Lysophosphatidylcholine acyltransferase 1 promotes epithelial-mesenchymal transition of hepatocellular carcinoma via the Wnt/ß-catenin signaling pathway.

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most malignant digestive tumors, and its insidious onset and rapid progression are the main reasons for the difficulty in effective treatment. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a key enzyme that regulates phospholipid metabolism of the cell membrane. However, the mechanism by which LPCAT1 regulates HCC metastasis remains unknown. This study aimed to explore its biological function and potential mechanisms concerning migration and invasion in HCC. MATERIALS AND METHODS: LPCAT1 expression in HCC tissues and its association with clinical outcomes were investigated by western blotting and bioinformatic methods, respectively. The role of LPCAT1 in migration and invasion was assessed via Transwell assays. The expression pattern of epithelial-mesenchymal transition (EMT) markers was quantified by western blotting. The biological behaviors of LPCAT1 in vivo were evaluated using xenograft tumor models and caudal vein metastatic models. Signaling pathways related to LPCAT1 were predicted using gene set enrichment analysis (GSEA) and further confirmed by western blotting. RESULTS: LPCAT1 expression was significantly upregulated in HCC tissues and indicated a poor prognosis of HCC patients. Several EMT-related markers were found to be regulated by LPCAT1. HCC cells overexpressing LPCAT1 exhibited remarkably high migration and invasion capacities, upregulated expression of mesenchymal markers and reduced E-cadherin expression. In vivo, LPCAT1 promoted HCC pulmonary metastasis. Furthermore, the Wnt/ß-catenin signaling pathway was confirmed to be activated by LPCAT1. CONCLUSIONS: LPCAT1 could serve as a promising biomarker of HCC and as a novel therapeutic target for the treatment of metastatic HCC.

In silico characterization of 1,2-diacylglycerol cholinephosphotransferase and lysophospha-tidylcholine acyltransferase genes in Glycine max L. Merrill.

The enzymes 1,2-diacylglycerol cholinephosphotrans-ferase (CPT) and lysophosphatidylcholine acyltransferase (LPCAT) are important in lipid metabolism in soybean seeds. Thus, understand-ing the genes that encode these enzymes may enable their modification and aid the improvement of soybean oil quality. In soybean, the genes encoding these enzymes have not been completely described; there-fore, this study aimed to identify, characterize, and analyze the in silico expression of these genes in soybean. We identified two gene models encoding CPT and two gene models encoding LPCAT, one of which presented an alternative transcript. The sequences were positioned on the physical map of soybean and the promoter regions were analyzed. Cis-elements responsible for seed-specific expression and responses to biotic and abiotic stresses were identified. Virtual expression analysis of the gene models for CPT and LPCAT indicated that these genes are expressed under different stress conditions, in somatic embryos during differentiation, in immature seeds, root tissues, and calli. Putative ami-no acid sequences revealed the presence of transmembrane domains, and analysis of the cellular localization of these enzymes revealed they are located in the endoplasmic reticulum.

Daily rhythms of glycerophospholipid synthesis in fibroblast cultures involve differential enzyme contributions.

Circadian clocks regulate the temporal organization of several biochemical processes, including lipid metabolism, and their disruption leads to severe metabolic disorders. Immortalized cell lines acting as circadian clocks display daily variations in [(32)P]phospholipid labeling; however, the regulation of glycerophospholipid (GPL) synthesis by internal clocks remains unknown. Here we found that arrested NIH 3T3 cells synchronized with a 2 h-serum shock exhibited temporal oscillations in a) the labeling of total [(3)H] GPLs, with lowest levels around 28 and 56 h, and b) the activity of GPL-synthesizing and GPL-remodeling enzymes, such as phosphatidate phosphohydrolase 1 (PAP-1) and lysophospholipid acyltransferases (LPLAT), respectively, with antiphase profiles. In addition, we investigated the temporal regulation of phosphatidylcholine (PC) biosynthesis. PC is mainly synthesized through the Kennedy pathway with choline kinase (ChoK) and CTP:phosphocholine cytidylyltranferase (CCT) as key regulatory enzymes. We observed that the PC labeling exhibited daily changes, with the lowest levels every ~28 h, that were accompanied by brief increases in CCT activity and the oscillation in ChoK mRNA expression and activity. Results demonstrate that the metabolisms of GPLs and particularly of PC in synchronized fibroblasts are subject to a complex temporal control involving concerted changes in the expression and/or activities of specific synthesizing enzymes.

Rhythms of glycerophospholipid synthesis in retinal inner nuclear layer cells.

The present study demonstrates that the biosynthesis of phospholipids in the inner nuclear layer cells of the chicken retina displays daily rhythms under constant illumination conditions. The vertebrate retina contains circadian oscillators and photoreceptors (PRCs) that temporally regulate its own physiology and synchronize the whole organism to the daily environmental changes. We have previously reported that chicken photoreceptors and retinal ganglion cells (RGCs) present significant daily variations in their phospholipid biosynthesis under constant illumination conditions. Herein, we demonstrate that cell preparations highly enriched in inner nuclear layer cells also exhibit a circadian-regulated phospholipid labeling after the in vivo administration of [(32)P]phosphate or [(3)H]glycerol both in animals maintained under constant darkness or light for at least 48h. In constant darkness, there was a significant incorporation of both precursors into phospholipids with the highest levels of labeling around midday and dusk. In constant light, the labeling of (32)P-phospholipids was also significantly higher during the day and early night whereas the incorporation of [(3)H]glycerol into phospholipids, that indicates de novo biosynthesis, was greater during the day but probably reflecting a higher precursor availability at those phases. We also measured the in vitro activity of phosphatidate phosphohydrolase and diacylglycerol lipase in preparations obtained from the dark condition. The two enzymes exhibited the highest activity levels late in the day. When we assessed the in vitro incorporation of [(14)C]oleate into different lysophospholipids from samples collected at different phases in constant darkness, reaction catalyzed by lysophospholipid acyltransferases II, labeling showed a complex pattern of daily activity. Taken together, these results demonstrate that the biosynthesis of phospholipids in cells of the chicken retinal inner nuclear layer exhibits a daily rhythmicity under constant illumination conditions, which is controlled by a circadian clock.

Synthesis of retinal ganglion cell phospholipids is under control of an endogenous circadian clock: daily variations in phospholipid-synthesizing enzyme activities.

Retinal ganglion cells (RGCs) are major components of the vertebrate circadian system. They send information to the brain, synchronizing the entire organism to the light-dark cycles. We recently reported that chicken RGCs display daily variations in the biosynthesis of glycerophospholipids in constant darkness (DD). It was unclear whether this rhythmicity was driven by this population itself or by other retinal cells. Here we show that RGCs present circadian oscillations in the labeling of [32P]phospholipids both in vivo in constant light (LL) and in cultures of immunopurified embryonic cells. In vivo, there was greater [32P]orthophosphate incorporation into total phospholipids during the subjective day. Phosphatidylinositol (PI) was the most 32P-labeled lipid at all times examined, displaying maximal levels during the subjective day and dusk. In addition, a significant daily variation was found in the activity of distinct enzymes of the pathway of phospholipid biosynthesis and degradation, such as lysophospholipid acyltransferases (AT II), phosphatidate phosphohydrolase (PAP), and diacylglycerol lipase (DGL) in cell preparations obtained in DD, exhibiting differential but coordinated temporal profiles. Furthermore, cultures of immunopurified RGCs synchronized by medium exchange displayed a circadian fluctuation in the phospholipid labeling. The results demonstrate that chicken RGCs contain circadian oscillators capable of generating metabolic oscillations in the biosynthesis of phospholipids autonomously.

Effect of surfactants and natural detergents on phosphatidylcholine synthesis in photoreceptor membranes.

The synthesis of phosphatidylcholine (PC) in rod outer segments (ROS) catalysed by lysophosphatidylcholine acyltransferase and phosphatidylethanolamine N-methyltransferase (PE N-MTase) was studied and the effects of natural (FA and lysophospholipids) and synthetic (Triton X-100, deoxycholate and CHAPS) surfactants was evaluated. In all experimental conditions used, incorporation of labelled oleate into lysophosphatidylcholine (lysoPC) was at least 40 times greater than oleate incorporation into any other lysophospholipid. Acylation of lysoPC was slightly affected by Triton X-100 and was totally inhibited in the presence of 10 mM sodium deoxycholate (NaDOC) or CHAPS. Below their critical micelle concentration (cmc) Triton X-100 and NaDOC stimulated acylation of all ROS lysophospholipids analysed. The activity of PE N-MTase was stimulated at detergent concentrations below the cmc and inhibited at concentrations above the cmc for all three detergents tested. The effect of FA with differing degree of unsaturation on PC synthesis was evaluated. Oleic acid (10 microM) inhibited methyl group incorporation into total PC, whereas from 100 microM onward, the methylating activity increased with preferential synthesis of PC. Docosahexaenoic acid, in turn, inhibited PE N-MTase activity at every concentration tested. These results suggest that PC synthesis in ROS membranes is modified by bioregulators and surfactants altering the physico-chemical state of the membrane.

Acyl-CoA:lysophosphatidylcholine acyltransferase activity in bovine retina rod outer segments.

In the present paper the properties of acyl-CoA:lysophosphatidylcholine acyltransferase activity associated with rod outer segments (ROS) have been studied. Under adequate experimental conditions, ROS acyl-CoA:lysophosphatidylcholine acyltransferase activity presented a maximum at pH 7.0. The enzyme was able to incorporate as much as 60% of the label offered as [1-14C]oleoyl-CoA into phosphatidylcholine after 5 min of incubation. The use of varying concentrations of oleoyl-CoA and 46 microM lysophosphatidylcholine gave an apparent K(m) value for oleoyl-CoA of 100 microM and a Vmax value of 153 nmol x h-1 x (mg protein)-1. The use of varying concentrations of lysophosphatidylcholine and 100 microM oleoyl-CoA gave an apparent K(m) value for lysophosphatidylcholine of 27 microM and a Vmax value of 155 nmol x h-1 x (mg protein)-1. The enzyme was inhibited by 25% when ROS membranes were incubated in the presence of 10 mM MgCl2. The acyltransferase was able to incorporate other acyl-CoAs (palmitoyl-CoA and arachidonoyl-CoA) into ROS phospholipids and to acylate other lysophospholipids but less efficiently than lysophosphatidylcholine. Lysophoshatidylcholine was preferentially acylated with arachidonic acid followed by oleic acid and, less efficiently, with palmitic acid. The high specific activity of acyl-CoA lysophosphatidylcholine acyltransferase found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to the ROS.