Glycogen synthase kinase-3β negatively regulates group IIA phospholipase A2 expression in human aortic smooth muscle and HepG2 hepatoma cells

AbstractThe present study shows that the IFN-γ-mediated upregulation of secretory phospholipase A2 of group IIA (sPLA2-IIA) in HASMC and HepG2 cells is synergistically increased after simultaneous inhibition of glycogen synthase kinase-3β (GSK-3β) by indirubin-3′-monoxime, 5-iodo or AR-A014418. The effect of GSK-3β inhibition was dose- and time-dependent and can be further augmented by its concomitant incubation with Clostridium difficile toxin B, an inhibitor of small Rho proteins, or H-1152, an inhibitor of Rho-associated kinase. Using AG-490 and caffeic acid phenethyl ester (CAPE), it is further demonstrated that the effect of GSK-3β inhibition on sPLA2-IIA expression depends on Janus kinase-2 and NF-κB-signaling

Expression and shedding of endothelial protein C receptor in prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>Increasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines.</p> <p>Results</p> <p>EPCR expression is up-regulated both at the mRNA and protein levels in invasive prostate DU-145 and PC-3 cells in comparison to normal prostate epithelial cells (PrEC) and less-invasive LNCaP cells. Release of soluble EPCR (sEPCR) is induced by 12-myristate 13-acetate, ionomycin, H₂O₂, and disruptor of lipid rafts in PrEC, DU-145, and PC-3 cells. Furthermore, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interleukin-6 or interferon-γ increase sEPCR release. In LNCaP cells, neither pharmacological agents nor IL-1β or TNF-α result in a significant increase of sEPCR release. The effects of IL-1β and TNF-α on EPCR shedding in DU-145 cells are mediated by MEK/ERK 1/2, JNK, and p38 MAPK signalling cascades. In PC-3 cells, however, the MEK/ERK 1/2 pathway is down-regulated and incubation with cytokines did not elevate the phosphorylated ERK-1/2 fraction as in the case of DU-145 cells. Treatment with 4-aminophenylmercuric acetate (APMA), an activator of metalloproteases, causes a disproportionately large increase of sEPCR release in DU-145 and PC-3 cells, compared to PrEC and LNCaP cells. Finally, an increased release of sEPCR mediated by APMA treatment is shown to be connected with reduced generation of activated protein C indicating the functionality of EPCR in these cells.</p> <p>Conclusions</p> <p>The study demonstrates a number of substantial differences in expression and shedding of EPCR in prostate cancer cell lines in comparison with normal cells that may be relevant for understanding the role of this receptor in carcinogenesis.</p

Involvement of Epigenetic Mechanisms in the Regulation of Secreted Phospholipase A2 Expressions in Jurkat Leukemia Cells

Epigenetic changes provide a frequent mechanism for transcriptional silencing of genes in cancer cells. We previously established that epigenetic mechanisms are important for control of group IIA phospholipase A2 (PLA2G2A) gene transcription in human DU-145 prostate cells. In this study, we analyzed the involvement of such mechanisms in the regulation of five sPLA2 isozymes and the M-type receptor of sPLA2 (sPLA2-R) in human leukemic Jurkat cells. These cells constitutively expressed sPLA2-IB, sPLA2-III, sPLA2-X, and sPLA2-R but not sPLA2-IIA and sPLA2-V. Transcription of sPLA2-IIA and sPLA2-V was, however, detected after exposure of cells to the DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC). Expression of sPLA2-IIA was further enhanced by additional exposure to interferon-γ and blocked by inhibitors of specificity protein 1, nuclear factor κB, and Janus kinase/signal transducer and activator of transcription-dependent pathways. Sequence analysis and methylation-specific polymerase chain reaction of bisulfite-modified genomic DNA revealed two 5′-CpG sites (-111 and -82) in the sPLA2-IIA proximal promoter that were demethylated after 5-aza-dC treatment. These sites may be involved in the DNA binding of specificity protein 1 and other transcription factors. Similar findings after treatment of human U937 leukemia cells with 5-aza-dC indicate that this mechanism of PLA2G2A gene silencing is not restricted to Jurkat and DU-145 cells. These data establish that regulation of sPLA2-IIA and sPLA2-V in Jurkat and other cells involves epigenetic silencing by DNA hypermethylation

Differential Expression of Secretory Phospholipases A2 in Normal and Malignant Prostate Cell Lines: Regulation by Cytokines, Cell Signaling Pathways, and Epigenetic Mechanisms

Upregulation of group IIA phospholipase A2 (sPLA2-IIA) correlates with prostate tumor progression suggesting prooncogenic properties of this protein. Here, we report data on expression of three different sPLA2 isozymes (groups IIA, V, and X) in normal (PrEC) and malignant (DU-145, PC-3, and LNCaP) human prostate cell lines. All studied cell lines constitutively expressed sPLA2-X, whereas sPLA2-V transcripts were identified only in malignant cells. In contrast, no expression of sPLA2-IIA was found in PrEC and DU-145 cells, but it was constitutively expressed in LNCaP and PC-3 cells. Expression of sPLA2-IIA is upregulated in PC-3 and in PrEC cells by IFN-γ in a signal transducer and activator of transcription-1-dependent manner, but not in LNCaP cells. Additional signaling pathways regulating sPLA2-IIA expression include cAMP/protein kinase A, p38 mitogen-activated protein kinase, protein kinase C, Rho-kinase, and mitogen-activated/extracellular response protein kinase / extracellular signal-regulated kinase. No deletions were revealed in the sPLA2-IIA gene from DU-145 cells lacking the expression of sPLA2-IIA. Reexpression of sPLA2-IIA was induced by 5-aza-2′-deoxycytidine demonstrating that DNA methylation is implicated in the regulation of sPLA2-II. Together, these data suggest that sPLA2-IIA and sPLA2-V, but not sPLA2-X, are differentially expressed in normal and malignant prostate cells under the control of proinflammatory cytokines; epigenetic mechanisms appear involved in the regulation of sPLA2-IIA expression, at least in DU-145 cells

Aberrant methylation of the M-type phospholipase A₂ receptor gene in leukemic cells

Abstract Background The M-type phospholipase A2 receptor (PLA2R1) plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS) or acute leukemia. Methods Sites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM) analysis was then carried out to quantify PLA2R1 methylation at 5`-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA. Results Expression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2´-deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% ± 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia. Conclusions The study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis.</p