Simultaneous silencing of lysophosphatidylcholine acyltransferases 1-4 by nucleic acid nanoparticles (NANPs) improves radiation response of melanoma cells.

Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.

Expression QTL analysis of glaucoma endophenotypes in the Norfolk Island isolate provides evidence that immune-related genes are associated with optic disc size.

Primary open-angle glaucoma (POAG) is influenced by both genetic and environmental factors. Despite significant progress in identifying genetic variants associated with POAG, there remains a substantial amount of unexplained heritability. Study design features that may enhance knowledge of the genetic architecture include focusing on multiple quantitative traits related to ocular disorders (i.e. endophenotypes), targeting genetic variants that directly influence gene expression (i.e. cis-eQTLs) and utilising genetically isolated populations to reduce genetic and environmental noise and thus enhance association signals. In this study we performed heritability and blood-based eQTL association analysis of five key POAG endophenotypes in 330 individuals from the Norfolk Island (NI) isolate. Results showed evidence of heritability for all five traits, with H2 estimates ranging from 0.35 for intraocular pressure (IOP) to 0.82 for central corneal thickness (CCT) (P < 0.05). The primary finding was for BTN3A2, whereby both cis-SNP and transcript were significantly associated with disc size within a conditional regression model. Specifically, this model included rs853676 (ß = 0.23,P = 0.008) and transcript (ß = 0.23, P = 0.03). We also observed a cis-SNP association between optic disc size and LPCAT2 independent of transcript (P = 0.0004). These genes have specific functions in immune system pathways and suggest a role for an inherited immune component of POAG risk. This study also demonstrates an alternate approach to understanding the functional genetic basis of POAG and ocular health more generally.

Effects of Subretinal Gene Transfer at Different Time Points in a Mouse Model of Retinal Degeneration.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is necessary for photoreceptors to generate an important lipid component of their membranes. The absence of LPCAT1 results in early and rapid rod and cone degeneration. Retinal degeneration 11 (rd11) mice carry a mutation in the Lpcat1 gene, and are an excellent model of early-onset rapid retinal degeneration (RD). To date, no reports have documented gene therapy administration in the rd11 mouse model at different ages. In this study, the AAV8 (Y733F)-smCBA-Lpcat1 vector was subretinally injected at postnatal day (P) 10, 14, 18, or 22. Four months after injection, immunohistochemistry and analysis of retinal morphology showed that treatment at P10 rescued about 82% of the wild-type retinal thickness. However, the diffusion of the vector and the resulting rescue were limited to an area around the injection site that was only 31% of the total retinal area. Injection at P14 resulted in vector diffusion that covered approximately 84% of the retina, and we found that gene therapy was more effective against RD when exposure to light was limited before and after treatment. We observed long-term preservation of electroretinogram (ERG) responses, and preservation of retinal structure, indicating that early treatment followed by limited light exposure can improve gene therapy effectiveness for the eyes of rd11 mice. Importantly, delayed treatment still partially preserved M-cones, but not S-cones, and M-cones in the rd11 retina appeared to have a longer window of opportunity for effective preservation with gene therapy. These results provide important information regarding the effects of subretinal gene therapy in the mouse model of LPCAT1-deficiency.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) upregulation in breast carcinoma contributes to tumor progression and predicts early tumor recurrence.

Breast cancer is the most common cancer in women worldwide. Aberrant lipid metabolism is an established hallmark of cancer cells. The recently isolated lysophosphatidylcholine acyltransferase 1 (LPCAT1), the most important enzyme in membrane biogenesis, has been currently implicated in cancer development and progression. The published literature lacks comprehensive reports on LPCAT1 expression in breast cancer and its impact on patients' outcome. We evaluated the immunohistochemical expression of LPCAT1 in 80 primary breast carcinomas, 24 metastatic lymph nodes, and 30 non-neoplastic breast tissue specimens and statistically analyzed the association between LPCAT1 expression and clinicopathological variables and patients' outcome. LPCAT1 protein was significantly upregulated in primary breast carcinoma and showed a significant ascending pattern being the lowest in normal breast tissues, relatively increased in fibrocystic disease, and the highest in primary carcinoma. LPCAT1 expression was significantly higher at tumor's advancing edge and correlated positively with tumor's grade and TNM stage. Compared to primary tumor, LPCAT1 expression was significantly lower in ductal carcinoma in situ and significantly higher in metastatic lymph nodes. LPCAT1 overexpression was significantly associated with increased proliferative activity, negative estrogen receptor (ER) and progesterone receptor (PR) status, positive human epidermal growth factor receptor 2 (HER2) status, as well as triple-negative and HER2 disease molecular subtypes. Multivariate analysis showed that advanced stage, high grade, and LPCAT1 overexpression were independent predictors of early tumor recurrence. We conclude that LPCAT1 is implicated in breast cancer pathogenesis, evolution, and progression and appears to play a potentially crucial role as a determinant of local invasiveness and metastasis. LPCAT1 is an independent predictor of early tumor recurrence of breast carcinoma and represents a novel prognostic biomarker that reflects underlying biological alterations and thus constitutes a potentially promising target for new therapeutic strategies.

Characterization of a lysophospholipid acyltransferase involved in membrane remodeling in Candida albicans.

Phospholipid remodeling involves phospholipase activity to remove acyl chains and acyltransferases to replace acyl chains. We here describe the characterization of a lysophospholipid acyltransferase in the opportunistic fungal pathogen, Candida albicans. Expression of this gene, C.a. LPT1, complemented the lysophospholipid acyltransferase defect in Saccharomyces cerevisiae strains lacking the homologous LPT1 gene. In vitro, lysophospholipid acyltransferase activity in these strains showed acyl-CoA substrate specificity, as measured by apparent Vmax/Km ratios, to be linolenoyl-CoA>oleoyl-CoA>linoleoyl-CoA>stearoyl-CoA. To address the physiological importance of C.a. LPT1, homozygous deletion strains were generated. Lysophospholipid acyltransferase activity with amine containing lysophospholipids was dramatically reduced while lysophosphatidylinositol and lysophosphatidic acid esterification was not significantly lowered. However, C.a. LPT1 over-expression yielded an increased amount of lysophosphatidic acyltransferase activity, suggesting a role in de novo phospholipid synthesis. LPT1 deletion strains showed slightly slowed growth in standard liquid media but no phenotype in media containing three antifungals that target sterols. To assess the role of C.a. Lpt1 in phospholipid remodeling, an in vivo, pulse-chase assay utilizing polysorbitan palmitate and mass spectrometry was developed. Cellular phospholipid composition became atypical with the provision of palmitate and gradually returned to the typical distribution when palmitate was removed. Deletion of C.a. LPT1 showed a modest yet significant effect on remodeling under these conditions.

High lysophosphatidylcholine acyltransferase 1 expression independently predicts high risk for biochemical recurrence in prostate cancers.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) has been suggested to play a role in cancer. To assess its role in prostate cancer, LPCAT1 expression was analyzed on a tissue microarray containing samples from 11,152 prostate cancer patients. In benign prostate glands, LPCAT1 immunostaining was absent or weak. In prostate cancer, LPCAT1 positivity was found in 73.8% of 8786 interpretable tumors including 29.2% with strong expression. Increased LPCAT1 expression was associated with advanced tumor stage (pT3b/T4) (p < 0.0001), high Gleason score (≥4 + 4) (p < 0.0001), positive nodal involvement (p = 0.0002), positive surgical margin (p = 0.0005), and early PSA recurrence (p < 0.0001). High LPCAT1 expression was strongly linked to ERG-fusion type prostate cancer. Strong LPCAT1 staining was detected in 45.3% of ERG positive but in only 16.7% of ERG negative tumors (p < 0.0001). Within ERG negative cancers, LPCAT1 staining was strongly increased within the subgroup of PTEN deleted cancers (p < 0.0001). Further subgroup analyses revealed that associations of high LPCAT1 expression with PSA recurrence and unfavorable tumor phenotype were largely driven by ERG negative cancers (p < 0.0001) while these effects were substantially mitigated in ERG positive cancers (p = 0.0073). The prognostic impact of LPCAT1 expression was independent of histological and clinical parameters. It is concluded, that LPCAT1 measurement, either alone or in combination, may be utilized for better clinical decision-making. These data also highlight the potentially important role of lipid metabolism in prostate cancer biology.

Effects of platelet-activating factor and its differential regulation by androgens and steroid hormones in prostate cancers.

BACKGROUND: Platelet-activating factor (PAF) is an arachidonic acid metabolite that plays an important role in cell proliferation, migration and neoangiogenesis, but whether it is involved in the progression of prostate cancer remains undiscovered. METHODS: Clinical prostate specimens were investigated with immunohistochemistry method and in vitro cell experiments referred to MTS cell proliferation assay, invasion and migration experiment, quantitative real-time RT-PCR assay, western blotting analysis and ELISA assay. RESULTS: Platelet-activating factor synthetase, lyso-PAF acetyl transferase (LPCAT1), increased significantly in castration-resistant prostate cancer (CRPC) specimens and CRPC PC-3 cells than that in controls. Intriguingly, PAF induced invasion and migration of PC-3 cells but not LNCaP cells. The PAF receptor antagonist inhibited proliferation of LNCaP and PC-3 cells. Dihydrotestosterone (DHT) treatment caused a decrease in LPCAT1 expression and PAF release in LNCaP cells, which could be blocked by androgen receptor antagonists. Finally, DHT increased LPCAT1 expression and PAF release in PC-3 cells in a Wnt/ß-catenin-dependent manner. CONCLUSION: For the first time, our data supported that PAF might play pivotal roles in the progression of prostate cancer, which might throw a new light on the treatment of prostate cancer and the prevention of the emergence of CRPC.

Efficient and rapid isolation and purification of mouse alveolar type II epithelial cells.

The alveolar surface is covered by an epithelium composed of 2 main cell types: type I and type II cells. Alveolar type II (ATII) cells have a distinct morphology with apical microvilli and characteristic lamellar bodies, which are the intracellular storage form of pulmonary surfactant. ATII cells play an important role in innate immunity and produce and secrete pulmonary surfactant. They proliferate to restore the epithelium after damage to the more sensitive type I cells. We developed an efficient and rapid method to isolate and purify ATII cells from mice. Alveolar epithelial cells were dissociated in the murine lung with dispase and lung tissue was gently minced with a GentleMACS Dissociator. ATII cell purification was performed using negative depletion with CD45 MicroBeads and positive selection for the epithelial-cell adhesion molecule (Ep-CAM) by magnetic labeling with Streptavidin MicroBeads in MACS LS columns. The purity of these cells as measured by flow cytometry was up to 92.1% and 91.1% for co-staining with Ep-CAM and cytokeratin and co-staining with Ep-CAM and SP-A, respectively. The resulting ATII cell population has a high purity, viability, and yield. The phenotype of isolated and cultured ATII cells was confirmed by electron micrographs, expression of surfactant proteins (SP-A, proSP-B, mature SP-B, proSP-C, SP-D), and lysophosphatidylcholine acyltransferase (LPCAT) by western blotting and immunocytofluorescence. This protocol is based on surface antigens and our data demonstrated that murine ATII cells can be rapidly isolated, efficiently purified, and effectively cultured.

Lithocholic acid disrupts phospholipid and sphingolipid homeostasis leading to cholestasis in mice.

UNLABELLED: Lithocholic acid (LCA) is an endogenous compound associated with hepatic toxicity during cholestasis. LCA exposure in mice resulted in decreased serum lysophosphatidylcholine (LPC) and sphingomyelin levels due to elevated lysophosphatidylcholine acyltransferase (LPCAT) and sphingomyelin phosphodiesterase (SMPD) expression. Global metabolome analysis indicated significant decreases in serum palmitoyl-, stearoyl-, oleoyl-, and linoleoyl-LPC levels after LCA exposure. LCA treatment also resulted in decreased serum sphingomyelin levels and increased hepatic ceramide levels, and induction of LPCAT and SMPD messenger RNAs (mRNAs). Transforming growth factor-ß (TGF-ß) induced Lpcat2/4 and Smpd3 gene expression in primary hepatocytes and the induction was diminished by pretreatment with the SMAD3 inhibitor SIS3. Furthermore, alteration of the LPCs and Lpcat1/2/4 and Smpd3 expression was attenuated in LCA-treated farnesoid X receptor-null mice that are resistant to LCA-induced intrahepatic cholestasis. CONCLUSION: This study revealed that LCA induced disruption of phospholipid/sphingolipid homeostasis through TGF-ß signaling and that serum LPC is a biomarker for biliary injury.

Phosphorylation of lysophosphatidylcholine acyltransferase 2 at Ser34 enhances platelet-activating factor production in endotoxin-stimulated macrophages.

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions under physiological and pathological conditions. We have recently identified two enzymes involved in PAF production: lysophosphatidylcholine acyltransferase-1 (LPCAT1) and LPCAT2. We found that LPCAT2 is highly expressed in inflammatory cells and is activated by lipopolysaccharide (LPS) treatment through Toll-like receptor 4. However, the molecular mechanism for the activation remains elusive. In this study, Phos-tag SDS-PAGE revealed the LPS-induced phosphorylation of LPCAT2. Furthermore, mass spectrometry and mutagenesis analyses identified Ser(34) of LPCAT2 as the phosphorylation site to enhance the catalytic activities. The experiments using inhibitors and siRNA against MAPK cascades demonstrated that LPCAT2 phosphorylation through LPS-TLR4 signaling may directly depend on MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2). These findings develop a further understanding of both PAF production and phospholipid remodeling triggered by inflammatory stimuli. Specific inhibition of the PAF biosynthetic activity by phosphorylated LPCAT2 will provide a novel target for the regulation of inflammatory disorders.

Peroxisome proliferators attenuate free arachidonic acid pool in the kidney through inducing lysophospholipid acyltransferases.

Attenuating effects of peroxisome proliferators on the concentration of free arachidonic acid by inducing 1-acyl-2-lysophospholipid acyltransferases in the kidney were studied. The administration of the three structurally dissimilar peroxisome proliferators, 2-(4-chlorophenoxy)-2-methylpropionic acid (clofibric acid), di(2-ethylhexyl)phthalate, and 2,2'-(decamethylenedithio)diethanol, to rats or mice considerably increased the activities of microsomal 1-acylglycerophosphoethanolamine acyltransferase (LPEAT), 1-acylglycerophosphoinositol acyltransferase (LPIAT), 1-acylglycerophosphoserine acyltransferase (LPSAT), and 1-acylglycerophosphocholine acyltransferase (LPCAT), and the mRNA level of LPCAT3, but not the mRNA level of LPCAT1, LPCAT4, or LPEAT1, in the kidney and the liver. The proportions of arachidonic acid in phospholipids in renal microsomes are rather high for the low proportion of arachidonic acid in free fatty acids in renal microsomes of control rats. The treatment of rats with clofibric acid attenuated the concentration and the proportion of free arachidonic acid to about a half; nevertheless the treatment lowered slightly the proportions of arachidonic acid in phospholipids other than phosphatidylcholine. These results indicate that peroxisome proliferators upregulate the four 1-acyl-2-lysophospholipid acyltransferases of the kidney and, and the induced 1-acyl-2-lysophospholipid acyltransferases seem to play a physiologically crucial contribution in attenuating the pool of free arachidonic acid in the kidney.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) overexpression in human colorectal cancer.

The alteration of the choline metabolite profile is a well-established characteristic of cancer cells. In colorectal cancer (CRC), phosphatidylcholine is the most prominent phospholipid. In the present study, we report that lysophosphatidylcholine acyltransferase 1 (LPCAT1; NM_024830.3), the enzyme that converts lysophosphatidylcholine into phosphatidylcholine, was highly overexpressed in colorectal adenocarcinomas when compared to normal mucosas. Our microarray transcription profiling study showed a significant (p < 10(-8)) transcript overexpression in 168 colorectal adenocarcinomas when compared to ten normal mucosas. Immunohistochemical analysis of colon tumors with a polyclonal antibody to LPCAT1 confirmed the upregulation of the LPCAT1 protein. Overexpression of LPCAT1 in COS7 cells localized the protein to the endoplasmic reticulum and the mitochondria and increased LPCAT1 specific activity 38-fold. In cultured cells, overexpressed LPCAT1 enhanced the incorporation of [(14)C]palmitate into phosphatidylcholine. COS7 cells transfected with LPCAT1 showed no growth rate alteration, in contrast to the colon cancer cell line SW480, which significantly (p < 10(-5)) increased its growth rate by 17%. We conclude that LPCAT1 may contribute to total choline metabolite accumulation via phosphatidylcholine remodeling, thereby altering the CRC lipid profile, a characteristic of malignancy.

Platelet-activating factor production in the spinal cord of experimental allergic encephalomyelitis mice via the group IVA cytosolic phospholipase A2-lyso-PAFAT axis.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) plays a critical role in inflammatory disorders including experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Although PAF accumulation in the spinal cord (SC) of EAE mice and cerebrospinal fluid of MS patients has been reported, little is known about the metabolic processing of PAF in these diseases. In this study, we demonstrate that the activities of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase (LysoPAFAT) are elevated in the SC of EAE mice on a C57BL/6 genetic background compared with those of naive mice and correlate with disease severity. Correspondingly, levels of groups IVA, IVB, and IVF cytosolic PLA(2)s, group V secretory PLA(2), and LysoPAFAT transcripts are up-regulated in the SC of EAE mice. PAF acetylhydrolase activity is unchanged during the disease course. In addition, we show that LysoPAFAT mRNA and protein are predominantly expressed in microglia. Considering the substrate specificity and involvement of PAF production, group IVA cytosolic PLA(2) is likely to be responsible for the increased PLA(2) activity. These data suggest that PAF accumulation in the SC of EAE mice is profoundly dependent on the group IVA cytosolic PLA(2)/LysoPAFAT axis present in the infiltrating macrophages and activated microglia.

The activities of monocyte lysophosphatidylcholine acyltransferase and coenzyme A-independent transacylase are changed by the inflammatory cytokines tumor necrosis factor alpha and interferon gamma.

Alteration of membrane phospholipid fatty acid compositions has been shown to be important for leukocyte inflammatory responses. Such modification of the molecular species of these lipid classes requires deacylation and reacylation reactions and for phosphatidylcholines, lysophosphatidylcholine acyltransferase (LPCAT) and a coenzyme A-independent transacylase (CoAIT) can each be involved. Since previous studies have shown a significant IFNgamma- and TNFalpha-induced modification of phosphatidylcholine species, we have examined whether these inflammatory cytokines alter the activity of reacylation enzymes in the human monocyte cell line MonoMac 6 (MM6). IFN-gamma caused a significant increase in the activity of the LPCAT and CoAIT enzymes in the microsomal fraction at concentrations and over a time-course consistent with an important role for these enzymes in the sensitization (priming) of monocytes. In contrast, TNFalpha was found to significantly increase the activity of the CoAIT by 50% over controls in MM6 cells after 30 min incubation with the cytokine, but decreased LPCAT activity by 65% after 24 h incubation. Such data imply that CoAIT is important for the remodelling of phospholipid composition, which is seen during the acute response of cells to TNFalpha. The results provide further information to emphasise the role of acyltransferases as part of the molecular mechanism underlying inflammation.

Characterization of a novel CI-976-sensitive lysophospholipid acyltransferase that is associated with the Golgi complex.

Recent studies have identified a novel lysophospholipid acyltransferase (LPAT) that is associated with the Golgi complex and that is sensitive to the previously characterized acyl-CoA cholesterol acyltransferase inhibitor, 2,2-methyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976). Here we show that besides acting on exogenous lysophospholipid (LPL) substrates, the CI-976-sensitive LPAT is also capable of reacylating endogenous Golgi LPL substrates, preferentially lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Moreover, using exogenous substrates, we find that the CI-976-sensitive LPAT is capable of using a variety of fatty acyl-CoA donors ranging in chain length from 10 to 20 carbons. Additional characterization demonstrates that the CI-976-sensitive LPAT is ubiquitously expressed in rat tissues, is tightly associated with Golgi membranes, and has a pH optimum between pH 7.0 and 8.0. These studies further define a unique LPC/LPE-specific LPAT from Golgi membranes that likely has a novel function in membrane trafficking.

The mitogen-induced lysophospholipid:acyl-CoA acyltransferase (LAT) expression in human T-lymphocytes is diminished by hydrocortisone.

One of the earliest changes observed in activated lymphocytes is the enhanced incorporation of unsaturated fatty acids into membrane phospholipids catalyzed by phospholipases and acyltransferases. This early membrane phospholipid remodeling has been shown to be independent from protein synthesis. We have investigated the oleic acid incorporation into phospholipids of activated T-lymphocytes within hours and present data that the sustained membrane phospholipid remodeling in activated T-lymphocytes was largely decreased by cycloheximide and actinomycin D treatment while neither protein synthesis inhibitor had an effect on the fatty acid incorporation into phospholipids in resting T-lymphocytes. Lisofylline, an inhibitor of lysophosphatidic acid:acyl-CoA acyltransferase, had no inhibitory activity, indicating that the membrane lipid remodeling was not due to fatty acid incorporation into de novo-synthesized phospholipids. The membrane phospholipid alteration induced by mitogens was also diminished by hydrocortisone (HC) in a concentration-dependent manner. The steroid hormone antagonist RU486 failed to reverse but potentiated this inhibitory activity of HC. HC did not affect the fatty acid uptake, and the decrease of fatty acid incorporation into phospholipids induced by HC was accompanied by an increase of fatty acid incorporation into triglycerides, indicating that the inhibitory activity of HC was specific for fatty acid incorporation into phospholipids catalyzed by lysophospholipidacyl-CoA acyltransferase (LAT). HC did not directly inhibit the LAT enzyme activity. From these data we conclude that LAT gene transcription is induced as an early event following T-cell activation. The inhibitory action of hydrocortisone may give new insights into the regulatory mechanisms involved in LAT expression.