Cathepsin G-induced malignant progression of MCF-7 cells involves suppression of PAF signaling through induced expression of PAFAH1B2.

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.

Lp-PLA2 Antagonizes Left Ventricular Healing After Myocardial Infarction by Impairing the Appearance of Reparative Macrophages.

BACKGROUND: Healing after myocardial infarction (MI) involves the biphasic accumulation of inflammatory Ly-6C(high) and reparative Ly-6C(low) monocytes/macrophages. Excessive inflammation disrupts the balance between the 2 phases, impairs infarct healing, and contributes to left ventricle remodeling and heart failure. Lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of the phospholipase A2 family of enzymes, produced predominantly by leukocytes, participates in host defenses and disease. Elevated Lp-PLA2 levels associate with increased risk of cardiovascular events across diverse patient populations, but the mechanisms by which the enzyme elicits its effects remain unclear. This study tested the role of Lp-PLA2 in healing after MI. METHODS AND RESULTS: In response to MI, Lp-PLA2 levels markedly increased in the circulation. To test the functional importance of Lp-PLA2, we generated chimeric mice whose bone marrow-derived leukocytes were Lp-PLA2-deficient (bmLp-PLA2 (-/-)). Compared with wild-type controls, bmLp-PLA2 (-/-) mice subjected to MI had lower serum levels of inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6, and decreased number of circulating inflammatory myeloid cells. Accordingly, bmLp-PLA2 (-/-) mice developed smaller and less inflamed infarcts with reduced numbers of infiltrating neutrophils and inflammatory Ly-6C(high) monocytes. During the later, reparative phase, infarcts of bmLp-PLA2 (-/-) mice contained Ly-6C(low) macrophages with a skewed M2-prone gene expression signature, increased collagen deposition, fewer inflammatory cells, and improved indices of angiogenesis. Consequently, the hearts of bmLp-PLA2 (-/-) mice healed more efficiently, as determined by improved left ventricle remodeling and ejection fraction. CONCLUSIONS: Lp-PLA2 augments the inflammatory response after MI and antagonizes healing by disrupting the balance between inflammation and repair, providing a rationale for focused study of ventricular function and heart failure after targeting this enzyme acutely in MI.

Elevated expression of lipoprotein-associated phospholipase A2 in calcific aortic valve disease: implications for valve mineralization.

OBJECTIVES: This study sought to document the presence and role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in calcific aortic valve disease (CAVD). BACKGROUND: CAVD is a chronic disorder characterized by pathological mineralization and remodeling. Studies have indicated that human CAVD tissues are infiltrated by lipids and that inflammation may play a role in the pathobiology. We hypothesized that Lp-PLA2 (encoded by the PLA2G7 gene) is expressed in CAVD and may play a role in the mineralization of valve interstitial cells. METHODS: We have documented the expression of the phospholipase A2 family of genes in aortic valves by using a transcriptomic assay. Messenger ribonucleic acid and protein expression were confirmed in aortic valves explanted from 60 patients by quantitative polymerase chain reaction and immunohistochemistry, respectively. The effect of lysophosphatidylcholine, the product of Lp-PLA2 activity, was documented on the mineralization of valve interstitial cell cultures. RESULTS: Transcriptomic analyses of CAVD and control nonmineralized aortic valves revealed that Lp-PLA2 was increased by 4.2-fold in mineralized aortic valves. Higher expression of Lp-PLA2 in stenotic aortic valves was confirmed by quantitative polymerase chain reaction, immunohistochemistry, and enzymatic Lp-PLA2 activity. The number of Lp-PLA2 transcripts correlated with several indexes of tissue remodeling. In vitro, lysophosphatidylcholine increased the expression of alkaline phosphatase, the ectonucleotide pyrophosphatase/phosphodiesterase 1 enzyme, sodium-dependent phosphate cotransporter 1 (encoded by the SLC20A1 gene), and osteopontin. We then showed that lysophosphatidylcholine-induced mineralization involved ectonucleotidase enzyme as well as apoptosis through a protein-kinase-A-dependent pathway. CONCLUSIONS: Together, these results demonstrated that Lp-PLA2 is highly expressed in CAVD, and it plays a role in the mineralization of valve interstitial cells. Further work is necessary to document whether Lp-PLA2 could be considered as a novel target in CAVD.

Serum amyloid A stimulates lipoprotein-associated phospholipase A2 expression in vitro and in vivo.

OBJECTIVES: Although lipoprotein-associated phospholipase A2 (Lp-PLA2) has been regarded as a biomarker and a causative agent for acute coronary events recently, the mechanism of the regulation of Lp-PLA2 has not been fully elucidated yet. This study was aimed to investigate the influence of serum amyloid A (SAA) on the expression of Lp-PLA2 in THP-1 cells and ApoE-deficient (ApoE(-/-)) mice. METHODS: THP-1 cells were stimulated by SAA and the mRNA and protein expression of Lp-PLA2 was detected. ApoE(-/-) mice were intravenously injected with murine SAA1 lentivirus. Formyl peptide receptor like-1 (FPRL1) agonist (WKYMVm) and inhibitor (WRW(4)), mitogen-activated protein kinases (MAPKs) inhibitors, and peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist and inhibitor were used to investigate the mechanism of regulation of Lp-PLA2. RESULTS: Recombinant SAA up-regulated Lp-PLA2 expression in a dose and time-dependent manner in THP-1 cells. Immunohistochemical analysis of aortic root of ApoE(-/-) mice also demonstrated that the expression of Lp-PLA2 was up-regulated significantly with SAA treatment. WRW(4) decreased SAA-induced Lp-PLA2 production; while WKYMVm could induce Lp-PLA2 expression. ERK1/2, JNK1/2, and p38 inhibition reduced SAA-induced Lp-PLA2 production. Furthermore, the results suggested the activation of PPAR-γ played a crucial role in this process. CONCLUSION: These results demonstrate that SAA up-regulates Lp-PLA2 production significantly via a FPRL1/MAPKs./PPAR-γ signaling pathway.

Oxidatively truncated phospholipids are required agents of tumor necrosis factor α (TNFα)-induced apoptosis.

TNFα generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the (hydro)peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNFα receptor signaling to mitochondrial damage and apoptotic death.

The human LIS1 is downregulated in hepatocellular carcinoma and plays a tumor suppressor function.

The human lissencephaly-1 gene (LIS1) is a disease gene responsible for Miller-Dieker lissencephaly syndrome (MDL). LIS1 gene is located in the region of chromosome 17p13.3 that is frequency deleted in MDL patients and in human liver cancer cells. However, the expression and significance of LIS1 in liver cancer remain unknown. Here, we investigated the expression of LIS1 in hepatocellular carcinoma (HCC) tissues by real-time PCR, Western blot, and immunohistochemistry. The results indicated that the mRNA and protein levels of LIS1 were downregulated in about 70% of HCC tissues, and this downregulation was significantly associated with tumor progression. Functional studies showed that the reduction of LIS1 expression in the normal human liver cell line QSG7701 or the mouse fibroblast cell line NIH3T3 by shRNA resulted in colony formation in soft agar and xenograft tumor formation in nude mice, demonstrating that a decrease in the LIS1 level can promote the oncogenic transformation of cells. We also observed that the phenotypes of LIS1-knockdown cells displayed various defective mitotic structures, suggesting that the mechanism by which reduced LIS1 levels results in tumorigenesis is associated with its role in mitosis. Furthermore, we demonstrated that ectopic expression of LIS1 could significantly inhibit HCC cell proliferation and colony formation. Our results suggest that LIS1 plays a potential tumor suppressor role in the development and progression of HCC.

PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids.

Platelet activating factor (PAF) and PAF-like lipids induce inflammatory responses in target cells. These lipid mediators are inactivated by PAF-acetylhydrolase (PAF-AH). The PAF signaling system affects the growth of hematopoietic CD34(+) cells, but roles for PAF-AH in this process are unknown. Here, we investigated PAF-AH function during megakaryopoiesis and found that human CD34(+) cells accumulate this enzymatic activity as they differentiate toward megakaryocytes, consistent with the expression of mRNA and protein for the plasma PAF-AH isoform. Inhibition of endogenous PAF-AH activity in differentiated megakaryocytes increased formation of lipid mediators that signaled the PAF receptor (PAFR) in fully differentiated human cells such as neutrophils, as well as megakaryocytes themselves. PAF-AH also controlled megakaryocyte alpha(IIb)beta(3)-dependent adhesion, cell spreading, and mobility that relied on signaling through the PAFR. Together these data suggest that megakaryocytes generate PAF-AH to modulate the accumulation of intracellular phospholipid mediators that may detrimentally affect megakaryocyte development and function.

Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression.

BACKGROUND: Neuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem.Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment. METHODS: We analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration) and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively. RESULTS: Doublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness. Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin. CONCLUSION: a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness;b) Retinoic acid treatment reduces migration and invasiveness of the more aggressive cell components of SK-N-SH cells;c) The cells that after retinoic acid exposure show migration and invasive capability may be identified on the basis of doublecortin expression.

The proangiogenic phenotype of tumor-derived endothelial cells is reverted by the overexpression of platelet-activating factor acetylhydrolase.

PURPOSE: We previously reported that human tumor-derived endothelial cells (TEC) have an angiogenic phenotype related to the autocrine production of several angiogenic factors. The purpose of the present study was to evaluate whether an enhanced synthesis of platelet-activating factor (PAF) might contribute to the proangiogenic characteristics of TEC and whether its inactivation might inhibit angiogenesis. EXPERIMENTAL DESIGN: To address the potential role of PAF in the proangiogenic characteristics of TEC, we engineered TEC to stably overexpress human plasma PAF-acetylhydrolase (PAF-AH), the major PAF-inactivating enzyme, and we evaluated in vitro and in vivo angiogenesis. RESULTS: TECs were able to synthesize a significantly enhanced amount of PAF compared with normal human microvascular endothelial cells when stimulated with thrombin, vascular endothelial growth factor, or soluble CD154. Transfection of TEC with PAF-AH (TEC-PAF-AH) significantly inhibited apoptosis resistance and spontaneous motility of TEC. In addition, PAF and vascular endothelial growth factor stimulation enhanced the motility and adhesion of TEC but not of TEC-PAF-AH. In vitro, TEC-PAF-AH lost the characteristic ability of TEC to form vessel-like structures when plated on Matrigel. Finally, when cells were injected s.c. within Matrigel in severe combined immunodeficiency mice or coimplanted with a renal carcinoma cell line, the overexpression of PAF-AH induced a significant reduction of functional vessel formation. CONCLUSIONS: These results suggest that inactivation of PAF, produced by TEC, by the overexpression of plasma PAF-AH affects survival, migration, and the angiogenic response of TEC both in vitro and in vivo.

Platelet-activating factor receptor (PAF-R) and acetylhydrolase (PAF-AH) are co-expressed in immature bovine trophoblast giant cells throughout gestation, but not at parturition.

Platelet-activating factor (PAF) was associated with successful implantation in the cow, trophoblast invasiveness and angiogenesis. Bovine placentation is characterized by the limited invasion of trophoblast giant cells (TGC) into the maternal caruncular epithelium. TGC exhibit both endocrine activity and properties of tumor cells and may thus be targets of and mediators for the action of PAF. We examined PAF-receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) gene expression and localized mRNA and corresponding proteins in bovine placentomes throughout gestation and at parturition. PAF-R and PAF-AH protein and mRNA were highly expressed and colocalized in immature TGC from early gestation until near term, while mature TGC were negative. After the onset of parturition both PAF-R and PAF-AH were expressed in the maternal stroma, predominantly endothelial cells. The expression of PAF-R and PAF-AH in immature but not mature TGC during gestation implicates a role for PAF in the differentiation, maturation and function of bovine placentomal TGC. Placentomal angiogenesis could be mediated by binding of PAF to PAF-R present in endothelial cells. The parturition-related "switch" of PAF-R and PAF-AH from TGC to the maternal stroma suggests that PAF may participate in the regulation of parturition and in prepartum tissue programming.

Adenovirus-mediated gene transfer and lipoprotein-mediated protein delivery of plasma PAF-AH ameliorates proteinuria in rat model of glomerulosclerosis.

Oxidative stress has been proposed to play a crucial role in glomerulosclerosis, although its in vivo demonstration has proved taxing given the difficulty of inducing gene expression in specific renal cells. In this study, we examined whether the liver-directed expression of plasma platelet-activating factor acetylhydrolase (PAF-AH) would affect the glomerular pathophysiology in Imai rats, an animal model for glomerulosclerosis. Adenovirus-mediated liver-directed gene delivery of human PAF-AH resulted in a significant increase in plasma PAF-AH activity, which was detected almost exclusively on HDL. Histological examination of rats overexpressing PAF-AH showed not only the deposition of PAF-AH in mesangial cells, but also a reduction in hydroxynonenal and matrix protein content in the glomeruli. In situ hybridization analysis was negative for human PAF-AH mRNA in the kidney, while injection of HDL abundant in PAF-AH resulted in the deposition of PAF-AH in mesangial cells. Urine protein levels did not increase in rats overexpressing PAF-AH, while those of control rats increased significantly with age. This study provides direct evidence of the in vivo role of an enzyme that degrades lipid peroxides during the progression of glomerulosclerosis. Adenovirus-mediated extrarenal gene expression and lipoprotein-mediated glomeruli-targeted protein delivery promise to be a novel therapeutic approach to glomerulosclerosis.

Pioglitazone induces plasma platelet activating factor-acetylhydrolase and inhibits platelet activating factor-mediated cytoskeletal reorganization in macrophage.

Platelet activating factor (PAF) is a key molecule for inflammation. To examine a role of peroxisome proliferator-activated receptor gamma (PPARgamma) in inflammatory reactions of atherosclerosis, we investigated the effects of 15-deoxy-(Delta12,14)-Prostaglandin J2 (15d-PGJ2) and pioglitazone, PPARgamma ligands, on plasma PAF-acetylhydrolase (PAF-AH) expression in THP-1 macrophages. PAF-AH mRNA and protein were up-regulated by the PPARgamma ligands. Prostaglandin F2alpha (PGF2alpha), a PARgamma inhibitor, abrogated the up-regulation of PAF-AH mRNA by pioglitazone, suggesting that PPARgamma activation is involved in the induction of PAF-AH by pioglitazone. As PAF promotes the cell motility with cytoskeletal reorganization, we investigated the effect of pioglitazone on PAF-mediated morphological changes in THP-1 macrophages. In the absence of pioglitazone, PAF promoted the elongation of actin cytoskeleton, which was inhibited by pretreatment with pioglitazone. In contrast, pioglitazone was not able to inhibit the morphological changes induced by C-PAF, a non-hydrolyzable PAF agonist. Thus, it is suggested that PAF-induced morphological changes could be inhibited by pioglitazone through PAF-AH, which rapidly hydrolyzed PAF. These data propose that PPARgamma/PAF-AH pathway is a clinical target for the prevention against atherosclerosis.