87 astroglioma cells. Moreover, CKII-selective inhibitor, emodin and apigenin suppressed PMA-induced PLD activation in a dose-dependent manner as well as basal PLD activity, suggesting the involvement of CKII in the activation of both PLD1 and PLD2. CKII was associated with PLD1 and PLD2 in co-transfection experiments. Furthermore, CKII induced serine/threonine phosphorylation of PLD2 in vivo, and the multiple regions of PLD2 were phosphorylated by CKII in vitro kinase assay using glutathione S-transferase-PLD2 fusion protein fragments. Elevated expression of CKII or PLD increased cell proliferation but pretreatment of cells with 1-butanol suppressed CKII-induced cell proliferation. These results suggest that CKII is involved in proliferation of U87 cells at least in part, through stimulation of PLD activity.

Phospholipase D activity is elevated in hepatitis C virus core protein-transformed NIH3T3 mouse fibroblast cells

Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and has also been overexpressed and hyperactivated in some human cancer cells. The aim of this study was to understand how PLD was regulated in the HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that PLD activity was elevated in the NIH3T3 cells overexpressing HCV core protein over the vector alone-transfected control cells, however, expression levels of PLD protein and protein kinase C (PKC) in the HCV core protein-transformed cells was similar to the control cells. Phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated PLD activity significantly more in the core protein-transformed cells, in comparison with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor and PKC translocation experiment showed that PKC-delta was mainly involved in the PMA- induced PLD activation in the core-transformed cells. Moreover, in cells overexpressing HCV core protein, PMA also stimulated p38 kinase more potently than that of the control cells, and an inhibitor of p38 kinase abolished PMA-induced PLD activation in cells overexpressing HCV core protein. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.

Phospholipase D is involved in oxidative stress-induced migration of vascular smooth muscle cells via tyrosine phosphorylation and protein kinase C

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD1, protein kinase C-a (PKC-a), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-a in VSMCs. Stimulation of the cells by H2O2 and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H2O2-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H2O2-stimulated PLD activation. The cells stimulated by oxidative stress (H2O2) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.

Phosphorylation of phospholipase D1 and the modulation of its interaction with RhoA by cAMP-dependent protein kinase

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.

Efffects of Fluoxetine on ATP-induced Calcium Signaling in PC12 Cells

Fluoxetine, a widely used anti-depressant compound, has several additional effects, including blockade of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells by using fura-2-based digital calcium imaging and assay for [3H]-inositol phosphates (IPs). Treatment with ATP (100microM) for 2 min induced [Ca2+]i increases. The ATP-induced [Ca2+]i increases were significantly decreased by removal of extracellular Ca2+ and treatment with the inhibitor of endoplasmic reticulum Ca2+ ATPase thapsigargin (1microM). Treatment with fluoxetine for 5 min blocked the ATP-induced [Ca2+]i increase concentration-dependently. Treatment with fluoxetine (30microM) for 5 min blocked the ATP-induced [Ca2+]i increase following removal of extracellular Ca2+ and depletion of intracellular Ca2+ stores. While treatment with the L-type Ca2+ channel antagonist nimodipine for 10 min inhibited the ATP-induced [Ca2+]i increases significantly, treatment with fluoxetine alone blocked the ATP-induced responses. Treatment with fluoxetine also inhibited the 50 mM K+-induced [Ca2+]i increases completely. However, treatment with fluoxetine did not inhibit the ATP-induced [3H]-IPs formation. Collectively, we conclude that fluoxetine inhibits ATP-induced [Ca2+]i increases in PC12 cells by inhibiting both an influx of extracellular Ca2+ and a release of Ca2+ from intracellular stores without affecting IPs formation.

Inhibition by Norfluoxetine, the Major Metabolite of Fluoxetine, of Voltage-Gated K+ Channels in Primary Cultured Rat Hippocampal Neurons / 대한마취과학회지

BACKGROUND: Fluoxetine (Prozac), a selective serotonin reuptake inhibitor, has been shown to be effective in the treatment of depression. We investigated the effects of norfluoxetine, the major active metabolite of fluoxetine, on voltage-gated K+ currents in primary cultured hippocampal neurons, and determined the potency and modes of actions of norfluoxetine. METHODS: Voltage-gated K+ currents were studied in primary cultured rat hippocampal neurons using the whole-cell configuration of the patch-clamp technique. Electrophysiological recordings were done in hippocampal neurons between 5-10 days in culture. Transient A-type K+ currents (KA) and delayed-rectifier K+ (KDR) currents were isolated from whole-cell K+ currents using a pulse protocol. RESULTS: Norfluoxetine accelerated the decay rate of whole-cell K+ currents, and thus decreased the current amplitude at the end of a pulse in a concentration-dependent manner. Norfluoxetine inhibited KA and KDR currents in a concentration-dependent manner with IC50's of 0.93 and 0.70micro M, respectively. Norfluoxetine also reduced the areas of KA currents and the steady-state KDR current over the range of test potentials, and the reduction was voltage-dependent (greater increase at more positive potentials). From the onset of the fractional block of KA currents by norfluoxetine during the initial 40 ms of a clamp step, we calculated k1 = 53.26/micro M.s for the association rate constant, and k2 = 70.24/s for the dissociation rate constant. The resulting apparent KD was 1.32micro M, which is similar to the IC50 value obtained from the concentration-response curve. CONCLUSIONS: Our results indicate that norfluoxetine, the major metabolite of fluoxetine, at therapeutic levels, produces a concentration- and voltage-dependent inhibition of KA and KDR currents in primary cultured hippocampal neurons. These effects could perturb the neuronal excitability in the hippocampus, and may contribute to the therapeutic antidepressant action of fluoxetine.

Electrophysiological and Morphological Classification of Inhibitory Interneurons in Layer II/III of the Rat Visual Cortex

Interneuron diversity is one of the key factors to hinder understanding the mechanism of cortical neural network functions even with their important roles. We characterized inhibitory interneurons in layer II/III of the rat primary visual cortex, using patch-clamp recording and confocal reconstruction, and classified inhibitory interneurons into fast spiking (FS), late spiking (LS), burst spiking (BS), and regular spiking non-pyramidal (RSNP) neurons according to their electrophysiological characteristics. Global parameters to identify inhibitory interneurons were resting membrane potential (> -70 mV) and action potential (AP) width ( 200 M omega) and the shorter P-T time (< 20 msec) than those of regular spiking pyramidal neurons. Confocal reconstruction of recorded cells revealed characteristic morphology of each subtype of inhibitory interneurons. Thus, our results provide at least four subtypes of inhibitory interneurons in layer II/III of the rat primary visual cortex and a classification scheme of inhibitory interneurons.

Comparative Analysis of Phospholipase D2 Localization in the Pancreatic Islet of Rat and Guinea Pig

To examine the localization pattern of phospholipase D2 (PLD2) in the pancreatic islet (the islet of Langerhans) depending on species, we conducted a morphological experiment in the rat and guinea pig. Since individual islets display a typical topography with a central core of B cell mass and a peripheral boundary of A, D, and PP cells, double immunofluorescent staining with a panel of antibodies was performed to identify PLD2-immunoreactive cells in the islets PLD2 immunoreactivity was mainly present in A and PP cells of the rat pancreatic islets. And yet, in the guinea pig, PLD2 immunoreactivity was exclusively localized in A cells, and not in PP cells. These findings suggest a possibility that PLD2 is mainly located in A cells of rodent pancreatic islets, and that the existence of PLD2 in PP cells is not universal in all species. Based on these results, it is suggested that PLD2 may play a significant role in the function of A and/or PP cells via a PLD-mediated signaling pathway.

Altered Cholecystokinin-induced Calcium Signal in Streptozotocin-induced Diabetic Rat Pancreatic Acini / 대한소화기학회지

BACKGROUND/AIMS: Pancreatic acini of streptozotocin (STZ)-induced diabetic rats release amylase less than normal acini on cholecystokinin (CCK) stimulation. Pancreatic enzyme secretion has been closely related to the intracellular calcium concentration ([Ca2+]i) of the acinar cell. In the present study, sequential changes of the intracellular calcium signal which probably underlie the altered enzyme secretion in response to CCK-8 were investigated using pancreatic acini from diabetic rats. METHODS: Diabetic rats were prepared by single intravenous injection of STZ (70 mg/kg). Stimulating experiments with CCK-8 were performed 7 days later. Pancreatic acini were isolated by collagenase digestion. Amylase release and [Ca2+]i were measured by colorimethod and calcium imaging, respectively. The geometry of intracellular calcium signal was analyzed. RESULTS: Normal acini exhibited concentration-dependent [Ca2+]i increase and regular oscillatory calcium signal on CCK-8 stimulation. Amylase release was also concentration-dependent. However, diabetic acini showed significantly less [Ca2+]i increase, prolonged time to peak [Ca2+]i, decreased calcium spikes number, and decreased amylase release compared with normal acini. The decreased [Ca2+]i in diabetic acini was restored significantly by insulin treatment. CONCLUSIONS: Relatively decreased amylase release in diabetic pancreatic acini in response to CCK, appears to be associated with altered calcium signal due to insulin deficiency.

Altered Secretory Pattern of Pancreatic Enzymes and Gastrointestinal Hormones in Streptozotocin-induced Diabetic Rats

This study was performed to investigate the pancreatic exocrine dysfunction in streptozotocin- induced diabetic rats. Changes in pancreatic enzymes secretion and in pancreatic enzymes content were observed. The output and the tissue content of amylase were significantly reduced in diabetic rats, while the output and the content of lipase were increased. Plasma secretin and cholecystokinin (CCK) concentrations of diabetic rats were significantly increased compared to those of normal rats. The altered pancreatic exocrine function was abolished by the exogenous insulin administration. The exogenous insulin also restored the increased plasma secretin and CCK concentrations. From the above results, it is suggested that, in streptozotocin-induced diabetic rats, anticoordinated changes in pancreatic enzymes secretion as well as pancreatic enzymes content are attributable to insulin deficiency and that the insulin deficiency is responsible for the increased plasma concentrations of both secretin and CCK. However, it is not clear whether the elevated plasma secretin and CCK concentrations played a direct role in changes of pancreatic exocrine function.