Phospholipase D1 Expression Research Articles

Phospholipase D2 drives cellular lipotoxicity and tissue inflammation in alcohol-associated liver disease

AimsExcessive alcohol consumption leads to alcoholic liver disease (ALD), a major contributing factor to cirrhosis and hepatocellular carcinoma. In the present study we investigated the involvement of phospholipase D2 (PLD2) in the pathogenesis of ALD. Methods and materialsALD was induced in mice by chronic and binge ethanol feeding (the NIAAA model). Cellular transcriptome was examined by RNA-seq. Key findingsAnalysis of RNA-seq datasets indicated that PLD2 expression was up-regulated in liver tissues and in hepatocytes during ALD pathogenesis. Exposure of hepatocytes to ethanol treatment led to an increase in PLD2 expression. Similarly, ethanol feeding in mice stimulated PLD2 expression in the liver. On the contrary, PLD2 knockdown in hepatocytes down-regulated expression of pro-inflammatory and pro-lipogenic genes and dampened lipid accumulation. Consistently, PLD2 knockdown in mice significantly ameliorated ALD pathogenesis as evidenced by reduced steatosis and hepatic inflamamation. RNA-seq identified several metabolic pathways that were influenced by PLD2 deficiency. SignificanceOur data demonstrate that PLD2 is a novel regulator of ALD and suggest that small-molecule PLD2 inhibitors can be considered as a reasonable strategy for ALD treatment.

PLD2 deletion ameliorates sepsis-induced cardiomyopathy by suppressing cardiomyocyte pyroptosis via the NLRP3/caspase 1/GSDMD pathway

ObjectiveSepsis-induced cardiomyopathy (SICM) is a life-threatening complication. Phospholipase D2 (PLD2) is crucial in mediating inflammatory reactions and is associated with the prognosis of patients with sepsis. Whether PLD2 is involved in the pathophysiology of SICM remains unknown. This study aimed to investigate the effect of PLD2 knockout on SICM and to explore potential mechanisms.MethodsThe SICM model was established using cecal ligation and puncture in wild-type and PLD2-knockout mice and lipopolysaccharide (LPS)-induced H9C2 cardiomyocytes. Transfection with PLD2-shRNA lentivirus and a PLD2 overexpression plasmid were used to interfere with PLD2 expression in H9C2 cells. Cardiac pathological alterations, cardiac function, markers of myocardial injury, and inflammatory factors were used to evaluate the SICM model. The expression of pyroptosis-related proteins (NLRP3, cleaved caspase 1, and GSDMD-N) was assessed using western blotting, immunofluorescence, and immunohistochemistry.ResultsSICM mice had myocardial tissue damage, increased inflammatory response, and impaired heart function, accompanied by elevated PLD2 expression. PLD2 deletion improved cardiac histological changes, mitigated cTNI production, and enhanced the survival of the SICM mice. Compared with controls, PLD2-knockdown H9C2 exhibits a decrease in inflammatory markers and lactate dehydrogenase production, and scanning electron microscopy results suggest that pyroptosis may be involved. The overexpression of PLD2 increased the expression of NLRP3 in cardiomyocytes. In addition, PLD2 deletion decreased the expression of pyroptosis-related proteins in SICM mice and LPS-induced H9C2 cells.ConclusionPLD2 deletion is involved in SICM pathogenesis and is associated with the inhibition of the myocardial inflammatory response and pyroptosis through the NLRP3/caspase 1/GSDMD pathway.

Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling.

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP2) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response.

Importance of PLD2 in an IL-23 driven psoriasiform dermatitis model and potential link to human psoriasis.

Phospholipase D2 (PLD2), a major isoform of the PLD family, has been reported to regulate inflammatory responses. Thus far, the relevance of PLD2 in psoriasis, an inflammatory skin disease, has not been explored. In the current study, we examined PLD2 expression in the skin of psoriasis patients and the role of PLD2 in an interleukin (IL)-23-induced mouse model of psoriasiform dermatitis. Both in situ hybridization and bulk RNA sequencing showed PLD2 gene expression is significantly higher in lesional relative to non-lesional skin of psoriasis patients or the skin of healthy subjects. PLD2 expression is also enriched in residual lesions from patients on biologic therapies. Murine in vivo studies showed that PLD2 deficiency significantly reduced psoriasiform inflammation in IL-23-injected ears, as reflected by decreases in ear thickness, expression of defensin beta 4A and the S100 calcium binding protein A7A, macrophage infiltrate, and expression of CXCL10 and IL-6. However, the expression of type 17 cytokines, IL-17A and IL-17F, were not reduced. Dual knockout of PLD1 and PLD2 offered little additional protection compared to PLD2 knockout alone in the IL-23 model. In addition, pharmacological inhibition with a pan-PLD1/PLD2 inhibitor also suppressed IL-23-induced psoriasiform dermatitis. Bone-marrow-derived macrophages from wild type (WT) and PLD2 knockout (KO) mice exhibited little difference in viability and sensitivity to lipopolysaccharide and/or interferon gamma, or resiquimod (R848). PLD2 deficiency did not alter the differentiation and function of Th17 cells in an ex vivo study with splenocytes isolated from WT and PLD2 KO mice. Overall, these data suggest that PLD2 may play a role in the pathophysiology of psoriasis. Reducing macrophage infiltrate and cytokine/chemokine production might contribute to an anti-inflammatory effect observed in PLD2 knockout mice. Further studies are required to better understand the mechanisms by which PLD2 contributes to skin lesions in psoriasis patients and psoriasiform dermatitis models.

Upregulated phospholipase D2 expression and activity is related to the metastatic properties of melanoma.

The incidence rates of melanoma have increased steadily in recent decades and nearly 25% of the patients diagnosed with early-stage melanoma will eventually develop metastasis, for which there is currently no fully effective treatment. The link between phospholipases and tumors has been studied extensively, particularly in breast and colon cancers. With the aim of finding new biomarkers and therapeutic options for melanoma, the expression of different phospholipases was assessed in 17 distinct cell lines in the present study, demonstrating that phospholipase D2 (PLD2) is upregulated in metastatic melanoma as compared to normal skin melanocytes. These results were corroborated by immunofluorescence and lipase activity assays. Upregulation of PLD2 expression and increased lipase activity were observed in metastatic melanoma relative to normal skin melanocytes. So far, the implication of PLD2 activity in melanoma malignancies has remained elusive. To the best of our knowledge, the present study was the first to demonstrate that the overexpression of PLD2 enhances lipase activity, and its effect to increase the proliferation, migration and invasion capacity of melanoma cells was assessed with XTT and Transwell assays. In addition, silencing of PLD2 in melanoma cells reduced the metastatic potential of these cells. The present study provided evidence that PLD2 is involved in melanoma malignancy and in particular, in its metastatic potential, and established a basis for future studies evaluating PLD2 blockade as a therapeutic strategy to manage this condition.

Repeated isoflurane exposures of neonatal rats contribute to cognitive dysfunction in juvenile animals: the role of miR-497 in isoflurane-induced neurotoxicity.

Isoflurane anesthesia at the period of brain development can lead to neurotoxicity and long-term cognitive impairment. This study aimed to investigate the role of miR-497 on isoflurane-induced neurotoxicity. Neonatal rats (P7) were subject to isoflurane for 2 h at P7, P9, and P11. MiR-497 and neuron apoptosis were evaluated in hippocampal tissue by qRT-PCR and western blot. Fear conditioning test and Morris water maze were performed to determine cognitive function. The cell viability of isolated hippocampal neuronal cells exposed to isoflurane was measured using MTT test. The regulation of phospholipase D1 (PLD1) by miR-497 in isolated hippocampal neuronal cells was evaluated by luciferase reporter assays and western blot. Immunohistochemistry and TUNEL staining were employed to examine the PLD1 expression and neuronal cell apoptosis in hippocampus of neonatal rats, respectively. Repeated isoflurane anesthesia led to neurons' apoptosis and long-term cognitive impairment. Isoflurane exposure led to apoptosis and viability reduction in hippocampal neuronal cells. MiR-497 was observed to be upregulated after isoflurane exposure both in vivo and in vitro. Knockdown of miR-497 attenuated isoflurane-induced neuronal cells apoptosis and viability reduction. Furthermore, PLD1 was predicted and then validated as a novel target of miR-497. miR-497 could negatively regulate PLD1 by binding to its 3'-untranslated region. Downregulation of PLD1 was also observed after isoflurane exposure in neonatal rat hippocampus and hippocampal primary neuronal cell cultures. Induction of miR-497 was involved in isoflurane anesthesia-induced cognitive impairment and neuronal cell apoptosis by targeting PLD1. miR-497 may be a novel potential mechanism in isoflurane-induced neurotoxicity so that our findings provide new insight into a better and understanding of the clinical application of isoflurane.

Hypoxia-induced cofilin 1 promotes hepatocellular carcinoma progression by regulating the PLD1/AKT pathway.

BackgroundHepatocellular carcinoma (HCC) is the fourth fatal malignant tumour type worldwide. However, the exact molecular mechanism involved in HCC progression remains unclear.MethodsThree pairs of HCC and matched portal vein tumour thrombus (PVTT) tissue samples were analysed by isobaric tags for relative and absolute quantification (iTRAQ) assay to investigate the differentially expressed proteins. Real‐time quantitative PCR, immunostaining, and immunoblotting were performed to detect cofilin 1 (CFL1) in HCC and non‐tumour tissues. CCK8 and EdU, and Transwell assays, respectively, determined cell proliferation, migration, and invasion of HCC cells. Further, subcutaneous and tail vein injection were performed in nude mice for investigating HCC growth and lung metastasis in vivo. Regulatory effect of hypoxia‐inducible factor‐1α (HIF‐1α) on CFL1 was confirmed by chromatin immunoprecipitation (ChIP) assay. Finally, interaction between CFL1 and phospholipase D1 (PLD1) was studied using immunoprecipitation (IP) assay.ResultsThe iTRAQ analysis identified expression of CFL1 to be significantly upregulated in PVTT than in HCC tissues. Increased expression of CFL1 was closely associated with unfavourable clinical features, and was an independent risk predictor of overall survival in HCC patients. The knockdown of CFL1 inhibited cell growth viability, invasiveness, and epithelial‐mesenchymal transformation (EMT) in HCC cells. Furthermore, CFL1 silencing significantly suppressed the growth and lung metastasis of HCC cells in nude mice. Next, HIF‐1α directly regulated CFL1 transcription by binding to the hypoxia‐responsive element (HRE) in the promoter. Moreover, we disclosed the interaction between CFL1 and PLD1 in HCC cells using IP assay. Mechanistically, CFL1 maintained PLD1 expression by repressing ubiquitin‐mediated protein degradation, thereby activating AKT signalling in HCC cells. Notably, the CFL1/PLD1 axis was found mediating the hypoxia‐induced activation of the AKT pathway and EMT.ConclusionThe analysis suggests that hypoxia‐induced CFL1 increases the proliferation, migration, invasion, and EMT in HCC by activating the PLD1/AKT pathway.

Inhibition of phospholipase D2 augments histone deacetylase inhibitor-induced cell death in breast cancer cells

BackgroundHistone deacetylase (HDAC) inhibitors are promising anticancer drugs but their effect on tumor treatment has been disappointing mainly due to the acquisition of HDAC inhibitor resistance. However, the mechanisms underlying such resistance remain unclear.MethodsIn this study, we performed Western blot, q-PCR, and promoter assay to examine the expression of HDAC inhibitor-induced phospholipase D2 (PLD2) in MDA-MB231and MDA-MB435 breast cancer cells. Apoptosis and proliferation were analyzed by flow cytometry. In addition to invasion and migration assay, angiogenesis was further measured using in vitro tube formation and chick embryo chorioallantoic membrane model.ResultsHDAC inhibitors including suberoylanilide hydroxamic acid (SAHA), trichostatin, and apicidin, induce expression of PLD2 in a transcriptional level. SAHA upregulates expression of PLD2 via protein kinase C-ζ in breast cancer cells and increases the enzymatic activity of PLD. The combination treatment of SAHA with PLD2 inhibitor significantly enhances cell death in breast cancer cells. Phosphatidic acid, a product of PLD activity, prevented apoptosis promoted by cotreatment with SAHA and PLD2 inhibitor, suggesting that SAHA-induced PLD2 expression and subsequent activation of PLD2 might confers resistance of breast cancer cells to HDAC inhibitor. The combinational treatment of the drugs significantly suppressed invasion, migration, and angiogenesis, compared with that of either treatment.ConclusionThese findings provide further insight into elucidating the advantages of combination therapy with HDAC and PLD2 inhibitors over single-agent strategies for the treatment of cancer.

MiR-122-5p and miR-326-3p promote cadmium-induced NRK-52E cell apoptosis by downregulating PLD1.

Cadmium is a toxic heavy metal distributed broadly in the environment and manufactory industry. Long-term exposure to cadmium, considered as a risk for kidney injury, leads to chronic kidney disease eventually. Phospholipase D1 (PLD1) promotes cell proliferation and inhibits apoptosis, and might be involved in cadmium-induced kidney injury. In this study, we used miRNA microarray assays and bioinformatics analysis to identify miRNAs, which may regulate PLD1 expression and exert an impact on cadmium-induced kidney injury. MiR-122-5p and miR-326-3p，selected as candidates, were explored for their regulatory functions in kidney injury, using NRK-52E cells. Both of these two miRNAs exhibited higher expression in kidneys of SD rats after exposure to cadmium for 6 weeks. Cadmium treatment also increased miR-122-5p and miR-326-3p and decreased PLD1 in NRK-52E cells. Both of miR-122-5p and miR-326-3p could downregulate PLD1 expression through targeting its 3'UTR and enhance cadmium-induced apoptosis, while inhibiting either of these two miRNAs could reverse such effects. In conclusion, our results suggest that miR-122-5p and miR-326-3p might enhance cadmium-induced NRK-52E cell apoptosis through downregulating PLD1 expression.

HIF1α/PLD2 axis linked to glycolysis induces T-cell immunity in oral lichen planus

BackgroundOral lichen planus (OLP) is a common T-cell-mediated immunological disease. Hypoxia-inducible factor 1 alpha (HIF1α) plays an integral role in the glycolytic metabolism that facilitates immune functions from boosting cellular proliferative capacity to driving T-cell differentiation. In general, phospholipase D2 (PLD2) is required for HIF1α regulation. However, the involvement of HIF1α and PLD2 in dysfunctional T-cell immunity of OLP remains elusive. MethodsHIF1α and PLD2 expression in OLP lesions were determined by qRT-PCR, immunohistochemistry and immunofluorescence staining, and correlation analysis was carried out between their expressions. HIF1α or PLD2 silencing in T cells was performed to investigate the glycolytic alteration. Then their involvement in T-cell immunobiology was evaluated by detecting cell proliferation, cell cycle, apoptosis, and effector subsets differentiation. Additionally, the modulation of HIF1α on PLD2 expression and the engagement of mTOR in this process were explored. ResultsHIF1α and PLD2 protein were highly expressed in OLP lesions and they were both observed in large numbers of local CD3+ T cells in OLP. Besides, HIF1α expression was positively correlated with PLD2 expression in OLP. Both HIF1α and PLD2 promoted T-cell proliferation and pro-inflammatory phenotype differentiation, which was associated with the upregulation of glycolysis mediated by HIF1α or PLD2. Moreover, HIF1α induced PLD2 expression in an mTOR-independent way. ConclusionsHIF1α/PLD2 axis was supposed to be critical regulatory signaling involved in the T-cell immunity of OLP.

Promotion of cell autophagy and apoptosis in cervical cancer by inhibition of long noncoding RNA LINC00511 via transcription factor RXRA-regulated PLD1.

An increasing number of studies have explored the relationship of long noncoding RNAs (lncRNAs) with cervical cancer, yet the role of LINC00511 in cervical cancer still remains elusive. The current dissertation was intended to explore the effect of LINC00511 on cervical cancer development by regulating phospholipase D1 (PLD1) expression through transcription factor retinoic X receptor alpha (RXRA). Differentially expressed lncRNA and messenger RNA related to cervical cancer were screened by microarray-based expression profiling. Cervical cancer and paracancerous tissues were harvested to determine the LINC00511 expression using reverse transcription-quantitative polymerase chain reaction and western blot analysis. The relationship among LINC00511, PLD1 promoter activity, and RXRA were determined via RNA immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assays. Proliferation, autophagy, and apoptosis of cervical cancer cells were detected with a series of experiments. Tumor xenograft in nude mice was employed to determine the influence of LINC00511 and PLD1 on tumor formation and growth of cervical cancer in vivo. LINC00511 might influence the occurrence of cervical cancer by upregulating PLD1 expression via recruiting transcription factor RXRA. LINC00511 and PLD1 expressions were remarkably high in cervical cancer tissues and cells. LINC00511 combined with RXRA, and overexpression of LINC00511 in cervical cancer cells elevated PLD1 expression. Si-LINC00511, si-RXRA or si-PLD1 triggered repression of proliferation and promotion of autophagy and apoptosis of cervical cancer cells. In vivo experiment, si-LINC00511, or si-PLD1 inhibited the tumorigenic ability of nude mice. Collectively, this study suggests that LINC00511 acts as an oncogenic lncRNA in cervical cancer via the promotion of transcription factor RXRA-regulated PLD1.

MicroRNA-638 expression change in osteosarcoma patients via PLD1 and VEGF expression.

The present study aimed to investigate the function and mechanism of microRNA-638 (miR-638) in osteosarcoma. MiR-638 expression change in patients with osteosarcoma was detected by reverse transcription-quantitative polymerase chain reaction. Expression of miR-638 was observed to be downregulated in patients with osteosarcoma compared with the control group. In vitro, overexpression of miR-638 induced apoptosis, and inhibited cell proliferation and invasion of osteosarcoma cells. Overexpression of miR-638 induced Bcl-2 associated X and caspase-3 protein expression, and suppressed cyclin D1, phospholipase D1 (PLD1) and vascular endothelial growth factor (VEGF) protein expression in osteosarcoma. The promotion of PLD1 decreased the effects of miR-638 on osteosarcoma cell proliferation. In summary, it was demonstrated that miRNA-638 expression change in patients with osteosarcoma and an in vitro model via PLD1 and VEGF expression and miRNA-638 may be potential clinical indicators of osteosarcoma.

Tumor cell-secreted PLD increases tumor stemness by senescence-mediated communication with microenvironment.

Cancer cells are in continuous communication with the surrounding microenvironment and this communication can affect tumor evolution. In this work, we show that phospholipase D2 (PLD2) was overexpressed in colon tumors and is secreted by cancer cells, inducing senescence in neighboring fibroblasts. This occurs through its lipase domain. Senescence induced by its product, phosphatidic acid, leads to a senescence-associated secretory phenotype (SASP) able to increase the stem properties of cancer cells. This increase in stemness occurs by Wnt pathway activacion. This closes a feedback loop in which senescence acts as a crosspoint for the generation of CSCs mediated by phospholipid metabolism. We also demonstrate the connexion of both phenomena in mouse models in vivo showing that a high PLD2 expression increased stemness and tumorigenesis. Thus, the patients with colon cancer show high levels of PLD2 and SASP factor genes expression correlating with Wnt pathway activation. Therefore, we demonstrate that tumor cell-secreted PLD2 contributes to tumor development by modifying the microenvironment, making it a possible therapeutic target for cancer treatment. This mechanism may also explain the high levels of Wnt pathway activation in colon cancer.

High Expression of PhospholipaseD2 Induced by Hypoxia Promotes Proliferation of Colon Cancer Cells through Activating NF- κ Bp65 Signaling Pathway.

Hypoxia is a typical feature of colon cancer occurrence and progression. We have reported that high expression and activity of PhospholipaseD2 (PLD2) induced by hypoxia in colon cancer cells. In order to further investigate the role of PLD2 in colon cancer under hypoxic conditions. MTT assay was used to detect the proliferation of human colon cancer cells (SW480 and SW620) under hypoxic conditions by decrease the PLD2 gene expression or inhibit the activity of PLD2. Expression level of p-P65/T-P65 and Cyclin D1 were detected in those cells treated as above through using western blot and RT-PCR analysis. Effect of NF-Bp65 inhibitor (BAY-117082) on the proliferation and expression level of Cyclin D1 and PLD2 of colon cancer cells under hypoxic conditions were further analysised. As a result, decreased the expression of PLD2 or inhibited the activity of PLD2 leaded to the proliferation of hypoxia colon cancer cells reduced, and along with the expression level of p-P65/T-P65 and Cyclin D1 reduced. However, inhibition the expression level of p-P65/T-P65 lead to the proliferation and expression of Cyclin D1 in those hypoxia colon cancer cells also reduced. In vivo growth decreased in response to PLD2 and NF-Bp65 inhibition. Our study indicates that high expression of PLD2 induced by hypoxia promotes the proliferation of colon cancer cells, and it may elevate the expression level of Cyclin D1 through activating NF-Bp65 signaling pathway. Inhibition of the PLD2 expression may provide a new clue for treatment for colon cancer.

Tissue microarray staining reveals PLD1 and Sp1 have a collaborative, pro-tumoral effect in patients with osteosarcomas

It has been reported that phospholipase D1 (PLD1) - a key enzyme involved in lipid metabolism - is important for the initiation and progression of various human solid cancers; however, its biological significance and regulation in human osteosarcomas remain elusive. In this study, We found that PLD1 and Specificity Protein 1 (Sp1) expression were elevated in 137 osteosarcoma specimens with immunohistochemical staining. Our results showed that both PLD1 and Sp1 were expressed at much higher rates in the cancerous tissue compared to adjacent normal tissue. A correlation analyze also indicated that PLD1 was significantly associated with lactate dehydrogenase expression (p = 0.041) and the Enneking stage (p = 0.000), while Sp1 was significantly associated with the nuclear grade (p = 0.024). Furthermore, survival analyses showed that elevated PLD1 confers a poor prognosis on patients with osteosarcomas, acting as an independent prognostic factor. Of note, we showed a positive correlation between PLD1 and Sp1 expression in the cancer tissues (r = 0.357; p < 0.001). High co-expression of the two molecules results in the worst prognosis for the patients, and can also be regarded as independent prognostic factor (p = 0.001; HR = 2.71; 95% CI 1.53–4.80).

PLD2 regulates microtubule stability and spindle migration in mouse oocytes during meiotic division.

Phospholipase D2 (PLD2) is involved in cytoskeletal reorganization, cell migration, cell cycle progression, transcriptional control and vesicle trafficking. There is no evidence about PLD2 function in oocytes during meiosis. Herein, we analyzed PLD2 expression and its relationship with spindle formation and positioning in mouse oocyte meiosis. High protein level of PLD2 was revealed in oocytes by Western blot, which remained consistently stable from prophase I with intact germinal vesicle (GV) up to metaphase II (MII) stage. Immunofluorescence showed that PLD2 appeared and gathered around the condensed chromosomesafter germinal vesicle breakdown (GVBD), and co-localized with spindle from pro-metaphase I (pro-MI) to metaphase I (MI) and at MII stage. During anaphase I (Ana I) to telophase I (Tel I) transition, PLD2 was concentrated in the spindle polar area but absent from the midbody. In oocytes incubated with NFOT, an allosteric and catalytic inhibitor to PLD2, the spindle was enlarged and center-positioned, microtubules were resistant to cold-induced depolymerization and, additionally, the meiotic progression was arrested at MI stage. However, spindle migration could not be totally prevented by PLD2 catalytic specific inhibitors, FIPI and 1-butanol, implying at least partially, that PLD2 effect on spindle migration needs non-catalytic domain participation. NFOT-induced defects also resulted in actin-related molecules’ distribution alteration, such as RhoA, phosphatidylinosital 4, 5- biphosphate (PIP2), phosphorylated Colifin and, consequently, unordered F-actin dynamics. Taken together, these data indicate PLD2 is required for the regulation of microtubule dynamics and spindle migration toward the cortex in mammalian oocytes during meiotic progression.

Phospholipase D1 deficiency in mice causes nonalcoholic fatty liver disease via an autophagy defect.

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of triglycerides (TG) as lipid droplets in the liver. Although lipid-metabolizing enzymes are considered important in NAFLD, the involvement of phospholipase D1 (PLD1) has not yet been studied. Here, we show that the genetic ablation of PLD1 in mice induces NAFLD due to an autophagy defect. PLD1 expression was decreased in high-fat diet-induced NAFLD. Subsequently, PLD1 deficiency led to an increase in hepatic TGs and liver weight. Autophagic flux was blocked in Pld1−/− hepatocytes, with decreased β-oxidation rate, reduced oxidation-related gene expression, and swollen mitochondria. The dynamics of autophagy was restored by treatment with the PLD product, phosphatidic acid (PA) or adenoviral PLD1 expression in Pld1−/− hepatocytes, confirming that lysosomal PA produced by PLD1 regulates autophagy. Notably, PLD1 expression in Pld1−/− liver significantly reduced hepatic lipid accumulation, compared with Pld1−/− liver. Thus, PLD1 plays an important role in hepatic steatosis via the regulation of autophagy.

PLD1 regulates adipogenic differentiation through mTOR - IRS-1 phosphorylation at serine 636/639.

Phospholipase D1 (PLD1) plays a known role in several differentiation processes, but its role in adipogenic differentiation remains unknown. In the present study, we identified PLD1 as a negative regulator of adipogenic differentiation. We showed that PLD activity was downregulated by both 3-Isobutyl-1-methylxanthine (IBMX) and insulin upon induction of differentiation in 3T3-L1 adipogenic cells. In line with this observation, PLD activity decreased in both high fat diet (HFD)-fed mice and ob/ob mice. We also found that differentiation of 3T3-L1 preadipocytes was enhanced by the depletion of PLD1 levels or inhibition of PLD1 activity by VU0155069, a PLD1-specific inhibitor. Conversely, treatment with phosphatidic acid (PA), a PLD product, and overexpression of PLD1 both caused a decrease in adipogenic differentiation. Moreover, the elevated differentiation in PLD1-knockdown 3T3-L1 cells was reduced by either PA treatment or PLD1 expression, confirming negative roles of PLD1 and PA in adipogenic differentiation. Further investigation revealed that PA displaces DEP domain-containing mTOR-interacting protein (DEPTOR) from mTORC1, which subsequently phosphorylates insulin receptor substrate-1 (IRS-1) at serine 636/639 in 3T3-L1 cells. Taken together, our findings provide convincing evidence for a direct role of PLD1 in adipogenic differentiation by regulating IRS-1 phosphorylation at serine 636/639 through DEPTOR displacement and mTOR activation.

위암치료제로서 rebamipide의 잠재적 효능

Rebamipoide는 위궤양과 위염치료에 쓰이는 위보호제로 임상에서 사용되고 있지만 위암에서의 역할에 대해서는 알려지바가 거의 없다. 헬리코박토 파일로리(Helicobacter pylori)의 주요독성인자인 CagA는 위암의 발생과 관련이 있다. CagA는 위암세포에서 NF&#x3BA;B의 활성을 증가시켜 PLD1 단백질의 발현을 증가시킴으로써 위암세포의 증식과 침윤을 유도한다. Rebamipide는 NF&#x3BA;B의 활성을 억제함으로써 H. pylori의 CagA에 의해 유도되는 PLD1의 발현을 저해하는것으로 규명되었다. 게다가, rebamipide는 H. pylori의 CagA에 의해 유도되는 &#x3B2;-catenin과 그 표적 유전자로서 인 암줄기세포 마커 단백질의 발현을 증가시킴으로써 위암줄기세포의 자가재생능력을 감소시킨다. 또한 rebamipide는 항암제 내성을 극복하는 화학감수성을 증가시켜서 위암생성을 감소시키는 것으로 나타났다. 그래서 rebamipide는 위암치료제로서의 잠재적 효능을 가지고 있는 약물로서의 가능성이 제시되고 있다. Rebamipide is a mucosal-protective antiulcer drug, but its mechanism of action in gastric cancer remains elusive. CagA, a major virulence factor of Helicobacter pylori (H. pylori), is associated with the risk of gastric cancer. CagA protein is injected into gastric epithelial cells and deregulates a variety of cellular signaling molecules. CagA from H. pylori induces phospholipase D1 (PLD1) expression through NF&#x3BA;B activation in gastric epithelial cells, followed by invasion and proliferation of gastric epithelial cancer cells. Infection with cagA-positive H. pylori and expression of CagA enhances the binding of NF&#x3BA;B to the PLD1 promoter. Rebamipide abolishes H. pylori cagA-induced PLD1 expression via inhibition of binding of NF&#x3BA;B to the PLD1 promoter and also inhibits PLD activity. Moreover, rebamipide abolishes H. pylori CagA-induced &#x3B2;-catenin and the expression of a target cancer stem cell (CSC) marker gene via upregulation of miRNA-320a and -4496, followed by attenuation of self-renewal capacity of H. pylori CagA-infected gastric CSCs. In addition, rebamipide increases the chemosensitivity of CagA-expressed gastric CSCs and suppresses gastric carcinogenesis. Thus, it is speculated that rebamipide might show a potent efficacy as chemotherapeutic drug against gastric cancer cells. In this review, we summarizes recent results regarding the novel insights for the efficacy of rebamipide in gastric cancer cells.

Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease.

Background. Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are chronically remittent and progressive inflammatory disorders. Phospholipase D2 (PLD2) is reported to be involved in the pathogenesis of several inflammatory diseases. However, the exact role of PLD2 in IBD is obscure. Methods. PLD2 expression was determined in peripheral blood cells and inflamed mucosa from patients with IBD by qRT-PCR. Colonic biopsies were also obtained from CD patients before and after infliximab (IFX) treatment to examine PLD2 expression. PLD2 selective inhibitor (CAY10594) was administrated daily by oral gavage in DSS-induced colitis mice. Bone marrow neutrophils from colitis mice were harvested to examine the migration using Transwell plate. Results. PLD2 was found to be significantly increased in peripheral blood cells and inflamed mucosa in patients with active IBD. Treatment with IFX could significantly decrease PLD2 expression in intestinal mucosa in patients with CD. Moreover, blockade of PLD2 with CAY10594 could markedly ameliorate DSS-induced colitis in mice and promote neutrophil migration. Conclusions. PLD2 plays a critical role in the pathogenesis of IBD. Blockade of PLD2 may serve as a new therapeutic approach for treatment of IBD.